# US 11,441,152 B2 · Methods for improving callus formation and regeneration in plants

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- Number: US 11,441,152 B2
- Document ID: US11441152B2
- Status: Granted
- Kind: B2
- Application number: 16/525,724
- Filed: 2019-07-30
- Published: 2020-01-30
- Granted: 2022-09-13
- Earliest claimed: 2018-07-30
- Assignees: UT-Battelle, LLC
- Applicants: UT-Battelle, LLC
- Inventors: Wellington Muchero; Gerald Tuskan; Jin-Gui Chen; Lee E. Gunter
- Web page: https://patentgrep.com/patent/US11441152B2

## CPC

- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This disclosure provides methods of improving callus formation in plants. This disclosure further provides genetically engineered plants with improved callus formation.

## Claims

### Claim 1

A genetically modified Populus plant, plant cell or plant tissue, wherein the expression of an endogenous suppressor of kinase 1 (SOK1) gene is altered in the Populus plant, plant cell or plant tissue, wherein the alteration comprises inactivation of the endogenous SOK1 gene in the Populus plant, plant cell or plant tissue, resulting in increased callus formation by the Populus plant cell, or increased callus formation in the Populus plant or plant tissue, and wherein the endogenous SOK1 gene encodes an enzyme with at least 98% sequence identity to SEQ ID NO: 23.

### Claim 2 (depends on 1)

The genetically modified Populus plant, plant cell or plant tissue of claim 1, wherein the inactivation of the endogenous SOK1 gene is achieved by introducing a nucleic acid inhibitor of the selected gene to the Populus plant, plant cell or plant tissue.

### Claim 3 (depends on 2)

The genetically modified Populus plant, plant cell or plant tissue of claim 2, wherein the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.

### Claim 4 (depends on 1)

The genetically modified Populus plant, plant cell or plant tissue of claim 1, wherein the inactivation of the endogenous SOK1 gene is achieved by genome editing, which is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, ZFNs system and homologous recombination.

### Claim 5 (depends on 4)

The genetically modified Populus plant, plant cell or plant tissue of claim 4, wherein the CRISPR-mediated genome editing comprises introducing into the Populus plant a first nucleic acid encoding a Cas9 nuclease, a second nucleic acid comprising a guide RNA (gRNA), wherein said gRNA is specific to the endogenous SOK1 gene.

### Claim 6

A method for increasing callus formation, comprising altering in a Populus plant, plant cell or plant tissue the expression of a SOK1 gene or a homolog thereof, wherein the alteration comprises inactivation of the endogenous SOK1 gene in the Populus plant, plant cell or plant tissue, resulting in increased callus formation by the Populus plant cell, or increased callus formation in the Populus plant or plant tissue, and wherein the endogenous SOK1 gene encodes an enzyme with at least 98% sequence identity to SEQ ID NO: 23.

### Claim 7 (depends on 6)

The method of claim 6, wherein the inactivation of the endogenous SOK1 gene is achieved by introducing a nucleic acid inhibitor of the selected gene to the Populus plant, plant cell or plant tissue.

### Claim 8 (depends on 7)

The method of claim 7, wherein the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.

### Claim 9 (depends on 6)

The method of claim 6, wherein the inactivation of the endogenous SOK1 gene is achieved by genome editing, which is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, ZFNs system and homologous recombination.

### Claim 10 (depends on 9)

The method of claim 9, wherein the CRISPR-mediated genome editing comprises introducing into the Populus plant a first nucleic acid encoding a Cas9 nuclease, a second nucleic acid comprising a guide RNA (gRNA), wherein said gRNA is specific to the endogenous SOK1.

### Claim 11 (depends on 1)

The genetically modified Populus plant, plant cell or plant tissue of claim 1, wherein said Populus is Populus trichocarpa.

### Claim 12 (depends on 6)

The method of claim 6, wherein said Populus is Populus trichocarpa.