System for gene expression in a plant, methods, plants comprising bromoform production and composition genes

Description

1 / 54 SYSTEM FOR GENE EXPRESSION IN A PLANT, METHODS, PLANTS COMPRISING BROMOFORM PRODUCTION GENES AND COMPOSITION FIELD OF THE INVENTION

[001] This disclosure is generally directed to transgenic plants. Specifically, the invention relates to transgenic plants for use in reducing greenhouse gas emissions from ruminant animals. BACKGROUND OF THE INVENTION

[002] Agriculture plays a fundamental role in food production worldwide and is a major factor in the gross domestic product of many countries. Livestock production is essential for generating high-quality protein foods and supplying food in regions where animal products are the main source of food. The environmental impacts of livestock production have been examined for decades, but recently methane emissions from enteric fermentation have been identified as a substantial source of greenhouse gases.

[003] Methane (CH4) is recognized as the second most important greenhouse gas emitted from anthropogenic sources and largely results from the microbial activity of methanogenic bacteria in the first chamber of the ruminant stomach (rumen). Ruminants are animals that have a multi-chambered stomach that relies on bacteria to break down plant-based food. In this digestive process, an adult cow can release up to 500 liters of methane into the atmosphere per day, contributing approximately 17% of total global anthropogenic methane emissions.

[004] Asparagopsis taxiformis is a species of red algae. Researchers have shown that feeding cows a diet Petition 870250094916, dated 10 / 17 / 2025, page 8 / 154 2 / 54 containing one to two percent of these algae reduced its methane emissions by more than 90%. As demonstrated later, this effect was obtained without compromising other fermentation parameters (i.e., volatile fatty acid production) and with A. taxiformis organic matter inclusion rates of up to 5%. This effect was due to the inhibition of methanogen fermentation in the rumen by brominated organic hydrocarbons, such as bromoform, present in A. taxiformis. However, bromoform-producing algae, such as A. taxiformis, grow relatively slowly in deep seawater and therefore their large-scale production is not profitable.

[005] Several attempts have been made to clone vanadate-dependent haloperoxidases (VHPOs) from algae and bacteria into various organisms, however, this has been difficult due to low yield and toxicity problems.

[006] Still, there is a need for an efficient system to feed the livestock and, at the same time, reduce methane emissions. SUMMARY OF THE INVENTION

[007] The following modalities and aspects are described and illustrated together with compositions and methods that are intended to be exemplary and illustrative, not limited to the scope. In several modalities, one or more of the problems described above have been reduced or eliminated, while other modalities are directed towards other advantages or improvements.

[008] The invention relates to the preparation of transgenic pasture and forage plants carrying bromoform-producing genes derived from red or brown algae, in order to reduce methane emissions generated by ruminants. Petition 870250094916, dated 10 / 17 / 2025, p. 9 / 154 3 / 54

[009] Advantageously, genetically modified bromoform-producing pasture plants can be sown in pasture fields for consumption by ruminant animals. Alternatively, feed can be prepared from genetically modified bromoform-producing plants and fed to ruminant animals.

[010] In some embodiments, the present invention provides a system for gene expression in a plant, including one or more recombinant nucleic acid molecules comprising one or more nucleotide sequences joined together, including two or more genes, wherein: each gene is functionally linked to a promoter for expression in the plant; and the two or more genes encode two or more enzymes involved in bromoform production and not encoded by the native plant.

[011] In some embodiments, the two or more genes are included in a single nucleic acid molecule. In some embodiments, the two or more genes are included in two or more nucleic acid molecules.

[012] In some forms, at least one of the two or more genes are from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus, Macrocystis pyrifera, Medicago sativa and Medicago truncatula.

[013] In some forms, at least two of the two or more genes are from at least one non-plant organism.

[014] In some embodiments, the two or more enzymes include at least one haloperoxidase.

[015] In some embodiments, the two or more enzymes include an NAD(P)H Oxidase (NOX). Petition 870250094916, dated 10 / 17 / 2025, p. 10 / 154 4 / 54

[016] In some embodiments, the two or more enzymes include at least one enzyme involved in fatty acid synthesis.

[017] In some embodiments, at least one of the enzymes involved in fatty acid synthesis is selected from Beta-ketoacyl-[acyl carrier protein] synthase III (FabH), phosphopantetheine transferase (sfp), acetyl-CoA carboxylase (ACCase) and malonyl CoA synthetase (MatB).

[018] In some embodiments, the two or more enzymes include haloperoxidase, NOX and at least one enzyme involved in fatty acid synthesis selected from FabH, sfp, ACCase and MatB.

[019] In some embodiments, the two or more enzymes include haloperoxidase, NOX, FabH, sfp and ACCase.

[020] In some modalities, two or more genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase and MatB.

[021] In some forms, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4 and at least one gene selected from FaBH, Sfp, ACCase and MatB.

[022] In some forms, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, FaBH, Sfp and ACCase.

[023] In some forms, haloperoxidase has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with respect to SEQ ID NO: 1 (Mbb1), SEQ ID NO: 3 (Mbb3) or SEQ ID NO: 4 (Mbb4); NOX has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with respect to SEQ ID NO: 2 (Mbb2); FabH has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with respect to SEQ ID NO: 5; sfp has at least 70, 75, 80, 85, 90, or 99% sequence identity with respect to SEQ ID NO: 6; Petition 870250094916, dated 10 / 17 / 2025, p. 11 / 154 5 / 54 and / or ACCase has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with respect to SEQ ID NO: 7.

[024] In some embodiments, the two or more genes are functionally linked to identical promoters. In some embodiments, the two or more genes are functionally linked to different promoters. In some embodiments, at least one of the promoters is a constitutive promoter. In some embodiments, at least one of the promoters is an inducible promoter. In some embodiments, the inducible promoter is a tissue-specific promoter, activated in plant organs that develop late in the plant life cycle, such as leaves or flowers. In some embodiments, at least one of the two or more genes also encodes a chloroplast transit peptide to direct enzymes to the chloroplast.

[025] In some embodiments, the plant is a pasture plant or a forage plant. In some embodiments, the plant is a grass, a cereal, or a legume. In some embodiments, the plant is selected from brachiaria grass (Brachiaria), alfalfa (Medicago sativa, alfalfa), ryegrass, tall fescue, dactylus, clover, oats, millet, kikuyu, chicory, corn, soybean, and forage plantain.

[026] In some forms, this application provides a composition that includes the system disclosed here.

[027] In some embodiments, this application provides a method for preparing a genetically modified plant or plant part; the method includes transforming a plant or plant part with the system described in this document, or with the composition presented here that includes the aforementioned system. Petition 870250094916, dated 10 / 17 / 2025, p. 12 / 154 6 / 54

[028] In some embodiments, the present application provides a genetically modified plant or plant part, including the system disclosed in this document or prepared by the method for obtaining a genetically modified plant or plant part described herein.

[029] In some embodiments, the present application provides a genetically modified plant or plant part, including one or more exogenous nucleotide sequences including two or more genes, wherein each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production and not encoded by the native plant.

[030] In some embodiments, the present application provides a genetically modified plant cell, including the system disclosed in this document or prepared by the method for obtaining a genetically modified plant or plant part described herein.

[031] In some embodiments, the present application provides a genetically modified plant cell, including one or more exogenous nucleotide sequences including two or more genes, wherein each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production and not encoded by the native plant.

[032] In some varieties, the plant is selected from brachiaria grass (Brachiaria), alfalfa (Medicago sativa, alfalfa), ryegrass, tall fescue, dactylus, clover, oats, millet, kikuyu, chicory, corn, soybean and forage plantain. Petition 870250094916, dated 10 / 17 / 2025, p. 13 / 154 7 / 54

[033] In some forms, the plant part is selected from seed, cell, leaf, stem, root, tuber, cutting, flower, bark, fruit, bulb, trichome or rhizome.

[034] In some embodiments, the present application provides a composition that includes the genetically modified plant or plant part disclosed in this document combined with a plant or plant part that is not capable of producing bromoform, wherein the composition includes up to about 10% of genetically modified plant or plant part.

[035] In some embodiments, the present application provides a method for reducing methane gas emissions generated by a ruminant animal, the method includes: production of a genetically modified seed of a pasture plant, the seed including two or more genes involved in the production of bromoform; Sowing the genetically modified seed in a field where a ruminant animal grazes; and cultivating a bromoform-producing pasture plant derived from the seed, causing the bromoform-producing pasture plant to be consumed by the ruminant animal.

[036] In some embodiments, the genetically modified seed is produced by the method for preparing a genetically modified plant or plant part disclosed in this document.

[037] In some forms, the ruminant animal is a cow. Petition 870250094916, dated 10 / 17 / 2025, page 14 / 154 8 / 54

[038] In some embodiments, the present application provides a method for preparing feed to reduce methane gas emissions generated by a ruminant animal, the method includes: production of a genetically modified seed of a forage plant, the seed including two or more genes involved in the production of bromoform; sowing of genetically modified seeds; cultivation of a bromoform-producing forage plant from seed; and preparation of a feed containing the bromoform-producing forage plant or a part thereof.

[039] In some embodiments, the genetically modified seed is produced by the method for preparing a genetically modified plant or plant part disclosed in this document.

[040] In some forms, the ruminant animal is a cow.

[041] Certain embodiments of this disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art based on the figures, descriptions, and claims included in this description. Furthermore, although specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.

[042] In addition to the example aspects and modalities described above, other aspects and modalities will become evident through reference to the figures and by studying the detailed descriptions that follow. Petition 870250094916, dated 10 / 17 / 2025, page 15 / 154 9 / 54 BRIEF DESCRIPTION OF THE DRAWINGS

[043] Figures 1A-1C show an RT-PCR analysis of tobacco plants transformed using specific primers to identify the integration and transcription of 7 or 4 bromoform-producing genes per transgenic plant (the primers used are for genes encoding resistance to kanamycin, spectinomycin, and Basta used for selection, see Table 2). Plants 1-3 show in planta transcription of the genes for resistance to kanamycin (Figure 1A), spectinomycin (Figure 1B), and Basta (Figure 1C), which provides evidence of the expression of all 7 genes: mbb1-4, sfp, fabH, and ACCase (from plasmids 35), involved in bromoform production. Plants 4-6 show in planta transcription of kanamycin resistance genes (Figure 1A) and spectinomycin resistance genes (Figure 1B) (but not the Bar gene (Basta resistance) (Figure 1C)), which provides evidence for the expression of 4 genes: mbb1-4 of plasmids 1+2.MW - molecular weight marker, wt - wild-type tobacco plants, NTC control without model. DETAILED DESCRIPTION OF THE INVENTION

[044] In the following description, various aspects of disclosure will be described. For explanatory purposes, specific configurations and details are presented to provide a complete understanding of the different aspects of disclosure. However, it will also be evident to a specialist in the field that disclosure can be practiced without specific details being presented in this document. Furthermore, well-known features may be omitted or simplified so as not to obscure disclosure.

