Nucleic acid molecule, expression cassette and methods for expressing a nucleic acid, for producing a plant, for controlling unwanted vegetation and for cultivating a plant.

Abstract

The present invention relates to a recombinant chimeric nucleic acid molecule comprising a nucleic acid sequence encoding a dual transit peptide operatively linked to a heterologous nucleic acid sequence encoding a polypeptide of interest which, when overexpressed in a plant, confers herbicide tolerance to said plant.

Description

1 / 153 “NUCLEIC ACID MOLECULE, EXPRESSION CASSETTE AND METHODS FOR EXPRESSING A NUCLEIC ACID, FOR PRODUCING A PLANT, FOR CONTROLLING UNWANTED VEGETATION AND FOR GROWING A PLANT” Field of Invention

[001] The present invention relates to the field of plant molecular biology, more specifically to the polypeptide of interest for the chloroplast and / or mitochondria, using novel dual transit peptides. Background of the Invention

[002] Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Due to their endosymbiotic origin, these organelles contain their own DNA, but only a few dozen genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the host's nuclear genome, with the product of their expression being directed to the corresponding organelle. Although protein targeting to mitochondria and chloroplasts is generally highly specific, a growing number of examples have been discovered where the same protein is imported into both organelles (Peelers and Small, Biochimica et Biophysica Acta 1541 (2001), pages 54-63). Among many other functions, these two organelles are best known for their roles in energy metabolism, notably respiration and photosynthesis.Since mitochondria and chloroplasts are composed of several thousand proteins encoded in the nucleus and imported into their respective organelles, it is assumed that targeting is generally highly specific to one organelle or the other. Even though there might be some proteins that could theoretically be shared by both organelles, most proteins have a specific function in the organelle to which they are targeted, such that targeting errors could occur. Petition 870250068507, dated 04 / 08 / 2025, page 13 / 329 2 / 153 could be counter-selected as an unnecessary loss of valuable proteins, or it could even be detrimental to the organelle (Peeters and Small, Biochimica et Biophysica Acta 1541 (2001), pages 54 to 63). Peeters and Small list in their review about 20 examples in eukaryotes (plants) where a gene produces products located in different parts of the cell. According to the authors, there are two basic ways in which a single gene can provide a product for both organelles. It can have dual targeting sequences, represented by a tandem mitochondrial and chloroplast targeting sequence at the N-terminal of the protein, interspersed with alternative transcription and / or translation initiations and / or alternative exon splicing. The second way to achieve dual targeting is to have a so-called “ambiguous” targeting presequence that is recognized as an import signal by mitochondria and chloroplasts.

[003] Genes reported to have naturally encoded transit peptide sequences at their N-terminus include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (RuBisCo), de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780; Schnell, DJ et al. (1991) J. Biol. Chem. 266 (5): 3335-3342; 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS), Archer et al. (1990) J. Bioenerg. and Biomemb. 22 (6): 789-810; tryptophan synthase, Zhao, J. et al. (1995) J. Biol. Chem. 270 (II): 6081-6087; plastocyanin, Lawrence et al. (1997) J. Biol. Chem. 272 (33): 20357-20363; chorismate synthase, Schmidt et al. (1993) J. Biol. Chem. 268 (36): 27477-27457; and light harvest chlorophyll a / b binding protein (LHBP), Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999.Although several transit peptides have been described, only a few have been successfully used in attempts to target chimeric molecules to chloroplasts and / or mitochondria in higher plants. The problem to be solved by the present invention is the precise and efficient targeting of proteins synthesized by ribosomes. Petition 870250068507, dated 04 / 08 / 2025, page 14 / 329 3 / 153 cytoplasmic to their appropriate intracellular locations. This is essential for higher transgenic plants where the transgenic product is required in an appropriate organelle or cellular compartment. The present invention provides novel dual-transit peptides that efficiently transport a heterologous polypeptide of interest to the chloroplast and / or mitochondria of transgenic higher plants. Brief Description of the Invention

[004] Consequently, a first embodiment of the present invention relates to a recombinant chimeric nucleic acid molecule comprising a nucleic acid sequence encoding a dual transit peptide linked to a heterologous nucleic acid sequence encoding a polypeptide of interest.

[005] Preferably, the said transit peptide is of the genus Amaranthus or of the genus Alopecurus.

[006] In another preferred embodiment, said double transit peptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or a variant or fragment thereof.

[007] In another preferred embodiment, said heterologous polypeptide of interest is a polypeptide that, when expressed in excess in a plant, confers herbicide tolerance to said plant.

[008] In another preferred embodiment, the said heterologous polypeptide of interest is from a prokaryote.

[009] More preferably, the prokaryote is of the genus selected from the group consisting of Escherichia, Rhodothermus, Petition 870250068507, dated 04 / 08 / 2025, page 15 / 329 4 / 153 Opitutus, Chloroflexus, Acinetobacter and Bacillus.

[010] Even more preferably, said polypeptide has PPO activity and confers tolerance to herbicides that inhibit PPO.

[011] In a particularly preferred embodiment, said polypeptide possessing PPO activity comprises the sequence SEQ ID NO: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, , 159, 160, 161, 162, 163, 164, 165,166, , 175, 176, 177, 179, 180, 181, 182,183, , 192, 193, 194, 195, 196, 197, 198,199, , 208, 209, 210, 211, 212, 213, 214, 215, 224, 225, 226, 227, 228, 229, 230, 231, 240, 241, 242, 243, 244, 245, 246, 247, 256, 257, 258, 259, 260, 261, 262, 263, 272, 273, 274, 275, 276, 277, 278, 279 288, 289, 290, 291, 292, 293, 294, 295, 304, 305, 306, 307, 308, 309, 310, 311, 320, 321, 322, 323, 324, 325, 326, 327, 336, 337, 338, 339, 340, 341, 342, 343, 352, 353, 354, 355, 356, 357, 358, 359, 368, 369, 370, 371, 372, 373, 374, 375, 384, 385, 386, 387, 388, 389, 390, 391, 400, 401, 402, 403, 404, 405, 406, 407, 416, 417, 418, 419, 420, 421, 422, 423, 432, 433, 434, 435, 436, 437, 448, 449, 458, 459, 460, 461, 462, 463, 464, 465, 516, 517, 518, 519, 520, 521, 522, 523, 532, 533, 534, 535 or a variant or 151, 152, 153, 154, 155, 156, 157, 158 167, 168, 169, 170, 171, 172, 173, 174 184, 185, 186, 187, 188, 189, 190, 191 200, 201, 202, 203, 204, 205, 206, 207 216, 217, 218, 219, 220, 221, 222, 223 232, 233, 234, 235, 236, 237, 238, 239 248, 249, 250, 251, 252, 253, 254, 255 264, 265, 266, 267, 268, 269, 270, 271 280, 281, 282, 283, 284, 285, 286, 287, 296, 297, 298, 299, 300, 301, 302, 303, 312, 313, 314, 315, 316, 317, 318, 319, 328, 329, 330, 331, 332, 333, 334, 335, 344, 345, 346, 347, 348, 349, 350, 351, 360, 361, 362, 363, 364, 365, 366, 367, 376, 377, 378, 379, 380, 381, 382, ​​383, 392, 393, 394, 395, 396, 397, 398, 399, 408, 409, 410, 411, 412, 413, 414, 415, 424, 425, 426, 427, 428, 429, 430, 431, 450, 451, 452, 453, 454, 455, 456, 457, 466, 467, 468, 511, 512, 513, 514, 515, 524, 525, 526, 527, 528, 529, 530, 531, fragments thereof. Petition 870250068507, dated 04 / 08 / 2025, p. 16 / 329 5 / 153

[012] In a particularly preferred embodiment, the recombinant chimeric nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding a polypeptide presented in SEQ ID NO: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624, or active variants and fragments thereof.

[013] In another aspect, the present invention relates to an expression cassette comprising the nucleic acid molecule of the present invention, operatively linked to a promoter that drives expression in a plant.

[014] In another aspect, the present invention relates to a plant cell comprising the expression cassette of the present invention.

[015] In another aspect, the present invention relates to a plant or part of a plant comprising a plant cell of the present invention.

[016] In another aspect, the present invention relates to a seed derived from a plant of the present invention.

[017] In another aspect, the present invention relates to a method for expressing a nucleic acid encoding a polypeptide of interest in a plant comprising (a) introducing into a plant cell the nucleic acid molecule of the present invention or the expression cassette of the present invention, and (b) regenerating a plant thereof comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention.

[018] In another aspect, the present invention relates to a method for producing a herbicide-tolerant plant comprising (a) Petition 870250068507, dated 04 / 08 / 2025, p. 17 / 329 6 / 153 introduce into a plant cell the nucleic acid molecule of the present invention or the expression cassette of the present invention, and (b) regenerate a plant therefrom comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention.

[019] In another aspect, the present invention relates to a method for controlling unwanted vegetation in a plant cultivation area, the method comprising the steps of: a) To provide at that location a herbicide-tolerant plant comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention. b) Apply an effective amount of herbicide to that location, where the effective amount of said herbicide does not kill or inhibit the growth of the herbicide-tolerant plant from a).

[020] In another aspect, the present invention relates to a method for growing the plant of the present invention while controlling weeds in the vicinity of said plant, said method comprising the steps of: a) to grow the said plant; and b) Apply a herbicide composition comprising a PPO-inhibiting herbicide to the plant and weeds, wherein the herbicide normally inhibits protoporphyrinogen oxidase, at a level of herbicide that would inhibit the growth of a corresponding wild-type plant.

[021] In another aspect, the present invention relates to a useful combination for weed control, comprising (a) a nucleic acid molecule of the present invention, whose polynucleotide is capable of being expressed in a plant to thereby provide tolerance to a PPO-inhibiting herbicide; and (b) a PPO-inhibiting herbicide. Petition 870250068507, dated 04 / 08 / 2025, page 18 / 329 7 / 153

[022] In another aspect, the present invention relates to a process for preparing a useful combination for weed control comprising (a) providing a nucleic acid molecule of the present invention, which nucleic acid molecule is capable of being expressed in a plant to thereby provide that plant with tolerance to a PPO-inhibiting herbicide; and (b) providing a PPO-inhibiting herbicide.

[023] In a preferred embodiment, said step of providing a nucleic acid molecule comprises providing a plant containing said nucleic acid molecule.

[024] In another preferred embodiment, the said step of providing a nucleic acid molecule comprises providing a seed containing the nucleic acid molecule.

[025] Preferably, the said process also includes a step of applying the PPO inhibitor herbicide to the seed.

[026] In another aspect, the present invention relates to the use of a useful combination for weed control, comprising (a) a nucleic acid molecule of the present invention, which nucleic acid molecule is capable of being expressed in a plant to thereby provide that plant with tolerance to a PPO inhibitor herbicide; and (b) a PPO inhibitor herbicide, to control weeds in a plant growing area. Brief Description of the Drawings

[027] Figure 1 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 113 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, the top 5 pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[028] Figure 2 shows spraying of T1 plants of Petition 870250068507, dated 04 / 08 / 2025, page 19 / 329 8 / 153 Arabidopsis comprising SEQ ID NO: 114 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom, there are 2 wild-type plants; the top 5 pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[029] Figure 3 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 115 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[030] Figure 4 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 116 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[031] Figure 5 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 117 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[032] Figure 6 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 118 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, Petition 870250068507, dated 04 / 08 / 2025, page 20 / 329 9 / 153 selected by confirming the presence of the AHAS resistance gene.

[033] Figure 7 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 119 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[034] Figure 8 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 120 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[035] Figure 9 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 121 with the indicated amounts of Saftufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[036] Figure 10 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 123 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[037] Figure 11 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 127 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after the Petition 870250068507, dated 04 / 08 / 2025, page 21 / 329 10 / 153 spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[038] Figure 12 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 128 with the indicated amounts of Saflufenacil. The photos were taken 8 days after treatment (days after spraying). From top to bottom contains 2 wild-type plants, 5 upper pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[039] Figure 13 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 130 with the indicated amounts of Saflufenacil. Photos were taken 8 days after treatment (days after spraying). From top to bottom, 2 plants are wild-type, the top 5 plants contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[040] Figure 14 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 133 with the indicated amounts of Saflufenacil. Photos were taken 8 days after treatment (days after spraying). From top to bottom, 2 plants are wild-type, the top 5 plants contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[041] Figure 15 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 134 with the indicated amounts of Saflufenacil. Photos were taken 8 days after treatment (days after spraying). From top to bottom, 2 plants are wild-type, the top 5 plants contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[042] Figure 16 shows spraying of T1 plants Petition 870250068507, dated 04 / 08 / 2025, page 22 / 329 11 / 153 Arabidopsis comprising SEQ ID NO: 126 with the indicated amounts of Saflufenacil. Photos were taken 8 days after treatment (days after spraying). From top to bottom, 2 plants are wild-type; the top 5 pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[043] Figure 17 shows spraying of Arabidopsis T1 plants comprising SEQ ID NO: 124 with the indicated amounts of Saflufenacil. Photos were taken 8 days after treatment (days after spraying). Top to bottom contain 2 wild-type plants, top 5 pots contain independent transgenic events (T1 plants, selected by confirmation of the presence of the AHAS resistance gene).

[044] Figure 18 shows Arabidopsis T2 plants treated with the indicated amounts of Saflufenacil. Panel A: Untransformed control; B: Transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 117; C: Transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 118; D: Transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 121; E: Transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 128.

[045] Figure 19 shows Arabidopsis T2 plants treated with the indicated amounts of Trifludimoxazine. Panel A: untransformed control; B: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 119; C: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 117; D: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 118; E: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 121; F: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 128. Petition 870250068507, dated 04 / 08 / 2025, page 23 / 329 12 / 153

[046] Figure 20 shows T0 soybean cuttings treated with the indicated amounts of Saflufenacil or Trifludimoxazine. Panel A: untransformed control; B: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 118 (Event 1); C: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 118 (Event 2); D: transformed with a nucleic acid encoding a polypeptide comprising SEQ ID NO: 118 (Event 3).

[047] Figure 21 AH shows non-transgenic and transgenic Arabidopsis thaliana treated with various PPO-inhibiting herbicides. Rates indicated are in g ai / ha. Photos taken 14 days after treatment. - 21 A: Treatment with the indicated amounts g ai / ha of Saflufenacil (A = wild type / non-transgenic; B = transgene SEQ ID NO: 121; C = transgene SEQ ID NO: 128; D = transgene SEQ ID NO: 117; E = SEQ ID NO: 118) - 21 B: Treatment with the indicated amounts g ai / ha of Trifludimoxazine (A = wild type / non-transgenic; B = transgene SEQ ID NO: 121; C = transgene SEQ ID NO: 128; D = transgene SEQ ID NO: 117; E = ID SEQ NO: 118; F = transgene SEQ ID NO: 119) - 21C: Treatment with the indicated amounts g ai / ha of Sulfentrazone (A = wild type / non-transgenic; B = transgene SEQ ID NO: 118) - 21 D: Treatment with the indicated amounts g ai / ha of Flumioxazin (A - wild type / non-transgenic; B = transgene SEQ ID NO: 118) - 21 E: Treatment with the indicated mixtures g ai / ha of Saflufenacil + Flumioxazin (A = wild type / non-transgenic; B = transgene SEQ ID NO: 118) - 21 F: Treatment with the indicated mixtures g ai / ha of Petition 870250068507, dated 04 / 08 / 2025, page 24 / 329 13 / 153 Saflufenacil + Trifludimoxazine (A = wild type / non-transgenic; B = transgene SEQ ID NO: 118) - 21 G: Treatment with the indicated mixtures g ai / ha of Saflufenacil + Sulfentrazone (A = wild type / non-transgenic; B = transgene SEQ ID NO: 118) - 21 H: Treatment with the indicated amounts g ai / ha of phenylpyridine (A = wild type / non-transgenic; B = transgene SEQ ID NO: 118).

[048] Figure 22 shows non-transgenic and transgenic Arabidopsis thaliana treated with various PPO herbicides with 4 treatments (1 - 4). (1) is the untreated check, 2 - 4 are the treated plants at descending application rates (2) being the highest and (4) being the lowest. See the evaluation in Table 6. Photographs taken 14 days after treatment (A: S3100; B: Saflufenacil; C: Trifludimoxazin; D: Saflufenacil + Trifludimoxazin; E: Flumioxazin)

[049] Figure 23 shows the T1 Generation PPO Herbicide Spray. Transgenic and non-transgenic Arabidopsis thaliana were sprayed with 10 (= 1), 50 (= 2) and 150 (= 3) g ai / ha of Saflufenacil as a post-application (4 - no treatment). Photos were taken 8 days after treatment (days after spraying). The 5 upper pots contain independent transgenic events, while the 2 lower pots, below the white line, contain non-transgenic plants: A: Transgene = SEQ ID NO:113 B: Transgene = SEQ ID NO:120 C: Transgene = SEQ ID NO:129. GENERAL DEFINITIONS

[050] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs and reagents described as such. It is also to be Petition 870250068507, dated 04 / 08 / 2025, page 25 / 329 14 / 153 understands that the terminology used herein is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. It should be noted that, as used herein and in the appended claims, the singular forms “a / an”, “and” and “the” include the plural reference, unless the context clearly dictates otherwise. Thus, for example, the reference to “a vector” is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth.

[051] The term “about” is used here to mean approximately, close to, around, or in the region of. When the term “about” is used in conjunction with a numerical gap, it modifies that gap by extending the limits above and below the stated numerical values. In general, the term “about” is used here to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (greater or less).

[052] As used here, the word “or” means any of the members of a particular list and also includes any combination of list members.

[053] “Expression cassette” as used herein means a linear or circular nucleic acid molecule. It encompasses DNA as well as RNA sequences that are capable of directing the expression of a particular nucleotide sequence in an appropriate host cell. In general, it comprises a promoter operationally linked to a polynucleotide of interest, which is – optionally – operationally linked to termination signals and / or other regulatory elements. The expression cassette of the present invention is characterized by comprising a transcription regulatory nucleotide sequence, as defined below. An expression cassette may also comprise sequences necessary for proper translation. Petition 870250068507, dated 04 / 08 / 2025, page 26 / 329 15 / 153 of the nucleotide sequence. The coding region typically codes for a protein of interest, but it can also code for a functional RNA of interest, for example, antisense RNA or an untranslated RNA, in either the sense or antisense direction. The expression cassette comprising the polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that occurs naturally but has been obtained in a recombinant form useful for heterologous expression. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using endogenous components in part.For example, an expression cassette can be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which is thus functionally linked and controlled by said promoter sequences. Similarly, a nucleic acid sequence to be expressed can be placed (or inserted) downstream of an endogenous promoter sequence, thus forming an expression cassette. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage (for example, the embryo-specific or embryo-specific promoters of the invention).In a preferred embodiment, such expression cassettes will comprise the transcription initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is preferably provided with a plurality of restriction sites for... Petition 870250068507, dated 04 / 08 / 2025, page 27 / 329 16 / 153 insertion of the gene of interest to be under the transcriptional regulation of regulatory regions. The expression cassette may additionally contain selectable marker genes. The cassette will include, in the 5'-3' direction of transcription, a transcription and translation initiation region, a DNA sequence of interest, and a transcription and translation termination region functional in plants. The termination region may be native with the transcription initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions and others described below (see also Guerineau 1991; Proudfoot 1991; Sanfacon 1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987). The expression cassette may also comprise a multiple cloning site.In this case, the multiple cloning site is preferably arranged to allow the operative binding of a polynucleotide to be introduced into the multiple cloning site with the transcription regulatory sequence. In addition to the components mentioned above, the expression cassette of the present invention may preferably comprise components required for homologous recombination, i.e., genomic sequences flanking a target locus. However, an expression cassette consisting essentially of the transcription regulatory nucleotide sequence, as defined below, is also contemplated.

[054] “Promoter” refers to a nucleotide sequence, usually upstream (5') of its coding sequence, that controls the expression of the coding sequence, providing recognition by RNA polymerase and other factors necessary for proper transcription. “Promoter” includes a minimal promoter which is a short DNA sequence comprising, in some cases, a TATA box and other sequences that serve to specify the transcription initiation site, to which are Petition 870250068507, dated 04 / 08 / 2025, page 28 / 329 17 / 153 regulatory elements for enhanced expression added. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements and is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter often referred to as enhancers. Therefore, an “enhancer” is a DNA sequence that can stimulate promoter activity and can be an innate promoter element or a heterologous element inserted to increase the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or inverted) and is able to function even when moved upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects.Promoters can be derived entirely from a native gene, or they can be composed of different elements derived from different promoters found in nature, or even be composed of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental conditions. The "initiation site" is the position around the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. Relative to this location, all other sequences of the gene and its control regions are numbered. Downstream sequences (i.e., other sequences that code for proteins in the 3' direction) are termed positive, while upstream sequences (mostly the control regions in the 5' direction) are termed negative.Promoting elements, such as a TATA element, that are inactive or have greatly reduced promoter activity in the absence of upstream activation, are said to be "minimal" or "core" promoters. Petition 870250068507, dated 04 / 08 / 2025, page 29 / 329 18 / 153 In the presence of an appropriate transcription factor, the minimal promoter functions to allow transcription. A “minimal” or “core” promoter thus consists only of all the basic elements necessary for the initiation of transcription, for example, a TATA box and / or an initiator.

[055] “Constitutive promoter” refers to a promoter that is capable of expressing the open reading frame (ORF) in all or nearly all plant tissues during all or nearly all stages of plant development. Each of the transcription activation elements does not exhibit absolute tissue specificity, but mediates transcriptional activation in most plant tissues at a level of at least 1% achieved in the plant tissue where transcription is most active. “Constitutive expression” refers to expression using a constitutive promoter.

[056] “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally and / or spatially regulated manner, and includes tissue-specific and inducible promoters. It includes natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered; numerous examples can be found in the compilation of Okamuro et al. (1989).Typical regulated promoters useful in plants include, but are not limited to, promoter-inducible promoters, tetracycline-inducible system-derived promoters, salicylate-inducible system-derived promoters, alcohol-inducible system-derived promoters, glucocorticoid-inducible system-derived promoters, pathogen-inducible system-derived promoters, and promoters derived from systems inducible by... Petition 870250068507, dated 04 / 08 / 2025, page 30 / 329 19 / 153 ecdysone. “Conditional expression” and “regulated expression” refer to expression controlled by a regulated promoter.

[057] “Inducible promoter” refers to regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen. As used herein, “transcriptional regulatory nucleotide sequence” refers to nucleotide sequences that influence the transcription, processing, or stability of RNA or translation of the associated (or functionally linked) nucleotide sequence to be transcribed. The transcriptional regulatory nucleotide sequence can have various locations relative to the nucleotide sequences to be transcribed. The transcriptional regulatory nucleotide sequence can be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the sequence to be transcribed (e.g., a coding sequence).The nucleotide sequences that regulate transcription can be selected from the group comprising enhancers, promoters, translational leader sequences, introns, 5' untranslated sequences, 3' untranslated sequences, and polyadenylation signal sequences. They include natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. As noted above, the term "transcriptional regulatory nucleotide sequence" is not limited to promoters. However, preferably, a transcriptional regulatory nucleotide sequence of the invention comprises at least one promoter sequence (e.g., a sequence located upstream of the transcription start of a gene capable of inducing transcription of downstream sequences).In a preferred embodiment, the transcription regulation nucleotide sequence of the invention comprises the promoter sequence of the corresponding gene and – optionally and preferably – the native 5' untranslated region of said gene. Furthermore, the 3' untranslated region and / or the polyadenylation region of the gene may also be utilized. Petition 870250068507, dated 04 / 08 / 2025, page 31 / 329 20 / 153

[058] As used herein, the term “cis regulatory element” or “promoter motif” refers to a cis-acting transcriptional regulatory element that confers an aspect of overall control of gene expression. A cis element can function to bind transcription factors, trans-acting protein factors that regulate transcription. Some cis elements bind more than one transcription factor, and transcription factors can interact at different affinities with more than one cis element. The promoters of the present invention desirably contain cis elements that can confer or modulate gene expression.Cis elements can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' or internal end of a promoter; DNA-binding protein analysis using DNAase I footprinting, methylation interference, mobility shift assays by electrophoresis, in vivo gene imprinting via ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis element motifs using conventional DNA sequence comparison methods. The fine structure of a cis element can also be studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods.Cis elements can be obtained by chemical synthesis or by isolation from promoters that include these elements, and can be synthesized with additional flanking nucleotides that contain restriction enzyme sites useful for facilitating subsequent manipulation.

