# US 11,441,153 B2 · Compositions and methods for improving crop yields through trait stacking > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,441,153 B2 - Document ID: US11441153B2 - Status: Granted - Kind: B2 - Application number: 16/276,620 - Filed: 2019-02-15 - Published: 2019-08-15 - Granted: 2022-09-13 - Earliest claimed: 2018-02-15 - Assignees: Monsanto Technology LLC - Applicants: Monsanto Technology LLC - Inventors: Charles R. Dietrich; Natalia Ivleva; Thomas L. Slewinski - Web page: https://patentgrep.com/patent/US11441153B2 ## CPC - C12N 15/8261: {with agronomic (input) traits, e.g. crop yield} - C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]} - C12N 15/8297: {Gibberellins; GA3} - Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present disclosure provides modified, transgenic, or genome edited/mutated corn plants that are semi-dwarf and have one or more improved ear traits relative to a control plant, such as increase in ear diameter, single kernel weight, ear fresh weight, ear area, ear volume, ear length, number of kernels per ear, and yield. The modified, transgenic, or genome edited/mutated corn plants comprise a transgene encoding one or more MADS-box polypeptides and have a reduced expression of one or more GA20 or GA3 oxidase genes. Also provided are methods for producing the modified, transgenic, or genome edited/mutated corn plants. ## Claims ### Claim 1 A transgenic corn plant or a plant part thereof comprising (i) a first recombinant expression cassette comprising a first heterologous plant-expressible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of one or more gibberellic acid 20 (GA20) oxidase genes, and ii) a second recombinant expression cassette comprising a DNA sequence encoding a MADS-box polypeptide having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 168; wherein the non-coding RNA comprises a targeting sequence that is (a) at least 95% complementary to at least 19 consecutive nucleotides of a first mRNA molecule encoding a first endogenous GA20 oxidase protein that has at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9, and (b) at least 95% complementary to at least 19 consecutive nucleotides of a second mRNA molecule encoding a second endogenous GA20 oxidase protein that has at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15; and wherein expression of said non-coding RNA and said MADS-box polypeptide in said transgenic corn plant results in a semi-dwarf phenotype, and an increase in one or more improved ear trait selected from the group consisting of ear diameter, single kernel weight, ear fresh weight, ear area, ear volume, ear length, kernels per ear, yield, grain yield estimate, broad acreage yield, ear dry weight, and ear tip void, as compared to a control wild-type corn plant grown under comparable conditions. ### Claim 2 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the transcribable DNA sequence encoding said non-coding RNA comprises a nucleotide sequence that is 100% identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 39. ### Claim 3 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the MADS-box polypeptide comprises the amino acid sequence as set forth in SEQ ID NOs: 168. ### Claim 4 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the DNA sequence encoding said MADS-box polypeptide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence as set forth in SEQ ID NO: 169. ### Claim 5 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the DNA sequence comprised in the second recombinant expression cassette is operably linked to a second heterologous plant-expressible promoter. ### Claim 6 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the first heterologous plant-expressible promoter is selected from the group consisting of a vascular promoter a leaf promoter and a constitutive promoter. ### Claim 7 (depends on 6) The transgenic corn plant or plant part thereof of claim 6, wherein the first heterologous plant-expressible promoter is said vascular promoter selected from the group consisting of a sucrose synthase promoter, a sucrose transporter promoter, a sucrose synthase-1 (Sh 1) promoter, a Commelina yellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV) large intergenic region (LIR) promoter, a maize streak geminivirus (MSV) coat polypeptide (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter, and a rice yellow stripe 2 (OsYSL2) promoter. ### Claim 8 (depends on 6) The transgenic corn plant or plant part thereof of claim 6, wherein the first heterologous plant-expressible promoter is said leaf promoter selected from the group consisting of a RuBisCO promoter, a pyruvate phosphate dikinase (PPDK) promoter, a fructose 1-6 bisphosphate aldolase (FDA) promoter, a Nadh-Gogat promoter, a chlorophyll a/b binding polypeptide gene promoter, a phosphoenolpyruvate carboxylase (PEPC) promoter and a