[045] The problem of methane emissions from ruminant animals has attracted much attention, and various solutions have been proposed. Petition 870250094916, dated 10 / 17 / 2025, page 16 / 154 10 / 54 developed. Many of the solutions were based on the fact that the red algae Asparagopsis taxiformis produces bromoform, which interferes with methane production in the cow's stomach. For example, one approach involved feeding cows Asparagopsis taxiformis, while another included transferring the genes encoding haloperoxidase from A. taxiformis, the main enzyme involved in bromoform production, into the genome of various organisms, such as yeasts and bacteria, to produce animal feed that includes these bromoform-producing organisms. Each of the approaches had some shortcomings, including costs, bromoform toxicity, and low yield.

[046] The present invention presents a novel approach to the problem, which involves transferring a combination of bromoform-producing genes from A. taxiformis and additional organisms into plants, thus enabling them to be sown in pasture fields and consumed by cows. One of the advantages of this method is that the method of supplying bromoform through grazing instead of feeding can allow a lower yield of bromoform to be sufficient to achieve the methane-inhibiting effect. A system for generating bromoform-producing plants

[047] Consequently, in some embodiments, a system for gene expression in a plant is provided, including one or more recombinant nucleic acid molecules that include one or more nucleotide sequences, including two or more genes, wherein: each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production that are not encoded by the native plant. Petition 870250094916, dated 10 / 17 / 2025, p. 17 / 154 11 / 54

[048] The phrase one or more nucleotide sequences, including two or more genes, is intended to clarify that the total number of genes included in the system is two or more (at least two), and that this total number of genes may be included in a single nucleotide sequence or in two or more nucleotide sequences. Similarly, it is clarified that the total of two or more genes may be included in one recombinant nucleic acid molecule, or in two or more recombinant nucleic acid molecules. Each nucleic acid molecule and each nucleotide sequence may include one gene, more than one gene, a part of a gene, no genes or parts thereof, or any combination of genes and / or parts thereof. In total, the recombinant nucleic acid molecule system includes at least two genes involved in bromoform production and encodes at least two enzymes involved in bromoform production.

[049] In some embodiments, the two or more genes are included in a single nucleic acid molecule. In some embodiments, the two or more genes are included in two or more nucleic acid molecules.

[050] The term native plant means the original plant, before adding the system of the invention. In some embodiments, “native plant” is a plant found in nature, without including any genetic modifications.

[051] The term operationally linked, as used here, means that the respective elements are arranged in such a way that there is a functional relationship between them. For example, referring to a gene that is functionally linked to a promoter means that the position and orientation of the gene and the promoter are appropriate, so that the promoter is driving gene expression. It is known that a promoter can be functionally linked to more than one Petition 870250094916, dated 10 / 17 / 2025, p. 18 / 154 12 / 54 gene. It is also recognized that a promoter can be functionally linked to the gene through a transactivating factor, so that the promoter drives the expression of the transactivating factor, which binds to a regulatory element that induces gene expression.

[052] In some embodiments, more than one gene is functionally linked to a single promoter. In some embodiments, each gene is functionally linked to a separate promoter.

[053] Bromoform (CHBra) is a brominated organic solvent, a colorless liquid at room temperature, with a high refractive index, very high density, and a sweetish odor, similar to that of chloroform. Bromoform is known as a methanogenesis inhibitor and is a common component of seaweed. The inhibition mechanism is believed to be through the reduction of the efficiency of cobalamin-dependent methyltransferase, interfering with the binding of vitamin B12, which is a crucial step in rumen methanogenesis.

[054] Several organisms are known as bromoform producers, including, but not limited to, species of algae Asparagopsis taxiformis (red sea plume or limu kohu, formerly A. sanfordiana), Asparagopsis armata, Chondrus crispus (Irish moss or carrageenan moss), and Macrocystis pyrifera (giant seaweed).

[055] A. taxiformis is a species of red algae widely found in tropical and warm temperate waters. Researchers have shown that feeding ruminants a diet containing only 0.2% A. taxiformis algae reduced their methane emissions by almost 99%. Other types of seaweed have been tested, and many have managed to reduce methane emissions by more than 90%. The active ingredient found in seaweed was Petition 870250094916, dated 10 / 17 / 2025, p. 19 / 154 13 / 54 bromoform. A. armata is also a species of red seaweed that has been shown to reduce methane production in dairy cows. C. crispus is a species of red algae also used as a source of the thickener carrageenan. Macrocystis pyrifera is a brown macroalga.

[056] In some forms, the two or more genes are three or more, four or more, five or more, six or more, or seven genes.

[057] In some forms, at least one of the two or more genes are from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus, Macrocystis pyrifera, Medicago sativa and Medicago truncatula.

[058] In some embodiments, at least two of the two or more genes are from at least one non-plant organism. In some embodiments, at least one of the non-plant organisms is an alga. In some embodiments, the alga is a red or brown alga. In some embodiments, the alga is a red alga. In some embodiments, the alga is selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus, and Macrocystis pyrifera. In some embodiments, the alga is Asparagopsis taxiformis.

[059] Several enzymes have been identified as being involved in bromoform production. These enzymes include haloperoxidases (such as vanadate-dependent haloperoxidase or VHPO) and NAD(P)H oxidase (NOX). Haloperoxidases catalyze the conversion of halide anions into hypohalous acid with hydrogen peroxide acting as an oxidant (Br-+H2O2 => HOBr+OH-). The hypohalous acid provides electrophilic halogen ions that halogenate hydrocarbon substrates to yield natural products such as bromoform (3HOBr+CH => CHBr3+3OH-). VHPOs use vanadium as a cofactor. A Petition 870250094916, dated 10 / 17 / 2025, page 20 / 154 14 / 54 The need for hydrogen peroxide in the peroxidase reaction links this pathway to the enzymatic production of reactive oxygen species (ROS) by NOXs. NOXs are membrane-bound enzymes that catalyze the transfer of electrons from the electron donor, NAD(P)H, via flavin and heme cofactors to molecular oxygen in order to generate ROS, such as hydrogen peroxide and superoxide anion (dismutated into hydrogen peroxide).

[060] Consequently, in some embodiments, the two or more enzymes include at least one haloperoxidase. In some embodiments, the two or more enzymes include an NOX. In some embodiments, the two or more enzymes include at least one haloperoxidase and an NOX.

[061] Furthermore, as demonstrated by Thapa 2020 (ACS (Chem Biol. 15(6): 1662-1670), intermediates of fatty acid biosynthesis, such as malonyl CoA, can provide substrates for VHPO enzymes and are therefore also considered to be involved in bromoform production. The enzymes involved in the relevant fatty acid synthesis reactions are encoded by the genes FabH (Beta-ketoacyl-[acyl carrier protein] synthase III), MatB (malonyl CoA synthetase), and Sfp (phosphopantetheine transferase). In summary, malonate and CoA-SH are condensed by MatB into malonyl-coenzyme A. Sfp modifies ACP (acyl carrier protein) to malonyl-S-ACP using malonyl-coenzyme A. After incubation with acetyl-CoA and FabH, it is converted into acetoacetyl-S-ACP. Acetoacetyl-S-ACP can be incubated with a haloperoxidase to produce bromoform.

[062] Malonyl CoA is most frequently produced from acetyl CoA by acetyl-CoA carboxylase (ACCase), rather than malonyl CoA synthetase. ACCase is a biotin-dependent carboxylase that catalyzes the irreversible carboxylation of acetyl-CoA. Petition 870250094916, dated 10 / 17 / 2025, p. 21 / 154 15 / 54 to produce malonyl-CoA through its two catalytic activities, biotin carboxylase and carboxyltransferase. The most important function of ACCase is to provide malonyl-CoA substrate for fatty acid biosynthesis.

[063] In some embodiments, the two or more enzymes are three or more, four or more, five or more, six or more, or seven enzymes.

[064] Consequently, in some embodiments, the two or more enzymes include at least one enzyme involved in fatty acid synthesis. In some embodiments, at least one of the enzymes involved in fatty acid synthesis is selected from Sfp, FabH, ACCase, and MatB.

[065] In some embodiments, the two or more enzymes include at least one haloperoxidase and at least one enzyme involved in fatty acid synthesis selected from FabH, sfp, ACCase and MatB.

[066] In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and at least one enzyme involved in fatty acid synthesis selected from FabH, sfp, ACCase and MatB.

[067] In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and FabH. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and sfp. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and ACCase.

[068] In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, FabH, and sfp. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, FabH, and ACCase. In some embodiments, the Petition 870250094916, dated 10 / 17 / 2025, p. 22 / 154 16 / 54 Two or more enzymes include at least one haloperoxidase, NOX, sfp, and ACCase.

[069] In some embodiments, the two or more enzymes include at least one haloperoxidase, FabH, sfp, and ACCase. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, FabH, sfp, and ACCase.

[070] In some embodiments, at least one of the haloperoxidases is selected from Mbb1, Mbb3 and Mbb4 and combinations thereof. In some embodiments, at least one of the haloperoxidases includes Mbb1, Mbb3 and Mbb4. In some embodiments, the NOX is Mbb2.

[071] In some game modes, Mbb1 has at least 70.75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 1. In some modes, Mbb1 has a sequence according to SEQ ID NO: 1.

[072] In some game modes, Mbb2 has at least 70.75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 2. In some game modes, Mbb2 has a sequence according to SEQ ID NO: 2.

[073] In some game modes, Mbb3 has at least 70.75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 3. In some game modes, Mbb3 has a sequence according to SEQ ID NO: 3.

[074] In some game modes, Mbb4 has at least 70,75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 4. In some game modes, Mbb4 has a sequence according to SEQ ID NO: 4.

[075] In some disciplines, FabH has at least 70.75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: Petition 870250094916, dated 10 / 17 / 2025, p. 23 / 154 17 / 54 5. In some modes, FabH has a sequence according to SEQ ID NO: 5.