[059] The “expression pattern” of a promoter (with or without enhancer) is the pattern of expression levels, which shows where in the plant and at what stage of development transcription is initiated by said promoter. The expression patterns of a set of promoters are said to be complementary when the expression pattern of a promoter shows little Petition 870250068507, dated 04 / 08 / 2025, page 32 / 329 21 / 153 overlap with the expression pattern of the other promoter. The expression level of a promoter can be determined by measuring the steady-state concentration of a standard transcribed reporter mRNA. This measure is indirect, since the concentration of reporter mRNA is dependent not only on its synthesis rate but also on the rate at which the mRNA is degraded. Therefore, the steady-state level is the product of synthesis rates and degradation rates. The degradation rate can, however, be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of the synthesis rates. When promoters are compared in this way, the techniques available to those skilled in the art are S1-RNase hybridization analysis, northern blots, and competitive RT-PCR.This list of techniques does not represent all available techniques, but describes commonly used procedures for analyzing transcriptional activity and mRNA expression levels. Analysis of transcription start sites in virtually all promoters has revealed that there is typically not a single base at which transcription begins, but rather a more or less clustered set of initiation sites, each representing several mRNA starting points. Since this distribution varies from promoter to promoter, the reporter mRNA sequences in each population would differ from one another. Because each mRNA species is more or less prone to degradation, a single degradation rate cannot be expected for different reporter mRNAs.It has been shown for several eukaryotic promoter sequences that the sequence surrounding the initiation site (“primer”) plays an important role in determining the level of RNA expression targeted by that specific promoter. This also includes some of the transcribed sequences. Direct fusion of the promoter with reporter sequences would therefore lead to suboptimal levels of transcription. One. Petition 870250068507, dated 04 / 08 / 2025, p. 33 / 329 22 / 153 A commonly used procedure for analyzing expression patterns and levels is through the determination of the 'steady state' level of protein accumulation in a cell. Commonly used candidates for reporter genes, known to those skilled in the art, are beta-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), and proteins with fluorescent properties, such as the green fluorescent protein (GFP) of Aequora victoria. In principle, however, many other proteins are suitable for this purpose, provided the protein does not interfere with the essential functions of the plant. For quantification and determination of location, several tools are suitable. Detection systems can be easily created or are available based on, for example, immunochemical, enzymatic, fluorescent detection and quantification.Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed strains with a chimeric promoter reporter construct can vary in their reporter gene expression levels. It is also frequently observed that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly attributed to “position effects,” although the molecular mechanisms underlying this inactivity are generally unclear.

[060] “Tissue-specific promoter” refers to regulated promoters that are not expressed in all plant cells, but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryos or cotyledons) or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as at the beginning or end of embryogenesis, during fruit ripening, in seed or fruit development, in leaves. Petition 870250068507, dated 04 / 08 / 2025, page 34 / 329 23 / 153 fully differentiated, or at the beginning of senescence. For the purposes of the present invention, “specific tissue” preferably refers to “seed-specific” or “seed-preferred” or embryo-specific or embryo-preferred.

[061] “Seed”, as used herein, preferably refers to whole seed tissues, endosperm and embryonic tissues, more preferably embryonic tissue. “Specific” within the meaning of the invention means that the polynucleotide of interest to be operationally linked to the transcription regulatory nucleotide sequence referred to herein will be predominantly expressed in the indicated tissues or cells when present in a plant. A predominant expression as understood herein is characterized by a statistically significantly greater amount of detectable transcription in said tissue or cells compared to other plant tissues. A statistically significant greater amount of transcription is preferably an amount being at least two times, three times, four times, five times, ten times, one hundred times, five hundred times or one thousand times the amount found in at least one of the other tissues with detectable transcription.Alternatively, it is expression in the indicated tissue or cell whereby the amount of transcription in other tissues or cells is less than 1%, 2%, 3%, 4%, or, more preferably, 5% of the total plant amount (whole plant). The amount of transcription correlates directly with the amount of transcripts (i.e., RNA) or polypeptides encoded by the transcripts present in a cell or tissue. Suitable techniques for measuring transcription based on RNA or polypeptides are well known in the art. Tissue or cell specificity alternatively, and preferably in addition to the above, means that expression is restricted or nearly restricted to the indicated tissue or cells, i.e., there is essentially no detectable transcription in other tissues. Almost. Petition 870250068507, dated 04 / 08 / 2025, page 35 / 329 24 / 153 restricted as intended herein means that nonspecific expression detectable in less than ten, less than five, less than four, less than three, less than two, or one other tissue(s). “Seed preferential” or “embryo preferential” in the context of this invention means the transcription of a nucleic acid sequence by a transcription regulatory element in such a way that the transcription of said nucleic acid sequence in seeds contributes to more than 50%, preferably more than 70%, most preferably more than 80% of the total amount of RNA transcribed from said nucleic acid sequence throughout the plant during any of its developmental stages.

[062] “Expression” refers to the transcription and / or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. For example, in the case of antisense constructs, expression may refer only to the transcription of antisense DNA. In addition, expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to protein production.

[063] Specific seed expression can be determined by comparing the expression of a nucleic acid of interest, for example, a reporter gene such as GUS, operatively linked to the expression control sequence in the following tissues and stages: 1) roots and leaves at the 5-leaf stage, 2) stem at the V-7 stage, 3) leaves, bark and silk at the flowering stage at first silk emergence, 4) spikelets / tassels at pollination, 5) ears or kernels at 5, 10, 15, 20 and 25 days after pollination. Preferably, the expression of the nucleic acid of interest can be determined only in ears or kernels at 5, 10, 15, 20, and 25 days after pollination in said assay, as shown in the attached Figures. The expression of the polynucleotide of interest can be determined by several well-known techniques, for example by Northern Blot or hybridization techniques in Petition 870250068507, dated 04 / 08 / 2025, page 36 / 329 25 / 153 situ as described in document WO 02 / 102970, and preferably by histochemical analysis of GUS as described in the accompanying Examples. Transgenic plants for seed-specific expression analysis may also be generated by techniques well known to those skilled in the art and as discussed elsewhere in this description.

[064] The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and their single- or double-stranded polymers, composed of monomers (nucleotides) containing a sugar, phosphate, and a base, which is a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence.Specifically, degenerate codon substitutions can be obtained, generating sequences in which the third position of one or more selected codons (or all) is replaced with mixed base and / or deoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini 1994). A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is involved in transferring information from DNA to proteins. The term “nucleotide sequence” refers to a polymer of DNA or RNA that can be single-stranded or double-stranded, optionally containing synthetic, unnatural, or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence” may be used interchangeably. Petition 870250068507, dated 04 / 08 / 2025, page 37 / 329 26 / 153 can also be used interchangeably with the gene, cDNA, DNA, and RNA encoded by a gene.

[065] The invention encompasses compositions of isolated or substantially purified nucleic acids or proteins. In the context of the present invention, an “isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by human intervention, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or protein, or its biologically active portion, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid is free of sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes protein or polypeptide preparations with less than about 30%, 20%, 10%, 5% (by dry weight) of contaminating protein.When the protein of the invention, or its biologically active portion, is produced recombinantly, preferably the culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of precursors. Petition 870250068507, dated 04 / 08 / 2025, page 38 / 329 27 / 153 chemicals or non-protein chemicals of interest. The nucleotide sequences of the invention include both naturally occurring sequences and mutant (variant) forms. Such variants will continue to possess the desired activity, i.e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence.

[066] The term “variant” in relation to a sequence (for example, a polypeptide or nucleic acid sequence such as – for example – a transcription regulatory nucleotide sequence of the invention) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences which, due to degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as, for example, polymerase chain reaction (PCR) and hybridization techniques.Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, using site-directed mutagenesis and open reading frames, encoding the native protein, as well as those encoding a polypeptide possessing amino acid substitutions relative to the native protein. Generally, the variants of the nucleotide sequence of the invention will have at least 40, 50, 60 to 70% variations, for example. preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, for example, 81% to 84%, at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity with the native nucleotide sequence (wild type or endogenous), that is, for example for SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 135,. Petition 870250068507, dated 04 / 08 / 2025, page 39 / 329 28 / 153 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 587, 588, 589 or 591.

[067] The nucleic acid molecules of the invention can be “optimized” for enhanced expression in plants of interest (see, for example, WO 91 / 16432; Perlak 1991; Murray 1989). In this way, open reading frames in genes or gene fragments can be synthesized using plant-preferred codons (see, for example, Campbell and Gowri, 1990, for a discussion of the use of host-preferred codons). Thus, nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthesized. That is, synthesized or partially optimized sequences can also be used. Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure, such as DNA scrambling.With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this way, recombinant polynucleotide libraries are generated from a population of sequence-related polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA scrambling are known in the art (see, for example, Stemmer 1994; Stemmer 1994; Crameri 1997; Moore 1997; Zhang 1997; Crameri 1998; and US 5,605,794, 6, 8, 10, and 12,837,458). Petition 870250068507, dated 04 / 08 / 2025, page 40 / 329 29 / 153

[068] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “sequence identity percentage”, and (e) “substantial identity”. (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a complete cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used herein, “comparison window” refers to a specified contiguous segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides long and may optionally be 30, 40, 50, 100 or more. Those skilled in the art understand that, to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

[069] Sequence alignment methods for comparison are well known in the art. Thus, the determination of the percentage of identity between any two sequences can be performed using a mathematical algorithm. Preferred, but not limiting, examples of such mathematical algorithms are the Myers and Miller algorithm, 1988; the local homology algorithm of Smith et al. 1981; the homology alignment algorithm of Needleman and Wunsch 1970; the Pearson similarity search method. Petition 870250068507, dated 04 / 08 / 2025, page 41 / 329 30 / 153 and Lipman 1988; the Karlin and Altschul, 1990 algorithm, modified as in Karlin and Altschul, 1993.

[070] Computational implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, Calif); the ALIGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software package, version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described (Higgins 1988, 1989; Corpet 1988; Huang 1992; Pearson 1994). The ALIGN program is based on the Myers-Miller algorithm, supra. The BLAST programs of Altschul et al., 1990, are based on the Karlin-Altschul algorithm, supra.Multiple alignments (i.e., of more than 2 sequences) are preferably performed using the Clustal W algorithm (Thompson 1994; e.g., in VetorNTI™ software, version 9: Invitrogen Inc.) with the BLOSUM62MT2 scoring matrix with the default settings (gap opening penalty 15 / 19, gap extension penalty 6.66 / 0.05, gap separation penalty 8; % identity for alignment delay 40: using specific residue gaps and hydrophilic residue gaps).

[071] The software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first involves identifying high-score sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold value T when aligned with a word of the same length in a database sequence. T is referred to as Petition 870250068507, dated 04 / 08 / 2025, p. 42 / 329 31 / 153 the threshold for neighborhood word scoring (Altschul 1990). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. Word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a matching residue pair; always > 0) and N (penalty score for non-matching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.The extension of word hits in each direction is interrupted when the cumulative alignment score falls by X amount from its maximum value reached, the cumulative score goes to zero or below due to the accumulation of one or more negative score residual alignments, or the end of any sequence is reached.

[072] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul (1993)). One similarity measure provided by the BLAST algorithm is the least-sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the least-sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[073] To obtain alignments with gaps for the purpose of Petition 870250068507, dated 04 / 08 / 2025, p. 43 / 329 32 / 153 comparison, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al., 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When using BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation (E) of 10, a cutoff point of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See http: / / www.ncbi.nlm.nih.gov. Alignment can also be performed manually by inspection.

[074] For the purposes of the present invention, the comparison of nucleotide sequences to determine the percentage of sequence identity with specific nucleotide sequences (for example, the promoter sequences disclosed herein) is preferably performed using the BlastN program (version 1.4.7 or later) with its predefined parameters (word length (W) of 11, an expectation (E) of 10, a cutoff point of 100, M = 5, N = -4 and a comparison of both strands) or any equivalent program. By “equivalent program” is meant any sequence comparison program that, for any two sequences in question, generates an alignment possessing identical nucleotide or amino acid residue matches and an identical percentage sequence identity when compared with the corresponding alignment generated by the preferred program. Petition 870250068507, dated 04 / 08 / 2025, page 44 / 329 33 / 153

[075] For the purposes of the present invention, the comparison of polypeptide or amino acid sequences to determine the percentage of identity / homology of sequences with specific polypeptide or amino acid sequences is preferably done using the BlastP program (version 1.4.7 or later) with its standard parameters (wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989); see http: / / www.ncbi.nlm.nih.gov) or any equivalent program. By “equivalent program” is meant any sequence comparison program that, for any two sequences in question, generates an alignment possessing identical nucleotide or amino acid residue matches and an identical percentage sequence identity when compared with the corresponding alignment generated by the preferred program. (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum match over a specified comparison window. When percentage sequence identity is used in reference to proteins, it is recognized that the positions of residues that are not identical often differ by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percentage sequence identity may be adjusted upward to correct for the conservative nature of the substitution.Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Methods for making this adjustment are well known to engineers. Petition 870250068507, dated 04 / 08 / 2025, page 45 / 329 34 / 153 on the subject. Typically, this involves scoring a conservative substitution as a partial mismatch, rather than a complete one, thus increasing the percentage of sequence identity. So, for example, when an identical amino acid receives a score of 1 and a non-conservative substitution receives a score of zero, a conservative substitution receives a score between zero and 1. The scoring of conservative substitutions is calculated, for example, as implemented in the PC / GENE program (Inteiligenetics, Mountain View, Calif.). (d) As used herein, “sequence identity percentage” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the sequence identity percentage. (e) (i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 38%, for example, 39%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, plus preferably at least 90%, 91%, 92%, 93% or 94%, and more preferably at least 95%, 96%, 97%, 98% or 99% identity of Petition 870250068507, dated 04 / 08 / 2025, p. 46 / 329 35 / 153 sequence, compared to a reference sequence using one of the alignment programs described using standard parameters. A person skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the matching identity of proteins encoded by two nucleotide sequences taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means a sequence identity of at least 38%, 50% or 60%, preferably at least 70% or 80%, more preferably at least 90%, 95%, and most preferably at least 98%.

[076] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize with each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1 °C to about 20 °C, depending on the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize with each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. (ii) The term “substantial identity” in the context of a peptide Petition 870250068507, dated 04 / 08 / 2025, page 47 / 329 36 / 153 indicates that a peptide comprises a sequence with at least 38%, for example, 39%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence within a specified comparison window. Preferably, optimal alignment is conducted using the Needleman and Wunsch (1970) homology alignment algorithm. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies created against the second peptide. Thus, one peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

[077] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and the sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence(s) relative to the reference sequence, based on the assigned program parameters.

[078] As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridization specifically to” refers to the binding, duplexing, or hybridization of a molecule only to a particular nucleotide sequence under stringent conditions. Petition 870250068507, dated 04 / 08 / 2025, p. 48 / 329 37 / 153 stringent, when that sequence is present in a complex mixture (e.g., whole cell) of DNA or RNA. “Substantially ligand(s)” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and encompasses small mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[079] “Stringing hybridization conditions” and “stringing hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent and differ under different environmental parameters. Tm is the temperature (below defined ionic strength and pH) at which 50% of the target sequence hybridizes with a perfectly matched probe. Specificity is typically a function of post-hybridization washes, with the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, Tm can be approximated by the Meinkoth and Wahl equation, 1984: Tm = 81.5 °C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. Tm is reduced by approximately 1 °C for each 1% mismatch; thus, the Tm, hybridization, and / or washing conditions can be adjusted to hybridize with sequences of the desired identity. For example, if sequences with > 90% identity are sought, Tm can be decreased by 10 °C. Generally, stringent conditions are selected to be approximately 5 °C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions may require hybridization and / or washing at 1, 2, 3 or 4 °C below the thermal melting point I; Petition 870250068507, dated 04 / 08 / 2025, p. 49 / 32938 / 153 Moderately stringent conditions may utilize hybridization and / or washing at 6, 7, 8, 9, or 10 °C below thermal melting point I; low stringent conditions may utilize hybridization and / or washing at 11, 12, 13, 14, 15, or 20 °C below thermal melting point I. Using the equation, the hybridization and washing compositions, and the desired T, those skilled in the art will understand that variations in the stringency of the hybridization and / or washing solutions are inherently described. If the desired degree of unpairing results in a T less than 45 °C (aqueous solution) or 32 °C (formamide solution), it is preferable to increase the SSC concentration so that a higher temperature can be used. An extensive guide to nucleic acid hybridization is found in Tijssen, 1993.Generally, highly stringent hybridization and washing conditions are selected to be approximately 5 °C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH.

[080] An example of highly stringent washing conditions is 0.15 M NaCl at 72 °C for about 15 minutes. An example of stringent washing conditions is a 0.2 x SSC wash at 65 °C for 15 minutes (see Sambrook, below, for a description of SSC buffer). Often, a high-stringence wash is preceded by a low-stringence wash to remove the background probe signal. An example of a medium-stringence wash for a duplex of, for example, more than 100 nucleotides, is 1 x SSC at 45 °C for 15 minutes.An example of low-stringency washing for a duplex of, for example, more than 100 nucleotides, is 4 to 6 X SSC at 40 °C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations below about 1.5 M, more preferably about 0.01 to 1.0 M, the concentration of Na+ ions (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 °C and at least about 60 °C for probes. Petition 870250068507, dated 04 / 08 / 2025, p. 50 / 329 39 / 153 long (e.g., > 50 nucleotides). Stringent conditions can also be obtained by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2X (or greater) than that observed for an unrelated probe in the particular hybridization assay indicates the detection of a specific hybridization. Nucleic acids that do not hybridize with each other under stringent 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 using the maximum codon degeneracy allowed by the genetic code.

[081] Highly stringent conditions are selected to be equal to Tm for a given probe. An example of highly stringent conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, for example, hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 °C and a wash in 0.1 x SSC at 60 °C to 65 °C. Examples of low stringent conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55 °C. Examples of moderately stringent conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 °C, and a wash in 0.5 X to 1 X SSC at 55 to 60 °C.

[082] The following are examples of sets of hybridization / washing conditions that can be used to clone nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: a reference nucleotide sequence preferentially hybridizes with 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 (very stringent conditions). Petition 870250068507, dated 04 / 08 / 2025, p. 51 / 329 40 / 153 low), more desirably 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 (low stringency conditions), more desirably 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 (moderate stringency conditions), preferably in 7% sodium dodecyl sulfate (SDS). 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in 0.1 X SSC, 0.1% SDS at 50 °C (high stringency conditions), more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 and 1 mM EDTA at 50 °C with washing in 0.1 X SSC, 0.1% SDS at 65 °C (very high stringency conditions).

[083] The terms “open reading frame” and “ORF” refer to the amino acid sequence encoded between the initiation and termination codons of translation of a coding sequence. The terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies the initiation and termination of the chain, respectively, of protein synthesis (mRNA translation).

[084] “Coding” or “Coding sequence” refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes non-coding sequences. It may constitute an “uninterrupted coding sequence,” i.e., lacking an intron, such as in a cDNA, or it may include one or more introns delimited by appropriate splice junctions. An “intron” is an RNA sequence that is contained in the primary transcript but is removed through RNA cleavage and rejoining within the cell to create mature mRNA that can be translated into a protein.

[085] “Operationally linked” or “functionally linked” preferably refers to the association of nucleic acid sequences into a single nucleic acid fragment, such that the function of one is affected by the other. Petition 870250068507, dated 04 / 08 / 2025, p. 52 / 329 41 / 153 other. For example, a regulatory DNA sequence is said to be “operatively linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated in such a way that the regulatory DNA sequence affects the expression of the coding DNA sequence (that is, that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operationally linked to regulatory sequences in either sense or antisense orientation.

[086] The terms “heterologous DNA sequence,” “exogenous DNA segment,” or “heterologous nucleic acid” generally refer to a sequence that originates from a source external to the specific host cell or, if from the same source, is modified from its original form. “Heterologous” generally refers to nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or similar means. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA scrambling. The terms also include multiple unnatural copies of a naturally occurring DNA sequence.Thus, the terms refer to a segment of DNA that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell's nucleic acid where the element is not normally found. Exogenous DNA segments are expressed to produce exogenous polypeptides. In the context of the present invention, a "heterologous nucleic acid sequence" encoding a polypeptide of interest is intended to be a sequence that is not naturally operatively linked to the coding sequence of the dual transit peptide of the invention, including multiple non-naturally occurring copies of the DNA sequence that... Petition 870250068507, dated 04 / 08 / 2025, page 53 / 329 42 / 153 occurs. Although this nucleotide sequence is heterologous to the coding sequence of the double transit peptide, it can be homologous or “native,” or heterologous, or “foreign,” to the plant host. In some cases, the transformed plant may have a change in phenotype.

[087] “Homologous to” in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by DNA-DNA or DNA-RNA hybridization under stringency conditions as is well understood by those skilled in the art (as described in Haines and Higgins (eds). Nucleic Acid Hybridization, IRL Press, Oxford. UK), or by comparing the sequence similarity between two nucleic acids or proteins.

[088] “Vector” defined to include, inter alia, any Agrobacterium plasmid, cosmid, phage or binary nucleic acid molecule in linear or circular double- or single-stranded form that may or may not be self-transmissible or mobilizable, and that can transform the prokaryotic or eukaryotic host by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[089] Specifically included are transport vectors, which are understood to be a vehicle of DNA capable, naturally or by design, of replicating in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotes (e.g., in higher plant cells, mammalian cells, yeast cells or fungal cells).

[090] Preferably, the nucleic acid in the vector is under the control of, and operatively linked to, an appropriate promoter or other regulatory elements for transcription in a host cell, such as a cell Petition 870250068507, dated 04 / 08 / 2025, page 54 / 329 43 / 153 microbial, for example, bacterial or plant. The vector can be a bi-functional expression vector that works in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory elements, and in the case of cDNA, it may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

[091] “Cloning vectors” typically contain one or a small number of restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in a determinable manner without loss of the vector’s essential biological function, as well as a marker gene that is suitable for use in identifying and selecting cells transformed with the cloning vector. Marker genes typically include genes that confer tetracycline resistance, hygromycin resistance, kanamycin resistance, streptomycin resistance, or ampicillin resistance.

[092] A “transgene” or “transgenic” refers to a gene that has been introduced into the genome by transformation and is stably or transiently maintained. Transgenes may include, for example, genes that are heterologous or homologous to the genes of a specific plant being transformed. In addition, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term “endogenous gene” refers to a gene native to its natural location in an organism’s genome. A “foreign” gene refers to a gene not normally found in the host organism but which is introduced by gene transfer.

[093] The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising Petition 870250068507, dated 04 / 08 / 2025, p. 55 / 329 44 / 153 transgenic cells are referred to as “transgenic organisms.” Examples of modes of plant and plant cell transformation include Agrobacterium-mediated transformation (De Blaere 1987) and particle bombardment technology (US 4,945,050). Whole plants can be regenerated from transgenic cells by modes well known to those skilled in the art (see, for example, Fromm 1990).

[094] “Transformed,” “transgenic,” and “recombinant” refer to a host organism such as a bacterium or plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art and disclosed (Sambrcok 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). For example, “transformed,” “transforming,” and “transgenic” plants or calluses have undergone the transformation process and contain a foreign gene integrated into their chromosome. The term “untransformed” refers to normal plants that have not undergone the transformation process. A polynucleotide or polypeptide is “recombinant” when it is artificial or manipulated, or is derived from an artificial or manipulated protein or nucleic acid.For example, a polynucleotide that is inserted into a vector or any other heterologous location, for example, in the genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as found in nature, is a recombinant polynucleotide. A polypeptide expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Similarly, a polynucleotide sequence that does not appear in nature, for example, a variant of a naturally occurring gene, is recombinant.

[095] “Transiently transformed” refers to cells into which transgenes and foreign DNA have been introduced (e.g., by methods Petition 870250068507, dated 04 / 08 / 2025, p. 56 / 329 45 / 153 as transformation mediated by Agrobacterium or biolytic bombardment), but not selected for stable maintenance.

[096] “Stablely transformed” refers to cells that have been selected and regenerated in a selection medium after transformation.

[097] “Chromosomally integrated” refers to the integration of a foreign gene or DNA construct into the host genome by covalent linkages. When genes are not “chromosomally integrated”, they may be “transiently expressed”. Transient gene expression refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of a plasmid or autonomously replicating expression cassette, for example, or as part of another biological system, such as a virus. “Genetically stable” and “heritable” refer to chromosomally integrated genetic elements that are stably maintained in the plant and stably inherited by offspring across successive generations.

[098] A “transgenic plant” is a plant with one or more plant cells containing an expression vector as defined below in the detailed description.

[099] “Primary transformant” and “T0 generation” refer to transgenic plants that are of the same genetic generation as the tissue that was initially transformed (i.e., not having undergone meiosis and fertilization since the transformation).

[100] “Secondary transformants” and the “T1, T2, T3 generations”, etc. refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They can be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or non-transformed plants. Petition 870250068507, dated 04 / 08 / 2025, page 57 / 329 46 / 153

[101] “Plant tissue” includes differentiated and undifferentiated tissues or plants, including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture, such as individual cells, protoplasts, embryos and callus tissue. Plant tissue may be in plants or in organs, tissues or cell cultures.

[102] The term “altered plant trait” means any phenotypic or genotypic alteration in a transgenic plant relative to the wild or non-transgenic plant host.

[103] The word “plant” refers to any plant, particularly agronomically useful plants (e.g., seed plants), and “plant cell” is a structural and physiological unit of the plant, comprising a cell wall, but may also refer to a protoplast. The plant cell may be in the form of a single isolated cell or a cultured cell, or as part of a higher organized unit, such as, for example, a plant tissue or a differentiated plant organ in a structure that is present at any stage of plant development. Such structures include one or more plant organs including, but not limited to, fruit, bud, stem, leaf, flower petal, etc.Preferably, the term "plant" includes whole plants, vegetative organs / structures of the aerial part (e.g., leaves, stems, and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (including embryo, endosperm, and seed coat), and fruits (the mature ovary), plant tissues (e.g., vascular tissue, parenchyma, collenchyma, and sclerenchyma and the like), and cells (e.g., guard cells, ovules, trichomes, and the like) and their progeny. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (plants). Petition 870250068507, dated 04 / 08 / 2025, page 58 / 329 47 / 153 monocotyledons and dicotyledons), gymnosperms, ferns and multicellular algae. Includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. Included in the scope of the invention are all genera and species of higher and lower plants of the plant kingdom. This also includes mature plants, seeds, buds and seedlings, and parts, propagation material (e.g. seeds and fruits) and cultures, e.g. cell cultures derived therefrom. Detailed Description of the Invention

[104] Accordingly, a first embodiment of the present invention relates to a recombinant chimeric nucleic acid molecule comprising a nucleic acid sequence encoding a dual transit peptide operatively linked to a heterologous nucleic acid sequence encoding a polypeptide of interest.