Myb gene promoter. ### Claim 9 (depends on 6) The transgenic corn plant or plant part thereof of claim 6, wherein the first heterologous plant-expressible promoter is said constitutive promoter selected from the group consisting of an actin promoter, a Cauliflower mosaic virus (CaMV) 35S promoter, a CaMV 19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a Figwort mosaic virus (FMV) promoter, a cytomegalovirus (CMV) promoter, a mirabilis mosaic virus (MMV) promoter, a peanut chlorotic streak caulimovirus (PCLSV) promoter, an Emu promoter, a tubulin promoter, a nopaline synthase promoter, an octopine synthase promoter, a mannopine synthase promoter, and a maize alcohol dehydrogenase. ### Claim 10 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the first heterologous plant-expressible promoter is a rice tungro bacilliform virus (RTBV) promoter. ### Claim 11 (depends on 10) The transgenic corn plant or plant part thereof of claim 10, wherein the RTBV promoter comprises a DNA sequence that is at least 95% identical to the nucleotide sequence as set forth in SEQ ID NO: 65 or SEQ ID NO: 66. ### Claim 12 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter is selected from the group consisting of a root-specific promoter, a meristem promoter, a seed or kernel promoter, and a constitutive promoter. ### Claim 13 (depends on 12) The transgenic corn plant or plant part thereof of claim 12, wherein the second heterologous plant-expressible promoter is said root-specific promoter comprising a DNA sequence that is at least 95% identical to the nucleotide sequence as set forth in SEQ ID NO: 170. ### Claim 14 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter comprises an Rcc3 gene promoter. ### Claim 15 (depends on 12) The transgenic corn plant or plant part thereof of claim 12, wherein the second heterologous plant-expressible promoter is said constitutive promoter selected from the group consisting of an actin promoter, a Cauliflower mosaic virus (CaMV) 35S promoter, a CaMV 19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a Figwort mosaic virus (FMV) promoter, a cytomegalovirus (CMV) promoter, a mirabilis mosaic virus (MMV) promoter, a peanut chlorotic streak caulimovirus (PCLSV) promoter, an Emu promoter, a tubulin promoter, a nopaline synthase promoter, an octopine synthase promoter, a mannopine synthase promoter, and a maize alcohol dehydrogenase. ### Claim 16 (depends on 1) A transgenic corn seed or a part thereof of the transgenic corn plant of claim 1, wherein the transgenic corn seed comprises the first and second recombinant expression cassettes. ### Claim 17 (depends on 1) A commodity product produced from the transgenic corn plant or a part thereof of claim 1, wherein the commodity product comprises the first and second recombinant expression cassettes. ### Claim 18 A recombinant DNA construct comprising (i) a first expression cassette comprising a first heterologous plant-expressible promoter operably linked to a transcribable DNA sequence encoding a non-coding RNA for suppression of one or more GA20 oxidase genes, and (ii) a second expression cassette comprising a second heterologous plant-expressible promoter operably to a DNA sequence encoding a MADS-box polypeptide having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 168; wherein the non-coding RNA comprises a targeting sequence that is (a) at least 95% complementary to at least 19 consecutive nucleotides of a first mRNA molecule encoding a first endogenous GA20 oxidase protein that has at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9, and (b) at least 95% complementary to at least 19 consecutive nucleotides of a second mRNA molecule encoding a second endogenous GA20 oxidase protein that has at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15; and wherein expression of said non-coding RNA and said MADS-box polypeptide in a corn plant results in a semi-dwarf phenotype, and an increase in one or more improved ear trait selected from the group consisting of ear diameter, single kernel weight, ear fresh weight, ear area, ear volume, ear length, kernels per ear, yield, grain yield estimate, broad acreage yield, ear dry weight, and ear tip void, as compared to a control wild-type corn plant grown under comparable conditions. ### Claim 19 (depends on 18) The recombinant DNA construct of claim 18, wherein the transcribable DNA sequence encoding said non-coding RNA comprises a nucleotide sequence that is 100% identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NO: 39. ### Claim 20 (depends on 18) The recombinant DNA construct of claim 18, wherein the MADS-box polypeptide comprises the amino acid sequence as set forth in SEQ ID NOs: 168. ### Claim 21 (depends on 18) The recombinant DNA construct of claim 18, wherein the DNA sequence encoding said MADS-box polypeptide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence as set forth in SEQ ID NO: 169. ### Claim 22 (depends on 18) The recombinant DNA construct of claim 18, wherein the first heterologous plant-expressible promoter is selected from the group consisting of a vascular promoter, a leaf promoter, and a constitutive promoter. ### Claim 23 (depends on 22) The recombinant DNA construct of claim 22, wherein the first heterologous plant-expressible promoter is said vascular promoter selected from the group consisting of a sucrose synthase promoter, a sucrose transporter promoter, a sucrose synthase-1 (Sh 1) promoter, a Commelina yellow mottle virus (CoYMV) promoter, a wheat dwarf geminivirus (WDV) large intergenic region (LIR) promoter, a maize streak geminivirus (MSV) coat polypeptide (CP) promoter, a rice yellow stripe 1 (YS1)-like promoter, and a rice yellow stripe 2 (OsYSL2) promoter. ### Claim 24 (depends on 22) The recombinant DNA construct of claim 22, wherein the first heterologous plant-expressible promoter is said leaf promoter selected from the group consisting of a RuBisCO promoter, a pyruvate phosphate dikinase (PPDK) promoter, a fructose 1-6 bisphosphate aldolase (FDA) promoter, a Nadh-Gogat promoter, a chlorophyll a/b binding polypeptide gene promoter, a phosphoenolpyruvate carboxylase (PEPC) promoter, and a Myb gene promoter. ### Claim 25 (depends on 22) The recombinant DNA construct of claim 22, wherein the first heterologous plant-expressible promoter is said constitutive promoter selected from the group consisting of an actin promoter, a Cauliflower mosaic virus (CaMV) 35S promoter, a CaMV 19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a Figwort mosaic virus (FMV) promoter, a cytomegalovirus (CMV) promoter, a mirabilis mosaic virus (MMV) promoter, a peanut chlorotic streak caulimovirus (PCLSV) promoter, an Emu 9 promoter, a tubulin promoter, a nopaline synthase promoter, an octopine synthase promoter, a mannopine synthase promoter, and a maize alcohol dehydrogenase. ### Claim 26 (depends on 18) The recombinant DNA construct of claim 18, wherein the first heterologous plant-expressible promoter is a rice tungro bacilliform virus (RTBV) promoter. ### Claim 27 (depends on 18) The recombinant DNA construct of claim 18, wherein the RTBV promoter comprises a DNA sequence that is at least 95% identical to the nucleotide sequence as set forth in SEQ ID NO: 65 or SEQ ID NO: 66. ### Claim 28 (depends on 18) The recombinant DNA construct of claim 18, wherein the second heterologous plant-expressible promoter is selected from the group consisting of a root-specific promoter, a meristem promoter, a seed or kernel promoter, and a constitutive promoter. ### Claim 29 (depends on 28) The recombinant DNA construct of claim 28, wherein the second heterologous plant-expressible promoter is said root-specific promoter comprising a DNA sequence that is at least 95% identical to the nucleotide sequence as set forth in SEQ ID NO: 170. ### Claim 30 (depends on 18) The recombinant DNA construct of claim 18, wherein the second heterologous plant-expressible promoter comprises an Rcc3 gene promoter. ### Claim 31 (depends on 28) The recombinant DNA construct of claim 28, wherein the second heterologous plant-expressible promoter is said constitutive promoter selected from the group consisting of an actin promoter, a Cauliflower mosaic virus (CaMV) 35S promoter, a CaMV 19S promoter, a plant ubiquitin promoter, a plant Gos2 promoter, a Figwort mosaic virus (FMV) promoter, a cytomegalovirus (CMV) promoter, a mirabilis mosaic virus (MMV) promoter, a peanut chlorotic streak caulimovirus (PCLSV) promoter, an Emu promoter, a tubulin promoter, a nopaline synthase promoter, an octopine synthase promoter, a mannopine synthase promoter, and a maize alcohol dehydrogenase. ### Claim 32 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the first heterologous plant-expressible promoter is a vascular promoter. ### Claim 33 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the first heterologous plant-expressible promoter is a leaf promoter. ### Claim 34 (depends on 1) The transgenic corn plant or plant part thereof of claim 1, wherein the first heterologous plant-expressible promoter is a constitutive promoter. ### Claim 35 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter is a root-specific promoter. ### Claim 36 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter is a meristem promoter. ### Claim 37 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter is a seed or kernel promoter. ### Claim 38 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the second heterologous plant-expressible promoter is a constitutive promoter. ### Claim 39 (depends on 5) The transgenic corn plant or plant part thereof of claim 5, wherein the first heterologous plant-expressible promoter is a vascular promoter, and the second heterologous plant-expressible promoter is a root promoter.