[076] In some modalities, the sfp has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO: 6. In some forms, the SFP has a sequence matching SEQ ID NO: 6.

[077] In some modalities, ACCase has at least 70.75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 7. In some modalities, the ACCase has a sequence according to SEQ ID NO: 7.

[078] In A. taxiformis, the relevant genes were identified and located at a single locus called the marine bromoform biosynthesis locus (GenBank accession number (MN966723 and MN893468). The genes identified at the Mbb locus include VHPOs - Mbb1, Mbb3, and Mbb4, as well as NOX - Mbb2. Note that Mbb2 is not the only NOX identified in A. taxiformis. Corresponding genes have also been identified in the algal species Chondrus crispus and include the VHPO genes CcVHPO1-5 and the NOX gene CcMbb2 (Thapa et al., 2020, ACS Chem Biol. 15(6): 1662-1670). The VHPOs of A. taxiformis and C. crispus identified above are bromine-specific.

[079] Thus, in some modalities, two or more genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase and MatB.

[080] Mbb1-4 were successfully expressed in E. coli., however, attempts to express Mbb2 in bacteria and yeast were unsuccessful and CcMbb2 was expressed in yeast. Furthermore, it is observed that Mbb3 did not produce bromoform and showed a low level of expression, and that CcVHPO4 and CcVHPO5 were not tested (Thapa et al. 2020, ACS Chem Biol. 15(6): 1662-1670). Petition 870250094916, dated 10 / 17 / 2025, p. 24 / 154 18 / 54

[081] In some forms, the two or more genes include at least mbb2 or CcMbb2.

[082] It is also noted that some of the genes, especially NOX and fatty acid synthesis enzymes, may be present endogenously in the plant and, therefore, it may sometimes be sufficient to provide only haloperoxidases or any other partial list of the genes above.

[083] In some forms, the two or more genes include the Mbb1 and Mbb2 genes.

[084] In some forms, the two or more genes include the Mbb3 and Mbb4 genes.

[085] In some forms, the two or more genes include the Mbb1, Mbb2, Mbb3 and Mbb4 genes.

[086] In some embodiments, the two or more genes include the Mbb1, Mbb3, Mbb4 genes and at least one gene selected from sfp, FabH and ACCase. In some embodiments, the two or more genes include the Mbb1, Mbb2, Mbb3, Mbb4 genes and at least one gene selected from sfp, FabH and ACCase.

[087] In some embodiments, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4 and sfp. In some embodiments, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4 and FabH. In some embodiments, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4 and ACCase.

[088] In some modalities, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, sfp and FabH. In some modalities, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, sfp and ACCase. In some modalities, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, FabH and ACCase. Petition 870250094916, dated 10 / 17 / 2025, p. 25 / 154 19 / 54

[089] In some embodiments, the two or more genes include Mbbl, Mbb3, Mbb4, sfp, FabH, and ACCase. In some embodiments, the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, sfp, FabH, and ACCase.

[090] In some embodiments, the Mbb1, Mbb2, Mbb3, and / or Mbb4 genes are from Asparagopsis taxiformis or encode the same enzymatic sequence encoded by the Mbb1, Mbb2, Mbb3, and / or Mbb4 genes of Asparagopsis taxiformis, respectively. In some embodiments, the sfp gene is from Medicago truncatula or encodes the same enzymatic sequence encoded by the sfp gene of Medicago truncatula. In some embodiments, the FabH gene is from Chondrus crispus or encodes the same enzymatic sequence encoded by the FabH gene of Chondrus crispus. In some embodiments, the ACCase gene is from Medicago sativa or encodes the same enzymatic sequence encoded by the ACCase gene of Medicago sativa. In some embodiments, at least one of the two or more genes is an ortholog from a different species of a gene indicated above.

[091] It is recognized that any of the genes of the invention may encode the same enzymatic sequence encoded by a reference gene (for example, the gene of the invention encodes the same enzymatic sequence encoded by the FabH gene of Chondrus crispus), but have a different or altered coding sequence compared to the respective gene. In some embodiments, the coding sequence is altered using codon optimization. In some embodiments, the coding sequence is altered using a codon usage scheme from a different organism. In some embodiments, the coding sequence is altered using a codon usage scheme from Medicago sativa. Petition 870250094916, dated 10 / 17 / 2025, p. 26 / 154 20 / 54

[092] In some embodiments, the Mbbl gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 1.

[093] In some embodiments, the Mbb2 gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 2.

[094] In some embodiments, the Mbb3 gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 3.

[095] In some embodiments, the Mbb4 gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 4.

[096] In some embodiments, the FabH gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 5.

[097] In some embodiments, the sfp gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 6.

[098] In some embodiments, the ACCase gene has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with a sequence encoding SEQ ID NO: 7.

[099] In some embodiments, the two or more genes are all functionally linked to identical promoters. In some embodiments, at least two of the two or more genes are functionally linked to promoters that are different from each other. In some embodiments, each of the two or more genes is functionally linked to a separate promoter. In some embodiments, more than one gene is functionally linked to a single promoter. Petition 870250094916, dated 10 / 17 / 2025, p. 27 / 154 21 / 54

[0100] One of the problems with bromoform production is that bromoform itself can be dangerous to the ozone layer. Thus, an advantage of the present invention is that it allows controlling the amount of bromoform produced in several ways, such as, for example, by controlling the expression of bromoform-producing genes, limiting them to specific developmental stages, specific states, or specific tissues. This can be achieved, for example, by using an inducible promoter, which limits the expression of these genes to a specific time or location.

[0101] The promoter can be any promoter suitable for expression in plants. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, at least one of the genes is functionally linked to an inducible promoter.

[0102] Non-limiting examples of a constitutive promoter include, for example, the 35S promoter of cauliflower mosaic virus, the NOS promoter of Agrobacterium and a promoter of housekeeping genes such as actin (e.g., promoter act8) or ubiquitin (promoter ubq10) etc.

[0103] Non-limiting examples of a tissue-specific promoter include, for example, the leaf-specific promoter of sedoheptulose-1,7-bisphosphatase (SBPase, for example, from B. distachyon) or the promoter of fructose-1,6-bisphosphate aldolase (FBPA) highly expressed in leaves.

[0104] The term constitutive promoter refers to a promoter that is always in an active state, that is, driving the transcription of a gene. It is generally not affected by regulatory factors, such as timing factors or gene-specific factors. Petition 870250094916, dated 10 / 17 / 2025, page 28 / 154 22 / 54 tissue. Examples of a constitutive promoter are promoters of maintenance genes.

[0105] The term inducible promoter or regulated promoter refers to a promoter that is, by default, in an inactive state, i.e., it does not direct transcription, and is activated in response to specific regulatory factors, for example, tissue-specific or developmentally regulated factors. Non-limiting examples of an inducible promoter include a tissue-specific promoter and a developmentally regulated promoter.

[0106] To keep bromoform production to a minimum, thereby reducing excess bromoform, which is harmful to both the plant and the environment, an inducible promoter can be used, so that the relevant genes are expressed only when needed.

[0107] For example, according to some embodiments, bromoform is only required when the animal grazes. Consequently, the expression of at least some of the bromoform-producing genes may be activated only when the plant reaches the appropriate stage of development and is ready to be consumed. Alternatively, bromoform production may be activated only when cows are grazing, triggered, for example, by the stress response caused by damage to the plant from the cow's bite.

[0108] Therefore, in some embodiments, the inducible promoter is a developmentally regulated promoter, induced by factors that are activated in late stages of plant development. In some embodiments, the inducible promoter is a tissue-specific promoter, activated in specific tissues that are Petition 870250094916, dated 10 / 17 / 2025, page 29 / 154 23 / 54 develop at late stages in the plant's life cycle, such as leaves or flowers.

[0109] In some embodiments, the inducible promoter is induced by factors that are activated due to consumption by a ruminant animal, such as stress-induced regulatory factors, for example, jasmonic acid. In some embodiments, the inducible promoter is a jasmonic acid promoter or a jasmonic acid responsive promoter.

[0110] One of the problems with gene targeting in plants is the suppression of exogenous genes by the plant's defense systems. One approach that can help overcome this problem is to target the action to an organelle in the plant cell, for example, a plastid such as the chloroplast. Targeting the chloroplast may have a double advantage: the first is the sequestration of exogenous genes, possibly preventing them from being silenced by the host plant; and the second is that fatty acid synthesis occurs in the chloroplast and may provide precursors for haloperoxidase.

[0111] Thus, in some embodiments, at least one of the two or more genes further encodes a plastid targeting motif or a chloroplast targeting motif, such as a chloroplast transit peptide. In some embodiments, the two or more genes further encode chloroplast transit peptides.

[0112] A chloroplast transit peptide is a short peptide that directs the transport of a protein into the chloroplast. The transit peptide is usually cleaved upon import. Chimeric chloroplast transit peptides are disclosed in document WO2012161982, which is incorporated herein by reference. Petition 870250094916, dated 10 / 17 / 2025, page 30 / 154 24 / 54

[0113] Another approach is to use the viral RNA silencing suppressor P19, which can prevent the silencing of exogenous genes.

[0114] Thus, in some forms, the system also includes a sequence that encodes the P19 viral RNA silencing suppressor gene.

[0115] It is recognized that the recombinant nucleic acid molecules of the invention may include further additional elements, such as transcription terminators, regulatory elements, selective markers, etc., as required for transformation and expression in plants. Non-limiting examples of selective markers include kanamycin resistance, spectinomycin resistance and / or resistance to the herbicide glufosinate or phosphinothricin (e.g., the Basta brand).

[0116] In some embodiments, one or more recombinant nucleic acid molecules are included in at least one vector suitable for plant transformation and / or plant expression. In some embodiments, the vector is a binary vector. In some embodiments, the binary vector system is a Ti plasmid-based system in which a gene of interest is cloned into the T-DNA region of the plasmid, and transformation is conducted in Agrobacterium containing a vir helper plasmid. In some embodiments, the vector is Ti plasmid-based. In some embodiments, the vector is a pAB vector. In some embodiments, the vector is a pPA binary vector.

[0117] The recombinant nucleic acid molecules of the invention can be prepared by any suitable genetic engineering method, including, for example, plasmid cloning, DNA synthesis, restriction digestion and ligation, T-DNA manipulation, clustered short palindromic repeat technology and Petition 870250094916, dated 10 / 17 / 2025, page 31 / 154 25 / 54 regularly interspaced (CRISPR), polymerase chain reaction (PCR), etc.