[105] As used herein, the term “dual transit peptide” refers to the N-terminal portion of a chloroplast and / or mitochondrial precursor protein and is instrumental in the specific recognition of the chloroplast and / or mitochondrial surface and in mediating the post-translational translocation of pre-proteins across the chloroplast envelope and into the various subcompartments within the chloroplast (e.g., stroma, thylakoid membrane and thylakoid) and / or mitochondria. Thus, as used herein, a polypeptide possessing “dual transit peptide activity” comprises a polypeptide that, when operatively bound to the N-terminal region of a protein of interest, facilitates the translocation of the polypeptide of interest into the chloroplast and / or mitochondria.

[106] The term “chimeric” sequence refers to a sequence that has two or more heterologous sequences linked together. As used herein, a “heterologous” double transit peptide comprises a transit peptide sequence that is foreign to the polypeptide of interest to which it is operatively linked. Petition 870250068507, dated 04 / 08 / 2025, p. 59 / 329 48 / 153

[107] In one embodiment, the dual transit peptides disclosed herein provide improved translocation compared with dual transit peptides derived, for example, from higher plant organisms. The dual transit peptides disclosed herein may result in at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or higher, or at least about 2 times, at least about 3 times, at least about 4 times, or more improvement in polypeptide translocation to the chloroplast and / or mitochondria, when compared with a reference transit peptide. An improvement can be measured in terms of the amount of polypeptide that is translocated to the chloroplast and / or mitochondria, the amount of active polypeptide that is translocated to the chloroplast and / or mitochondria, or both.An improvement can also be measured in terms of an improvement in the phenotype of an organism transformed with the target protein of interest in the chloroplast and / or mitochondria. For example, when the dual transit peptide of the invention is used to target a herbicide resistance protein to the plant chloroplast and / or mitochondria, an improvement in activity can be measured in terms of an improvement in herbicide resistance.

[108] The inventors of the present invention have surprisingly discovered that linking the N-terminal double transit peptide of a type 2 protoporphyrinogen oxidase (PPO-2) from plant species that are considered to be “weeds” to polypeptides of interest that are heterologous to the transit peptide enhances the translocation of said polypeptides of interest to the chloroplast and / or mitochondria.

[109] The website http: / / weedscience.org / provides a comprehensive list of these so-called weed species, some of which may be Petition 870250068507, dated 04 / 08 / 2025, page 60 / 329 49 / 153 herbicide-resistant or tolerant. Those skilled in the art will recognize that PPO-2 polypeptide dual transit peptides from various weed species, as well as dual transit peptides with a high similarity to the PPO-2 polypeptide dual transit peptide, are covered by the present invention.

[110] Specifically, these weed species include Abutilon theophrasti, Acalypha australis, Ageratum conyzoides, Agrostis stolonifera, Alistna canalicuiatum, Alisma plantago-aquatica, Alopecurus aequalis, Alopecurus japonicas, Alopecurus myosuroides, Amaranthus albus, Amaranthus blitoides, Amaranthus blitum (ssp. oleraceus), Amaranthus cruentus, Amaranthus hybridus (syn: quitensis), Amaranthus palmeri, Amaranthus powellii, Amaranthus retroflexus, Amaranthus spinosus, Amaranthus tuberculatus (= A. rudis), Amaranthus viridis, Ambrosia artemisiifolia, Ambrosia trifida, Ammannia auriculata, Ammannia coccinea, Anthemis arvensis, Anthemis cotuia, Apera spicaventi, Arabidopsis thaliana, Arctotheca calendula, Arenaria serpyllifolia, Atriplex patula, Avena fatua, Avena sterilis, Avena sterilis ssp.Ludoviciana, Bacopa rotundifolia, Beckmannia syzigachne, Bidens pilosa, Bidens subalternans, Bidens tripartite, Bifora radians, Blyxa aubertii, Brachiaria eruciformis, Brachypodium distachyon, Brassica rapa (= B. campestris), Brassica tournefortii, Bromus diandrus, Bromus diandrus ssp. rigidus (= B. rigidus), Bromus japonicas, Bromus rubens, Bromus secalinus, Bromus sterilis, Bromus tectorum, Buglossoides arvensis (=Lithospermum arvense), Camelina microcarpa, Capsella bursapastoris, Carduus nutans, Carduus pycnocephalus, Centaurea cyanus, Centaurea solstitialis,. Chenopodium album, Chenopodium album var. striatum (=C. strictum var. glaucophyllum). Chenopodium ficifolium, Chenopodium polyspermum, Chenopodium simplex (=C. hybridum), Chíoris barbata = (C. inflate), Chloris elata, Chloris truncate, Chloris virgate, Chrysanthemum coronarium, Cirsium Petition 870250068507, dated 04 / 08 / 2025, p. 61 / 329 50 / 153 arvense, Clidemia hirta, Commelina diffusa, Convolvulus arvensis, Conyza bonariensis, Conyza Canadensis, Conyza sumatrensis, Crassocephalum crepidioides, Crepis tectorum, Crypsis schoenoides, Cuphea carthagenenis, Cuscuta pentagona (=C, campestris), Cynodon hirsutus, Cynosurus echinatus, Cyperus brevifolius, Cyperus compressus, Cyperus difformis, Cyperus esculentus, Cyperus iria, Cyperus odoratus, Damasonium minus, Datura stramonium, Daucus carota, Descurainia Sophia, Digitaria ciliaris, Digitaria insularis, Digitaria ischaemum, Digitaria sanguinalis, Diplotaxis erucoides, Diplotaxis tenuifolia, Dopatrium junceum, Echinochloa colona, ​​Echinochloa crusgalli var. crus-galli, Echinochloa crus-galli var. formosensis, Echinochloa crusgaili var. zelayensis, Echinochloa crus-vonis, Echinochloa erecta, Echinochloa oryzoides, Echinochloa phyltopogon (=E. oryzicola), Echium plantagineum, Ehrharta longiflora, Elatine triandra var.pedicellata, Eleocharis acicuiaris, Eleusine indica, Epilobium ciliatum, Epilobium tetragonum, Erigeron phtiadelphicus, Eriochloa punctate, Erucaria hispanica, Erysimum repandum, Euphorbia heterophylla, Fimbristylis miiiacea, Fumaria densiflora, Galeopsis tetrahit, Galinsoga ciliate, Galium aparine, Galium spurium, Galium tricornutum, Gamochaeta pensylvanica, Hedyotis verticillata, Helianthus annuus, Hirschfeldia incana, Hordeum murinum ssp., Glaucum, Hordeum murinum ssp., Leporinum, Hydrilla verticillata, Ischaemum rugosum, Iva xanthifolia, Ixophorus unisetus, Kochia scoparia, Lactuca serriola, Lamium amplexicaule, Landoltia punctate, Lepidium virginicum, Leptochloa chinensis, Leptochloa panicoides, Leptochloa scabra, Leptochloa virgate, Limnocharis fiava, Limnophila erecta, Limnophila sessiliflora, Lindernia dubia (= Lindernia dubia var. major), Lindernia micrantha, Lindernia procumbens, Lolium perenne, Lolium perenne ssp., Multrflorum, Lolium persicum, Lolium rigidum, Ludwigia prostrata, Matricaria discotdea, Matricaria recutita (= M. chamomilla), Mazus fauriei, Mazus pumiius, Mesembryanthemum crystailinum, Mitracarpus hirtus, Monochoria korsakowii, Monochoria vaginalis,. Petition 870250068507, dated 04 / 08 / 2025, p. 62 / 329 51 / 153 Myosoton aquaticum, Nassella neesiana, Nasseila trichotoma, Nesiia panicuiata, Oryza sativa var. sylvatica, Panicum capilare, Panicum dichotomiflorum, Papaver rhoeas, Parthenium hysterophorus, Pentzia suffruticosa, Phalaris brachystachys, Phalaris minor, Phalaris paradoxa, Picris hieracioides, Plantago lagopus, Plantago lanceolate, Poa annua, Polygonum aviculare, Polygonum: convolvulus (=Fallopia convolvulus), Polygonum hydropiper, Polygonum lapathifolium, Polygonum pensylvanicum, Polygonum persicaria, Polypogon fugax, Polypogon monspeliensis, Portulaca oleracea, Ranunculus acris, Raphanus raphanistrum, Raphanus sativus, Rapislrum rugosum, Rorippa indica, Rostraria smyrnacea (= Lophochloa smyrnacea), Rotala indica var. uliginosa, Rotala pusiita, Roltboelüa cochinchinensis (= R.exaltata), Rumex dentatus, Sagittaria guyanensts, Sagittaria montevidensis, Sagittaria pygmaea, Sagittaria trifolia, Salsofa tragus, Schoenoplectus fluviatilis, Schoenoplectus juncoides, Schoenoplectus mucronatus (= Scirpus mucronatus), Schoenoplectus wallichii, Sclerochloa dura, Sclerochloa kengiana, Senecio vernalts, Senecio vulgaris, Setaria faberi, Selaria pumila (=S. glauca), Setaria verticillata, Setaria viridis, Setaria viridis var. major (= var. robusta-alba, var. robustapurpurea), Sida spinose, Silene gallica, Sinapis alba, Sinapis arvensis, Sisymbrium orientate, Sisymbrium theilungii, Snowdenia polystachya, Solanum americanum, Solanum nigrum, Solanum ptycanthum, Soliva sessilis, Sonchus asper, Sonchus oleraceus, Sorghum bicolor, Sorghum bicolor ssp. drummondii (= Sorghum Sudanese), Sorghum halepense, Spergula arvensis, Sphenoclea zeylanica, Sporobolus fertilis, Stellaria media, Thlaspi arvense, Tripleurospermum perforatum (= T.inodorum), Urochloa panicoides, Urochloa plantaginea (= Brachiaria piantaginea), Urtica urens, Vaccaria hispanica, Vicia sativa, Vulpia bromoides, Xanthium strumarium, Youngia japonica.

[111] In a preferred embodiment, said dual transit peptide is of the genus Amaranthus or of the genus Alopecurus. Petition 870250068507, dated 04 / 08 / 2025, page 63 / 329 52 / 153

[112] Dual transit peptides from other polypeptides, other PPO-2 type polypeptides or PPO-2 from other plant species that have a certain degree of homology with the dual transit peptide PPO-2 from the genus Amaranthus or the genus Alopecurus, may be considered useful and thus encompassed by the present invention.

[113] Consequently, in another preferred embodiment, said double transit peptide comprises the amino acid sequence of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or a variant or fragment thereof.

[114] By “fragment” is meant a portion of the polynucleotide or a portion of the amino acid sequence and, consequently, the protein it encodes. Polynucleotide fragments may encode protein fragments that retain the activity of the dual transit peptide and are thus able to facilitate the translocation of a polypeptide of interest to the chloroplast and / or mitochondria of a plant. Alternatively, polynucleotide fragments that are useful as a hybridization probe generally do not encode fragment proteins that retain biological activity. Thus, fragments of a nucleotide sequence can vary from at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 nucleotides or up to the full-length double transit peptide.A polynucleotide fragment encoding a biologically active portion of a dual transit peptide will encode at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 60, 65, 70, 75, 80, 85. Petition 870250068507, dated 04 / 08 / 2025, page 64 / 329 53 / 153 contiguous amino acids, or up to the total number of amino acids present in any of the following sequence IDs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98.

[115] The term “variant” in relation to a sequence (e.g., a polypeptide or nucleic acid sequence such as – for example, a transcription regulation nucleotide sequence of the invention) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include sequences that, due to degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques such as, for example, polymerase chain reaction (PCR) and hybridization techniques.Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, using site-directed mutagenesis and open reading frames, encoding the native protein, as well as those encoding a polypeptide possessing amino acid substitutions relative to the native protein, for example, the chimeric polypeptide according to the present invention as disclosed herein.

[116] Generally, the nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, for example, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, for example, 81% to 84%, at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% of nucleotide “sequence identity” to the nucleotide sequences established for Petition 870250068507, dated 04 / 08 / 2025, page 65 / 329 54 / 153 a SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 135, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504,505, 506, 507, 508, 509, 510, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 587, 588, 589, or 591. The % identity of a polynucleotide is determined by GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5, and a gap extension penalty = 0.3. Unless otherwise indicated, the query sequence is at least 45 nucleotides long and the GAP analysis aligns the two sequences in a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides long and the GAP analysis aligns the two sequences in a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides long, and the GAP analysis aligns the two sequences along a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences along their entire length.

[117] In addition, the nucleotide sequence variants of the invention will have at least 30, 40, 50, 60 to 70%, for example, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, for example, 81% to 84%, at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide “sequence identity” to the nucleotide sequence encoding a double transit peptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, Petition 870250068507, dated 04 / 08 / 2025, page 66 / 329 55 / 153 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98. The percentage of identity of a polynucleotide is determined by analysis. GAP (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5 and a gap extension penalty = 0.3. Unless otherwise specified, the query sequence is at least 45 nucleotides long and the GAP analysis aligns the two sequences in a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides long and the GAP analysis aligns the two sequences in a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides long and the GAP analysis aligns the two sequences along a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences along their entire length.

[118] With respect to a defined polypeptide, it will be appreciated that the % of identity higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % of identity, it is preferred that the PPO polypeptide of the invention comprise an amino acid sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more Petition 870250068507, dated 04 / 08 / 2025, page 67 / 329 56 / 153 preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 137, 138, 139, 140, 149, 150, 151, 152, 153, 154, 155,156, 165, 166, 167, 168, 169, 170, 171,172, 181, 182, 183, 184, 185, 186, 187,188, 197, 198, 199, 200, 201, 202, 203,204, 213, 214, 215, 216, 217, 218, 219,220, 229, 230, 231, 232, 233, 234, 235,236, 245, 246, 247, 248, 249, 250, 251,252, 261, 262, 263, 264, 265, 266, 267,268, 277, 278, 279, 280, 281, 282, 283,284, 293, 294, 295, 296, 297, 298, 299,300, 309, 310, 311, 312, 313, 314, 315,316, 325, 326, 327, 328, 329, 330, 331,332, 341,342, 343, 344, 345, 346, 347, 348, 357, 358, 359, 360, 361, 362, 363,364, 373, 374, 375, 376, 377, 378, 379,380, 389, 390, 391, 392, 393, 394, 395,396, 141, 142, 143, 144, 145, 146, 147, 148, 157, 158, 159, 160, 161, 162, 163,164, 173, 174, 175, 176, 177, 178, 179,180, 189, 190, 191, 192, 193, 194, 195, 196, 205, 206, 207, 208, 209, 210, 211, 212, 221, 222, 223, 224, 225, 226, 227, 228, 237, 238, 239, 240, 241, 242, 243, 244, 253, 254, 255, 256, 257, 258, 259, 260, 269, 270, 271, 272, 273, 274, 275, 276, 285, 286, 287, 288, 289, 290, 291, 292, 301, 302, 303, 304, 305, 306, 307, 308, 317, 318, 319, 320, 321, 322, 323, 324, 333, 334, 335, 336, 337, 338, 339, 340, 349, 350, 351, 352, 353, 354, 355, 356, 365, 366, 367, 368, 369, 370, 371, 372, 381, 382, ​​​​383, 384, 385, 386, 387,388, 397, 398, 399, 400, 401, 402, 403, 404, Petition 870250068507, dated 04 / 08 / 2025, p. 68 / 329 57 / 153 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, or 535.

[119] By “variant” polypeptide is meant a polypeptide derived from the protein of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 137, 138, 139, 140, 141, 142, 143, 144, 153, 154, 155, 156, 157, 158, 159, 160, 169, 170, 171, 172, 173, 174, 175, 176, 185, 186, 187, 188, 189, 190, 191, 192, 201, 202, 203, 204, 205, 206, 207,208, 217, 218, 219, 220, 221, 222, 223,224, 233, 234, 235, 236, 237, 238, 239,240, 249, 250, 251, 252, 253, 254, 255,256, 265, 266, 267, 268, 269, 270, 271,272, 281, 282, 283, 284, 285, 286, 287,288, 297, 298, 299, 300, 301, 302, 303,304, 313, 314, 315, 316, 317, 318, 319,320, 329, 330, 331, 332, 333, 334, 335,336, 345, 346, 347, 348, 349, 350, 351,352, 361, 362, 363, 364, 365, 366, 367,368, 377, 378, 379, 380, 381, 382, 383,384, 393, 394, 395, 396, 397, 398, 399,400, 145, 146, 147, 148, 149, 150, 151,152, 161, 162, 163, 164, 165, 166, 167,168, 177, 178, 179, 180, 181, 182, 183,184, 193, 194, 195, 196, 197, 198, 199,200, 209, 210, 211, 212, 213, 214, 215,216, 225, 226, 227, 228, 229, 230, 231,232, 241, 242, 243, 244, 245, 246, 247, 248, 257, 258, 259, 260, 261, 262, 263, 264, 273, 274, 275, 276, 277, 278, 279, 280, 289, 290, 291, 292, 293, 294, 295, 296, 305, 306, 307, 308, 309, 310, 311, 312, 321, 322, 323, 324, 325, 326, 327, 328, 337, 338, 339, 340, 341, 342, 343, 344, 353, 354, 355, 356, 357, 358, 359, 360, 369, 370, 371, 372, 373, 374, 375, 376, 385, 386, 387, 388, 389, 390, 391, 392, 401, 402, 403, 404, 405, 406, 407, 408, Petition 870250068507, dated 04 / 08 / 2025, p. 69 / 329 58 / 153 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, or 535, by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal ends of the full-length double-transit peptide; deletion or addition of one or more amino acids at one or more sites in the full-length double-transit peptide; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result, for example, from genetic polymorphism or human manipulation. Methods for such manipulations are generally known in the art.

[120] Additionally, the various dual-transit peptides disclosed herein can be modified to improve and / or alter the translocation of the polypeptide of interest to the chloroplast. For example, the dual-transit peptide may contain additional regions that alter or improve interactions with cytosolic factors that facilitate the passage of ribosome precursors to the chloroplast surface. See, for example, Hiltbrunner et al. (2001) Journal of Cell Biology 154: 309-316, Jackson-Constan et al. (2001) Biochimica et Biophysica Acta 1541: 102-113, both of which are incorporated herein by reference. Other regions may be employed to increase the efficiency of organelle import. See, for example, May et al. (2000) Plant Cell 12: 5364, Qbadou et al. (2006) EMBO Journal 25: 1837-1837 and Sohrt et al. (2000) Journal of Cell Biology 148: 1213-1221, incorporated here by reference.These regions can be native (derived from a region of the PPO-2 polypeptide) or heterologous to the operationally linked PPO-2 dual transit peptide. Assays to determine the efficiency with which the dual transit peptide sequences of the invention target a protein of interest for a chloroplast. Petition 870250068507, dated 04 / 08 / 2025, page 70 / 329 59 / 153 are known. See, for example, Mishkind et al. (1985) J of Cell Biol 100: 226-234, which is incorporated here by reference in its entirety. A reporter gene such as glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), or green fluorescent protein (GFP) is operatively linked to the dual transit peptide sequence. This fusion is placed behind the control of a suitable promoter, linked to a transformation vector, and transformed into a plant or plant cell. After a suitable time period for expression and localization in the organelle, the organelle fraction is extracted and reporter activity is analyzed. The ability of the isolated sequences to reach and deliver the reporter protein to the chloroplast and / or mitochondria can be compared with other known dual transit peptide sequences (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780).Protein import can also be verified in vitro by adding proteinases to the isolated chloroplast fraction. Proteins that have been successfully imported into the chloroplast are resistant to externally added proteinases, while proteins that remain in the cytosol and / or mitochondria are susceptible to digestion. Protein import can also be verified by the presence of functional protein in the chloroplast and / or mitochondria using standard molecular detection techniques, evaluating the resulting phenotype of the expression of a target protein from chloroplasts and / or mitochondria, or by microscopy.

[121] The dual transit peptides disclosed herein target the desired protein of interest to the chloroplast and / or mitochondria and can facilitate the translocation of the protein to the organelle. This is accompanied by the cleavage of the transit peptide from the mature polypeptide or protein at the appropriate transit peptide cleavage site by a chloroplast processing proteinase. Consequently, a dual transit peptide further comprises a suitable cleavage site for the correct processing of the pre-protein to the mature polypeptide contained in the chloroplast. Petition 870250068507, dated 04 / 08 / 2025, page 71 / 329 60 / 153

[122] Any polynucleotide of interest (i.e., the “polypeptide of interest”) may be used with the dual transit peptide coding sequences described herein. Such polynucleotides / polypeptides of interest include, but are not limited to, herbicide tolerance coding sequences, insecticide coding sequences, nematicide coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences that modify plant characteristics such as yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and / or utilization, and oil content and / or composition.More specific polynucleotides of interest to the present invention include, but are not limited to, genes that improve crop yield, polypeptides that improve crop desirability, genes that encode proteins that confer resistance to abiotic stress, such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those that confer resistance to toxins, such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects and nematodes and the development of diseases associated with these organisms. It is recognized that any polypeptides of interest can be operatively linked to the coding sequences of the dual transit peptide of the invention and expressed in a plant, provided that the polypeptide encoded by the polynucleotide is functional in chloroplasts and / or mitochondria.

[123] Exemplary polynucleotides of interest that can be operationally linked to the dual transit peptides of the present invention include polynucleotides encoding: polypeptides possessing pesticidal and / or insecticidal activity, such as other toxic proteins from Bacillus thuringiensis (described in US Patents 5,366,892; 5,747,450, Petition 870250068507, dated 04 / 08 / 2025, page 72 / 329 61 / 153 5,737,514, 5,723,756, 5,593,881 and Geiser et al., (1986) Gene 48: 109). lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825), pentin (described in US Patent No. 5,981,722) and the like: desirable characteristics for disease or herbicide resistance (e.g., fumonisin detoxification genes (US Patent No. 5,792,931), avirulence and disease resistance genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (EPSPS), described in US Patents 4,940,935 and 5,188,642, or the glyphosate N-acetyltransferase (GAT) gene, described in Castle et al., (2004) Science, 304: 1151-1154 and in US Patent Application Nos.20070004912, 20050246798 and 20050060767; glufosinate resistance (e.g., phosphinothricin acetyltransferase PAT and BAR genes described in U.S. Patents 5,561,236 and 5,276,268); herbicide resistance including sulfonylureas. DHT (2,4-D) and PPO herbicides (e.g., glyphosate acetyltransferase, aryloxyacetoate dioxygenase, acetolactate synthase, and protoporphyrinogen oxidase): a cytochrome P450 or a variant thereof that confers resistance or tolerance to herbicides, inter alia, HPPD herbicides (U.S. Patent Application Ser. No. 12 / 156,247; U.S. Patents Nos. 6,380,465; 6,121,512; 5,349,127; 6,649,814; and 6,300,544; and PCT Patent Application No. WO 2007000077); and desirable characteristics for process or processing products, such as high oil content (e.g., U.S. Patent No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (US Patent No.5,952,544; WO 94 / 11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE); and polymers or bioplastics (e.g., US 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase and. Petition 870250068507, dated 04 / 08 / 2025, p. 73 / 329 62 / 153 acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs)); disclosures are incorporated herein by reference.

[124] In a preferred embodiment, said heterologous polypeptide of interest is a polypeptide that, when overexpressed in a plant, confers herbicide tolerance to said plant. A “herbicide resistance protein” or a protein resulting from the expression of a “nucleic acid molecule encoding herbicide resistance” includes proteins that confer on a cell the ability to tolerate a higher concentration of a herbicide than cells that do not express the protein or to tolerate a certain concentration of a herbicide for a longer period of time than cells that do not express the protein.Herbicide resistance traits can be introduced into plants by genes encoding herbicide resistance that inhibit the action of protoporphyrinogen oxidase (PPO), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate acetyltransferase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), hydroxyphenylpyruvate dioxygenase (HPPD), phytoene denaturase (PD) and dicamba degradation enzymes as disclosed in WO 02 / 068607, or phenoxyacetic acid and phenoxypropionic acid-derived degradation enzymes as disclosed in WO 2008141154 or WO 2005107437.

[125] In a particularly preferred embodiment, said polypeptide of interest which can be operatively linked to the dual transit peptides of the present invention has PPO activity and confers tolerance to PPO-inhibiting herbicides. Protoporphyrinogen oxidase (herein referred to as PPO; EC: 1.3.3.4) is a key enzyme in the biosynthesis of protoporphyrin IX, and has been used for selective weed control. Petition 870250068507, dated 04 / 08 / 2025, page 74 / 329 63 / 153 since the 1960s. PPO catalyzes the last common step in chlorophyll and heme biosynthesis, which is the oxidation of protoporphyrinogen IX to protoporphyrin IX. (Matringe et al. 1989. Btochem. 1.260: 231).

[126] The PPO polypeptide ortholog sequences of interest that are covered by the present invention are disclosed in documents WO 2012 / 080975, WO 2013 / 189984, WO 2015 / 022636, WO 2015 / 022640, WO 2015 / 022639, and WO 2015 / 092706, the contents of which are incorporated herein by reference in their entirety.

[127] “Orthologs” refers to genes from different organisms that originated through speciation, and may also be derived from a common ancestral gene. It is well known in the art that paralogs and orthologs may share distinct domains that harbor suitable amino acid residues at certain locations, such as binding pockets for particular substrates or binding motifs for interaction with other proteins.

