# US 2025/0146010 A1 · Compositions and methods for soybean plant transformation

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2025/0146010 A1
- Document ID: US20250146010A1
- Status: Published application
- Kind: A1
- Application number: 18/867,924
- Filed: 2023-07-11
- Published: 2025-05-08
- Earliest claimed: 2022-07-12
- Assignees: Inari Agriculture Technology, Inc.
- Applicants: Inari Agriculture Technology, Inc.
- Inventors: TEJINDER KUMAR MALL; CHRISTOPHER ALEXANDER BAGLEY; RHODA ARABA TAWIAH BREW-APPIAH; LARISSA ARRAIS GUIMARAES
- Web page: https://patentgrep.com/patent/US20250146010A1

## CPC

- C12N 15/8267: {Seed dormancy, germination or sprouting}
- C07K 14/415: from plants
- C12N 9/1022: {transferring aldehyde or ketonic groups (2.2)}
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8274: {for herbicide resistance}
- C07K 2319/00: Fusion polypeptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Chimeric GRF-GIF polypeptides that can be used to improve the capacity of plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants are disclosed. Also disclosed are plant cells comprising the chimeric polypeptides and related methods for improving the capacity of the plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants.

## Claims

### Claim 1

A composition comprising:
(i) a first recombinant polynucleotide comprising: (a) polynucleotide encoding a chimeric GRF4-GIF1 (cGRF4-GIF1) polypeptide, optionally wherein the GRF4-GIF1 polypeptide comprises a chimeric dicot plant GRF4-GIF1 (cdpGRF4-dpGIF1) polypeptide or a chimeric GmGRF4-GmGIF1 (cGmGRF4-GmGIF1) polypeptide, said cGmGRF4-GmGIF1 polypeptide comprising from its amino terminus (N-terminus) to its carboxy terminus (C-terminus) a GmGF4 polypeptide comprising an amino acid sequence having at least 92%, 95%, 98%, or 99% sequence identity across the entire length of SEQ ID NO: 1 and a GmGIF1 polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of SEQ ID NO: 2; and (b) a terminator which is operably linked to said polynucleotide; and
(ii) a second recombinant polynucleotide comprising a promoter which is operably linked to a polynucleotide encoding an acetohydroxyacid synthase (AHAS) which confers resistance to an AHAS inhibitor and a terminator which is operably linked to said polynucleotide.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the GmGF4 polypeptide comprises an amino acid sequence having at least 98% or 99% sequence identity across the entire length of SEQ ID NO: 1 and the GmGIF1 polypeptide comprises an amino acid sequence having at least 98%, or 99% sequence identity across the entire length of SEQ ID NO: 2.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the GF4, dpGF4, or GmGF4 polypeptide comprises a QLQ and a WRC domain, optionally wherein QLQ domain comprises the amino acid sequence of SEQ ID NO: 4 and/or wherein the WRC domain comprises the amino acid sequence of SEQ ID NO: 5.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the GIF1, dpGIF1, or GmGIF1 polypeptide comprises an SNH domain, optionally wherein the SNH domain comprises the amino acid sequence of SEQ ID NO: 6.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the cGmGRF4-GmGIF1 polypeptide comprises an amino acid sequence having at least 95%, 98%, or 99% sequence identity across the entire length of SEQ ID NO: 3.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the GF4, dpGF4, or cGmGRF4-GmGIF1 polypeptide encoded by the polynucleotide further comprises a spacer peptide which operably links the C-terminus of said GmGF4 polypeptide to the N-terminus of said GmGIF1 polypeptide, optionally wherein the spacer peptide comprises at least two amino acids comprising alanine, glycine, or comprises a combination of at least two glycine residues and one serine residue.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the first recombinant polynucleotide comprises a recombinant DNA molecule or a recombinant RNA molecule.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the recombinant DNA molecule further comprises a promoter which is operably linked to the polynucleotide encoding the cGmGRF4-GmGIF1 polypeptide.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the second recombinant polynucleotide further comprises an expression cassette comprising a promoter which is operably linked to a polynucleotide encoding an RNA or protein of interest and a terminator which is operably linked to said polynucleotide, optionally wherein the RNA or protein of interest comprise one or more gene editing molecules and/or optionally wherein the second recombinant polynucleotide further comprises one or more DNA elements which provide for bacterially mediated transfection or transformation of a plant cell.

### Claim 10 (depends on 9)

The composition of claim 9, wherein the polynucleotide encodes an RNA comprising a guide RNA (gRNA) which is directed to a target DNA sequence found in a soybean genome and optionally wherein the target DNA sequence is exclusively found in a soybean genome.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

A bacterial cell comprising the composition of any one of claims 1 to 10, optionally wherein the bacterial cell is an Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., or Phyllobacterium sp. cell.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

A soybean plant cell comprising the composition of any one of claims 1 to 10.

### Claim 13 (depends on 12)

The soybean plant cell of claim 12, wherein the second recombinant polynucleotide is stably incorporated into the genome of the soybean plant cell.