[0118] In some embodiments, a composition is provided that includes the system disclosed in this document. In some embodiments, the composition also includes reagents suitable for transforming plants. Suitable reagents are well known in the art and are also disclosed in the Examples.

[0119] In some embodiments, a composition is provided that includes the system of the invention for use in processing a plant. In some embodiments, the use of a composition that includes the system of the invention for processing a plant is provided. Methods for preparing bromoform-producing plants

[0120] In some embodiments, a method is provided for preparing a genetically modified plant or plant part, the method includes transforming a plant or plant part with the system described in this document or with the composition disclosed above.

[0121] The definitions and modalities cited above, which may be relevant to the modalities of the preparation method, also apply here and vice versa. Some particularly relevant modalities may be indicated or explicitly repeated.

[0122] The transformation of plants with the nucleic acids of the invention can be carried out by any suitable method known in the art. Non-limiting examples of methods include agrobacterium-mediated transformation and / or transformation by the floral dip method (for example, as described in WO 2018 / 178975). Petition 870250094916, dated 10 / 17 / 2025, p. 32 / 154 26 / 54

[0123] In some embodiments, the method includes transforming the system of the invention into plant leaves. In some embodiments, the method includes transforming the system of the invention into plant seeds.

[0124] In some embodiments, the system of the invention includes more than one (at least two) recombinant nucleic acid molecules, and at least two of the recombinant nucleic acid molecules are transformed together in the plant. In some embodiments, the system of the invention includes more than one recombinant nucleic acid molecule, and at least two of the recombinant nucleic acid molecules are transformed separately in the plant. In some embodiments, the system of the invention includes more than one recombinant nucleic acid molecule, and all recombinant nucleic acid molecules are transformed together (co-transformed) in the plant.

[0125] It is recognized that the genes of the invention can be inserted together or separately into the plant by any suitable method, including the use of any genome editing system or method, including systems using engineered nucleases selected from the group consisting of: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system and any combination thereof.

[0126] In some embodiments, the method also includes a step of cultivating a plant from the part of the plant transformed by the system of the invention. In some embodiments, the method also includes a step of obtaining a part of the cultivated plant. Petition 870250094916, dated 10 / 17 / 2025, page 33 / 154 27 / 54

[0127] For example, the leaves of a plant can be transformed with the system of the invention, and a plant is grown from the transformed leaves. Seeds can then be obtained from the grown transformed plant to generate additional transformed plants.

[0128] In some embodiments, the method also includes a step of selection of transformed plants. Selection may be conducted by any suitable method known in the art, such as including in the transformed vectors in the plant one or more genes that encode selectable markers, such as resistance to kanamycin, resistance to spectinomycin and / or resistance to the herbicide glufosinate or phosphinothricin (for example, the Basta brand), and transformant cultivars in the presence of appropriate selection. Plants and plant parts genetically modified to produce bromoform.

[0129] In some embodiments, a genetically modified plant or plant part is provided including the system disclosed in this document or prepared by the method for preparing a genetically modified plant or plant part described herein.

[0130] In some embodiments, a genetically modified plant or plant part is provided, including one or more exogenous nucleotide sequences including two or more genes, wherein: each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production and not encoded by the native plant.

[0131] In some embodiments, the present application provides a genetically modified plant cell, including the system disclosed in this document or prepared by the method for obtaining Petition 870250094916, dated 10 / 17 / 2025, page 34 / 154 28 / 54 of a genetically modified plant or plant part described herein.

[0132] In some embodiments, the present application provides a genetically modified plant cell, including one or more exogenous nucleotide sequences including two or more genes, wherein each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production and not encoded by the native plant.

[0133] It is known that the genetically modified plants or plant parts disclosed in this document are intended to produce bromoform or to be capable of developing into plants or plant parts that produce bromoform. However, since at least some of the genes involved in bromoform production may be tissue-specific or developmentally regulated, bromoform production may be limited to certain plant tissues or parts. Consequently, the term plant part, as used in this document, encompasses either a plant part that produces bromoform after being transformed with the constructs used (and provided with a source of bromine), or a plant part that may not produce bromoform after being transformed but may develop into a bromoform-producing plant or plant part. Examples for the latter case may include a cell or a seed.

[0134] The terms genetically modified and transgenic are used interchangeably herein and refer to plants or plant parts that include exogenous nucleotide sequences encoding bromoform-producing genes, as described in this document. Petition 870250094916, dated 10 / 17 / 2025, page 35 / 154 29 / 54

[0135] The term exogenous nucleotide sequence, as used herein, means a nucleotide sequence that is not part of the native plant.

[0136] In some embodiments, a genetically modified plant or plant part is provided including two or more genes, wherein: each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in bromoform production.

[0137] The definitions and modalities mentioned above that may be relevant to genetically modified plant modalities also apply here and vice versa. Some particularly relevant modalities may be indicated or explicitly repeated.

[0138] The expression one or more exogenous nucleotide sequences including two or more genes is understood to mean that there may be one exogenous sequence that includes two or more genes, or there may be more than one exogenous sequence when all the exogenous sequences together include two or more genes. When there is more than one exogenous nucleotide sequence, each of the exogenous sequences may contain no genes, contain one gene, or contain more than one gene, provided that together they include two or more genes.

[0139] In some embodiments, the recombinant nucleic acid molecules are extrachromosomal. In some embodiments, the recombinant nucleic acid molecules are integrated into a plant chromosome.

[0140] In some embodiments, the plant is a pasture plant or a forage plant. In some embodiments, the plant is a pasture plant. In some embodiments, the plant is a forage plant. Petition 870250094916, dated 10 / 17 / 2025, page 36 / 154 30 / 54

[0141] The term pasture plant or forage plant is used here to refer to plants that are planted or sown in pasture fields for animals to graze on.

[0142] Non-limiting examples of pasture plants that are grasses include Agrostis spp. (curved grasses), Agrostis capillaris (common curved grass), Agrostis stolonifera (creeping curved grass), Andropogon hallii (blue sand grass), Arrhenatherum elatius (false oat grass), Bothriochloa bladhii (Australian blue grass), Bothriochloa pertusa (hurricane grass), Brachiaria spp. (brachiaria grass), Brachiaria decumbens (Suriname grass), Brachiaria humidicola (koronivia grass), Brachiaria ruziziensis or any hybrids of Brachiaria, Bromus spp. (brome grass), Cenchrus ciliaris (buffel grass), Chloris gayana (Rhodes grass), Cynodon dactylon (bermuda grass), Dactylis glomerata (orchard grass), Echinochloa piramidalis (antelope grass), Entolasia imbricata (bungoma grass), Festuca spp. (Festuca), Festuca arundinacea (tall fescue), Festuca pratensis (meadow fescue), Festuca rubra (red fescue), Heteropogon contortus (black speargrass), Hymenachne amplexicaulis (West Indian swamp grass), Hyparrhenia rufa (jaragua), Leersia hexandra (andrequicé grass), Lolium spp.(ryegrass), Lolium multiflorum (Italian ryegrass), Lolium perenne (perennial ryegrass), Megathyrsus maximus (Mombasa grass), Melinis minutiflora (Montessava grass), Paspalum conjugatum (Carabao grass), Paspalum dilatatum (Dallis grass), Phalaris arundinacea (Yellowgrass), Phleum pratense (Timothy), Poa spp. (Bluegrass, Meadow grasses), Poa arachnifera (Texas bluegrass), Poa pratensis (Kentucky bluegrass), Poa trivialis (rough bluegrass), Setaria sphacelata (African grass), Themeda triandra (Kangaroo grass) and Thinopyrum intermedium (intermediate wheat). Petition 870250094916, dated 10 / 17 / 2025, page 37 / 154 31 / 54

[0143] Non-limiting examples of pasture plants that are legumes include Arachis pintoi (forage peanut), Astragalus cicer (milk vetch), Chamaecrista rotundifolia (roundleaf vetch), Clitoria ternatea (butterfly pea), Kummerowia (annual lespedeza), Kummerowia stipulacea (Korean clover, Korean lespedeza), Kummerowia striata (Japanese clover, common lespedeza), Lotus corniculatus (bird's-foot clover), Macroptilium atropurpureum (purple bean), Macroptilium bracteatum (red bean), Medicago spp. (medicago), Medicago sativa (alfalfa, alfalfa), Medicago truncatula (barrel medic), Melilotus spp. (sweet clover), Neonotonia wightii (perennial soybean), Onobrychis viciifolia (common sainfoin), Stylosanthes spp. (stylo), Stylosanthes humilis (Townsville stylo), Stylosanthes scabra (shrub stylo), Trifolium spp. (clovers), Trifolium hybridum (also similar to clover), Trifolium incarnatum (crimson clover), Trifolium pratense (red clover), Trifolium repens (white clover), Vicia spp.(vetch), Vicia articulata (garob), Vicia ervilia (bitter vetch), Vicia narbonensis (narbonense vetch), Vicia sativa (common vetch, tara), Vicia villosa (velosa vetch) and Vigna parkeri (creeping vetch).

[0144] The term forage plant is used in this document to refer to plants that can be used to prepare feed for livestock. Non-limiting examples of forage plants include alfalfa, barley, duckweed, birdsfoot trefoil, brassicas (such as cabbage, rapeseed, rutabaga and turnip), clover, grasses such as Bermuda grass, false oats, fescue, ryegrass, maize, millet, oats, sorghum, soybeans and wheat.

[0145] The terms pasture plant and forage plant are not mutually exclusive, as many plants fall under both definitions. Petition 870250094916, dated 10 / 17 / 2025, p. 38 / 154 32 / 54

[0146] In some forms, the plant is selected from among a grass, a cereal and a legume.

[0147] Examples of grasses include brachiaria grass, ryegrass, tall fescue, dactylus and kikuyu; examples of cereals include corn, oats and millet; and examples of legumes include alfalfa (Medicago sativa, lucerne), clover and soybeans.

[0148] In some varieties, the plant is selected from among Brachiaria grass, alfalfa (Medicago sativa, alfalfa), ryegrass, tall fescue, dactylus, clover, oats, millet, kikuyu, chicory, corn, soybean and forage plantain.

[0149] In some forms, the plant part is selected from seed, cell, leaf, stem, root, tuber, cutting, flower, bark, fruit, bulb, trichome, rhizome and any suitable part of a plant.

[0150] In some embodiments, the plant part is selected from a seed or a cell. In some embodiments, the plant part is an explant derived from any plant tissue.