[128] In a preferred embodiment, the said heterologous polypeptide of interest with PPO activity is from a prokaryote, preferably bacteria.

[129] More preferably, the prokaryote, preferably a bacterium, is of the genus selected from the group consisting of Escherichia, Rhodothermus, Opitutus, Chloroflexus, Acinetobacter and Bacillus.

[130] In a particularly preferred embodiment, said polypeptide possessing PPO activity comprises the sequence SEQ ID NO: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, Petition 870250068507, dated 04 / 08 / 2025, page 75 / 329 64 / 153 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, or a variant or fragment thereof.

[131] The definitions of “variant” and “fragment”, which are given ABOVE in the context of the description of the dual transit peptides of the present invention, apply in the same way to the said PPO polypeptides of interest.

[132] In a particularly preferred embodiment, the chimeric nucleic acid molecules of the present invention comprise a nucleic acid sequence encoding a polypeptide presented in SEQ ID NO: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624, or active variants and fragments thereof. Petition 870250068507, dated 04 / 08 / 2025, p. 76 / 329 65 / 153

[133] The recombinant chimeric nucleic acid molecules of the present invention are provided in expression cassettes for expression in the plant of interest.

[134] Thus, in another aspect, the present invention relates to an expression cassette comprising the nucleic acid molecule of the present invention, operatively linked to a promoter that drives expression in a plant.

[135] The cassette will include regulatory sequences operationally linked to the chimeric nucleic acid molecules of the invention. The term “regulatory element,” as used herein, refers to a polynucleotide capable of regulating the transcription of an operatively linked polynucleotide. It includes, but is not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs. By “operatively linked” we mean a functional linkage between a promoter and a second sequence, in which the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. Generally, operatively linked means that the nucleic acid sequences being linked are contiguous and, where necessary to link two protein-coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be co-transformed into an organism.Alternatively, the additional gene(s) may be provided in multiple expression cassettes. This expression cassette is provided with a plurality of restriction sites for the insertion of chimeric nucleic acid molecules under the transcriptional regulation of regulatory regions. The expression cassette may also contain selectable marker genes.

[136] The expression cassette of the present invention will include in the 5'-3' direction of transcription, a transcription and translation initiation region (i.e., a promoter), a chimeric nucleic acid molecule of the invention and Petition 870250068507, dated 04 / 08 / 2025, page 77 / 329 66 / 153 a functional transcription and translation termination region (i.e., termination regions) in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and / or to the chimeric nucleic acid molecules of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. When the promoter is “foreign” or “heterologous” to the plant host, it is desired that the promoter not be found in the native plant into which the promoter is introduced. When the promoter is “foreign” or “heterologous” to the chimeric nucleic acid molecules of the invention, it is intended that the promoter not be the native or naturally occurring promoter for the operatively linked chimeric nucleic acid molecules of the invention. As used herein, a chimeric gene comprises a coding sequence operatively linked to a transcription initiation region that is heterologous to the coding sequence.

[137] Although it may be preferable to express the chimeric nucleic acid molecules of the invention using heterologous promoters, native promoter sequences may be used. Such constructs will alter the expression levels of the encoded polypeptide of interest in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

[138] The termination region may be native to the transcription initiation region, may be native to the operationally linked sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the chimeric nucleic acid molecules of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthetase and nopaline synthetase termination regions. See also Guerineau et al. (1991) Mol. Genet Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674: Petition 870250068507, dated 04 / 08 / 2025, p. 78 / 329 67 / 153 Sanfacon et al. (1991) Genes Dev. 5: 141-149; Moggen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91; 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639. When appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, genes may be synthesized using plant-preferred codons for better expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of the use of host-preferred codons. Methods are available in the state of the art for synthesizing plant-preferred genes. See, for example, US Patents Nos. 5,380,831, and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference.

[139] Additional sequence modifications are known to increase gene expression in a host cell. These include the elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence can be adjusted to average levels for a given host cell, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin mRNA secondary structures. Nucleotide sequences to increase gene expression can also be used in plant expression vectors. These include maize introns Adhl, gene intronl (Callis et al.Genes and Development 1: 1183-1200, 1987), and leader sequences (W sequence) of tobacco mosaic virus (TMV), maize chlorotic trait virus, and alfalfa mosaic virus (Gallie et al. Nucleic Acid Res. 15: 8693-8711, 1987 and Skuzeski et al. Plant Mol. Biol. 15: 65-79, 1990). The first intron of the maize shrunken-1 locus has been shown to increase gene expression in chimeric gene constructs. As Pat. US 5,424,412. Petition 870250068507, dated 04 / 08 / 2025, page 79 / 329 68 / 153 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106: 929-939, 1994) also showed that introns are useful for regulating gene expression on a tissue-specific basis. To further enhance or optimize gene expression of the chimeric nucleic acid molecule, the plant expression vectors of the invention may also contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems may then exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.

[140] The expression cassettes of the present invention may additionally contain 5' leader sequences in the construction of the expression cassette. These leader sequences may act to enhance translation. Translation leaders are known in the art and include: picorna virus leaders, for example, EMCV leader (5' non-coding region of Encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Natl. Acad. ScL USA 86: 6126-6130); potyvirus leaders, for example, TEV (Tobacco Etch Virus) leader (Gallie et al. (1995) Gene 165 (2): 233-238). MDMV (Maize Dwarf Mosaic Virus) leader (Virology 154: 9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated mRNA leader of alfalfa mosaic virus coat protein (AMV 4 RNA) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus (TMV) leader (Gallie et al. (1989) in Molecular RNA Biology. ed. Cech (Liss, New York), pp.237-256); and leader of maize chlorotic spot virus (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also, Delia Cioppa et al. (1987) Plant Physiol. 84: 965-968. Other known methods to improve translation can also be used, for example, introns and the like.

[141] In preparing the expression cassette, the various Petition 870250068507, dated 04 / 08 / 2025, p. 80 / 329 69 / 153 DNA fragments can be manipulated to provide DNA sequences in the appropriate orientation and, as appropriate, in the appropriate reading frame. For this purpose, adapters or ligands can be used to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove superfluous DNA, remove restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitutions, for example, transitions and transversions, may be involved.

[142] A number of promoters can be used in the practice of the invention. Promoters can be selected based on the desired outcome. Nucleic acids can be combined with tissue-preferred constitutive promoters or other promoters for expression in plants. Such constitutive promoters include, for example, the nuclear promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and US Patent No. 6,072,050; the CaMV 35S core promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-58); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (US Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, US Patents Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[143] Tissue-preferred promoters can be used to target the expression of enhanced chimeric polypeptide within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. The Petition 870250068507, dated 04 / 08 / 2025, page 81 / 329 70 / 153 tissue preferred promoters include Yamamoto et al. (1997) Plant J. 12 (2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7): 792-803; Hansen et al. (1997) Mol. Gen Genet. 254 (3): 337-343; Russell et al. (1997) Transgenic Res. 6 (2): 157-168; Rinehart et al. (1996) Plant Physiol. 112 (3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112 (2): 525-535; Canevascini et al. (1996) Plant Physiol. 112 (2) 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35 (5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181 -196; Orozco et al. (1993) Plant Mol Biol. 23 (6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4 (3) 495-505. Such promoters can be modified, if necessary, for weak expression.

[144] Methods for chloroplast transformation are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. ScL USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method is based on the delivery of DNA gum particles containing a selectable marker and targeting the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be achieved by transactivation of a transgene carrying the silent plastid by tissue-preferred expression of a nuclear-encoded, plastid-directed RNA polymerase. Such a system was reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305. Nucleic acids of interest to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for differences in codon utilization between the plant nucleus and this organelle.Thus, nucleic acids of interest can be synthesized using preferred chloroplast codons. See, for example, U.S. Patent No. 5,380,831, incorporated herein by reference.

[145] The expression cassettes of the invention may include another selectable marker gene for the selection of transformed cells. The genes Petition 870250068507, dated 04 / 08 / 2025, page 82 / 329 71 / 153 selectable markers, including those of the present invention, are used for the selection of transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicide compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotechnology 3: 506-511; Christophers et al. (1992) Proc. Natl. Acad. ScL USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72: Reznikoff (1992) Mol Microbiol 6: 2419-2422; Barkley et al (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722: Deuschle et al (1989) Proc. Natl Acad. AcL USA 86: 5400-5404; Fuerst et al (1989) Proc. Natl Acad.ScL USA 86:2549–2553; Deuschle et al (1990) Science 248: 480-483; Gossen (1993) Doctoral Thesis da. University of Heidelberg; Reines et al (1993) Proc. Natl Acad. ScL USA 90:1917–1921; Labow et al. (1990) Mol Cell Biol 10:3343–3356; Zambretti et al (1992) Proc. Natl Acad. ScL USA 89:3952–3956; Bairn et al (1991) Proc. Natl Acad. ScL USA 88:5072–5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol Struc. Biol 10:143–162; Degenkolb et al (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al (1988) Biochemistry 27: 1094-1104; Bonin (1993) Doctoral Thesis. University of Heidelberg; Gossen et al (1992) Proc. Natl Acad. ScL USA 89: 5547-5551; Oliva et al (1992) Antimicrob. Chemother Agents. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology. Vol. 78 (Springer-Verlag, Berlin); Gill et al (1988) Nature 334: 721-724. Such disclosures are incorporated herein by reference. The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene may be used in the present invention. Petition 870250068507, dated 04 / 08 / 2025, p. 83 / 329 72 / 153

[146] The invention further provides an isolated recombinant expression vector comprising the expression cassette containing a chimeric polypeptide nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a PPO-inhibiting herbicide compared with a wild-type variety of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been ligated. One type of vector is a “plasmid,” which refers to a loop of circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors).Other vectors (e.g., non-episomic mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell and are thus replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors useful in recombinant DNA techniques are frequently in the form of plasmids. In this descriptive report, “plasmid” and “vector” may be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention aims to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[147] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for Petition 870250068507, dated 04 / 08 / 2025, page 84 / 329 73 / 153 expression of nucleic acid in a host cell, meaning that recombinant expression vectors include one or more regulatory sequences, selected based on the host cells to be used for expression, that are operationally linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired level of polypeptide expression, etc.The expression vectors of the invention can be introduced into host cells to produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., chimeric polypeptides, fusion polypeptides, etc.).

[148] In a preferred embodiment of the present invention, chimeric polypeptides are expressed in plant cells and plants such as unicellular plant cells (such as algae) (See Falciatore et al., 1999. Marine Biotechnology 1 (3): 239-251 and references thereto) and plant cells of higher plants (e.g., spermatophytes, such as cultivated plants). A chimeric polynucleotide can be introduced into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agro-infection, biolistics and the like.

[149] Suitable methods for transforming or transfecting host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) Petition 870250068507, dated 04 / 08 / 2025, page 85 / 329 74 / 153 and other laboratory manuals, such as Methods in Molecular Biology, 1995. Vol. 44. Agrobacterium Protocols, ed: Gartland and Davey, Humana Press, Totowa, New Jersey. Since increased tolerance to PPO-inhibiting herbicides is a general trait desired in a wide variety of plants such as corn, wheat, rye, oats, triticale, rice, barley, soybeans, peanuts, cotton, rapeseed and canola, cassava, peppers, sunflowers and marigolds, solanaceous plants such as potatoes, tobacco, eggplant and tomatoes, Vicia species, peas, alfalfa, shrubby plants (coffee, cocoa, tea), Salix species, trees (oil palm, coconut), perennial grasses and forage crops, these cultivated plants are also preferred target plants for genetic engineering as another embodiment of the present invention. In a preferred embodiment, the plant is a cultivated plant.Forage crops include, but are not limited to, wheatgrass, canarygrass, bromegrass, wild rye grass, bluegrass, orchardgrass, alfalfa, sage, bird's foot clover, alsike clover, red clover, and sweet clover.

[150] In one embodiment of the present invention, the transfection of a chimeric polynucleotide into a plant is achieved by Agrobacterium-mediated gene transfer. A transformation method known to those skilled in the art is the immersion of a flowering plant in an Agrobacterium solution, wherein the Agrobacterium contains the nucleic acid of the chimeric polypeptide, followed by reproduction of the transformed gametes. Agrobacterium-mediated plant transformation can be performed using, for example, the Agrobacterium tumefaciens GV3101 (pMP90) strain (Koncz and Schell. 1986. Mol. Gen. Genet. 204: 383-396) or LBA4404 (Clontech). The transformation can be carried out by conventional transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13: 4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology). Petition 870250068507, dated 04 / 08 / 2025, p. 86 / 329 75 / 153 Manual, 2nd Ed. - Dordrecht: Kluwer Academic Publ., 1995. - in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4, Glick, Bernard R. and Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 p., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989. Plant Cell Report 8: 238-242; De Block et al., 1989. Plant Physiol. 91: 694-701). The use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Canola selection is typically performed using kanamycin as a selectable plant marker. Agrobacterium-mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13: 282-285.Additionally, soybean transformation can be achieved using, for example, a technique described in European Patent No. 0424 047, US Patent No. 5,322,783, European Patent No. 0397 687, US Patent No. 5,376,543, or US Patent No. 5,169,770. Corn transformation can be achieved by particle bombardment, polyethylene glycol-mediated DNA uptake, or through the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” by Springer Vertag: New York (1993) ISBN 3-540-978267). A specific example of corn transformation is found in US Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93 / 07256.

[151] According to the present invention, the introduced chimeric polynucleotide can be stably maintained in the plant cell if it is incorporated into a non-chromosomal autonomous replicon or integrated into plant chromosomes. Alternatively, the introduced chimeric polynucleotide can be present in an extra-chromosomal non-replicating vector and be transiently expressed or transiently active. Petition 870250068507, dated 04 / 08 / 2025, page 87 / 329 76 / 153

[152] In the homologous recombination vector, the chimeric polynucleotide can be flanked at its 5' and 3' ends by an additional nucleic acid molecule to allow homologous recombination to occur between the exogenous chimeric polynucleotide carried by the vector and an endogenous gene in a microorganism or plant. The additional flanking chimeric nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundred base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, for example, Thomas, KR and Capecchi, MR, 1987, Cell 51: 503 for a description of homologous recombination vectors or Strepp et al., 1998, PNAS, 95 (8): 4368-4373 for cDNA-based recombination in Physcornitrella patens).However, since the chimeric polynucleotide typically differs from the PPO gene by very few amino acids, a flanking sequence is not always necessary. The homologous recombination vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol-mediated DNA), and the cells in which the introduced chimeric polynucleotide has homologously recombined with the endogenous gene are selected using techniques known in the state of the art.

[153] In another embodiment, recombinant microorganisms containing selected systems that allow regulated expression of the introduced gene can be produced. For example, the inclusion of a chimeric polynucleotide in a vector placing it under the control of the lac operon allows expression of the chimeric polynucleotide only in the presence of IPTG. Such regulatory systems are well known in the art.

[154] Another aspect of the invention relates to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used from Petition 870250068507, dated 04 / 08 / 2025, p. 88 / 329 77 / 153 interchangeable form herein. It is understood that such terms refer not only to the cell in question, but also apply to the progeny or potential progeny of such a cell. As certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may, in fact, not be identical to the progenitor cell, but is still included within the scope of the term as used herein. A host cell may be any prokaryotic or eukaryotic cell. For example, a chimeric polynucleotide may be expressed in bacterial cells such as C. glutamicum, insect cells, fungal cells or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells), algae, ciliates, plant cells, fungi or other microorganisms such as C. glutamicum. Other suitable host cells are known to those skilled in the art.

[155] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a chimeric polynucleotide. Accordingly, the invention further provides methods for producing chimeric polypeptides using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a chimeric polypeptide has been introduced, or into which a gene encoding a chimeric or wild-type polypeptide has been introduced into the genome) in a suitable medium until the chimeric polypeptide is produced. In another embodiment of the invention, the method further comprises isolating chimeric polypeptides from the medium or from the host cell. Another aspect of the invention relates to isolated chimeric polypeptides and their biologically active moieties.An “isolated” or “purified” polypeptide, or a biologically active portion thereof, is free of any cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when synthesized. Petition 870250068507, dated 04 / 08 / 2025, page 89 / 329 78 / 153 chemically. The term “substantially free of cellular material” includes chimeric polypeptide preparations in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the term “substantially free of cellular material” includes preparations of a chimeric polypeptide with less than about 30% (by dry weight) of non-chimeric polypeptide material (also referred to herein as “contaminant polypeptide”), more preferably less than about 20% of non-chimeric polypeptide material, even more preferably less than about 10% of non-chimeric polypeptide material, and most preferably less than about 5% of non-chimeric polypeptide material.

[156] When the chimeric polypeptide, or its biologically active portion, is recombinantly produced, it is also preferably substantially free of culture medium, that is, the culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation. The term “substantially free of chemical precursors or other chemicals” includes chimeric polypeptide preparations in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In preferred embodiments, the isolated polypeptides, or their biologically active portions, do not contain contaminating polypeptides from the same organism from which the chimeric polypeptide is derived.Typically, such polypeptides are produced by recombinant expression of, for example, a chimeric polypeptide in plants other than, or in microorganisms such as C. glutamicum, ciliates, algae, or fungi.

[157] In another aspect, the present invention relates to a plant cell comprising the expression cassette of the present invention. Petition 870250068507, dated 04 / 08 / 2025, page 90 / 329 79 / 153 Preferably, the expression cassette comprises a chimeric nucleic acid molecule comprising (a) the sequence with SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 135 or a fragment or variant as defined herein, (b) a polynucleotide comprising at least 60 consecutive nucleotides of any of a); and (c) a polynucleotide complementary to the polynucleotide of any of a) through b).

[158] In another preferred embodiment, the expression cassette of said plant cell comprises a chimeric nucleic acid molecule encoding a polypeptide presented in SEQ ID NO: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624, or variants and fragments thereof.

[159] Preferably, the expression of the chimeric nucleic acid molecule of the invention in the plant cell results in increased resistance or tolerance to a herbicide compared with a wild-type variety of the plant cell.

[160] In another aspect, the present invention relates to a plant or part of a plant comprising a plant cell of the present invention.

[161] In specific embodiments, the plants and / or plant parts have stably incorporated at least one of the chimeric nucleic acid molecules of the present invention or a variant or fragment thereof.

[162] Thus, plants, plant cells, plant parts and seeds comprising at least one polynucleotide comprising a dual transit peptide coding sequence operatively linked to a heterologous polynucleotide encoding a Petition 870250068507, dated 04 / 08 / 2025, page 91 / 329 80 / 153 polypeptide of interest, wherein the dual transit peptide comprises any of the following SEQ ID numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or variants and fragments thereof.

[163] Plants, plant cells and seeds comprising chimeric nucleic acid molecules having sequences as presented in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 135 are also provided.

[164] Plants, plant cells and seeds comprising chimeric nucleic acid molecules encoding a polypeptide presented in SEQ ID NO: 112, 113, 114, 115, 116, 117, 118 are also provided. 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624, or active variants and fragments thereof.

[165] The term “plant” is used in its broadest sense as it refers to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, etc., as well as clones, bulbs and plant parts used for asexual propagation (e.g., cuttings, tubers, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term “plant” also encompasses whole plants, ancestral and progeny plants and plant parts, including seeds, shoots, Petition 870250068507, dated 04 / 08 / 2025, page 92 / 329 81 / 153 stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, peduncles, stamens, anthers, stigma, pistil, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell and any other known organs, tissues and cells of a plant, and tissues and organs, wherein each of the above-mentioned comprises the gene / nucleic acid of interest. The term “plant” also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the above-mentioned comprises the gene / nucleic acid of interest.

[166] The plants of the invention include all plants belonging to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including forage or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, rapeseed, nano]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara. Petition 870250068507, dated 04 / 08 / 2025, p. 93 / 329 82 / 153 spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g., Lycopersicon escutentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotytoma spp., Malus spp., Malplghia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Blackberry, Banana spp., Nicotiana spp., Otea spp., Opuntia spp., Ornithopus spp., Oryza spp. (for example, Oryza sativa, Oryza latifolia), Panicum miiiaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phataris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Sotanum spp. (for example, Soianum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecaie rimpaui, Triticum spp. (for example, Triticum aestivum, Triticum durum, Triticum turgidum, Triticum. Petition 870250068507, dated 04 / 08 / 2025, p. 94 / 329 83 / 153 hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeoium minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, celery, flax, cabbage (collard greens and kale), lentil, rapeseed, okra, onion, potato, rice, soybean, strawberry, beet, sugarcane, sunflower, tomato, pumpkin, tea and algae, among others. According to a preferred embodiment of the present invention, the plant is a cultivated plant. Examples of cultivated plants include, inter alia, soybeans, sunflowers, canola, alfalfa, rapeseed, cotton, tomatoes, potatoes, or tobacco. Preferably, the plant is a monocotyledon, such as sugarcane. Also preferably, the plant is a cereal, such as rice, corn, wheat, barley, millet, rye, sorghum, or oats.

[167] In another aspect, the present invention relates to a seed derived from a plant of the present invention.

[168] In another aspect, the present invention relates to a method for expressing a nucleic acid encoding a polypeptide of interest in a plant comprising (a) introducing into a plant cell the nucleic acid molecule of the present invention or the expression cassette of the present invention, and (b) regenerating a plant from the same comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention.

[169] The term “expression / expressing” or “gene expression” means the transcription of a specific gene or genes or a specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or a gene construct into structural RNA (rRNA, tRNA) or mRNA, with or without subsequent translation of the latter into a protein. The process includes DNA transcription and Petition 870250068507, dated 04 / 08 / 2025, page 95 / 329 84 / 153 processing of the resulting mRNA product.

[170] To obtain the desired effect, for example, plants that are tolerant or resistant to herbicides, it must be understood that at least one nucleic acid is “overexpressed” by methods and means known to those skilled in the art.

[171] The term “increased expression” or “overexpression” as used here means any form of expression that is additional to the original wild-type expression level. Methods for increasing the expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids that serve as promoter or enhancer elements can be introduced at an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to positively regulate the expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion and / or substitution (see, Kmiec, US 5,565,350; Zariing et al., WO 9322443), or isolated promoters can be introduced into a plant cell at the appropriate orientation and distance from a gene of the present invention in order to control gene expression.

[172] If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3' end of a polynucleotide coding region. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added may be derived, for example, from nopaline synthetase or octopine synthetase genes or, alternatively, from another plant gene, or less preferably from any other eukaryotic gene. Petition 870250068507, dated 04 / 08 / 2025, page 96 / 329 85 / 153

[173] An intron sequence can also be added to the 5' untranslated region (UTR) or to the coding sequence of the partial coding sequence to increase the amount of mature message that accumulates in the cytosol. The inclusion of splicable introns in the transcription unit in expression constructs in plants and animals has increased gene expression at mRNA and protein levels up to 1000 times (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1: 1183-1200). This increase in gene expression is typically greater when placed near the 5' end of the transcription unit. Using the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron is known in the art. For general information, see: The Maize Handbook. Chapter 116. Freeling and Walbot. Eds., Springer. NY (1994).

[174] The terms “introduction” or “transformation,” as referred to herein, encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with a gene construct of the present invention and an entire plant regenerated from it. The specific tissue chosen will vary depending on the clonal propagation systems available to, and most suitable for, the specific species being transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, calluses, existing meristematic tissue (e.g., apical meristem, axillary buds, and roots), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).The polynucleotide can be introduced transiently or stably into a host cell and can be retained non-integrated, for example, as a plasmid. Alternatively, it can be integrated into the host genome. The resulting transformed plant cell can then be used. Petition 870250068507, dated 04 / 08 / 2025, page 97 / 329 86 / 153 to regenerate a transformed plant in a manner known to those skilled in the art.