### Claim 14 (depends on 12)

The soybean plant cell of claim 12, wherein the first recombinant polynucleotide is transiently expressed in the soybean plant cell and/or is not stably incorporated into the genome of the soybean plant cell.

### Claim 15 (depends on 12)

The soybean plant cell of claim 12, wherein the soybean plant cell comprises elite soybean germplasm and/or soybean germplasm which is recalcitrant to regeneration.

### Claim 16 (depends on 12)

The soybean plant cell of claim 12, wherein expression of the chimeric polypeptide increases the regeneration capacity of the soybean plant cell in comparison to a control soybean plant cell lacking the chimeric polypeptide.

### Claim 17 (depends on 12)

The soybean plant cell of claim 12, wherein the percentage of transgenic or genome edited shoots recovered from the soybean plant cell in a transformation or genome editing procedure is increased by up to about 9-fold in comparison to a control soybean plant cell lacking the chimeric polypeptide.

### Claim 18 (depends on 12)

The soybean plant cell of claim 12, further comprising a genome editing system.

### Claim 19 (depends on 18)

The soybean plant cell of claim 18, wherein the genome editing system comprises a CRISPR-based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor DNA template polynucleotide.

### Claim 20 (depends on 19)

The soybean plant cell of claim 19, wherein the CRISPR-based system comprises (i) an RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease; and (ii) a guide RNA or a polynucleotide encoding the gRNA, optionally wherein the gRNA is directed to a target DNA sequence which is exclusively found in a soybean genome.

### Claim 21 (depends on 12)

A soybean plant, tissue, organ, callus, or cell culture comprising the soybean plant cell of claim 12.

### Claim 22 (depends on 12)

A method of producing a regenerable plant structure, the method comprising:
culturing the soybean plant cell of claim 12 in the presence of an acetohydroxyacid synthase (AHAS) inhibitor at a concentration sufficient to select for a regenerable plant structure comprising the second recombinant polynucleotide, thereby producing the regenerable plant structure.

### Claim 23 (depends on 22)

The method of claim 22, wherein the first and/or second recombinant polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

### Claim 24 (depends on 22)

The method of claim 22, wherein the second recombinant polynucleotide is stably incorporated into the genome of the soybean plant cell.

### Claim 25 (depends on 22)

The method of claim 22, wherein the first recombinant polynucleotide is transiently expressed in the soybean plant cell and/or is not stably incorporated into the genome of the soybean plant cell.

### Claim 26 (depends on 22)

The method of claim 22, wherein the soybean plant cell comprises a regeneration-recalcitrant germplasm.

### Claim 27 (depends on 22)

The method of claim 22, wherein the regenerable plant structure comprises a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots.

### Claim 28 (depends on 12, 22)

The method of claim 22, further comprising introducing the composition comprising the first recombinant polynucleotide and the second recombinant polynucleotide into a soybean plant cell to obtain the soybean plant cell of claim 12 that is cultured.

### Claim 29 (depends on 28)

The method of claim 28, wherein the introducing comprises bacterial-mediated transformation or biolistic-mediated transformation.

### Claim 30 (depends on 22)

The method of claim 22, wherein expression of the polypeptide results an increase in the percentage of transgenic or genome edited shoots recovered from the soybean plant cell in a transformation or genome editing procedure by up to about 9-fold in comparison to a control soybean plant cell lacking the chimeric polypeptide.

### Claim 31 (depends on 22)

The method of claim 22, further comprising introducing a genome editing system into the soybean plant cell.

### Claim 32 (depends on 31)

The method of claim 31, wherein the genome editing system comprises a CRISPR-based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor template polynucleotide.

### Claim 33 (depends on 32)

The method of claim 32, wherein the CRISPR-based system comprises (i) an RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease; (ii) a guide RNA or a polynucleotide encoding the gRNA; and optionally (ii) a donor.

### Claim 34 (depends on 22)

The method of claim 22, wherein the regenerable structure comprises a shoot of 1 to 2, 3, or 4 centimeters (cm) in length.

### Claim 35 (depends on 34)

The method of claim 34, wherein the culturing further comprises transferring the shoot to a rooting media.

### Claim 36 (depends on 34)

The method of claim 34, further comprising obtaining a plantlet comprising a shoot and roots from the shoot which was transferred to the rooting media.

### Claim 36 (depends on 22)

The method of claim 22, where the AHAS inhibitor comprises a sulfonylurea, imidazolinone, triazoloyrimidine, or triazolinone herbicide.

### Claim 37 (depends on 36)

The method of claim 36, wherein the sulfonylurea herbicide is bensulfuron-methyl, chlorsulfuron, ethametsulfuron-methyl, foramsulfuron, halosulfuron, mesosulfuron-methyl, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, or triflusulfuron-methyl.

### Claim 38 (depends on 36)

The method of claim 36, wherein the imidazolinone herbicide is imazapyr, imazapic, imazethapyr, imazamox, imazamethabenz, or imazaquin.