[0151] In some embodiments, at least two of the two or more genes are from a different species than the plant species. In some embodiments, at least one of the two or more genes is from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus, Macrocystis pyrifera, Medicago sativa, and Medicago truncatula. In some embodiments, at least 2, 3, 4, 5, 6, or 7 of the two or more genes are from at least one organism selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus, Macrocystis pyrifera, Medicago sativa, and Medicago truncatula.

[0152] In some forms, at least one of the two or more genes are from a non-plant organism. In some forms, Petition 870250094916, dated 10 / 17 / 2025, p. 39 / 154 33 / 54 at least 2, 3, 4 or 5 of the two or more genes are from at least one non-plant organism. In some embodiments, the non-plant organism is an alga. In some embodiments, the alga is a red or brown alga. In some embodiments, the alga is a red alga. In some embodiments, the alga is selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus Crispus and Macrocystis pyrifera. In some embodiments, the alga is Asparagopsis taxiformis.

[0153] In some embodiments, the genetically modified plant or part of the plant is obtained directly by transformation using the system of the invention.

[0154] In some embodiments, the plant or part of the plant, including the system of the invention, is not obtained directly by transformation with the system of the invention, but is derived from a plant or part of the plant that has been transformed with the system of the invention, or from a descendant of the transformed plant.

[0155] An additional way to reduce the amount of bromoform, besides that discussed above, is to dilute or mix the genetically modified plants or plant parts with plants or plant parts that do not produce bromoform, such as plants or plant parts that do not include the system of the invention. For example, genetically modified seeds of the invention can be mixed with non-genetically modified seeds to provide the amount of bromoform needed for the ruminant animal.

[0156] Thus, in some embodiments, a composition is provided that includes a mixture of genetically modified plants or plant parts of the invention (such as, for example, seeds) with plants or plant parts that are not capable of producing Petition 870250094916, dated 10 / 17 / 2025, page 40 / 154 34 / 54 bromoform. In some forms, the composition includes only parts of the plant, such as seeds, and no additional ingredients.

[0157] In some embodiments, up to about 5%, 10%, 15%, 20%, 25% or 30% w / w of the composition are genetically modified plants or plant parts of the invention. Methods for reducing methane emissions

[0158] The plants or plant parts of the invention, including the bromoform-producing genes described herein, are intended for consumption by ruminant animals, such as cows. There are two general orientations for this purpose: preparing feed for livestock based on the transformed plants or sowing transformed pasture plants in pasture fields, to be consumed by ruminant animals.

[0159] Consequently, in some embodiments, a method is provided to reduce methane gas emissions generated by a ruminant animal, the method includes: - production of a genetically modified seed of a pasture plant, the seed including two or more genes involved in the production of bromoform; - sowing genetically modified seed in a field where a ruminant animal grazes; and - Cultivation of a bromoform-producing pasture plant from seed, resulting in the bromoform-producing pasture plant being consumed by ruminant animals.

[0160] In some embodiments, the method also includes a step of adding a bromine source to the bromoform-producing pasture plant. In some embodiments, the method does not include the addition. Petition 870250094916, dated 10 / 17 / 2025, p. 41 / 154 35 / 54 from a bromine source to the bromoform-producing pasture plant.

[0161] A suitable source of bromine may include solutions containing bromine when bromine is in any suitable form, such as bromine or bromide, for example, a calcium bromide solution. The concentration of bromine (e.g., bromide) in the bromine-containing solution may be about 1-100, 5-100, or 5-50 ppm. The source of bromine may be added in a suitable manner, including spraying, irrigation, or immersion of plant parts (such as leaves or roots) in a bromine-containing solution.

[0162] In some embodiments, the genetically modified seed is produced by the method for preparing a genetically modified plant or plant part disclosed in this document.

[0163] In some embodiments, bromoform-producing plants include the system disclosed in this document.

[0164] Ruminants (suborder Ruminantia) or ruminant animals (those that ruminate) are herbivorous ungulate mammals that graze or consume leaves and shoots and obtain nutrients from plant-based foods by fermenting them in a compartmentalized stomach before digestion, primarily through microbial action. The process, which occurs in the front part of the digestive system and is therefore called pre-gastric fermentation, typically requires that the ingested and fermented food (known as ruminated food) be regurgitated and chewed again. The process of repeated chewing of ruminated food to further break down plant matter and stimulate digestion is called rumination. The digestion of food in the rumen (the first chamber and the main site of microbial fermentation) is carried out Petition 870250094916, dated 10 / 17 / 2025, page 42 / 154 36 / 54 mainly by the ruminal microflora, which contains dense populations of various species of bacteria, protozoa, and occasionally yeasts and other fungi. The hydrolysis of cellulose results in sugars, which are subsequently fermented into acetate, lactate, propionate, butyrate, carbon dioxide, and methane. Methane is produced by a type of archaea, called methanogen, within the rumen, and this methane is released into the atmosphere. The rumen is the main site of methane production in ruminants.

[0165] In some forms, the ruminant animal is selected from cattle, a domesticated bovine (such as a cow) or wild bovine, a goat, a sheep, a giraffe, a deer, a gazelle and an antelope.

[0166] In some forms, the ruminant animal is a cow.

[0167] In some embodiments, a method is provided for preparing feed to reduce methane gas emissions from a ruminant animal; the method includes: - production of a genetically modified seed of a forage plant, the seed including two or more genes involved in the production of bromoform; - sowing of genetically modified seeds; - cultivation of forage plants that produce bromoform from seed; and - preparation of a feed containing the bromoform-producing forage plant or a part thereof.

[0168] The definitions and modalities cited above, which may be relevant to the modalities of the method, also apply here and vice versa. Some particularly relevant modalities may be indicated or explicitly repeated. Petition 870250094916, dated 10 / 17 / 2025, p. 43 / 154 37 / 54

[0169] In some embodiments, the genetically modified seed is produced by the method for preparing a genetically modified plant or plant part disclosed in this document.

[0170] In some embodiments, bromoform-producing plants include the system disclosed in this document.

[0171] In some forms, the ruminant animal is a cow.

[0172] It should be noted that the preparation of plant-based animal feed is known in the art. Preparation can be carried out by wet or dry processing methods, including methods such as grinding, dry rolling, lamination, bursting, micronization, pressure cooking, roasting, extrusion, pelleting, etc. Various nutritional ingredients can be added to the plant raw material.

[0173] In some forms, the ruminant animal is selected from cattle, a domesticated bovine (such as a cow) or wild bovine, a goat, a sheep, a giraffe, a deer, a gazelle and an antelope.

[0174] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by a professional with average knowledge of the art to which the invention pertains.

[0175] The term a and an refers to one or more of a (i.e., at least one, or one or more) of the grammatical object of the article. As an example, an element means one element or more than one element.

[0176] The term "about" when referring to a measurable value, such as a quantity, a proportion, and the like, should encompass variations of ±10% of the stated value, as these variations are also suitable for carrying out the disclosed invention. Any Petition 870250094916, dated 10 / 17 / 2025, p. 44 / 154 38 / 54 numerical values ​​appearing in the request should be interpreted as if they were preceded by “approximately”, unless otherwise indicated.

[0177] The term nucleotide sequence or nucleic acid sequence, as used herein, refers to a sequence of nucleotides and may include different types of nucleotides, such as DNA nucleotides, RNA nucleotides, and synthetic nucleotides.

[0178] The term nucleic acid molecule is a molecule that includes at least one nucleotide sequence. A nucleic acid molecule can be linear, circular, or branched, and the nucleotides can be modified or unmodified. In some embodiments, a nucleic acid molecule is a nucleic acid vector (usually a DNA vector) that includes elements such as genes, promoters, ligands, etc.

[0179] The term recombinant, as used in this document with reference to a nucleic acid molecule or sequence, refers to a nucleic acid molecule or sequence that cannot be directly isolated from a natural organism and that includes a combination of nucleotide sequences that are not present together in the same molecule in a natural organism.

[0180] The term gene, as used in this document, refers to a portion of a nucleic acid sequence that is transcribed into RNA and optionally translated into a protein. Typically, a gene is functionally linked to a promoter that directs transcription and possibly to additional regulatory elements. In some embodiments, more than one gene is functionally linked to a single promoter, as in a prokaryotic operon. A gene may or may not include introns. Petition 870250094916, dated 10 / 17 / 2025, p. 45 / 154 39 / 54

[0181] The term bp, as used in this document, means base pair or base pairs.

[0182] The term aa, as used in this document, means amino acid or amino acids.

[0183] Although certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described in this document. Numerous modifications, alterations, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the principles and scope of the present invention, as described by the following claims.