[175] The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods can be used to introduce the gene of interest into a suitable ancestral cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that enhance the uptake of free DNA, injection of DNA directly into the plant, particle bombardment, or transformation using viruses or pollen and micro-projection. Methods can be selected from the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., Nature 296 (1982) 72-74; Negrutiu I et al.(1987) Plant Mol Biol 8: 363-373); protoplast electroporation (Shillito RD et al. (1985) Bio / Technol 3. 1099-1102); microinjection into plant material (Crossway A et al. (1986) Mol. Gen Genet 202: 179-185); bombardment of particles coated with DNA or RNA (Klein TM et al. (1987) Nature 327: 70); virus infection (non-integrative) and similar. Transgenic plants, including transgenic crop plants, are preferably produced by Agrobacterium-mediated transformation. An advantageous transformation method is in planta transformation. For this purpose, it is possible, for example, to allow agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. According to the invention, it has proven particularly convenient to allow a suspension of transformed agrobacteria to act on the intact plant or, at least, on the primordia of the flowers.The plant is subsequently cultivated until seeds from the treated plant are obtained (Clough and Bent, . Petition 870250068507, dated 04 / 08 / 2025, page 98 / 329 87 / 153 Planta J. (1998) 16, 735-743). Methods for Agrobacterium-mediated rice processing include well-known methods for rice processing, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993); Hiei et al. (Plant J 6 (2): 271-282, 1994), whose disclosures are incorporated herein by reference as if fully presented. In the case of maize processing, the preferred method is as described in Ishida et al. (Nat. Biotechnol 14 (6): 745-50, 1996) or Frame et al. (Plant Physiol 129 (1): 13-22, 2002), whose disclosures are incorporated here by reference as if fully presented. The aforementioned methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, vol. 1. Engineering and Utilization, eds. SD Kung and R. Wu.Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or construct to be expressed are preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in a known manner for the transformation of plants, such as model plants like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant) or crop plants such as, for example, tobacco plants, for example by immersing leaves or chopped leaves in an agrobacterial solution and then cultivating them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia as FFWhite, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, vol. 1, Engineering and Utilization, eds. SD Kung and R. Wu, Academic Press, 1993, pp. 15-38. Petition 870250068507, dated 04 / 08 / 2025, p. 99 / 329 88 / 153

[176] In addition to the transformation of somatic cells, which then need to be regenerated into intact plants, it is also possible to transform plant cells and, in particular, those that develop into gametes. In this case, the transformed gametes accompany the natural development of the plant, giving rise to transgenic plants. Thus, for example, Arabidopsis seeds are treated with agrobacterium and seeds are obtained from developing plants, of which a certain proportion is transformed and therefore transgenic [Feldman. KA and Marks MD (1987). Mol Gen Genet 208: 274-289; Feldmann K (1992)., In: C Koncz, NH Chua and J Shell, eds. Methods in Arabidopsis Research. World Scientific. Singapore, pp. 274-289.Alternative methods are based on the repeated removal of inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can similarly be obtained at a later time (Chang, 1994; Plant J. 5: 551-558; Katavic, 1994. Genet. 245: 363-370). However, a particularly effective method is the vacuum infiltration method with its modifications, such as the floral immersion method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthotd, N (1993). CR Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method, the developing floral tissue is briefly incubated with an agrobacterial suspension treated with a surfactant [Clough, SJ and Bent AF (1998) The Plant J. 16. 735-743].A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by their growth under the selective conditions described above. Furthermore, stable plastid transformation is advantageous because plastids are maternally inherited, with most crops reducing or eliminating the risk of transgene flow through pollen. Genome transformation of... Petition 870250068507, dated 04 / 08 / 2025, p. 100 / 329 89 / 153 chloroplast transformation is generally achieved by a process that was schematically presented in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly, the sequences to be transformed are cloned together with a selectable marker gene among flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site-specific integration into the plastome. Plastic transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastic in basic research and plant biotechnology. J Mol Biol. September 21, 2001; 312 (3): 425-38 or Maliga. P (2003) Progress towards commercialization of plastic transformation technology. Biotechnol Trends. 21, 20-28.Further biotechnological advances have recently been reported in the form of marker-free plastid transformants, which can be produced by a transient co-integrated marker gene (Klaus et al., 2004, Nature Biotechnology 22 (2). 225-229). Genetically modified plant cells can be regenerated by all methods with which the skilled in the art are familiar. Suitable methods can be found in the publications mentioned above by SD Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[177] Generally after transformation, plant cells or cell clusters are selected for the presence of one or more markers that are encoded by plant-expressible genes, co-transferred with the gene of interest, after which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from non-transformed plants. For example, seeds obtained in the manner described above can be planted and, after an initial growth period, subjected to appropriate selection by spraying. Another possibility Petition 870250068507, dated 04 / 08 / 2025, page 101 / 329 Method 90 / 153 involves cultivating the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent, so that only the transformed seeds can develop into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker, such as those described above.

[178] After DNA transfer and regeneration, the putatively transformed plants can also be evaluated, for example, using Southern blotting, for the presence of the gene of interest, copy number and / or genomic organization. Alternatively or additionally, the expression levels of the newly introduced DNA can be monitored using Northern and / or Western blotting, both techniques being well known to those skilled in the art.

[179] The generated transformed plants can be propagated by a variety of means, such as clonal propagation techniques or classical breeding. For example, a first-generation transformed plant (or T1) can itself be selected as homozygous second-generation transformants (or T2), and the T2 plants can then be further propagated through classical breeding techniques. The generated transformed organisms can take a variety of forms. For example, they can be chimeric cells of transformed and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and non-transformed tissues (e.g., in plants, a transformed rootstock grafted onto a non-transformed scion).

[180] In another aspect, the present invention relates to a method for producing a herbicide-tolerant plant comprising (a) introducing into a plant cell the nucleic acid molecule of the present invention or the expression cassette of the present invention, and (b) regenerating a Petition 870250068507, dated 04 / 08 / 2025, p. 102 / 329 91 / 153 plant derived therefrom, comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention.

[181] In another aspect, the present invention relates to a method for controlling unwanted vegetation in a plant cultivation area, the method comprising the steps of: (c) To provide at that location a herbicide-tolerant plant comprising the nucleic acid molecule of the present invention or the expression cassette of the present invention, d) Apply an effective amount of herbicide to that location, wherein the effective amount of said herbicide does not kill or inhibit the growth of the herbicide-tolerant plant from a).

[182] The term “control of unwanted vegetation” should be understood to mean the killing of weeds and / or retarding or inhibiting the normal growth of weeds. Weeds, in the broadest sense, are understood to mean all those plants that grow in locations where they are undesirable, for example, plant cultivation sites (crops). The weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum.Monocotyledonous weeds include, but are not limited to, weeds from the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleocharis, Scirpus. Petition 870250068507, dated 04 / 08 / 2025, page 103 / 329 92 / 153 Paspalum, Ischaemum, Sphenocea, Dactytoctenium, Agrostis, Alopecurus, and Apera. Furthermore, the weeds of the present invention may include, for example, cultivated plants that are growing in an undesirable location. For example, a volunteer corn plant that is in a field comprising predominantly soybean plants may be considered a weed if the corn plant is not desired in the soybean field.

[183] ​​Additionally, in certain embodiments, the chimeric nucleic acid molecules of the present invention can be combined with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the nucleic acids of the present invention can be combined with any other polynucleotides encoding polypeptides possessing pesticidal and / or insecticidal activity, such as, for example, the toxin proteins of Bacillus thuringiensis (described in US Patents Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109).

[184] By way of example, the polynucleotides that can be combined with the chimeric nucleic acid molecules of the present invention include nucleic acids that encode polypeptides that confer resistance to pests / pathogens, such as viruses, nematodes, insects or fungi and the like. Exemplary polynucleotides that can be combined with nucleic acids of the invention include polynucleotides encoding: polypeptides possessing pesticidal and / or insecticidal activity, such as other toxic proteins of Bacillus thuringiensis (described in US Patents 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881 and Geiser et al. (1986) Gene 48: 109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825), pentin (described in US Patent No. 5,981,722) and the like; desirable characteristics for disease or herbicide resistance (e.g., detoxification genes of Petition 870250068507, dated 04 / 08 / 2025, page 104 / 329 93 / 153 fumonisin (US Pat. No. 5,792,931): avirulence and disease resistance genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. 78: 1089): acetolactate synthase (ALS) mutants leading to herbicide resistance, such as S4 and / or Hra mutations, glyphosate resistance (e.g., 5-enol-pyrovil-shikimate-3-phosphate synthase (EPSPS) gene, described in US Pat. Nos. 4,940,935 and 5,188,642, or the glyphosate N-acetyltransferase (GAT) gene, described in Castle et al., (2004) Science, 304: 1151-1154, and in US Patent Applications Nos. 20050246798, and 20050060767); glufosinate resistance (e.g., phosphinothricin acetyltransferase PAT and BAR genes, described in U.S. Patents 5,561,236 and 5,276,268); herbicide resistance including sulfonylurea, DHT (2,4-D), and PPO herbicides (e.g., glyphosate acetyltransferase, aryloxyalkanoate dioxygenase, acetolactate synthase, and protoporphyrinogen oxidase); a cytochrome P450 or a variant thereof that confers herbicide resistance or tolerance, inter alia, HPPD herbicides (U.S. Patent Application No. 12 / 156,247; U.S. Patents Nos. 6,380,465; 6,121,512; 5,349,127); 6,649,814; and 6,300,544; and PCT Patent Application No. WO 2007000077); and desirable characteristics for process or processing products such as high oil (e.g., U.S. Patent No. 6,232,529): modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94 / 11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Patent No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthetase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs)): disclosures of which are incorporated herein by reference. Petition 870250068507, dated 04 / 08 / 2025, p. 105 / 329 94 / 153

[185] Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls, or otherwise adversely modifies plant growth. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is meant an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a wild-type plant, plant tissue, plant cell, or similar host cell, but said amount does not so severely kill or inhibit the growth of herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention. Typically, the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those skilled in the art.Herbicidal activity is exhibited by herbicides useful for the present invention when applied directly to the plant or to the plant site at any growth stage or before planting or emergence. The observed effect depends on the plant species to be controlled, the plant growth stage, the application parameters of dilution and spray droplet size, the particle size of the solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers used, the soil type and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, it is preferable to apply the herbicide post-emergence to relatively immature undesirable vegetation to achieve maximum weed control.

[186] By a plant that is “herbicide-tolerant” or “herbicide-resistant,” it is meant that a plant is tolerant or resistant to by Petition 870250068507, dated 04 / 08 / 2025, page 106 / 329 95 / 153 minus a herbicide at a level that would normally kill or inhibit the growth of a normal or wild-type plant. By “wild-type protein or herbicide-tolerant chimeric polypeptide protein” or “herbicide-resistant chimeric or wild-type polypeptide protein,” it is intended that such chimeric polypeptide exhibit higher PPO activity, relative to the PPO activity of a wild-type chimeric polypeptide, when in the presence of at least one herbicide that is known to interfere with PPO activity and at a concentration or level of herbicide that is known to inhibit the PPO activity of the wild-type chimeric polypeptide protein. Furthermore, the PPO activity of such a herbicide-resistant or herbicide-tolerant chimeric polypeptide protein may be referred to herein as “herbicide-tolerant” or “herbicide-resistant” PPO activity.

[187] Generally, if the PPO-inhibiting herbicides (also referred to as compounds A) and / or the herbicide compounds B, as described herein, which can be used in the context of the present invention, are capable of forming geometric isomers, for example E / Z isomers, it is possible to use both the pure isomers and their mixtures in compositions useful for the present invention. If the PPO-inhibiting herbicides A and / or the herbicide compounds B, as described herein, have one or more chiral centers and, as a consequence, are present as enantiomers or diastereomers, it is possible to use both the pure enantiomers and diastereomers and mixtures thereof in compositions according to the invention. If the PPO-inhibiting herbicides A and / or the herbicide compounds B as described herein possess ionizable functional groups, they can also be employed in the form of their agriculturally acceptable salts.Generally, suitable compounds include salts of these cations and acid addition salts of acids whose cations and anions, respectively, do not have an adverse effect on the activity of the active compounds. Preferred cations are alkali metal ions, preferably lithium. Petition 870250068507, dated 04 / 08 / 2025, page 107 / 329 96 / 153 sodium and potassium, alkaline earth metals, preferably calcium and magnesium, and transition metals, preferably manganese, copper, zinc and iron, also ammonium and substituted ammonium in which one to four hydrogen atoms are substituted by C1-C4 alkyl, C1-C6 hydroxy alkyl, C1-C4 alkoxy, C1-C4 alkyl, C1-C4 hydroxy alkoxy alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium. tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2-hydroxyethylammonium (olamine salt), 2-(2-hydroxyeth-1-oxy)ethyl-1-ylammonium (diglycolamine salt), di(2-hydroxyeth-1-yl)ammonium (diolamine salt), tris(2-hydroxyethyl)ammonium (trolamine salt), tris(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), in addition to phosphonium ions, sulfonium ions, preferably tri(alkyl C1-C4)sulfonium, such as trimethylsulfonium, and sulfoxonium ions, preferably tri(alkyl C1-C4)sulfoxonium, and finally the salts of polybasic amines such as N,N-bis(3-aminopropyl)methylamine and diethylenetriamine.The anions of useful acid addition salts are mainly chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and also the anions of C1-C4 alkanoic acids, preferably formate, acetate, propionate, and butyrate.

[188] PPO inhibitor herbicides A and / or herbicide compounds B, as described herein, having a carboxyl group, may be employed in the form of an acid, in the form of an agriculturally suitable salt, as mentioned above, or in the form of an agriculturally acceptable derivative, for example as amides, such as mono- and dialkylamides or C1-C6 arylamides, as esters, for example as allyl esters, propargyl esters, C1-C4 alkyl esters, alkoxyalkyl esters, tefuryl Petition 870250068507, dated 04 / 08 / 2025, page 108 / 329 97 / 153 ((tetrahydrofuran-2-yl)methyl) esters and also as thioesters, for example, as C1-C10 alkylthio esters. Preferred C1-C6 mono- and dialkylamides are methyl and dimethylamides. Preferred arylamides are, for example, anilides and 2-chloroanilides. Preferred alkyl esters are, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl), meptyl (1-methylheptyl), heptyl, octyl or iso-octyl (2-ethylhexyl) esters. Preferred C1-C4 alkyl alkoxy esters are linear or branched chain C1-C4 ethyl alkoxy esters, for example, 2-methoxyethyl, 2-ethoxyethyl, 2-butoxyethyl (butoxyl), 2-butoxypropyl or 3-butoxypropyl. An example of a linear or branched chain C1-C4 alkylthio ester is the ethylthio ester.

[189] Examples of PPO-inhibiting herbicides that can be used according to the present invention are acifluorfen, acifluorfen-sodium, aclonifen, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxifen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumictorac-pentyl, flumioxazin, fluoroglucofen, fluoroglucofen-ethyl, flutiacet, flutiacet-methyl, fomesafen, halosafen, lactofen, oxadiargil, oxadiazon, oxyfluorfen, pentoxazone, profluazol, piraclonil, piraflufen, piraflufen-ethyl, saflufenacil, sulfentrazone, tidiazimin, tiafenacil, chlornitrofen, fiumipropin, fluoronitrophen, flupropacil, furitoxifen, nitrofluorofen, ethyl [3-[2-chloro-4fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]2-pyridyloxy]acetate (CAS 353292-31-6;(CAS) 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1-carboxamide (CAS 452099-05-7), Ntetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione (CAS; Petition 870250068507, dated 04 / 08 / 2025, page 109 / 329 98 / 153 451484-50-7), 1,5-dim ethyl l-6-thioxo-3-(2,2,7-trif I u oro-3-oxo-4-(prop-2-ini I )-3,4dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazine dione (C,4-C 125883672-4), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-inyl-3,4-dihydro-2H-benzo[1,4]oxazin-6yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS 1001 ),-3908 1-Methyl-6trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-inyl-3,4-dihydro-2Hbenzo[1,4]oxazin-6-yl)-1 H-pyrimidine-2,4-dione, methyl (E)-4-[2-chloro-5-[4-chloro-5(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3], 3-[7-Chloro-5-fluoro-2-(trifluomethyl)-1 H-benzimidazol-4-yl]-1 methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4), emulating formula III in which R30e R31é independent urn do outro F, Cl or CN; R32é O or S; R33é H, F, Cl CH3ou OCH3; R34th CH or N; R35é O or S; R36 is H, CN, CH3, CF3, OCH3, OC2H5, SCH3, SC2H5, (CO)OC2H5, or CH2R38, wherein R38 is F, Cl, OCH3, SCH3, SC2H5, CH2F, CH2Br, or CH2OH; and R37 is (C1-C6 alkyl)amino, (C1-C6 dialkyl)amino, (NH)OR39, OH, OR40 or SR40 wherein R39 is CH3, C2H5 or phenyl; and R40 is independent of each other alkyl Ci-Ce, alkenyl Ci-Ce, Petition 870250068507, dated 04 / 08 / 2025, page 110 / 329 99 / 153 alkyla C3-C6, haloalquila C1-C6, alkoxy C1-C6 alkyla C1-C6, alkoxy C1-C6 alkoxy Ci-Ce alkyla C1-C6, cyanoalquila C1-C6, alkoxy Ci-C4-carbonyl-alkyla C1-C4, alkyl Ci-C4-carbonyl-amino, alquilsulfinil Ci-Ce-alquila Ci-Ce, alquil Ci-Ce-sulfonylalquila Ci-Ce, dialkoxy Ci-Ce- alquila Ci-Ce, alquil Ci-Ce-carboniloxi-alquila Ci-Ce, phenyl-carbonyl-alquila Ci-Ce, tri(alquil Ci-C3)-silyl-alquila Ci-Ce, tri(alquila Ci-Ca)silyl-alquenyla Ci-Ce, tri(alquil Ci-C3)-silyl-alquinyl Ci-Ce, tri(alquil Ci-C3)-silylalkoxy Ci-Ce-alquila Ci-Ce, dimethylamino, tetrahidropyranyl, tetrahidrofuranylalquila Ci-C3, phenyl Ci-Ce-alkoxy-alquila Ci-Ce, phenyl-alquila Ci-C3, pyridyl-alquila Ci-C3, pyridila, phenyl, that pyridilas and phenyls, independently of the other two, are replaced by one of the five substitutions selected from the group that consists of halogen, alquila Ci-C3, or haloalquila Ci-C2;C3-Ce cycloalkyl, or C3-Ce cycloalkyl-Ci-C4 cycloalkyl, wherein the cycloalkyls, independently of each other, are substituted by one to five substituents selected from the group consisting of halogen, Ci-C3 alkyl, and Ci-C2 haloalkyl; including their agriculturally acceptable alkali metal salts or ammonium salts. [i90] Preferred PPO-inhibiting herbicides that can be used according to the present invention: Acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, butafenacil, carfentrazone-ethyl, cinidone-ethyl, flufenpyr-ethyl, flumiclorac-pentyl, flumioxazin, fluoroglycophen-ethyl, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxadiazon, oxyfluorfen, pentoxazone, piraflufen-ethyl, saftufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(i-methyl-e-trifluoromethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2pyridyloxy]acetate (CAS353292-3i-e; S-3i00), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-iH-pyrazol-i-carboxamide (CAS 452098-92-9), Ntetrahydrofurfuryl-3-(2,e-dichloro-4-trifluoromethylphenoxy)-5-methyl-iH-pyrazol-i Petition 870250068507, dated 04 / 08 / 2025, page 111 / 329 100 / 153 carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-enyl)-3,4-dihydro-2Hbenzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5jtriazinan-2,4-dione (CAS 451484-50-7), 1,5-dimethyl-6-thioxo-3-(2,2,7-irifluoro-3-oxo-4-(prop-2-enyl)-3,4dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinan-2,4-dione (CAS 125883672-4), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS 1300118-96-0); 1-Methyl-6trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione (CAS 1304113-05-0), 3-[7-Chloro-5-fluoro-2(trifluoromethyl)-l H-benzimidazol-4-yl]-1 -methyl-6-(trifiuoromethyl)-1 H-pyrimidine-2,4dione (CAS 212754-02-4); Uracils of formula 111.1 (corresponding to uracils of formula III, where R30 is F, R31 is Cl, R32 is O; R33 is H; R34 is CH; R35 is O and R37 is OR40) „36 Γ1N R36 is OCH3, OC2H5, SCH3 or SC2H5; and R40 is C1-C6 alkyl, C2-C6 alkenyl, C3-C6 alkynyl, C1Ce haloalkyl, C1-C6 C1-Ce alkoxy-alkyl, C1-Ce alkoxy-C1-Ce alkoxy-alkyl, C1-C3 cyanoalkyl, C1-C3 phenylalkyl, C1-C3 pyridylalkyl, C3-C6 cycloalkyl or C1-C4 Cs-Ce cycloalkyl, wherein the cycloalkyls are substituted or unsubstituted by one to five substituents selected from the group consisting of halogen, C1-C3 alkyl, and C1-C2 haloalkyl; Petition 870250068507, dated 04 / 08 / 2025, p. 112 / 329 101 / 153 uracils of formula 111.2 (corresponding to uracils of formula III, where R30 is F, R31 is Cl, R32 is O; R33 is H; R34 is N; R35 is O and R37 is OR40) R40 being C1-Ce alkyl) 'Ci-C6-alkyl III.2

[191] Particularly preferred PPO-inhibiting herbicides that can be used according to the present invention are: Acifluorfen, acifluorfen-sodium, butafenacil, carfentrazone-ethyl, cinidone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saftufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS353292-31 6; S-3100), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6yl]-1,5-dimethyl-6-thioxo-[1,3,5jtriazinan-2,4-dione (CAS 451484-50-7), and 2(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7tetrahydro-isoindole-1,3-dione (CAS 1300118-96-0);1-Methyl-6-trifluoromethyl-3(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1Hpyrimidine-2,4-dione (CAS 1304113-05-0); Uracils of formula III.1.1 (corresponding to uracils of formula III, where R30 is F, R31 is Cl, R32 is O; R33 is H; R34 is CH; R35 is O, R36 is OCH3 and R37 is OR40) Petition 870250068507, dated 04 / 08 / 2025, page 113 / 329 102 / 153 where R40 is a C1-Ce alkyl, C2-C6 alkenyl, C3-C6 alkynyl, C1-Ce haloalkyl, C1-Ce alkoxy-Ce-Ce alkyl, C1-Ce alkoxy-Ce-Ce alkoxy-Ce-Ce alkyl, C1-C3 cyanoalkyl, C1-C3 phenyl alkyl, C1-C3 pyridyl alkyl, C3-C6 cycloalkyl or C1-C4 cycloalkyl-Cs-Ce alkyl, wherein the cycloalkyls are substituted or unsubstituted by one to five substituents selected from the group consisting of halogen, C1-C3 alkyl, and C1-C2 haloalkyl; It is preferably CH3, CH2CH2OC2H5, CH2CHF2, cyclohexyl, (1-methylcyclopropyl)methyl or CH2(pyridine-4-yl); uracils of formula 111.2.1 (corresponding to uracils of formula III, where R30 is F, R31 is Cl, R32 is O; R33 is H; R34 is N; R35 is O and R37 is OR40 R40 being CH3) uracils of formula III.2.2 (corresponding to uracils of formula III, where R30 is F, R31 is Cl, R32 is O; R33 is H; R34 is N; R35 is O and R37 is OR40 R40 being C2H5) F3C OC2H5 III.2.2,

[192] PPO-inhibiting herbicides are particularly preferred as PPO-inhibiting herbicides 1 to A. 15 listed below in Table 1: Petition 870250068507, dated 04 / 08 / 2025, p. 114 / 329 103 / 153 Table 1 A.1 Acifluorfen A.2 Butaphenacil A.3 Carphentrazone-ethyl A.4 Cinidone-ethyl A.5 Flumioxazine A.6 Flutiacet-methyl A.7 Fomesafen A.8 Lactofen A.9 Oxadiargyl A.10 Oxyfluorfen A.11 Saflufenacil A.12 Sulfetrazone A.13 ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS353292-31-6) A.14 1,5-dimethyl-6-thioxo-3-(2,2,7-irifluoro-3-oxo-4-(prop-2-enyl)-3,4-dihydro-2Hbenzo[b][1,4]oxazin-6-yl)-1,3,5-triazinano-2,4-dione (trifludimoxazine) A.15 Conforming phenylpyridines disclosed in WO 2016 / 1

[193] The PPO-inhibiting herbicides described above are useful for carrying out the present invention and are often best applied in conjunction with one or more other herbicides to achieve control of a wider variety of undesirable vegetation. For example, PPO-inhibiting herbicides can also be used in conjunction with additional herbicides to which the crop plant is naturally tolerant, or to which it is resistant through the expression of one or more additional transgenes as mentioned above, or to which it is resistant via mutagenesis and reproduction methods, described below. When used in conjunction with other targeting herbicides, the PPO-inhibiting herbicides to which the plant of the present invention has become resistant or tolerant can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides, or applied sequentially with the other herbicide or herbicides.

