# US 12,760,311 B2 · Enhanced plant regeneration and transformation by using GRF1 booster gene

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- Number: US 12,760,311 B2
- Document ID: US12760311B2
- Status: Granted
- Kind: B2
- Application number: 17/622,018
- Filed: 2020-06-29
- Published: 2022-07-28
- Granted: 2026-10-13
- Earliest claimed: 2019-06-28
- Assignees: KWS SAAT SE & Co. KGaA
- Applicants: KWS SAAT SE & Co. KGaA
- Inventors: Jixiang Kong; David Pacheco Villalobos; Susana Martin-Ortigosa
- Web page: https://patentgrep.com/patent/US12760311B2

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C07K 14/415: from plants
- 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

The present invention relates to the field of plant breeding and biotechnology and in particular to the generation of plants from cells and other tissues. More particularly, the invention provides methods and means for improving plant regeneration, especially from transformed or genetically modified plant cells using GRF1 booster gene.

## Claims

### Claim 1

A method for modifying the genome of a Zea mays plant cell, comprising the steps:
(a) introducing into the Zea mays plant cell an expression cassette comprising a polynucleotide encoding a growth-regulating factor 1 (GRF1) polypeptide, mRNA encoding a GRF1 polypeptide, or GRF1 polypeptide(s); and
(b) cultivating the Zea mays plant cell of (a) or a plant cell derived from the plant cell of (a) under conditions where in the Zea mays plant cell the GRF 1 polypeptide is expressed from the expression cassette, GRF 1 polypeptide is translated from introduced mRNA, GRF 1 polypeptide is expressed from the endogenous gene, or GRF 1 polypeptide(s) are present;
(c) modifying the genome of the Zea mays plant cell of (b) by means of a single stranded DNA break (SSB) or double stranded DNA break (DSB) inducing enzyme or a base editor enzyme which recognizes a predetermined site in the genome of said Zea mays plant cell, and optionally by means of a repair nucleic acid molecule, wherein the modification of said genome at said predetermined site is selected from:
i) a replacement of at least one nucleotide;
ii) a deletion of at least one nucleotide;
iii) an insertion of at least one nucleotide; or
iv) any combination of i)-iii); and
wherein step (c) is conducted simultaneously with step (a) and/or (b), before step (a), between step (a) and (b) or after step (b),
wherein the polynucleotide encoding the GRF 1 polypeptide comprises the coding nucleotide sequence of SEQ ID NO: 1,
wherein the presence of the GRF1 polypeptide in the Zea mays plant cell results in at least a two-fold increase in transformation efficiency relative to an otherwise identical Zea mays plant cell that does not comprise the GRF 1 polypeptide,
wherein step (a) further comprises introducing an expression cassette comprising a polynucleotide encoding a Wuschel2 (WUS2) polypeptide, a mRNA encoding a WUS2 polypeptide, or WUS2 polypeptide(s), and
wherein co-expression of the GRF1 polypeptide and the WUS2 polypeptide results in enhanced regeneration capability relative to regeneration capability resulting from co-expression of the WUS2 polypeptide and a Zea mays Plethora 7 (ZmPLT7) polypeptide.

### Claim 2 (depends on 1)

A method of producing a genetically modified Zea mays plant, comprising the steps:
(a) modifying the genome of a Zea mays plant cell according to the method of claim 1, and
(b) regenerating from the Zea mays plant cell of (a) or from a Zea mays plant cell derived from the Zea mays plant cell of (a) a Zea mays plant comprising in at least one cell the modification of the genome.

### Claim 3 (depends on 1)

The method of claim 1, wherein the GRF1 polypeptide comprises a QLQ domain and a WRC domain, wherein the QLQ domain finds a match of at least 90% coverage at or near the N-terminus of the GRF1 polypeptide and the WRC domain finds a match of at least 90% C-terminally located to the QLQ domain in the GRF1 polypeptide.

### Claim 4 (depends on 3)

The method of claim 3, wherein the QLQ and WRC domains are located in the N-terminal half of the GRF1 polypeptide.

### Claim 5 (depends on 1)

The method of claim 1, wherein the GRF1 polypeptide comprises the motif [D]-[P]-[E]-[P]-[G]-[R]-[C]-[R]-[R]-[T]-[D]-[G]-[K]-[K]-[W]-[R]-[C]-[AS]-[RK]-[ED]-[A]-[AH]-[PQS]-[D]-[S]-[K]-[Y]-[C]-[E]-[KR]-[H]-[M]-[H]-[R]-[G]-[R]-[N]-[R] (SEQ ID NO: 25) with a maximum number of two mismatches, wherein the motif consists of any of the amino acid sequences SEQ ID NO: 26 to SEQ ID NO: 35, and/or wherein the motif contains a sub-region of amino acid stretch matching the WRC domain.

### Claim 6 (depends on 1)

The method of claim 1, wherein the GRF1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

### Claim 7 (depends on 1)

The method of claim 1, wherein introducing into a Zea mays plant cell the expression cassette comprising a polynucleotide encoding a GRF1 polypeptide results in a stable integration thereof into the genome of the Zea mays plant cell, or wherein introducing into a Zea mays plant cell the expression cassette comprising a polynucleotide encoding a GRF1 polypeptide, mRNA encoding GRF1 polypeptide, or GRF1 polypeptide(s) or inducing in a Zea mays plant cell the enhanced expression level of an endogenous gene encoding a GRF1 polypeptide results in a transient occurrence of GRF1 polypeptide(s) in the Zea mays plant cell or in a progeny cell thereof.

### Claim 8 (depends on 1)

The method of claim 1, wherein the polynucleotide encoding the GRF1 polypeptide is in operative linkage to at least one regulatory sequence suitable for expression of the GRF1 polypeptide in a plant cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Zea mays plant cell of step (a) is a cell of a somatic tissue, callus tissue, a meristematic tissue, an embryonic tissue, a protoplast, gametophyte, pollen, ovule or microspore.