[0184] The following examples are presented to illustrate more fully some embodiments of the invention. However, they should in no way be interpreted as limiting the broad scope of the invention. A person skilled in the art can easily conceive of many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0185] The invention will now be illustrated with the following non-limiting examples. EXAMPLES Table 1: Protein sequences. Protein Name ID Sequence Protein Sequence Mbb1 1 mtdtqnpnraevafkvrvsaaelarargspahlsnnsesrfrnpdgtr sllanftkglphiketalvesaidydrfvraidsgdprdfadlplgpq gveprftsgiasdpevgtrawesggaglvfdlegpdaqavtmppapel dsdelvaevtecywmsllrdvpfptfesnshiqaaaesinntqwikfk Petition 870250094916, dated 10 / 17 / 2025, page 46 / 154 40 / 54 nnppahltaaersrlrgpvttanvfrgitpgdevgpylsqfllvgttg iangnevgdgfiqyggmrmdqrvrvakphidymttfgayldvqnaanv sgrelykeeeprfrfihtprdlatyvhfdalyqaylnaciilldigap fdsgipfqldndidkqqgfatfggphilslvtevatralkavrfqkfn vhrrlrpeaigarvdrycatkapefagaaklsealdkellqkvhdhnk kqnllsdrgnprandfnpdgevsegnllmpmafpegspmhpaygagha tvagacvtvlkaffdggyrlpfcyitdedgtglqaveidepltvdgel nkicsnisigrnwagvhyftdyiesirigeeiaigilqeqkltfsenf smtlnkfdgstiri Mbb2 2 makqkslftllsatleshlsthafqllaliiyalanalmfvwgahdef hhhtnannlrwyiciargagytlnlntalvillaarlfatylretplq hilpldksfpafhivvaytiaaavvihasfhlawlvaydmwetgmwgf tmsaatgvvllvvfivmfisampkyrkkhfrifylihsvgallffgll vfhgmynrvpetykwiaapliiytidrvlrrykistaeleltgehssl kgsdilelrvpkpfdyqagqyaevcvksinsewhpftiassphedsmc fyikalgdwttnlrdafearvendlyeplkvqirgpfgapaqhvsgyc rvvlisggvgstpfaaickhlhhlnksenhskaleshasskrmsqvqw rirdaisilfdvslddsrndeaanqqrrqqladmlnmspkgdvhnvsf krtqskrsddfdsvedskqmnfslcrhdskssfnlddcsdairyynrp sfqffslstrhvinlyeyrtrllaflhttrftfallltlvarivilcivsifnlghiglynshidatwviatnsilglilgvallttilleisfmr mrffyrvwrcvdffiflpitflcnisnfaswnghaqpkflifldliiv lpimllllchrmyrsvgsrnllddtaecrqgckcnktipdvdfvwttp rdsddewlrnelyplatgtelrlhryvtrenmadledperfittanag rpqwdeifaqiaeqtpshskigvffcgphpmgaavqksmrkvevmsnl rgsylrktesavlvddlilrdegevkllreygcnirfvfreenfs Mbb3 3 mtpptrrqtalvkriaaadlaanrhhphhvrnadettyklpnhvcsyi saftkglphhldgrlvsphhfqlfihavrtghvkdiaaiplgpphsss frsgiakdaavrgwesmaagntfdlqgpdaqavtmppcprlnshelvt Petition 870250094916, dated 10 / 17 / 2025, page 47 / 154 41 / 54 emtelyymallrdvpfthfnkhhlvqsavydmnhthwtksnrcclprp errhrrskfttdnifrgvtpgditgpyisqfllvgtkgladvheeteg yvqygatridqrvrtvqpkidymttwaayldvqngadlrrhsayaepp nyrfictprdlatyvhydalyqaylnaciilldinapydsglpftadd avdkqqgfatfgaphvltlvtevatralkavrfqkyavhrrlrpeavg glleqyryggghsdlayiirpirhladslssdlmsmvakengrqnsl irdnghprshdegsddthllsmayaegspmhpsygaghatvagacvti lkaffdhtfklpfayvsssdgrklktvklskkltvedelnklaanisi grswagvhyysdyvesirlgeevaigmlkeqkltysekftmtipkfdg sviei Mbb4 4 maeerrqnaleiriqaaklakkrdhpthkangdedrypetligsftkg lpheketgllsnpadfadfvraintgaikdmrrlkgidedprfisgi askgdkhpfadtrawesmaagltydlegpdaqavtmppapkldsdelv teitesywmallrdvpfteferdgntaaaaasisrtrwvqyneqpsqr pstltdeeiarlrgpytkknvfrgvtngenvgpylsqfllvgtkgigd aqqvsdgyvqyggmrmdqrvrvavpkrdymttwaswldvqnagdlrgr eiyddtpfrfittprdlatwvhfdalyqaylnaciilldikapfdph ipfqadddvdkqqgfatfggphilslctevatralkavrfqkynlhrr lrpeaigglverfkktngdpkfapvkklvndldgdmlrrveqhnceqn klsddgharredyspegessqsyllpmafpegspmhpsygaghatvag acvtvlkaffdheyeldfcyvpttdgkrlekwninekltvegelnklcanisigrnwagvhyysdyfesikvgeeiaigilqqqkltygedffmtl pkfdgekiri FabH 5 missilqgarilatgsaipdvcinnehlskivetsnewivtrtgieer riltgqnesiidlasskralekismnpleidliilatsspddlfgs agqlqakigaskavafdltaacgflqvlqtqnivqnivqvg adalskwtdwsdrstcilfgdaagaaiiqacseengvlgfqlntngn ksnqlsipyqthsyhldnhkslnlfqgrfqyitmngqevykfavskvp tsiaqclnslniskdeinwllhqancrilyavadrlgidyskliynl Petition 870250094916, of 17 / 10 / 2025, p. 48 / 154 42 / 54 dkygntsaasiplaldealakkqikyndiiviagfgagltwgttvikw kc Sfp 6 msstrykipwltmniycfgrnlttascclppvqlpthkeahfwyvlpq evkctnllnryfeilspcekenvlrmrgeelkksallaralvrttlar yqtncqinptslkfrkndygkpevdwqytddrslpplhfnishtssli acgvtvgspigidveekqrrlkndilafarryfsphevemlahivdpe lrrqefiklwtlkeayvkalgrgfsaspfntftirlrdhvvkggihvp pdviskeaeitvepsgdlknlssnwhfvlleligshyaaiciekdntn adkgsipvnliirktipfvedgcisgtdsaevigglskllvc ACCase 7 masvgrgngylnsvlpsrhpatttevdeycnalggnkpihsilianng maavkfirsvrswayetfgtekaillvamatpedmrinaehiriadqf vevpggtnnnnyanvqlileiaeithvdavwpgwghasenpelpdalk akgivflgppaismaalgdkigssliaqaaevptlpwsgshvkippes dlitipdeiyraacvytteeaiascqvvgypamikaswggggkgirkv hnddevralfkqvqgevpgspifimkvasqsrhlevqlicdqhgnfaa lhsrdcsvqrrhqkiieegpitvappetvkeleqaarrlaksvnyvga atveylysmetgeyyflelnprlqvehpvtewiaeinlpaaqvavgmg iplwqipeirrfygmehgggndgwkktsvlatpfdfdeaqstkpkghc vavrvtsedpddgftptggkvqelsfkskpnvwayfsvksgggihefs dsqfghvfafgesralaianmvlglkeiqirgeirtnvdytidllnas dyrdnkihtgwldsriamrvraerppwylsvvggalykasassaalvsdyvgylekgqippkhislvhsqvslsiegskytidmvrggpgsyklkl nqseieaeihtlrdggllmqldgnshviyaeeeaagtrllidgrtcll qndddpskligetpckllrylvaddsqidadtpyaevevmkmcmplls pasgiihfrmaegqamqageliakldlddgsavrkaepftgsfpilgp ptaisgkvhqkcaaslnaarmilagyehnidevvvksllncldspelp flqwqecfavlatrlpkdlrneleakykefeiisssqtidfpakllka ileahlsscpenekgalerlvepltslvksyeggreshahkivqslfe eylsveelfsdniqadvierlrlqykkdllkivdivlshqgvksknkl Petition 870250094916, dated 10 / 17 / 2025, page 49 / 154 43 / 54 ilrlmdklvypnpaayrdqlirfsqlnhivyselalkasqlleqtkls elrssiarslselemftedgenidtpkrksaindrmedlvsaplaved alvglfdhsdhtlqrrvvetyirrlyqpylvkdsirmqwhrsgliatw efleeyverkngvedktlvekhsekkwgvmvvikslqflpaiisaalr eatnnfhdplksgsgdssnhgnmmhiglvginnqmsllqdsgdedqaq eridklakilreqeigsiihaagvgdisciiqrdegrapmrhsfhwss eklyyveeplllhlepplsiyleldklkcyenirytpsrdrqwhlytv vdtkpqpiqrmflrtlirqpttnegyssyqrldaetsrtqlamsytsr sifrslmgameelelnshnttiksehahmylyiireqqiddlvpyskk inieagqeettveaileelaqeihssvgvrmhrlgvfvweiklwitac gqangawrvivnnvtghtctvhiyremedatthkvvyssvtvkgplhg vpvnenyqplggidrkrlaarknsttycydfplafqtsleqswsiqqt giqrandkdllkvtelkfsekagswgtslvpaerlpglndvgmvawlm emctpkfpsgrtilvvsndvtfkagsfgpredaffravtdlacakkip liylaansgarlgvaeevkacfkvgwseeskpehgfqyvyltpedyar igssvmahelklesgetrwvidtivgkedglgvenlsgsgaiagaysr ayketftltyvtgrtvgigaylarlgmrciqrldqpiiltgfsalnkl lgrevysshmqlggpkimatngvvhltvsddlegvssilkwlsyvpsh vggalpivkpldppereveylpenscdpraaisgtldvngkwlggifd kdsfvetlegwartvvtgraklggipvgivavetqtvmqiipadpgqldshervvpqagqvwfpdsatktaqaildfnreelplfiianwrgfsgg qrdlfegilqagstivenlrtykqpifvyipmmgelrggawvvvdsri nsdhiemyaertakgnvlepegmieikfrtrellecmrrldqqlinlk eklseaksnkdygaydslqqqirfrekqllplytqiatkfaelhdtsl rmkakgvirevldwrksrsvfyqrlhrrigehslinivrdaagdqlsy vsamnllkewylnsdiakgredawlddeaffrwrddpanyedklkelr vqrlllqltnigdsaldlqalpqglaallskleassrdkliselrkvl g Table 2: Primers. Petition 870250094916, dated 10 / 17 / 2025, page 50 / 154 44 / 54 Primer 1 ID seq Primer 2 ID seq Size (bp) Validation primers Mbbl gcctgaatgtgtgtgaattg ctctcg 8 tgacgcacaatcccac tatc 9 643 Mbb2 cttgtccaaagggagaat atgctgc 10 gatccgcatcctcg 111 Mbbc ccctggaggtcgaaggta ttacc 12 tgacgcacaatcccac tatc 13 478 Mbb4 actgtcaagctttggtgc tgg 14 gatccgcatcctttcg cttc 15 722 FabH cttgtaggatttgttctg tatgaactggg 16 agattcgtc Sfp cattcttacgaaacttaa ggctagttggg 18 agatcgaagacgattt tctgggt 19 568 ACCase gccatatcttgtcaaaga tagcatcagg 20 ctctgtatgatacagc tatatctccaacacc 21 450 Kan gaacaagatggattgcaaga gc 367 gagtc aggc 786 Spect ctcttcttgctgttcatc tttatg 38 ccttagtaagaagaat agcaagatc 39 333 Basta ctacgactggacggccg 40 gggggcatatccgagc g 41 187 Petition 870250094916, of 17 / 10 / 2025, p. 51 / 154 45 / 54 Expression primers Mbb1 atgactgatacacagaac cctaatagagcc 22 gcctgaatgtgtgaat tgctctcg 23 533 Mbb2 atggctaagcagaagtcc ctc 24 cttgtccaaagggaga atatgctgc 25 303 Mbb3 atgaccccccccgc6 tattacc 27 368 Mbb4 atggctgaagaacgtaga cag 28 actgtcaagctttggt gctgg 29 420 FabH atgatttcaagtattttg caaggggcc 30 cttgtaggatttgttc tgtatgaactggg 31 340 Sfp atgtccagtaccagatc cattcttacgaaactt aaggctagttggg 33 414 ACCase gccatatcttgtcaaaga tagcatcagg 34 gccatatcttgtcaaa gatagcatcagg 35 450 Left: canamicin; Spetc: Spectinomycin Example 1: Gene cloning of bromoformium-producing algae