[194] Suitable components for mixtures are, for example, selected from herbicides of classes b1) to b15) Petition 870250068507, dated 04 / 08 / 2025, page 115 / 329 104 / 153 B) Herbicides of classes b1) to b15): B1) Lipid biosynthesis inhibitors; b2) Acetolactate synthase inhibitors (ALS inhibitors); b3) Photosynthesis inhibitors; b4) Protoporphyrinogen-IX oxidase inhibitors; b5) Bleaching herbicides; b6) Enolpyruvyl shikimate-3-phosphate synthase inhibitors (EPSP inhibitors); b7) Glutamine synthetase inhibitors; b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors); b9) Mitosis inhibitors; b10) Inhibitors of very long-chain fatty acid synthesis. (VLCFA inhibitors); b11) inhibitors of cellulose biosynthesis; b12) uncoupling herbicides; b13) auxin herbicides; b14) auxin transport inhibitors; and b15) other herbicides selected from the group consisting of bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumiluron, dalapon, dazomet, difenzoquat, difenquat-methylsulfate, dimetipine, DSMA, dimron, endotal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosaminoammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, metiozolin (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dimron, methyl iodide, MSMA, oleic acid, oxaziclomefon, pelargonic acid, piributicarb, quinocyamine, triaziftam, tridipane and 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS 499223-49-3) and salts and esters thereof; including its salts or agriculturally acceptable derivatives. Petition 870250068507, dated 04 / 08 / 2025, page 116 / 329 105 / 153

[195] Examples of herbicides B that can be used in combination with the herbicides that inhibit PPO according to the present invention are: b1) from the group of lipid biosynthesis inhibitors: Herbicides-ACC such as aloxidim, aloxidim-sodium, butroxidim, clethodim, clodinafop, clodinafop-propargyl, cycloxidim, cihalofop, cihalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-phenoxaprop, fenoxaprop, fluazifop, fluazifop-butyl, fluazifop-P, fiuazifop-P-butyl, haloxifope, haloxifopmethyl, haloxifope-P, haloxifope-P-methyl, metamifop, pinoxaden, profoxidim, propaquizafop, quizalofop, quizalofop-e, quizafop, quizafop, quizalofop, quizafop, quizalophop-P-ethyl, quizalophop-P-tefuryl, sethoxidim, tepraloxidim, tralcoxidim, 4-(4'Chloro-4-cyclopropyl-2'-fluoro[1,1 '-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2(CH)(CH) 1312337-72-6); 4-(2',4'-dichloro-4-cyclopropyl[1,1'-biphenyl]3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3}; 4(4'-Chloro-4-fluoro-2,1'-ethyl '-biphenyl]-3-yl)-5-hydroxy-2,2,6, 6-tetramethyl-2H-pyran3(6H)-one (CAS 1033757-93-5);4-(2',4'-Dichloro-4-ethyl [1,1'-biphenyl]-3-yl)-2,2,6,6tetramethyl-2H-pyran-3,5 (4H, 6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4'chloro-4-cyclopropyl-2'-fluoro [1,1'-biphenyl-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2Hpyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2',4'-dichloro-4-cyclopropyl-[1,1'biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4'chloro-4-ethyl-2'-trifluoro[1,1 '-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-3,6dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); methyl ester of acid 4-(4'-chloro-4-cyclopropyl-2'-fluoro[1,1 '-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6tetramethyl-5-oxo-2H-pyran-3-yl carbonic (CAS); 137-1351-3 methyl ester of 4-(2',4'-dichloro-4-cyclopropyl-[1,1 '-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl5-oxo-2H-pyran-3-yl carboxylic acid;4-(4'-Chloro-4-ethyl-2'fluoro[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl acid methyl ester; Petition 870250068507, dated 04 / 08 / 2025, page 117 / 329 106 / 153 carbonic acid (CAS 1312340-83-2); methyl ester of 4-(2',4-Dichloro-4-ethyl[1,1'biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid (CAS 1033760-58-5); and non-ACC herbicides such as benfuresate, butylate, cycloate, dalapon, dimepiperate, EPTC, esprocarb, etofumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, TCA, thiobencarb, thiocarbazil, trialate and vernolate; b2) from the ALS inhibitor group: sulfonilureias tais como amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-metil, clorimuron, clorimuron-etil, clorsulfuron, cinossulfuron, cIclosulfamuron, etametsulfuron, etametsulfuron-metil, etoxisulfuron, flazasulfuron, flucetosulfuron, flupirsulfuron, flupirsulfuron-metil-sódio, foramsulfuron, halosulfuron, halosulfuron-metil, imazosulfuron, iodosulfuron, iodossulfuron-metil-sódio, iofensulfuron, iofensulfuron-sódio, mesosulfuron, metasulfuron, metsulfuron, metsulfuron-metil, nicosulfuron, ortossulfamuron, oxasulfuron, primisulfuron, primisulfuron-metil, propirisulfuron, prosulfuron, pirazosulfuron, pirazosulfuron-etil, rimsulfuron, sulfometuron, sulfometuron-metil, sulfosulfuron, tifensulfuron, tifensulfuron-metil, triasulfuron, tribenuron, tribenuron-metil, triftoxissulfuron, triflusulfuron, trifusulfuron-metil e tritosulfuron, imidazolinonas tais como imazametabenz, imazametabenz-metil, imazamox, imazapic, imazapir, imazaquin e imazetapir,Triazolopyrimidine and sulfonanilide herbicides, such as chloransulam, chloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsulam, pirimisulfan and piroxsulam, pyrimidinylbenzoates such as bispiribac, bispiribac-sodium, piribenzoxim, piriftalid, pirimino-bac, piriminobac-methyl, piritiobac, piritiobacsodium, 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]benzoic acid-1-methylethyl ester (CAS 420138-41-6), propyl ester of 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]benzoic acid (CAS 420138-40-5), N-(4Petition 870250068507, dated 04 / 08 / 2025, page 118 / 329, 107 / 153 bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenomethanamine (CAS 420138-01-8), sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, tiencarbazone and tiencarbazone-methyl; and triafamonone; Among these, a preferred embodiment of the invention relates to compositions comprising at least one imidazolinone herbicide; b3) from the group of photosynthesis inhibitors: amicarbazone, photosystem II inhibitors, e.g. triazine herbicides, including chlorotriazine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametrine, atrazine, chloridazone, cyanazine, desmethrin, dimethamethrin, hexaxinone, metribuzim, prometon, promethrin, propazine, simazine, simethrin, terbumeton, terbuthylazine, terbutrin and triethazine, aryl urea such as chlorobromurone, chlorotolurone, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, metabenzthiazuron, metobenzuron, methoxuron, monolinuron, neburon, siduron, tebuthiuron and thiadiazuron, carbamates of phenyl such as desmedifam, karbutilat, fenmedifam, fenmedifam-ethyl, nitrile herbicides such as bromofenoxim, bromoxynil and their salts and esters, ioxynil and their salts and esters, uracils such as bromacil, lenacil and terbacil, and bentazon and bentazon-sodium, pyridate, pyridafol, pentanechlor and propanil, and photosystem I inhibitors such as diquat, diquat-dibromide, paraquat,Paraquat dichloride and paraquat dimethyl sulfate. Among these, a preferred embodiment of the invention relates to compositions comprising at least one arylurea herbicide. Similarly, among these, a preferred embodiment of the invention relates to compositions comprising at least one triazine herbicide. Similarly, among these, a preferred embodiment of the invention relates to compositions comprising at least one nitrile herbicide. Petition 870250068507, dated 04 / 08 / 2025, p. 119 / 329 108 / 153 b4) of the protoporphyrinogen-IX oxidase inhibitor group: acifluorfen, acifluorfen-sodium, azafenidine, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, clomethoxyfen, cinidone-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycophen, fluoroglycophen-ethyl, flutiacet, flutiacet-methyl, fomesafen, halosafen, lactofen, oxadiargil, oxadiazon, oxyfluorfen, pentoxazone, profluazol, piraclonil, piraflufen, piraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimine, thiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy] acetate (CAS 353292-31-6; S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)5-methyl-1H-pyrazol-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazol1-carboxamide (CAS 452099-05-7), N-tetra-hydrofurfuryl-3-(2-chloro-6-fluoro-4trifluoromethylphenoxy)-5-methyl-1H-pyrazol-1-carboxamide (CAS 452100-03-7), 3-[7fluoro-3-oxo-4-(prop-2-enyl)-3,4-dihydro-2H-benzo [1,4] . oxazin-6-yl]-1,5-dimethyl-6thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4(prop-2-enyl)-3,4-dihydro-2H-benzo[b][1,4] oxazin-6-yl)-1,3,5-triazinan-2,4-dione (CAS 1258836-72-4), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4] oxazin-6-yl)-4,5,6,7-tetrahydro-isoindol-1,3-dione, 1 -Methyl-6-trifluoromethyl-3(2,2,7-trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1Hpyrimidin-2,4-dione (CAS 1304113-05-0), methyl(E)-4-[2-chloro-5-[4-chloro-5(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoato [CAS 948893-00-37], .and 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4);, b5) from the group of bleaching herbicides: PDS inhibitors: beflubutamid, diflufenican, fluridone, flurochloridone, flurtamone, norflurazone, picolinafen and 4-(3-trifluoromethylphenoxy)-2 Petition 870250068507, dated 04 / 08 / 2025, page 120 / 329 109 / 153 (4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors: benzobicyclone, benzofenap, clomazone, isoxaflutole, mesotrione, pyrasulfotole, pyrazolinate, pyrazoxifene, sulcotrione, tefuryltrione, tembotrione, topramezone and bicyclopyrone, bleaching agents, unknown target: aclonifene, amitrole and flumeturon; b6) from the EPSP synthase inhibitor group: glyphosate, glyphosate-isopropylammonium, glyphosate-potassium and glyphosatotrimesio (sulfosate); b7) from the group of glutamine synthetase inhibitors: bilanophos (bialafos), bilanofo-sodium, glufosinate, glufosinate-P and glufosinate-ammonium; b8) from the group of DHP synthase inhibitors: asulam; b9) from the group of mitosis inhibitors: Group K1 compounds: dinitroanilines such as benfluralin, butralin, dinitramine, etalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates such as amiprofos, amiprofos-methyl and butamifos, benzoic acid herbicides such as chlortal, chlortal-dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propizamid and tebutam; Group K2 compounds: chlorprofan, profam and carbamide, among these, Group K1 compounds, in particular dinitroanilines, are preferred; b10) from the VLCFA inhibitor group: Chloroacetamides such as acetochlor, alachlor, butachlor, dimetachlor, dimethenamide, dimethenamide-P, metazachlor, metoiachlor, metolachlor-S, petoxamid, pretilachlor, propachlor, propisochlor and teniclor, oxacetanilides such as flufenacet and mefenacet, acetanilides such as difenamide, naproanilide and napropamide, tetrazolinones such as fentrazamide, and other herbicides such as Petition 870250068507, dated 04 / 08 / 2025, page 121 / 329 110 / 153 compounds anilofos, cafenstrol, fonoxasulfone, ipfencarbazone, piperofos, piroxasulfonates and isoxazoline of formulas 11.1. II.2, II.3, II.4, II.5, II.6. II.7, II.8 and II.9; II 1 II.2 H,C H,C F3C NL n-ch I1.3 HjC H,C The isoxazoline compounds of formula (l)l are known in the art, for example, from WO 2006 / 024820, WO 2006 / 037945, WO 2007 / 071900 and WO 2007 / 096576; Among VLCFA inhibitors, preference is given to chloroacetamides and oxyacetamides; b11) from the group of cellulose biosynthesis inhibitors: chlorttiamid, diclobenil, flupoxam, indaziflam, triaziflam, isoxaben and 1-cyclohexyl-5-pentafluorophenyloxy-14-[1,2,4,6]triazenin-3-ylamine; b12) from the group of uncoupling herbicides: dinoseb, dinoterb and DNOC and their salts: b13) from the auxin herbicide group: 2,4-D and its salts and esters such as clacifos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts, such as aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, benazoline, Petition 870250068507, dated 04 / 08 / 2025, page 122 / 329 111 / 153 benazoline-ethyl, chlorambene and its salts and esters, clomeprop, cyclopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr, fluroxypyr-butomethyl, fluroxypyr-methyl, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, TBA (2,3,6) and its salts and esters and triclopyr and its salts and esters; b14) from the group of auxin transport inhibitors: diflufenzopyr, diflufenzopyr-sodium, naptalam and naptalam-sodium; b15) from the group of other herbicides: bromobutide, chlorfurenol, chlorfurenol-methyl, cinmethylin, cumiluron, cyclopyrimorate (CAS 499223-49-3) and its salts and esters, dalapon, dazomet, difenzoquat, difenzoquat-methylsulfate, dimethipine, DSMA, dimron, endotal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-misopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosaminoammonium, indanofan, indaziflam, maleic hydrazide, mefludide, metam, methiozoline (CAS 403640-27-7), methyl azide, methyl bromide, methyldimron, methyl iodide, MSMA, acid oleic acid, oxaziclomefona, pelargic acid, piributicarb, quinoclolamina, triaziflam, and tridipano.

[196] Preferred herbicides B that can be used in combination with PPO-inhibiting herbicides according to the present invention are: b1) from the group of lipid biosynthesis inhibitors: clethodim, clodinafop-propargyl, cycloxidim, cihalofop-butyl, diclofopmethyl, fenoxaprop-P-ethyl, fluazifop-P-butyl, haloxifope-P-methyl, metamifop, pinoxaden, profoxidim, propaquizafop, quizalofop-P-ethyl, quizalofop-P-tefuril, setoxidim, tepraloxidim, tralcoxidim, 4-(4'-Chloro-4-cyclopropyl-2'-fluoro[1,1'biphenyl)-3-yl)-5-hidroxi-2,2,6,6-tetramethyl-2H-pyran-3(6H)-ona (CAS 1312337-72 Petition 870250068507, 04 / 08 / 2025, pág. 123 / 329 112 / 153 6); 4-(2',4'-Dichloro-4-cyclopropyl [1,1 '-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetra-methyl-2Hpyrano-3(6H)-one (CAS 1312337-45-3); 4-(4'-chloro-4-ethyl-2'-fluoro [1,1-biphenyl]-3yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran 3 (6H)-one (CAS 1033757-93-5); 4(2',4'-Dichloro-4-ethyl [1,1'-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5 (4H, 6H)dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4'-chloro-4-cyclopropyl-2'-fluoro [1,1'biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2',4'-dichloro-4-cyclopropyl-[1,1'-biphenyl]-3-IL)-3,6-dihydro-2,2,6,6tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4′-chloro-4-ethyl-2′-fluoro [1,1′-biphenyl-3yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5(Acetyloxy)-4-(2',4'-dichloro-4-ethyl [1,1 '-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl2H-pyran-3-one (CAS 1033760-55-2);éster metílico do ácido 4-(4'-Cloro-4ciclopropil-2'-fluoro [1,1 '-bifenil]-3-il)-5,6-dihidro-2,2,6,6-tetrametil-5-oxo-2Hpiran-3-il) carbônico (CAS 1312337-51-1); éster metílico do ácido 4-(2',4'Dicloro-4-ciclopropil-[1,1 '-bifenil]-3-il)-5,6-dihidro-2,2,6,6-tetrametil-5-oxo-2Hpirano-3-il carbônico; methyl ester of 4-(4'-Chloro-4-ethyl-2'-fluoro [1,1'biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-il carbonic acid (CAS; 1312340-83-2); methyl ester of 4-(2',4'-dichloro-4-ethyl [1,1 '-biphenyl]-3-yl)-5,6dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-il carbonic acid (CAS 1033760-58-5), benfuresate, dimepiperate, EPIC, esprocarb, ethofumesate, molinate, orbencarb, prosulfocarb, thiobencarb, and trialate; b2) the group of ALS inhibitors; amidosulfuron, azimsulfuron, bensulfuron-methyl, bispiribac-sodium, chlorimuron-ethyl, chlorsulfuron, chloransulam-methyl, cyclosulfamuron, diclosulam, etametsulfuron-methyl, ethoxysulfuron, flazasulfuron, florasulam, flucarbazonasodium, flucetosulfuron, flumetsulam, flupirsulfon-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazametabenz-methyl, imazamox, imazapic, imazapyr, imazaquin, imazetapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosuffuron, metosulam, Petition 870250068507, dated 04 / 08 / 2025, page 124 / 329 113 / 153 metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, penoxsulam, primisutfuron-methyl, propoxycarbazon-sodium, propyrisulfuron, prosulfuron, pyrazolosulfuron-ethyl, pyribenzoxim, pyrimidiphane, pyriftalid, pyriminobac-methyl, pyridiobac-sodium, piroxsulam, rimsulfuron, sulfometuron-methyl, sulfosulfuron, tiencarbazon-melil, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron and triafamone; b3) from the group of photosynthesis inhibitors: ametryn, amicarbazone, atrazine, bentazone, sodium bentazone, bromoxynil and its salts and esters, chloridazone, chlorotoluron, cyanazine, desmedifan, diquat-dibromide, diuron, fluometuron, hexazinone, ioxynil and its salts and esters, isoproturon, lenacil, linuron, metamitron, methabenzthiazuron, metribuzin, paraquat, paraquat dichloride, fenmedifan, propanil, pyridate, simazine, terbutrin, terbuthylazine and thidiazuron; b4) from the group of protoporphyrinogen-IX oxidase inhibitors: acifluorfen, acifluorfen-sódio, azafenidina, bencarbazona, benzfendizona, butafenacil, carfentrazona-etil, cinidon-etil, flufenpir-etil, flumiclorac-pentil, flumioxazina,fluoroglicofen-etil, fluticet-metil, fomesafen, lactofen, oxadiargil, oxadiazona, oxifluorfen, pentoxazona, piraflufen-etil, saflufenacil, sulfentrazona, etil [3-[2-cloro-4-fluoro-5-(1-metil-6-trifluormetiletil)2,4-dioxo-1,2,3,4-tetrahidropirimidin-3-il) fenoxi]-2-piridiloxi] acetato (CAS 353292-31 -6;S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-Dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), Nethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100 03-7), 3-[7fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4)Petição 870250068507, but 04 / 08 / 2025, page 125 / 329; 114 / 153 (prop-2-enyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinan-2,4-dione (CAS 1258836-72-4), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2Hbenzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione; 1 -Methyl-6trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione e 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1Hbenzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754 02-4); b5) of group two clearing herbicides: aclonifen, beflubutamide, benzobicyclone, clomazone, diflufenican, fluorochloridone, flurtamon, isoxaflutole, mesotrione, norflurazone, picolinafen, pyrasulfotol, pyrazolinate, sulcotrione, tefuryltrione, tembotrione, topramezone, bicidopyrone, . 4-(3-trifluoromethylphenoxy)-2-(4 -trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), amitrol and flumeturon; b6) of the group two inhibitors of EPSP synthesize: glyphosate, glyphosate-isopropylammonium, glyphosate-potassium and grifosatotrimesio (sulfosate); b7) from the group of glutamine synthase inhibitors: glufosinate, glufosinate-p, glufosinate-ammonium; b8) from the group of DHP synthase inhibitors: asulam; b9) from the group of mitosis inhibitors: benfluralin, dithiopyr, etatfluralin, oryzalin, pendimethalin, thiazopyr and trifluralin; b10) from the group of VLCFA inhibitors: compounds acetochlor, alachlor, anilophos, butachlor, cafenstrole, dimethenamid, dimethenamid-P, fentrazamide, flufenacet, mefenacet, metazaclor, metolachlor, S-metolachlor, naproanilide, napropamide, pretilachlor, fenoxasulfone, ipfencarbazone, pyroxasulfone and isoxazoline of formulas II.1, II.2, II.3, II.4, II.5, II.6, II.7. II.8 and II.9 Petition 870250068507, dated 04 / 08 / 2025, pp. 126 / 329 115 / 153 as mentioned above; b11) from the group of cellulose biosynthesis inhibitors: diclobenil, flupoxam, isoxaben and 1-cyclohexyl-5-pentafluorophenyloxy-14-[1,2,4,6]-thiatriazine-3-ylamine; b13) from the auxin herbicide group: 2,4-D and its salts and esters; salts and esters (CAS 943832-60-8), MCPA and its salts and esters, MCPB and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac and triclopyr and their salts and esters; b14) from the group of auxin transport inhibitors: diflufenzopyr and diflufenzopyr sodium; b15) from the group of other herbicides: bromobutide, cinmethylline, cumyluron, cyclopyrimorate (CAS 49922349-3) and its salts and esters, dalapon, diphenzoquat, diphenylquat-methylsulfate, DSMA, Dimron (daimuron), flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flomprop-M-methyl, indanophan, indaziflam, metam, methyl bromide, MSMA, oxaziclomephone, piributicarb, triaziflam and tridifam.

[197] Particularly preferred herbicides B that can be used in combination with PPO-inhibiting herbicides according to the present invention are: b1) from the group of lipid biosynthesis inhibitors: clodinafop-propargyl, cycloxidim, cyhalofop-butyl, fenoxaprop-P-ethyl, pinoxaden, profoxidim, tepraloxidim, tralkoxidim, 4-(4'-Chloro-4-cyclopropyl-2'fluoro[1,1'-biphenyl)-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3 (6H)-one (CAS 1312337-72-6); 4-(2',4'-Dichloro-4-cyclopropyl [1,1'-biphenyl]-3-yl)-5-hydroxy-2,2,6,6 Petition 870250068507, dated 04 / 08 / 2025, page 127 / 329 116 / 153 tetra-methyl-2H-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4'-chloro-4-ethyl-2'-fluoro [1,1-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran 3(6H)-one (CAS 1033757-93-5); 4-(2',4'-Dichloro-4-ethyl [1,1 '-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2Hpyran-3,5 (4H, 6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4′-chloro-4cyclopropyl-2′-fluoro [1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2',4'-dichloro-4-cyclopropyl-[1,1'-biphenyl]3-IL)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4′-chloro-4ethyl-2′-fluoro [1,1′-biphenyl-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2',4'-dichloro-4-ethyl [1,1 '-biphenyl]-3-yl)-3,6-dihydro2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); methyl ester of acid 4(4'-Chloro-4-cyclopropyl-2'-fluorine [1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5oxo-2H-pyran-3-yl) carbonic acid (CAS); methyl ester of acid 4(2',4'-Dichloro-4-cyclopropyl-[1,1 '-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo2H-pyran-3-yl carbonic;4-(4'-Chloro-4-ethyl-2'-fluoro [1,1'biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS; 1312340-83-2); 4-(2',4'-dichloro-4-ethyl [1,1'-biphenyl]-3-yl)-5,6dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1033760-58-5), esprocarb, prosulfocarb, thiobencarb and triallate; b2) from the ALS inhibitor group; bensulfuron-methyl, bispiribac-sodium, cyclosulfamuron, diclosulam, flumetsulam, flupyrsulfon-methyl-sodium, foramsulfuron, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, nicosulfuron, penoxsulam, propoxycarbazon-sodium, propyrilsulfuron, pyrazolosulfuron-ethyl, piroxsulam, rimsulfuron, sulfosulfuron, tiencarbazon-melil, tritosulfuron and triafamona; b3) from the group of photosynthesis inhibitors: ametrin, atrazine, diuron, fluometuron, hexazinone, isoproturon, Petition 870250068507, dated 04 / 08 / 2025, pages 128 / 329 117 / 153 lenacil, linuron, metribuzin, paraquat, paraquat dichloride, propanil, terbutrin and terbuthylazine; b4) from the group of protoporphyrinogen-IX oxidase inhibitors: acifluorfen, acifluorfen-sodium, butafenacil, carfentrazone-ethyl, cinidone-ethyl, flumioxazine,fluticet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6trifluoromethyl-ethyl)2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl) phenoxy]-2-pyridyloxy] acetate (CAS 353292-31-6; S-3100), 3-[7-fluoro-3-oxo-4-(prop-2-enyl)-3,4-dihydro2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione, 1,5dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-enyl)-3,4-dihydro-2Hbenzo[b][1,4]oxazin-6-yl)-1,3,5-triazinan-2,4-dione (CAS 1258836-72-4) e 2(2,2,7-Trifluoro-3-oxo-4-prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7tetrahydro-isoindole-1,3-dione e 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4prop-2-enyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione; b5) of group two clearing herbicides: clomazone, diflufenican, flurochloridone, isoxaflutole, mesotrione, picolinafen, sulcotrione, tefuriltrione, tembotrione, topramezone, bicidopyrone, amitrol and flumeturon; b6) from the EPSP synthase inhibitor group: glyphosate, glyphosate-isopropylammonium, and griphosate-trimesium (sulfosate); b7) from the group of glutamine synthetase inhibitors: glufosinate, glufosinate-P and glufosinate-ammonium; b9) from the group of mitosis inhibitors: pendimethalin and trifluralin; b10) from the VLCFA inhibitor group: compounds acetochlor, cafenstrole, dimethenamide-P, fentrazamide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, phenoxaluflone, ipfencarbazone and pyroxaluflone; similarly, preference is given to Petition 870250068507, dated 04 / 08 / 2025, pages 129 / 329 118 / 153 compounds of formulas II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9 as mentioned above; b11) from the group of cellulose biosynthesis inhibitors: isoxabene; b13) from the auxin herbicide group: 2,4-D and its salts and esters, such as clacyfos and aminocyclopyrachlor and their salts and esters, aminopyralid and its salts and esters, clopyralid and its salts and esters, dicamba and its salts and esters, fluroxypyr-meptyl, quinclorac and quinmerac; b14) from the group of auxin transport inhibitors: diflufenzopyr and diflufenzopyr sodium; b15) from the group of other herbicides: Dimron (= daimuron), indanofan, indaziflam, oxaziclomephone, piributicarb, triaziflam and tridifam.

[198] In addition, it may be useful to apply PPO-inhibiting herbicides, when used in combination with a compound B described SUPRA, in combination with protective agents. Protective agents are chemical compounds that prevent or reduce damage to beneficial plants without having a large impact on the herbicidal action of herbicides against undesirable plants. They can be applied before sowing (e.g., in seed, sprout or seedling treatments) or in pre-emergence or post-emergence application to the beneficial plant.

[199] In addition, protective agents C, PPO-inhibiting herbicides and / or herbicides B may be applied simultaneously or in succession.

[200] Suitable protective agents are, for example, (quinol-8-oxy)acetic acids, 1-phenyl-5-haloalkyl-1H-1,2,4-triazole-3-carboxylic acids, 1-phenyl-4,5-dihydro-5-alkyl-1H-pyrazole-3,5-dicarboxylic acids, 4,5-dihydro-5,5-diaryl-3-isoxazolecarboxylic acid, dichloroacetamides, alpha Petition 870250068507, dated 04 / 08 / 2025, page 130 / 329 119 / 153 oxyminophenylacetonitriles, acetophenonoximes, 4,6-dihalo-2-phenylpyrimidines, N-[[4-(aminocarbonyl)phenyl]sulfonyl]-2-benzoic amides, 1,8-naphthalic anhydride, 2-halo-4-(haloalkyl)-5-thiazole-carboxylic acids, phosphorthiolates and N-alkyl-O-phenylcarbamates and their agriculturally acceptable salts and their agriculturally acceptable derivatives, such as amides, esters and thioesters, provided they have an acid group.

[201] Examples of preferred C-protective agents are benoxacor, cloquintocet, cietrinil, ciprosulfamide, dichlormid, dicyclonone, dietolate, fenclorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxifen, mefenpir, mefenate, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro [4,5] decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3oxazolidine (R-29148, CAS 52836-31-4).

[202] Especially preferred C-protective agents are benoxacor, cloquintocet, ciprosulfamide, dichlormide, finchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro [4,5]decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

[203] Particularly preferred C-protective agents are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, naphthalic anhydride, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4,5]decane (MON4660, CAS 71526-07-3), and 2,2,5-trimethyl-3(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

[204] Also preferred C protective agents are benoxacor, cloquintocet, cyprosulfamide, dichlormide, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, 4-(dichloroacetyl)-1-oxa-4-azaspiro [4,5] decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R29148, CAS 52836-3-4).