[0186] Gene transcripts encoding mbb1-mbb4 (Asparagopsis taxiformis), FabH (Chondrus crispus), sfp (Medicago truncatula), and ACCase (Medicago sativa) were synthesized using codons from Medicago sativa and amplified by PCR with the indicated primers. The amplified DNA was cloned into binary vectors containing a selective marker (spectinomycin, kanamycin, or basta) and transformed into Agrobacterium host cells. Gene screening was performed by colony PCR. Petition 870250094916, dated 10 / 17 / 2025, page 52 / 154 46 / 54 in transformed cells cultured on rifampicin and kanamycin (agrobacterial and vector-selectable antibiotics). Several clones were characterized by whole-plasmid sequencing as having complete coding sequences. Example 2: In-plant screening of plants expressing bromoform

[0187] The genes were grouped into two different combinations, as follows: Group 1 (2 plasmids with 4 genes) Plasmid 1: mbb1 + mbb2 (selective marker - Spectinomycin) Plasmid 2: mbb3 + mbb4 (selective marker - Kanamycin) Group 2 (3 plasmids with 7 genes) Plasmid 3: sfp + mbb1 + mbb2 (Spectinomycin selective marker) Plasmid 4: FabH + mbb3 + mbb4 (selective marker - Kanamycin) Plasmid 5: ACCase (selective marker - Basta)

[0188] The different coding regions were cloned into binary pAB vectors under the control of the constitutive promoters CaMV35S, act8 or ubq10 and terminators HSP, EFIA2 or GmPPO1, respectively, and different combinations of plasmids 1-5 (including each plasmid from 1-4 alone and combinations including 1+2, 3+4, 1+2+5 and 3+4+5) were transformed into plants of Arabidopsis using Agrobacterium according to the flower immersion method, as essentially described in document WO 2018 / 178975. In summary, the plants were grown in soil composed of 75% peat, 25% perlite and routinely irrigated with water supplemented with fertilizer (e.g., Shefer 5.3.8, ICL Israel) as per the manufacturer's instructions, when necessary. The plants began to flower after 3-4 weeks and were ready... Petition 870250094916, dated 10 / 17 / 2025, page 53 / 154 47 / 54 for transformation. Agrobacterium cultures transformed with the previously mentioned plasmids were suspended in suspension buffer (5% sucrose and 0.03% Silwet L-77), and the flowers were immersed in the mixture for 1 minute. After 5-6 weeks of continuous growth, when the plants dried, the seeds were collected and kept in a cool, dry place for 2 weeks or until used. The successful transformation of the plants is shown in Figure 1. Table 3: Genetic structure. Promoter Gene Terminator act8 Ms.mbb1 HSP CaMV35S Ms.mbb2 Gm CaMV35S Ms.mbb3 HSP act8 Ms.mbb4 GmPPO1 ubq10 Ms.FabH EIFA2 ubq10 Ms.sfp EIFA2 CaMV35S Ms.ACCase HSP

[0189] Transformed seeds from generation T1 were selected with a selection of glufosinate ammonium (Basta, Bayer), kanamycin, spectinomycin, or a combination of these selections, as per the manufacturer's instructions. Plants were irrigated with 10 ppm of CaBr2. Selected resistant plants are tested for bromoform production three weeks after germination, as described in Example 7 below. Petition 870250094916, dated 10 / 17 / 2025, page 54 / 154 48 / 54

[0190] Plants growing on selectable markers, confirming successful transformation with bromoform-producing genes, are expected to produce bromoform. Example 3: Bromoform-producing tobacco plants

[0191] Agrobacterium tumefaciens, strain GV3101, was used to transform tobacco plants (Nicotiana tabacum, cv. Little Dutch) with the above plasmids (combinations of plasmids 1-5, including 1+2, 3+4, 1+2+5 and 3+4+5). Each plasmid described above was independently transformed into bacteria by electroporation. For the expression of various genes in tobacco cells and plants, cotransformation with mixed bacteria was used. Tobacco leaf explants were immersed in the A. tumefaciens suspension for 10 minutes, followed by placement in Petri dishes in solid co-culture medium (Gamborg B5 basal salts with vitamins, 0.1 mM acetosyringone, 2 mg / ml BAP, 0.2 mg / ml NAA, 0.8% vegetable agar) and incubation at 25°C for 2 to 3 days in a dark environment.After co-culture, the explants were transferred to solidified shoot induction media (Gamborg B5 basal salts with vitamins, 2 mg / L BAP, 0.2 mg / L NAA, 250 mg / L cefotaxime, selective antibiotics according to plasmid; 100 mg / L kanamycin, 50 mg / L spectinomycin, 2 mg / L bast, 0.8% vegetable agar) and incubated at 25°C, 16h light / 8h dark. Every 14 days the explants were transferred to fresh media. Fully developed surviving shoots were planted in root induction medium (Gamborg B5 basal salts with vitamins, 2 mg / L IBA, 250 mg / L cefotaxime, selective antibiotics according to plasmid; 100 mg / L kanamycin, 50 mg / L spectinomycin, 2 mg / L basta, 0.8% vegetable agar) and cultivated until root development. After acclimation in propagators, the transgenic plants were transplanted to... Petition 870250094916, dated 10 / 17 / 2025, page 55 / 154 49 / 54 potted soil and kept in a greenhouse. The successful transformation of the plants is shown in Figure 1. The plants were irrigated with 10 ppm of CaBr2. Selected resistant plants at three weeks of age are tested for bromoform production, as described in Example 7 below. Example 4: Bromine absorption test by wild-type tobacco plants

[0192] Wild-type tobacco plants were used to test bromine uptake through the root system in soil. Tobacco plants were grown in soil composed of 75% peat, 25% perlite and routinely irrigated with water supplemented with fertilizer (e.g., Shefer 5.3.8, ICL Israel) and regularly watered. From one month of age, irrigation with CaBr2 at a concentration of 5 ppm or 50 ppm was carried out twice a week for 3 consecutive weeks, and control plants continued to be irrigated without CaBr2. Three 3 cm2 discs were collected from each plant and used for bromine content analysis to assess bromine uptake. Table 4: Bromine content analysis. Extract Treatment Extract weight (mg) Br (mg / kg) 1 control 100.6 0.0 2 control 100.2 0.0 3 5ppm 100.3 19.9 4 5ppm 103 9.7 5 50ppm 100.3 10.0 Petition 870250094916, dated 10 / 17 / 2025, page 56 / 154 50 / 54 6 50ppm 101.9 1265.9

[0193] The results presented in Table 4 indicate that plants can absorb bromine from the soil, which can accumulate in their leaves. The accumulation of bromine, even at these high concentrations, does not cause any detectable damage to the plants or to their respective growth and development. Example 5: Bromoform-producing alfalfa plants

[0194] The above coding regions are cloned into pPA binary vectors, as described above, and combinations of vectors 1-5, including 1+2, 3+4, 1+2+5 and 3+4+5, were used to transform alfalfa plants (Medicago sativa spp.) by Agrobacterium-mediated transformation. Seeds are collected from the transformants, planted and selected to detect the presence of the construct.

[0195] The seeds are superficially sterilized by immersion in 70% ethanol for 30 seconds. After removal of the ethanol, the seeds are treated with 20% sodium hypochlorite and thoroughly washed with sterile water 3 times. Individual seeds are then placed in magenta boxes containing MS basal medium (Murashige and Skoog 1962), 30 g / L sucrose and 0.8% vegetable agar. Cotyledons and fully developed leaves from plants 2 to 3 weeks old are removed and cut in half on moist filter paper.

[0196] Approximately 100 leaf and cotyledon explants are transferred to an A. tumefaciens suspension (OD 0.5 to 600 nm) in infection medium (MS basal medium (Murashige and Skoog 1962), 30 g / L sucrose and 346 μM acetosyringone) at pH 5.8 and incubated for 20 minutes at room temperature. The explants are transferred to filter paper to avoid Petition 870250094916, dated 10 / 17 / 2025, page 57 / 154 51 / 54 excessive bacterial growth - during access to co-culture and transferred to co-culture medium (Gamborg B5 medium (Gamborg 1984), 30 g / L sucrose, 2 mg / L 2,4-D, 0.1 mg / L kinetin and 346 μM acetosyringone) for 3 days in a dark environment.

[0197] Seven days after inoculation, the explants are washed with sterile water to remove bacteria and briefly dried on filter paper. 10-12 explants are transferred to callus induction medium (Gamborg B5 medium, 30 g / L sucrose, 0.8% vegetable agar, 2 mg / L 2,4-D, 0.1 mg / L kinetin, 500 mg / L cefotaxime sodium and selection reagent (according to the constructor 50 mg / L kanamycin / 4 mg / L PPT / 25 mg / L spectinomycin)). The explants are transferred to fresh media every 14 days until somatic embryos form. Mature dark green embryos are placed in plant development medium (Gamborg B5 medium, 30 g / L sucrose, 0.8% vegetable agar, 0.1 mg / L myo-inositol, 500 mg / L cefotaxime sodium, and selection reagent (according to the constructor: 50 mg / L kanamycin / 4 mg / L PPT / 25 mg / L spectinomycin)). Calluses are transferred to fresh medium every 14 days until shoots are formed. Developed green plantlets are transferred to rooting medium (Gamborg B5 medium, 30 g / L sucrose, 0.8% vegetable agar, 0.1 mg / L myo-inositol, 500 mg / L cefotaxime sodium). After acclimation in propagators, the transgenic plants are transplanted into potting soil and kept in a greenhouse.

[0198] The T1 generation transformed seeds are germinated in the selection (as described above) according to the manufacturer's instructions. 3-4 days after germination, the plants are irrigated with 5 ppm or 50 ppm of CaBr2. Selected resistant plants are Petition 870250094916, dated 10 / 17 / 2025, page 58 / 154 52 / 54 tested for bromoform production three weeks after germination.

[0199] Plants growing on selectable markers, confirming successful transformation with bromoform-producing genes, are expected to also produce bromoform. Example 6: Generation of a genetically modified plant from Brachiaria

[0200] The above plasmids (combinations of plasmids 1-5, including 1+2, 3+4, 1+2+5 and 3+4+5) are transformed into Brachiaria (brachiaria grass) by Agrobacterium-mediated transformation, and the seeds are collected from the transformants, planted and screened for the presence of the construct.

[0201] The seeds are surface sterilized for 20 minutes in 20% sodium hypochlorite and washed 3 times carefully with sterile water. The disinfected seeds are placed in solid agar medium containing 5 g / L sucrose and 6 g / L agar for 5 hours after immersion in sterile distilled water overnight at room temperature and in the dark. Embryo explants are isolated using a scalpel blade.

[0202] Approximately 150 embryos are transferred to the A. tumefaciens suspension (OD 0.5 to 600 nm) in infection medium (basal medium). MS (Murashige and Skoog 1962), 30 g / L sucrose, 346 μM acetosyringone, and 1 mM dithiothreitol) at pH 5.8 and sonicated for seconds. After sonication, the explants are incubated for 2 hours at room temperature and transferred to co-culture medium (MS basal medium, 30 g / L sucrose, 100 mg / L casein hydrolysate, 2 mg / L 2,4-D, 0.2 mg / L BAP, and 346 μM acetosyringone) with a single layer of sterile filter paper autoclaved for 3 days in the dark. Petition 870250094916, dated 10 / 17 / 2025, p. 59 / 154 53 / 54

[0203] After co-culture, the base of each embryonic axis is embedded in callus induction medium (MS basal medium, 30 g / L sucrose, 0.8% vegetable agar, 300 mg / L casein hydrolysate, 2 mg / L 2,4-D, 0.2 mg / L BAP, 500 mg / L cefotaxime sodium and selection reagent (according to the construct: 50 mg / L kanamycin / 4 mg / L PPT / 25 mg / L spectinomycin)). Explants are transferred to fresh media every 14 days. Embryonic calli are transferred to regeneration medium (MS basal medium, 30 g / L sucrose, 0.8% vegetable agar, 300 mg / L casein hydrolysate, 100 mg / L myo-inositol, 4 mg / L kinetin, 500 mg / L cefotaxime sodium and selection reagent (according to the construct: 50 mg / L kanamycin / 4 mg / L PPT / 25 mg / L spectinomycin)). The developed shoots are rooted in the presence of IAA mg / L and 15 g / L sucrose. After acclimation in propagators, the transgenic plants are transplanted into potting soil and kept in a greenhouse.

[0204] Plants growing on selectable markers, confirming successful transformation with bromoform-producing genes, are expected to also produce bromoform. Example 7: Bromoform production test

[0205] Transformed plant leaves, optionally after irrigation with bromine (e.g., CaBr2), as detailed above, are harvested, ground, and extracted. The extracts are analyzed for their bromoform-producing capacity by testing bromoform and its precursors by GC / LC-MS, as follows: ~500 mg of plant leaves are weighed into an HS-20 flask, and the flask is cryogenically frozen in a beaker containing acetone and dry ice; the leaves are then ground to a fine powder and 13 ml of H2O, 5.0 g of NaCl, and 10 µl of DCB (1,4-dichlorobenzene) (~0.13 mg / ml) are added to the flask; the flask Petition 870250094916, dated 10 / 17 / 2025, page 60 / 154 54 / 54 is tightly sealed and inserted into a headspace gas chromatography / mass spectrometry (GC / MS) analysis system. Petition 870250094916, dated 10 / 17 / 2025, page 61 / 154

Claims

1 / 6 Claims 1. A system for gene expression in a plant, comprising one or more recombinant nucleic acid molecules comprising one or more nucleotide sequences, together comprising two or more genes, characterized in that: each gene is functionally linked to a promoter for expression in the plant; and the two or more genes encode two or more enzymes involved in bromoform production and not encoded by the native plant.

2. System of claim 1, characterized in that the two or more genes are included in a single nucleic acid molecule.

3. System of claim 1, characterized in that the two or more genes are included in two or more nucleic acid molecules.

4. A system of any one of claims 1-3, characterized in that at least one of the two or more genes are from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Chondrus crispus, Macrocystis pyrifera, Medicago sativa, and Medicago truncatula.

5. A system of any one of claims 1-4, characterized in that at least two of the two or more genes are from at least one non-plant organism.

6. System of any one of claims 1-5, characterized in that the two or more enzymes comprise at least one haloperoxidase. Petition 870250094916, dated 10 / 17 / 2025, page 62 / 154 2 / 6 7. System of any one of claims 1-6, characterized in that the two or more enzymes comprise an NAD(P)H Oxidase (NOX).

8. A system of any one of claims 1-7, characterized in that the two or more enzymes comprise at least one enzyme involved in fatty acid synthesis.

9. System of claim 8, characterized in that at least one enzyme involved in fatty acid synthesis is selected from Beta-ketoacyl-[acyl carrier protein] synthase III (FabH), phosphopantetheine transferase (sfp), acetyl-CoA carboxylase (ACCase) and malonyl CoA synthetase (MatB).

10. System of claim 9, characterized in that the two or more enzymes comprise haloperoxidase, NOX and at least one enzyme involved in fatty acid synthesis, selected from FabH, sfp, ACCase and MatB.

11. System of claim 10, characterized in that the two or more enzymes comprise haloperoxidase, NOX, FabH, sfp and ACCase.

12. System of any one of claims 1-11, characterized in that two or more genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase and MatB.

13. System of claim 12, characterized in that the two or more genes comprise Mbb1, Mbb2, Mbb3, Mbb4 and at least one gene selected from FaBH, Sfp, ACCase and MatB.

14. System of claim 13, characterized in that the two or more genes comprise Mbb1, Mbb2, Mbb3, Mbb4, FaBH, Sfp and ACCase. Petition 870250094916, dated 10 / 17 / 2025, p. 63 / 154 3 / 6 15. System of any one of claims 10-14, characterized in that: haloperoxidase has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 1 (Mbb1), SEQ ID NO: 3 (Mbb3), or SEQ ID NO: 4 (Mbb4); NOX has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 2 (Mbb2); FabH has at least 70, 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 5; The SFP has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO: 6; and / or ACCase has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO:

7.

16. A system matching any one of claims 1-15, characterized in that two or more genes are functionally linked to identical promoters.

17. A system matching any one of claims 1-15, characterized in that two or more genes are functionally linked to different promoters.

18. System of any of the claims 1-17, characterized in that at least one of the promoters is a constitutive promoter.

19. System of any of the claims 1-17, characterized in that at least one of the promoters is an inducible promoter.

20. System of claim 19, characterized in that the inducible promoter is a tissue-specific promoter, activated in plant organs that develop late in the plant's life cycle, such as leaves or flowers.

21. System of any of claims 1-20, characterized in that at least one of the two or more genes Petition 870250094916, of 10 / 17 / 2025, page 64 / 154 4 / 6 further encodes a chloroplast transit peptide to direct enzymes to the chloroplast.

22. System of any one of claims 1-21, characterized in that the plant is a pasture plant or a forage plant.

23. System of any one of claims 1-22, characterized in that the plant is a grass, a cereal or a legume.

24. System of any one of claims 1-23, characterized in that the plant is selected from brachiaria grass (Brachiaria), alfalfa (Medicago sativa, alfalfa), ryegrass, tall fescue, dactylus, clover, oats, millet, kikuyu, chicory, corn, soybean and forage plantain.

25. Composition characterized in that it comprises the system of any one of claims 1-24.

26. A method for preparing a genetically modified plant or plant part, characterized in that it includes transforming a plant or plant part with the system of any one of claims 1 to 24 or with the composition of claim 25.

27. A genetically modified plant cell, characterized in that it comprises the system of any one of claims 1-24 or prepared by the method of claim 26.

28. A genetically modified plant or plant part producing bromoform, characterized in that it comprises the system of any one of claims 1 to 24 or prepared by the method of claim 26. Petition 870250094916, dated 10 / 17 / 2025, page 65 / 154 5 / 6 29. A genetically modified plant or plant part that produces bromoform, characterized in that it comprises one or more exogenous nucleotide sequences comprising two or more genes, wherein each gene is functionally linked to a promoter for expression in the plant; and the two or more genes together encode two or more enzymes involved in the production of bromoform and not encoded by the native plant.

30. A genetically modified plant or plant part that produces bromoform, characterized in that the plant part is a plant cell.

31. A plant or part of a plant producing bromoform of any of the claims 28-30, characterized in that the plant is selected from brachiaria grass (Brachiaria), alfalfa (Medicago sativa, alfalfa), ryegrass, tall fescue, dactylus, clover, oats, millet, kikuyu, chicory, corn, soybean and forage plantain.

32. A plant or part of a plant producing bromoform of any of claims 28 to 31, characterized in that the part of the plant is selected from seed, cell, leaf, stem, root, tuber, cutting, flower, bark, fruit, bulb, trichome and rhizome.

33. A composition comprising the bromoform-producing plant or plant part of any of claims 28 to 32 mixed with a non-bromoform-producing plant or plant part, characterized in that the composition comprises up to about 10% of bromoform-producing plant or plant part.

34. A method intended to reduce methane gas emissions from a ruminant animal, characterized in that it comprises: Petition 870250094916, dated 10 / 17 / 2025, page 66 / 154 6 / 6 - production of a genetically modified seed of a pasture plant, the seed comprising two or more genes involved in bromoform production; - sowing the genetically modified seed in a field where a ruminant animal grazes; and - cultivation of a bromoform-producing pasture plant derived from the seed, causing the bromoform-producing pasture plant to be consumed by the ruminant animal.

35. A method intended for the preparation of feed to reduce methane gas emissions from a ruminant animal, characterized in that it comprises: - production of a genetically modified seed of a forage plant, the seed comprising two or more genes involved in the production of bromoform; - sowing of the genetically modified seed; - cultivation of a bromoform-producing forage plant derived from the seed; and - preparation of a feed containing the bromoform-producing forage plant or a part thereof.

36. Method of claim 34 or 35, characterized in that the genetically modified seed is produced by the method of claim 26.

37. Method, according to any one of claims 34-36, characterized in that the ruminant animal is a cow. Petition 870250094916, dated 10 / 17 / 2025, p. 67 / 154

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