[205] Particularly preferred C protection agents, the Petition 870250068507, dated 04 / 08 / 2025, page 131 / 329 120 / 153 which, as component C, are constituents of the composition according to the invention are the protective agents C as defined above; in particular, the plant protection agents C.1-C12 listed below in Table 2: Table 2 Protective agent C C.1 Benoxacor C.2 Cloquintocet C.3 Cyprosulfanamide C.4 Dichlormid C.5 Fenchlorazole C.6 Fenclorim C.7 Furilazole C.8 Isoxadifen C.9 Mefenpyr C.10 Naphthalic acid anhydride C.11 4-(dichloroacetyl)-1-oxa-4-azaspiro [4,5] decane (MON4660, CAS 71526-07-3) C.12 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-3-4)

[206] PPO-inhibiting herbicides (A compounds) and active compounds B of groups b1) to b15) and active compounds C are known herbicides and protective agents, see, for example, The Compendium of Pesticide Common Names (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000 volume 86, Meister Publishing Company, 2000; B. Hock. C. Fedtke. RR Schmidt, Herblzide [Herbicides], Georg Thieme Verlag. Stuttgart 1995; WH Ahrens, Herbicide Handbook, 7th edition. Weed Science Society of America, 1994; and KK Hatzios, Herbicide Handbook, Supplement to the 7th edition. Weed Science Society of America, 1998. 2,2,5-Trimethyl-3(dichloroacetyl)-1,3-oxazolidine [CAS No. 52836-31-4] is also referred to as R29148. 4-(Dichloroacetyl)-1-oxa-4-azaspiro[4,5]decane (CAS No. 71526-07-3] is also referred to as AD-67 and MON 4660.

[207] The assignment of active compounds to their respective mechanisms of action is based on current knowledge. If several mechanisms of action apply to an active compound, this substance has been assigned to only one mechanism of action. Petition 870250068507, dated 04 / 08 / 2025, page 132 / 329 121 / 153

[208] Active compounds B and C having a carboxyl group can be employed in the form of the acid, in the form of a suitable salt as mentioned above, or in the form of an agriculturally acceptable derivative in the compositions according to the invention.

[209] In the case of dicamba, suitable salts include those in which the counter-ion is an agriculturally acceptable cation. For example, suitable dicamba salts are dicamba-sodium, dicamba-potassium, dicamba-methylammonium, dicamba-dimethylammonium, dicamba-isopropylammonium, dicamba-diglycolamine, dicamba-olamine, dicamba-diolamine, dicamba-trolamine, dicamba-N,N-bis-(3-aminopropyl)methylamine and dicamba-diethylenetriamine. Examples of suitable esters are dicamba-methyl and dicamba-butoyl.

[210] Suitable salts of 2,4-D are 2,4-D-ammonium, 2,4-D-dimethylammonium, 2,4-D-diethylammonium, 2,4-D-diethanetammonium (2,4-D-diolamine), 2,4-D-triethanolammonium, 2,4-D-isopropylammonium, 2,4-D-triisopropanolammonium, 2,4-D-heptylammonium, 2,4-D-dodecylammonium, 2,4-D-tetradecylammonium, 2,4-D-triethylammonium, 2,4-D-tri-(2-hydroxypropyl)ammonium, 2,4-D-tris(isopropyl)ammonium, 2,4-D-trolamine, 2,4-D-lithium, 2,4-D-sodium. Examples of suitable 2,4-D esters are 2,4-D-butyl, 2,4-D-2-butoxypropyl, 2,4-D-3-butoxypropyl, 2,4-D-butyl, 2,4-D-methyl, 2,4-D-doctyl, 2,4-D-pentyl, 2,4-D-propyl, 2,4-D-tefuryl, and clacifos. Suitable 2,4-DB salts are, for example, 2,4-DB-sodium, 2,4-DB-potassium, and 2,4-DB-dimethylammonium. Suitable 2,4-DB esters are, for example, 2,4-DB-butyl and 2,4-DB-isoctyl.

[211] Suitable salts of dichlorprop are, for example, dichlorpropsodium, dichlorprop-potassium and di-chlorprop-dimethylammonium. Examples of suitable esters of dichlorprop are dichlorprop-butotyl and dichlorprop-isoctyl.

[212] Suitable salts and esters of MCPA include MCPA-butotil, MCPA-butyl, MCPA-dimethylammonium, MCPA-diolamine, MCPA-ethyl, MCPA-thioethyl, MCPA-2-ethylhexyl, MCPA-isobutyl, MCPA-isoctyl, MCPA-isopropyl, MCPA-isopropylammonium, MCPA-methyl, MCPA-olamine, MCPA-potassium, MCPA-sodium and Petition 870250068507, dated 04 / 08 / 2025, page 133 / 329 122 / 153 MCPA-trolamine. A suitable salt of MCPB is sodium MCPB. A suitable ester of MCPB is ethyl MCPB.

[213] Suitable salts of clopiralid are clopiralid-potassium, clopiralid-olamine and clopiralid-tris-(2-hydroxypropyl)ammonium. An example of suitable esters of clopiralid is clopiralid-methyl.

[214] Examples of a suitable fluroxypyr ester are fluroxypyrmeptyl and fluroxypyr-2-butoxy-1-methylethyl, wherein fluroxypyr-meptyl is preferred.

[215] Suitable salts of picloram are picloramdimethylammonium, picloram-potassium, picloram-triisopropanolammonium, picloram-triisopropylammonium and picloram-trolamine. A suitable ester of picloram is picloram-isooctyl.

[216] A suitable triclopyr salt is triclopyr-triethiammonium. Suitable esters of triclopyr are, for example, triclopyr-ethyl and triclopyr-butotil.

[217] Suitable salts and esters of chloramben include chloramben-ammonium, chloramben-diolamine, chloramben-methyl, chloramben-methylammonium and chloramben-sodium. Suitable salts and esters of 2,3,6-TBA include 2,3,6-TBA-dimethylammonium, 2,3,6-TBA-lithium, 2,3,6-TBA-potassium and 2,3,6-TBA-sodium.

[218] Suitable salts and esters of aminopyralid include aminopyralid-potassium and aminopyralid-tris(2-hydroxypropyl)ammonium.

[219] Suitable glyphosate salts are for example glyphosate-ammonium, glyphosate-diammonium, glyphosate-dimethylammonium, glyphosate-isopropylammonium, glyphosate-potassium, glyphosate-sodium, glyphosate-trimesium as well as ethanolamine and diethanolamine salts, preferably glyphosate-diammonium, glyphosate-isopropylammonium and glyphosate-trimesium (sulfosate).

[220] A suitable glufosinate salt is, for example, glufosinate-ammonium.

[221] A suitable glufosinate-P salt is, for example, Petition 870250068507, dated 04 / 08 / 2025, page 134 / 329 123 / 153 glufosinate-P-ammonium.

[222] Suitable salts and esters of bromoxynil are, for example, bromoxynil-butyrate, bromoxynil-hepatoate, bromoxynil-octanoate, bromoxynil-potassium and bromoxynil-sodium.

[223] Suitable salts and esters of ioxonyl are, for example, ioxonyl-octanoate, ioxonyl-potassium and ioxonyl sodium.

[224] Suitable mecoprop salts and esters include mecopropbutotil, mecoprop-dimethylammonium, mecoprop-diolamine, mecoprop-ethadyl, mecoprop-2-ethylhexyl, mecoprop-isoctyl, mecoprop-methyl, mecoprop-potassium, mecoprop-sodium and meooprop-trolamine.

[225] Suitable mecoprop-P salts are, for example, mecoprop-P-butotil, mecoprop-P-dimethylammonium, mecoprop-P-2-ethylhexyl, mecoprop-P-isobutyl, mecoprop-P-potassium and mecoprop-P-sodium.

[226] A suitable salt of diflufenzopyr is, for example, diflufenzopyr-sodium.

[227] A suitable salt of naptalam is for example naptalamsodium.

[228] Suitable salts and esters of aminocyclopyrachlor are, for example, aminocyclopyrachlor-dimenylammonium, aminocyclopyrachlor-methyl, aminocyclopyrachlor-triisopropanolammonium, aminocyclopyrachlor-sodium and aminocyclopyrachlor-potassium.

[229] A suitable salt of quinclorac is, for example, quincloracdimethylammonium.

[230] A suitable salt of quinmerac is, for example, quinmeracdimethylammonium.

[231] A suitable salt of imazamox is, for example, imazamoxamonium.

[232] Suitable imazapic salts are for example imazapic Petition 870250068507, dated 04 / 08 / 2025, page 135 / 329 124 / 153 ammonium and imazapic-isopropylammonium.

[233] Suitable salts of imazapyr are, for example, imazapyrammonium and imazapyr-isopropylammonium.

[234] A suitable salt of imazaquine is, for example, imazaquine-ammonium.

[235] Suitable salts of imazethapyr are, for example, imazethapyr-ammonium and imazethapyr-isopropylammonium.

[236] A suitable salt of topramezone is, for example, topramezone-sodium.

[237] The preferred embodiments of the invention mentioned below should be understood as being preferred independently of each other or in combination with each other.

[238] According to a preferred embodiment of the invention, the composition comprises as component B at least one, preferably exactly a herbicide B.

[239] According to another preferred embodiment of the invention, the composition comprises at least two, preferably exactly two, herbicides B that are different from each other.

[240] According to another preferred embodiment of the invention, the composition comprises at least three, preferably exactly three, herbicides B that are different from each other.

[241] According to another preferred embodiment of the invention, the composition comprises, as component A, at least one, preferably exactly, a PPO inhibitor herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292- Petition 870250068507, dated 04 / 08 / 2025, page 136 / 329 125 / 153 -6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 125883672-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), and as component B at least one, preferably exactly one, herbicide B.

[242] According to another preferred embodiment of the invention, the composition comprises as component A, at least one, preferably, exactly a PPO inhibitor herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 35329231-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 125883672-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), and at least two, preferably exactly two, herbicides B different from each other.

[243] According to another preferred embodiment of the invention, the composition comprises as component A, at least one, preferably, exactly a PPO inhibitor herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 35329231-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836 Petition 870250068507, dated 04 / 08 / 2025, page 137 / 329 126 / 153 72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), and at least three, preferably exactly three, different herbicides B.

[244] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one herbicidal compound of group b1), in particular selected from the group consisting of clethodim, clodinafop-propargyl, cycloxydim, cihalofop-butyl, fenoxaprop-P-ethyl, fluazifop, pinoxaden, profoxydim, quizalofop, sethoxydim, tepraloxidim, tralcoxidim, esprocarb, prosulfocarb, thiobencarb and trialate.

[245] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS Petition 870250068507, dated 04 / 08 / 2025, page 138 / 329 127 / 153 1258836-72-4), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound from group b2), in particular, selected from the group consisting of bensulfuron-methyl, bispiribacosodium, cloransulam-methyl, cyclosulfuron, diclosulam, flumetsulam, flupyrsulfuronmethyl-sodium, foramsulfuron, halosulfuron-methyl, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, mesosulfuron-methyl, metazosulfuron, nicosulfuron, piroxsulam, propoxycarbazon-sodium, pyrazossulfuron-ethyl, piritiobac-sodium, piroxsulam, rimsulfuron, sulfosulfuron, tiencarbazon-methyl, tifensulfuron-methyl, trifloxysulfuron and tritosulfuron.

[246] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), saflufenacil especially preferred, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b3), in particular, selected from the group consisting of ametryn, atrazine, bentazon, bromoxynil, diuron, fluometuron, hexazinone, isoproturon, linuron, metribuzin, paraquat, paraquat-dichloride, promethrin, propanil, terbutrin and terbuthylazine.;

[247] According to another preferred embodiment of Petition 870250068507, dated 04 / 08 / 2025, pp. 139 / 329 128 / 153 invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound from group b4), in particular, selected from the group consisting of acifluorfen, acifluorfen-sodium, azafenidine, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, clomethoxyfen, cinidone-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin,fluoroglycofen, fluoroglycofen-ethyl, fluticet, fluticet-methyl, falesafen, halosafen, lactofen, oxadiargyl, oxadiazone, oxyfluorfen, pentoxazone, profluazole, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimine, thiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1methyl-6-trifluoromethyl-ethyl)2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2pyridyloxy]acetate (CAS 353292-31-6;S-3100); N-ethyl-3-(2-cloro-6-fluoro-4trifluorometilfenoxi)-5-metil-1H-pyrazole-1-carboxamide (CAS 452099-05-7), Ntetrahydrofurfuryl-3-(2-cloro-6-fluoro-4-trifluorometilfenoxi)-5-metil-1H-pyrazole-1-carboxamide (CAS 452100 03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro2H-benzo [1,4] oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5] triazinan-2,4-dione; 1,5dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-inyl)-3,4-dihydro-2HPetição 870250068507, de 04 / 08 / 2025, pág. 140 / 329; 129 / 153 benzo[b][1,4] oxazin-6-yl)-1,3,5-triazinan-2,4-diona (CAS 1258836-72-4), 2(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7tetrahidro-isoindol-1,3-diona; 1 -Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dionate, methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy] 3-methoxy-butane-2-enoate [CAS 948893-00-3], and 3-[7-Chloro-5-fluoro-2(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4dionate (CAS 212754-02-4).

[248] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound from group b5), in particular, selected from the group consisting of clomazone, diflufenican, flurochloridone, isoxaflutole, mesotrione, picolinafen, sulcotrione, tefuriltrione, tembotrione, topramezone, biciclopyrone, amitrol and flumeturon.;

[249] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6 Petition 870250068507, dated 04 / 08 / 2025, page 141 / 329 130 / 153 trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b6), in particular, selected from the group consisting of glyphosate, glyphosate-isopropylammonium and glyphosate-trimesium (sulfate).

[250] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound from group b7), in particular, selected from the group consisting of gfufosinate, glufosinate-P and glufosinate-ammonium.;

[251] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6 Petition 870250068507, dated 04 / 08 / 2025, page 142 / 329 131 / 153 trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b9), in particular, selected from the group consisting of pendimethalin and trifluralin.

[252] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound from group b10), in particular, selected from the group consisting of acetochlor, cafenstrol,dimethenamide-P, fentrazamide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, fenoxasulfone and piroxasulfone. Similarly, preference is given to compositions comprising, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate, Petition 870250068507, dated 04 / 08 / 2025, page 143 / 329 132 / 153 (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), saflufenacil especially preferred, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b10), in particular, particularly selected from the group consisting of compounds of isoxazoline of formulas II. 1. II.2, II.3, II.4, II.5, II.6. II.7, II.8 and II.9.

[253] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), saflufenacil especially preferred, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b13), in particular, selected from the group consisting of 2,4-D and their salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts, such as aminopyralid-tris(2-hydroxypropyl)ammonium and its salts, clopyralid and its salts and esters, dicamba and its salts and esters, fluroxypyr-meptyl, quinclorac and quinmerac.;

[254] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, Petition 870250068507, dated 04 / 08 / 2025, page 144 / 329 133 / 153 oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100); 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), especially preferably saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b14), in particular, selected from the group consisting of diflufenzopyr and diflufenzopyr-sodium.

[255] According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, flutiacet-methyl, fomesafen, lactofen, oxadiargil, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)-phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100);1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), saflufenacil especially preferred, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), at least one and especially exactly one active herbicidal compound of group b15), in particular, selected from the group consisting of dimron (= daimuron), indafen, indaziflam, oxaziclomephone and triaziflam.;

[256] The term “binary compositions” includes compositions containing one or more, for example 1, 2 or 3, active compounds of the PPO inhibitor herbicide and one or more, for example 1, 2 or 3, herbicides B.

[257] In binary compositions comprising at least one PPO-inhibiting herbicide as component A and at least one herbicide B, the Petition 870250068507, dated 04 / 08 / 2025, p. 145 / 329 134 / 153 The weight ratio of active compounds A:B is generally in the range of 1:1000 to 1000:1, preferably in the range of 1:500 to 500:1, in particular in the range of 1:250 to 250:1 and particularly preferably in the range of 1:75 to 75:1.

[258] In another aspect, the present invention relates to a method for cultivating the plant of the present invention while controlling weeds in the vicinity of said plant, said method comprising the steps of: c) grow plant; and (d) Apply a herbicide composition comprising a PPO-inhibiting herbicide to the plant and weeds, wherein the herbicide normally inhibits protoporphyrinogen oxidase, to a level of herbicide that would inhibit the growth of a corresponding wild-type plant.

[259] In another aspect, the present invention relates to a useful combination for weed control, comprising (a) a nucleic acid molecule of the present invention, whose polynucleotide is capable of being expressed in a plant to thereby provide tolerance to a PPO-inhibiting herbicide; and (b) a PPO-inhibiting herbicide.

[260] In another aspect, the present invention relates to a process for preparing a useful combination for weed control comprising (a) providing a nucleic acid molecule of the present invention, which nucleic acid molecule is capable of being expressed in a plant to thereby provide that plant with tolerance to a PPO-inhibiting herbicide; and (b) providing a PPO-inhibiting herbicide.

[261] In a preferred embodiment, said step of providing a nucleic acid molecule comprises providing a plant containing said nucleic acid molecule.

[262] In another preferred embodiment, the said stage of Petition 870250068507, dated 04 / 08 / 2025, page 146 / 329 135 / 153 providing a nucleic acid molecule includes providing a seed containing the nucleic acid molecule.

[263] Preferably, the said process also includes a step of applying the PPO inhibitor herbicide to the seed.

[264] In another aspect, the present invention relates to the use of a useful combination for weed control, comprising (a) a nucleic acid molecule of the present invention, which nucleic acid molecule is capable of being expressed in a plant to thereby provide that plant with tolerance to a PPO inhibitor herbicide; and (b) a PPO inhibitor herbicide, to control weeds in a plant growing area.

[265] In other respects, a method is provided for the treatment of a plant of the present invention.

[266] In some embodiments, the method involves contacting the plant with an acceptable agronomic composition.

[267] In another aspect, the present invention provides a method for preparing a descendant seed. The method comprises planting a seed of or capable of producing a plant of the present invention. In one embodiment, the method further comprises growing a descendant plant from the seed; and harvesting a descendant seed from the descendant plant. In other embodiments, the method further comprises applying a herbicidal composition to the descendant plant.

[268] In another embodiment, the invention relates to harvestable parts of the transgenic plant according to the present invention. Preferably, the harvestable parts comprise the chimeric nucleic acid molecule or chimeric polypeptide of the present invention. The harvestable parts may be seeds, roots, leaves and / or flowers comprising the chimeric nucleic acid molecule or chimeric polypeptide or parts thereof. Preferred parts of soybean plants are soybean grains comprising the Petition 870250068507, dated 04 / 08 / 2025, p. 147 / 329 136 / 153 chimeric nucleic acid molecule or chimeric polypeptide.

[269] In another embodiment, the invention relates to products derived from a plant according to the present invention, parts thereof or harvestable parts thereof. A preferred plant product is forage, seed flour, oil or seeds coated with seed treatment. Preferably, the flour and / or oil comprises the chimeric polypeptide nucleic acids or chimeric polypeptides of the present invention.

[270] In another embodiment, the invention relates to a method for producing a product, which method comprises a) growth of the plants of the invention or obtainable by the methods of the invention and b) production of said product from or by the plants of the invention and / or parts thereof, for example, seeds of those plants.

[271] In another embodiment, the method comprises the steps of a) to grow the plants of the invention, b) remove the harvestable parts as defined above from the plants and c) to produce said product from or using parts harvested from the invention.

[272] The product can be produced on-site where the plant was grown, the plants and / or their parts can be removed from the site where the plants were grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if possible in repeated cycles, and the product is made from the harvestable parts of the plant. The plant growth step can be performed only once each time the methods of the invention are carried out, allowing the product production steps to be repeated, for example, by repeated removal of harvestable parts from the Petition 870250068507, dated 04 / 08 / 2025, page 148 / 329 137 / 153 plants of the invention and, if necessary, further processing of these parts to arrive at the product. It is also possible that the plant growth step of the invention be repeated and plants or harvestable parts be stored until the product is then produced once for the accumulated plants or plant parts. Furthermore, the steps of cultivating the plants and producing the product can be carried out with an overlap in time, even simultaneously to a large extent or sequentially. Generally, the plants are cultivated for some time before the product is produced.

[273] In one embodiment, the products produced by the aforementioned methods of the invention are plant products such as, but not limited to, a foodstuff, food ingredient, food supplement, feed supplement, fiber, cosmetic and / or pharmaceutical. Foodstuffs are considered to be compositions used for nutrition and / or nutritional supplementation. Animal feed and animal feed supplements, in particular, are considered foodstuffs.

[274] In another embodiment, inventive methods for production are used to produce agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins and the like. Examples Example 1: Cloning expression cassettes

[275] All nucleic acid coding sequences encoding polypeptides comprising SEQ ID NO: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624 are synthesized and cloned by Geneart (Geneart AG, Regensburg, Germany). Rationally designed mutants are synthesized by... Petition 870250068507, dated 04 / 08 / 2025, page 149 / 329 138 / 153 Geneart. Random libraries of PPO genes are synthesized by Geneart. Plasmids are isolated from E. coli TOP10 by performing a plasmid minipreparation and confirmed by DNA sequencing. Example 2: Genetic engineering manipulation of herbicide-tolerant plants containing expression cassettes of the present invention.

[276] Herbicide-tolerant soybean (Glyceine max), corn (Zea mays) and canola (Brassica napus or Brassica rapa var. or Brassica campestris L.) plants are produced by a method as described by Olhoft et al. (US Patent 2009 / 0049567). For transformation of soybean or Arabidopsis thaliana, expression cassettes / constructs comprising chimeric nucleic acid molecules encoding polypeptides comprising SEQ ID NO: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, Seeds 615, 616, 617, 618, 619, 620, 621, 622, 623, or 624 are cloned using standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) into a binary vector containing a resistance marker gene cassette (AHAS) and a chimeric polypeptide sequence (labeled as GOI) between the ubiquitin promoter (PcUbi) and the nopalin synthase (NOS) terminator sequence. For maize transformation, wild-type or chimeric polypeptide sequences are cloned using standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) into a binary vector containing a resistance marker gene cassette (AHAS) and a chimeric polypeptide sequence (labeled as GOI) between the maize ubiquitin promoter (ZmUbi) and the nopalin synthase (NOS) terminator sequence. The binary plasmids are introduced into Agrobacterium tumefaciens for plant transformation.Plasmid constructs are introduced into... Petition 870250068507, dated 04 / 08 / 2025, page 150 / 329 139 / 153 Soybean axillary meristem at the primary node of shoot explants via Agrobacterium-mediated transformation. After inoculation and co-culture with Agrobacterium, the explants are transferred to shoot introduction medium without selection for one week. The explants are subsequently transferred to a shoot induction medium with imazapyr (Arsenal) at 1 to 3 μM for 3 weeks to select for transformed cells. Explants with healthy callus pads / shoots at the primary node are then transferred to shoot elongation medium containing imazapyr at 1 to 3 μM until a shoot elongates or the explant dies. Transgenic seedlings are rooted, subjected to TaqMan analysis for the presence of the transgene, transferred to soil, and grown to maturity in a greenhouse. The transformation of corn plants is done by a method described by McElver and Singh (WO 2008 / 124495).Plant transformation vector constructs containing chimeric polypeptide sequences are introduced into immature maize embryos via Agrobacterium-mediated transformation.

[277] Transformed cells are selected in selection medium supplemented with 0.5-1.5 μM imazethapyr for 3-4 weeks. Transgenic plantlets are regenerated in plant regeneration media and subsequently rooted. Transgenic plantlets are subjected to TaqMan analysis for the presence of the transgene before being transplanted into potting mix and grown to maturity in a greenhouse. Arabidopsis thaliana are transformed with wild-type or chimeric polypeptide sequences by the floral immersion method as described by McElver and Singh (WO 2008 / 124495). Transgenic Arabidopsis plants are subjected to TaqMan analysis for analysis of the number of integration loci. The transformation of Oryza sativa (rice) is done by protoplast transformation as described by Peng et al. (US 6653529). Transgenic soybean, corn, and rice plants T0 or T1 containing chimeric polypeptide sequences are Petition 870250068507, dated 04 / 08 / 2025, page 151 / 329 140 / 153 tested for improved herbicide tolerance in greenhouse and mini-plot studies with the following herbicides: trifludimoxazine, saflufenacil, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron, as a negative control. Example 3: Tissue culture conditions.

[278] An in vitro tissue culture mutagenesis assay was developed to isolate and characterize plant tissues (e.g., maize tissue, rice tissue) tolerant to protoporphyrinogen oxidase inhibitor herbicides (trifludimoxazin, saflufenacil, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as a negative control). The assay utilizes somaclonal variation found in in vitro tissue culture. Spontaneous mutations derived from somaclonal variation can be increased by chemical mutagenesis and subsequent gradual selection at increasing herbicide concentrations.

[279] The present invention provides tissue culture conditions to encourage the growth of friable calluses, embryogenic maize or rice calluses that can be regenerated. The calluses are initiated from 4 different maize or rice cultivars, encompassing the varieties Zea mays and japonica (Taipei 309, Nipponbare, Koshihikari) and indica (Indica 1), respectively. The seeds are surface sterilized in 70% ethanol for approximately 1 min followed by 20% Clorox commercial bleach for 20 minutes. The seeds are washed with sterile water and plated on callus induction medium. Several callus induction media are tested. The ingredient lists for the tested media are presented in Table 3. Table 3 Ingredient Supplier R001M R025M R026M R327M R008M MS711R Vitamins B5 Sigma 1.0 Petition 870250068507, dated 04 / 08 / 2025, p. 152 / 329 141 / 153 Ingredient Supplier R001M R025M R026M R327M R008M MS711R Vitamins N6 Phytotech 1.0 X 1.0 X L-proline Sigma 2.9 g / L 0.5 g / L 1.2 g / L Cosamic acids BD 0.3 g / L Hydrolysis 0.3 g / L, 23 g / L of casein Sigma 1.0 g / L Phytotech L-Asp monohydrate 150 mg / L Nicotinic acid Sigma 0.5 mg / L Pyridoxine HCl Sigma 0.5 mg / L Thiamine HCl Sigma 1.0 mg / L Myo-inositol Sigma 100 mg / L Sigma MES 500 mg / L 500 mg / L 500 mg / L 500 mg / L 500 mg / L 500 mg / L Maltose VWR 30 g / L 30 g / L 30 g / L 30 g / L Sorbitol Duchefa 30 g / L Sucrose VWR 10 g / L 30 NA NA 50 μg / L Duche 2,4-D Sigma 2.0 mg / L 1.0 mg / L MgCl2.6H2O VWR 750 mg / L pH 5.8 5.8 5.8 5.8 5.8 5.7 Gelrita Duchefa 4 g / L 2.5 g / L Agarose type 1 Sigma 7.0 g / L 10 g / L Autoclave 15 min 15 min 15 min 15 min 15 min 20 min Cetina Sigma 2.0 mg / L 2.0 mg / L NAA Duchefa 1.0 mg / L 1.0 mg / L ABA Sigma 5.0 mg / L Cefotaxima Duchefa 0.1 g / L 0.1 g / L Duchefa 0.1 g / L Vancocine 0.1 g / L 0.1 g / L 0.1 g / L Dissulfate G418 Sigma 20 mg / L 20 mg / L 20 mg / L

[280] The R001M callus induction medium is selected after testing numerous variations. Cultures are kept in the dark at 30 °C. Embryogenic callus is subcultured in fresh medium after 10 to 14 days. Example 4: Selection of herbicide-tolerant callus.

[281] Once the tissue culture conditions were determined, further selection conditions were established by analyzing tissue survival in death curves with trifludimoxazine, saflufenacil, phenylpyridines, flumioxazin, butafenacil, acifluorfen, lactofen, Petition 870250068507, dated 04 / 08 / 2025, page 153 / 329 142 / 153 bifenox, sulfentrazone, and the photosynthesis inhibitor diuron, as negative controls. Careful consideration of herbicide accumulation in the tissue, as well as its persistence and stability in cells and culture medium, was performed. Through these experiments, a sublethal dose was established for the initial selection of mutated material. After establishing the initial dose of trifludimoxazine, saflufenacil, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and the photosynthesis inhibitor diuron as negative controls in the selection media, tissues are selected in a stepped manner by increasing the concentration of the PPO inhibitor in each transfer until cells that grew vigorously in the presence of toxic doses are recovered. The resulting calluses are subsequently subcultured every 3-4 weeks to R001M with a selective agent. More than 26.000 calluses are subjected to selection for 4-5 subcultures until the selective pressure is above toxic levels, as determined by death curves and observations of continued culture. Alternatively, liquid cultures are started from calluses in MS711R with slow agitation and weekly subcultures. Once the liquid cultures are established, the selection agent is added directly to the flask in each subculture. After 2-4 cycles of liquid selection, the cultures are transferred to filters in solid R001M medium for further growth. Example 5: Plant Regeneration.

[282] The tolerant tissue is regenerated and characterized molecularly for the presence of chimeric nucleic acid molecules and / or biochemically for altered enzymatic activity in the presence of the selective agent. In addition, genes directly and / or indirectly involved in tetrapyrrole biosynthesis and / or metabolic pathways are also sequenced to characterize mutations. Finally, enzymes that alter fate (e.g., metabolism, translocation, transport) are also sequenced. Petition 870250068507, dated 04 / 08 / 2025, page 154 / 329 143 / 153 to characterize mutations. After herbicide selection, calluses are regenerated using a medium regime of R025M for 10–14 days, R026M for approximately 2 weeks, R327M until well-formed shoots are developed, and R008S until shoots are well-rooted for transfer to the greenhouse. Regeneration is carried out in light. No selection agent is included during regeneration. Once strong roots are established, M0 regenerants are transplanted to the greenhouse in square or round pots.

[283] Transplants are kept under a clear plastic cup until they are adapted to greenhouse conditions. The greenhouse is set to a 27°C / 21°C (80°F / 70°F) day / night cycle with 600W high-pressure sodium lights supplementing the light to maintain a 14-hour day. Plants are watered as needed, depending on the climate, and fertilized daily. Example 6: Sequence Analysis.

[284] Leaf tissue is collected from clonal plants separated for transplantation and analyzed as individuals. Genomic DNA is extracted using a Wizard® 96 Magnetic DNA Plant System kit (Promega, US Patent Nos. 6,027,945 and 6,368,800) as directed by the manufacturer. The isolated DNA is amplified by PCR using the appropriate forward and reverse primer.

[285] PCR amplification is performed using Hotstar Taq DNA Polymerase (Qiagen) using the adherence thermocycling program as follows: 96 °C for 15 min, followed by 35 cycles (96 °C, 30 sec; 58 °C - 0.2 °C per cycle, 30 sec; 72 °C, 3 min and 30 sec), 10 min at 72 °C. PCR products are checked for concentration and fragment size by agarose gel electrophoresis. Dephosphorylated PCR products are analyzed by direct sequencing using PCR primers (DNA Landmarks, or Entelechon). Chromatogram trace files (.scf) are Petition 870250068507, dated 04 / 08 / 2025, page 155 / 329 144 / 153 analyzed for mutations relative to the wild-type gene using NTI Advance 10™ vector (Invitrogen). Based on sequence information, mutations are identified in several individuals. Sequence analysis is performed on representative chromatograms and in the corresponding AlignX alignment with default settings and edited to call out secondary peaks. Example 7: Demonstration of herbicide tolerance.

[286] Transgenic T0 or T1 plants of soybean, corn, canola and rice varieties containing the expression cassettes of the present invention encoding polypeptides comprising the SEQ ID NOs sequences: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616,Plants 617, 618, 619, 620, 621, 622, 623, or 624 are tested for improved herbicide tolerance in greenhouse and mini-plot studies with the following herbicides: trifludimoxazin, saflufenacil, phenylpyridine, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, S-3100, thiafenacil, and mixtures thereof, and the photosynthesis inhibitor diuron as a negative control. For pre-emergence treatment, the herbicides are applied directly after sowing using fine-spray nozzles. The containers are gently irrigated to promote germination and growth and subsequently covered with clear plastic covers until the plants have rooted. This covering causes uniform germination of the test plants, unless this has been impaired by the herbicides. For post-emergence treatment, test plants first grow to a height of 3 to 15 cm, depending on the plant's growth habit, and only then are they treated with herbicides.For this purpose, the test plants are either sown directly and grown in the same containers, or they are first grown separately and transplanted into the test containers a few days before treatment. Petition 870250068507, dated 04 / 08 / 2025, p. 156 / 329 145 / 153

[287] For T0 plant testing, cuttings can be used. In the case of soybean plants, an optimal sprout for the cutting is about 7.5 to 10 cm tall, with at least two nodes present. Each cutting is taken from the original transformant (mother plant) and dipped in rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. Wild-type cuttings are also taken simultaneously to serve as controls. The cuttings are kept in the bio-dome for 5-7 days and then transplanted into pots and then acclimatized in the growth chamber for another two days. Subsequently, the cuttings are transferred to the greenhouse, acclimatized for approximately 4 days and then subjected to spray tests as indicated. Depending on the species, the plants are kept at 10 to 25 °C or 20 to 35 °C. The test period extends for 3 weeks.During this time, the plants are cared for and their response to individual treatments is evaluated. Herbicide damage assessments are performed 2 and 3 weeks after treatment. Plant damage is classified on a scale of 0% to 100%, with 0% being no damage and 100% being complete death. The results are shown in Figure 20.

[288] Transgenic Arabidopsis thaliana plants are tested for improved tolerance to trifludimoxazin, saflufenacil, phenylpyridine, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, S-3100, thiafenacil, and mixtures thereof, and photosynthesis inhibitor diuron as a negative control, in 48-well plates. Consequently, T2 seeds are surface-sterilized by agitation for 5 min in ethanol + water (70 + 30 by volume), washing once with ethanol + water (70 + 30 by volume) and twice with sterile deionized water. The seeds are resuspended in 0.1% agar dissolved in water (w / v). Four to five seeds per well are plated on solid nutrient medium, consisting Petition 870250068507, dated 04 / 08 / 2025, page 157 / 329 146 / 153 in a half-strength Murashige nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497). The compounds are dissolved in dimethyl sulfoxide (DMSO) and added to the medium before solidification (final DMSO concentration 0.1%). Multi-well plates are incubated in a growth chamber at 22 °C, 75% relative humidity and 110 μmol Phot * m-2* s-1 with a light:dark photoperiod of 14:10 h. Growth inhibition is evaluated seven to ten days after sowing, compared to wild-type plants.

[289] Additionally, transgenic Arabidopsis T1 plants are tested for improved herbicide tolerance in greenhouse studies with the following herbicides: trifludimoxazin, saflufenacil, phenylpyridine, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, S-3100, thiafenacil, and mixtures thereof, and photosynthesis inhibitor diuron as a negative control. The results are shown in Table 4 and Figures 1 to 17. Table 4 SEQ ID NO. Construction Saflufenacil Trifludimoxazine Tolerance Factor 117 hemG_tpFNR 332 4433 119 hemG_tpsAMATU_PP02 1 1 118 hemG_tpIAMATU_PP02 2200 9167 114 hemF_tpFNR 1 1 116 hemF_tpsAMATU_PP02 1 1 115 hemF_tpIAMATU_PP02 1 1 123 RHOM_PPO_wt_tpFNR 1 1 127 RHOM_PPO_wt_tpAMATU_PP02 1 1 124 RHOMA_PPO_F420V_tpFNR 1 1 121 OPITE_PPO_wt_tpFNR 14 9 128 CHLSP_PPO_wt_tpFNR 640 1500 Petition 870250068507, dated 04 / 08 / 2025, page 158 / 329 147 / 153

[290] In addition, transgenic Arabidopsis T2 plants are tested for improved tolerance to trifludimoxazine and saflufenacil in greenhouse studies. The results are presented in Figures 18 and 19.

[291] Additionally, several transgenic Arabidopsis plants were treated with various PPO-inhibiting herbicides in greenhouse studies. The results are presented in Table 5, Table 6 and Figures 21 to 23. Table 5 Evaluation of herbicide lesions 14 days after herbicide treatment. 100 = total plant lesion, 0 = no lesion observed compared to the untreated area. Wild type / non-transgenic SEQ ID NO. 119 SEQ ID NO. 119 SEQ ID NO. 117 SEQ ID NO. 117 SEQ ID NO. 118 SEQ ID NO. 118 SEQ ID NO. 121 SEQ ID NO. 121 SEQ ID NO. 128 SEQ ID NO. 128 ABICQRGHDE g ai / ha Saflufenacil + 1% MSO 300 100 100 100 95 97 68 48 100 100 97 97 150 100 100 100 99 98 58 65 100 100 97 97 50 100 100 100 94 100 55 5 100 100 93 97 Trifludimoxazine + 1% MSO 150 100 100 100 98 97 58 10 100 100 93 97 75 100 100 100 99 93 50 23 100 100 93 95 25 100 100 100 90 92 45 20 100 100 85 85 Sulfentrazone + 1% MSO 1120 100 98 98 48 40 100 100 95 95 560 100 85 99 40 18 100 100 83 93 280 100 89 83 25 15 100 100 73 70 Flumioxazin + 1% MSO 420 100 97 95 60 28 100 100 93 100 280 100 94 95 60 28 100 100 94 99 140 100 90 94 48 23 100 100 100 93 Saflufenacil + Trifludimoxazine + 1% MSO 300+150 100 100 100 100 97 65 63 100 100 98 97 150+75 100 100 100 95 97 60 58 100 100 95 98 50+25 100 100 100 97 97 58 53 100 100 93 98 Saflufenacil + Sulfentrazone + 1% MSO 300+1120 100 100 99 83 60100 100 98 99 150+560 100 99 99 63 38 100 100 94 98 50+280 100 97 97 58 28 100 100 95 97 Saflufenacil + Flumioxazin + 300+420 100 95 99 63 43 100 100 99 98 150+280 100 99 95 65 55 100 100 100 98 Petition 870250068507, dated 04 / 08 / 2025, page 159 / 329 148 / 153 Wild type / non-transgenic SEQ ID NO. 119 SEQ ID NO. 119 SEQ ID NO. 117 SEQ ID NO. 117 SEQ ID NO. 118 SEQ ID NO. 118 SEQ ID NO. 121 SEQ ID NO. 121 SEQ ID NO. 128 SEQ ID NO. 128 1% MSO 50+140 100 98 99 55 23 100 100 100 99 200 100 100 98 78 80 100 100 93 97 Phenylpyridine + 1% trace 100 100 99 100 78 73 100 100 97 97 50 100 98 95 70 65 100 100 93 90 Table 6 Evaluation of herbicide lesions 14 days after herbicide treatment. 100 = no damage observed compared to the untreated test. SEQ ID NO. 120 SEQ ID NO. 120 SEQ ID NO. 118 SEQ ID NO. 118 Wild type / non-transgenic Number of applications (2 - 4) and Rate (g ai / ha) JMQR S3100 + MSO (2) 200 + 1% 80 89 65 53 100 (3) 100 + 1% 95 85 65 63 100 (4) 50 + 1% 75 97 65 60 100 Saflufenacil + MSO (2) 300 + 1% 85 88 65 60 100 (3) 150 + 1% 83 93 63 45 100 (4) 50 + 1% 80 95 63 38 100 Trifludimoxazine + MSO (2) 150 + 1% 98 93 65 50 100 (3) 75 + 1% 94 98 63 28 100 (4) 25 + 1% 80 94 60 18 100 Saflufenacil + Trifludimoxazine + MSO (2) 300 + 150 + 1% 95 98 65 70 100 (3) 150 + 75 + 1% 88 85 68 40 100 (4) 50 + 25 + 1% 73 70 70 40 100 Flumioxazine + MSO (2) 420 + 1% 95 98 70 35 100 (3) 280 + 1% 85 98 65 60 100 (4) 140 + 1% 95 75 60 63 100 Petition 870250068507, dated 04 / 08 / 2025, page 160 / 329 149 / 153 Example 8: Herbicide selection using tissue culture.

[292] The medium is selected for the use-kill curves developed as specified above. Different techniques are used for selection. A stepwise selection is applied, or an immediate lethal level of herbicide is applied. In both cases, all calluses are transferred for each new round of selection. Selection is for 4-5 culture cycles with 3-5 weeks for each cycle. Calluses are placed on nylon membranes to facilitate transfer (200 micron pore sheets, Biodesign, Saco, Maine). The membranes are cut to fit 100x20 mm Petri dishes and are autoclaved before use. 25-35 calluses (average weight / callus being 22 mg) are used in all plates. In addition, a set of calluses is subjected to selection in liquid culture medium with weekly subcultures followed by further selection in semi-solid medium.Mutant lines are selected using trifludimoxazine, saflufenacil, flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and the photosynthesis inhibitor diuron as negative controls. The efficiency of obtaining mutants is high, based on the percentage of calluses that gave rise to a mutant line that can be regenerated or the number of lines, as determined by gram of tissue used. Example 9: Transformation of whole maize plants and tolerance testing to PPO inhibitors.

[293] Immature embryos are transformed according to the procedure described in Peng et al. (WO 2006 / 136596). Plants are tested for the presence of T-DNA by Taqman analysis, with the terminator being the target, which is present in all constructs. Apparently healthy plants are sent to the greenhouse for hardening and subsequent spray testing. Plants are transplanted individually into MetroMix 360 soil in 4” pots. Once in the greenhouse Petition 870250068507, dated 04 / 08 / 2025, page 161 / 329 In a 150 / 153 light cycle (27°C / 21°C day / night cycle with a 14-hour duration supported by 600W high-pressure sodium lamps), the plants were able to grow for 14 days. They were then sprayed with a treatment of 25 to 200 g ai / ha of saflufenacil + 1.0% v / v methylated seed oil (MSO) and / or 25 to 200 g ai / ha of trifludimoxazin with 1% MSO. Other PPO-inhibiting herbicides were also similarly tested to confirm cross-resistance: flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and the photosynthesis inhibitor diuron as a negative control. Herbicide lesion assessments were performed at 7, 14, and 21 days after treatment. Herbicide damage assessments are performed at 2, 7, 14, and 21 days post-spraying to look for damage at new growth points and overall plant health. Top survivors are transplanted into containers filled with MetroMix 360 for seed production. Example 10: Soybean transformation and PPO inhibitor tolerance test.

[294] Jake cv. soybean is transformed as previously described by Siminszky et al., Phytochem Rev. 5: 445-458 (2006). After regeneration, the transformants are transplanted into soil in small pots, placed in growth chambers (16 h day / 8 h night: 25 °C day / 23 °C night: 65% relative humidity; 130-150 µE m-2 s-1) and subsequently tested for the presence of T-DNA by Taqman analysis. After a few weeks, healthy, single-copy positive transgenic plants are transplanted into larger pots and allowed to grow in the growth chamber. An ideal shoot for cutting is about 3-4 centimeters tall, with at least two nodes present. Each cutting is taken from the original transformant (mother plant) and dipped in rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. The mother plant is then taken to... Petition 870250068507, dated 04 / 08 / 2025, p. 162 / 329 151 / 153 maturity in the greenhouse and harvested for seed. Wild-type cuttings are also taken simultaneously to serve as negative controls. The cuttings are kept in the bio-dome for 5-7 days and then transplanted into 3-inch pots and then acclimated in the growth chamber for another two days. Subsequently, the cuttings are transferred to the greenhouse, acclimated for approximately 4 days, and then sprayed with a treatment of 0-200 g ai / ha of saflufenacil plus 1% MSO and / or 25-200 g ai / ha of trifludimoxazin plus 1% MSO. Other PPO-inhibiting herbicides are also tested similarly to confirm cross-resistance: flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and the photosynthesis inhibitor diuron, as negative controls. Assessments of herbicide damage were performed 2, 7, 14, and 21 days after treatment. The results are shown in Figure 20. Table 7 Phenotypic Classification (phytotoxicity) of surviving plants: 1. No obvious damage (no phytotoxicity); 2. Minor leaf damage, plant will survive; 3. Moderate leaf damage, plant will survive; 4. Severe leaf damage, plant will survive; 5. No surviving plants - all plants have died / are dying.

[295] Next, a definition of the injury classifications measured above is presented: Table 8 Classification Description of the lesion 0 No lesion 1 Minimal lesion, only a few spots of leaf damage or chlorosis. 2 Minimal lesion with slightly stronger chlorosis. The spots Petition 870250068507, dated 04 / 08 / 2025, page 163 / 329 152 / 153 Classification: Description of the lesion: General growth points remain undamaged. 3: Slightly stronger lesion in secondary leaf tissue, but primary leaf and growth points are still undamaged. 4: The overall morphology of the plant is slightly different, some chlorosis and necrosis in secondary growth points and leaf tissue. Stems are intact. Regeneration is highly likely within 1 week. 5: The overall morphology of the plant is clearly different, some chlorosis and necrosis in some leaves and growth points, but the primary growth point is intact. Stem tissue is still green. Regeneration is highly likely within 1 week. 6: Strong lesions can be seen in young leaf growth. The plant has a high probability of surviving only through regeneration at different growth points. Most leaves are chlorotic / necrotic, but stem tissue is still green.It may regenerate, but with noticeable lesions. 7 Most active growth points are necrotic. There may be a single growth point that survives and may be partially chlorotic or green and partially necrotic. Two leaves may still be chlorotic with some green; the rest of the plant, including the stem, is necrotic. 8 The plant will likely die and all growth points are necrotic. One leaf may still be chlorotic with some green. The rest of the plant is necrotic. 9 The plant is dead. *Not tested. Example 14: Transient protein expression in tobacco leaves.

[296] Transient expression of chimeric polypeptide sequences (e.g. SEQ ID NOs: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623 or 624) can be done as described previously (Voinnet O., et al., 2003, The Plant Journal 33. 949-956). In summary, the cloning of the transformation of GOI and Petition 870250068507, dated 04 / 08 / 2025, p. 164 / 329 153 / 153 Agrobacterium (strain: GV2260) is prepared as described in EXAMPLE 5. Young leaves of Nicotiana benthamiana are infiltrated with a suspension of transgenic Agrobacterium (OD600 of 1.0) harboring binary vector constructs containing a GOI gene controlled by a promoter and terminator sequence. 48 to 72 hours after infiltration, leaf disc punctures (0.75 cm in diameter) are transferred to 6-well plates with medium (half-strength Linsmaier-Skoog (Linsmaier and Skoog (1965) Physiol. Plant. 18: 100-127) nutrient solution or water) containing herbicide of interest at different concentrations. Multi-well plates are incubated in a growth chamber at 22 °C, 75% relative humidity and 110 μmol Phot * m-2* s-1 with a 14:10 h light: dark photoperiod. Example 15: Demonstration of herbicide tolerance of transiently transformed tobacco leaf discs.

[297] Leaf discs, generated as described in EXAMPLE 14, expressing a GOI-encoded protein are subjected to enhanced herbicide treatment tolerance analysis. For herbicide damage analysis, chlorophyll fluorescence is identified as an indicative marker (Dayan and Zaccaro (2012) Pest. Biochem. Physiol. 102: 189-197). In addition to monitoring the herbicide effect by visual inspection, the photosynthetic yield of photosystem II is measured using a MAXI PAM imaging machine (IMAGINE-PAM M-Series, Walz, Effeltrich, Germany) 48 hours after the start of herbicide treatment. PSII yield is measured according to the manufacturer's instructions. Tolerance factors are calculated based on the IC50 values ​​of PSII yield inhibition of transformed versus vector-free transformed leaf discs.The IC50 of PSII yield inhibition on vector-free transformed leaf discs treated with saflufenacil or trifludimoxazin for 48 h was measured with 1.1 x 10-7M or 1 x 10-8M, respectively. Petition 870250068507, dated 04 / 08 / 2025, page 165 / 329

Claims

1 / 3 Claims 1. A recombinant chimeric nucleic acid molecule, characterized by comprising: a nucleic acid sequence with SEQ ID NO: 105 encoding a polypeptide with SEQ ID NO: 118;or a nucleic acid sequence with SEQ ID NO: 107 encoding a polypeptide with SEQ ID NO: 120, wherein the nucleotides at positions 1-294 of SEQ ID NO: 105 and the nucleotides at positions 1-321 of SEQ ID NO: 107 encode a dual transit peptide and the nucleotides at positions 295-837 of SEQ ID NO: 105 and the nucleotides at positions 322-864 of SEQ ID NO: 107 encode a heterologous polypeptide of interest, wherein the nucleic acid region encoding the dual transit peptide is operationally linked to the nucleic acid region encoding the heterologous polypeptide of interest, and wherein said heterologous polypeptide of interest is from a prokaryote that, when overexpressed in a plant, confers herbicide tolerance to said plant, and wherein said polypeptide possesses PPO activity and confers tolerance to PPO-inhibiting herbicides.

2. MOLECULE, according to claim 1, characterized by said polypeptide having PPO activity comprising the sequence having the SEQ ID NO: 320, 321, 451 or 586.

3. EXPRESSION CASSETTE, characterized by comprising the nucleic acid molecule, as defined in any one of claims 1 to 2, operatively linked to a promoter that drives expression in a plant.

4. METHOD FOR EXPRESSING A NUCLEIC ACID, which encodes a polypeptide of interest in a plant, characterized by comprising: (a) introducing into a plant cell the nucleic acid molecule, as defined in either of claims 1 to 2, or the expression cassette, as defined in claim 3, and (b) regenerating a plant from the same comprising the nucleic acid molecule, as defined in either of claims 1 to 2, or the expression cassette, as defined in claim 3.

5. METHOD FOR PRODUCING A HERBICIDE-TOLERANT PLANT, characterized by comprising: (a) introducing into a plant cell the nucleic acid molecule, as defined in either of claims 1 to 2, or the expression cassette, as defined in claim 3, and (b) regenerating a plant from the same comprising the nucleic acid molecule, as defined in either of claims 1 to 2, or the expression cassette, as defined in claim 3.

6. METHOD FOR CONTROLLING UNDESIRABLE VEGETATION, in a plant growing area, characterized in that the method comprises the steps of: (a) providing in that area a herbicide-tolerant plant comprising the nucleic acid molecule, as defined in either of claims 1 to 2, or the expression cassette, as defined in claim 3; (b) applying to that area an effective amount of a herbicide, wherein the effective amount of said herbicide does not kill or inhibit the growth of the herbicide-tolerant plant of (a).

7. METHOD FOR GROWING A PLANT comprising a plant cell comprising the expression cassette as defined in claim 3, and comprising the expression cassette as defined in claim 3, while controlling weeds in the vicinity of said plant, characterized in that said method comprises the steps of: (a) growing said plant; and (b) applying a herbicide composition comprising a PPO-inhibiting herbicide to the plant and to the weeds, wherein the herbicide normally inhibits protoporphyrinogen oxidase, at a level of the herbicide that would inhibit the growth of a corresponding wild-type plant.

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