# US 2019/0169624 A1 · Low inoculum, long co-culture agrobacterium-mediated transformation of plants

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- Number: US 2019/0169624 A1
- Document ID: US20190169624A1
- Status: Published application
- Kind: A1
- Application number: 16/071,420
- Filed: 2017-01-19
- Published: 2019-06-06
- Earliest claimed: 2016-01-19
- Assignees: Ohio State Innovation Foundation
- Applicants: Ohio State Innovation Foundation
- Inventors: John James FINER; Zhifen ZHANG
- Web page: https://patentgrep.com/patent/US20190169624A1

## CPC

- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein is a method for producing genetically modified plants comprising inoculating a plant, plant cell, plant seed, or plant tissue with an Agrobacterium containing a nucleic acid of interest at an inoculum density of about 10-10,000 CFU/ml; and culturing the Agrobacterium-inoculated plant, plant cell, plant seed, or plant tissue for a period of from 7 days to 28 days, wherein said culturing results in a genetically modified plant, plant cell, plant seed, or plant tissue. Also disclosed are genetically modified plants, plant cells, plant seeds, or plant tissue based on the methods disclosed herein, as well as their progeny, and propagation material related thereto.

## Claims

### Claim 1

A method for producing a genetically modified plant comprising
(a) inoculating a plant, plant cell, plant seed, or plant tissue with an Agrobacterium containing a nucleic acid of interest at an inoculum density of about 10-10,000 CFU/ml;
(b) culturing the Agrobacterium-inoculated plant, plant cell, plant seed, or plant tissue for a period of from 7 days to 28 days, wherein said culturing results in a genetically modified plant, plant cell, plant seed, or plant tissue.

### Claim 2 (depends on 1)

The method of claim 1, wherein the inoculum density is about 10-1,000 CFU/ml.

### Claim 3 (depends on 1)

The method of claim 1, wherein the inoculum density is about 10-100 CFU/ml.

### Claim 4 (depends on 1)

The method of claim 1, wherein culturing occurs from a period of 10 days to 21 days.

### Claim 5 (depends on 1)

The method of claim 1 wherein the plant is a monocot selected from the group consisting of maize, sorghum, triticale, barley, oats, rye, wheat, onions and rice.

### Claim 6 (depends on 1)

The method of claim 1 wherein the plant is a dicot selected from the group consisting of soybean, alfalfa, tobacco, brassicas, sunflower, cucurbits, potatoes, peppers and tomatoes.

### Claim 7 (depends on 1)

The method of claim 1, wherein the plant is a conifer selected from the group consisting of pine, spruce and fir.

### Claim 8 (depends on 1)

The method of claim 1, wherein the plant is a whole plant.

### Claim 9 (depends on 1)

The method of claim 1, wherein the plant tissue or cell originates from leaves, roots, shoots, seedlings, callus, cell suspension, cotyledon, leaf, root, egg, pollen, or microspores.

### Claim 10 (depends on 1)

The method of claim 1, wherein the plant cell or plant tissue is a meristem or inflorescence.

### Claim 11 (depends on 1)

The method of claim 1, wherein a genetically modified plant is recovered from the plant, plant cell, or plant tissue from step b).

### Claim 12 (depends on 1)

The method of claim 1, wherein the Agrobacterium is genetically modified.

### Claim 13 (depends on 1)

The method of claim 1, wherein an expression vector comprises the nucleic acid of interest.

### Claim 14 (depends on 1)

The method of claim 1, wherein the nucleic acid of interest comprises a selectable marker.

### Claim 15 (depends on 14)

The method of claim 14, wherein the selectable marker is antibiotic resistance.

### Claim 16 (depends on 14)

The method of claim 14, wherein the selectable marker is herbicide resistance.

### Claim 17 (depends on 1)

The method of claim 1, wherein the nucleic acid of interest comprises a screenable marker.

### Claim 18 (depends on 17)

The method of claim 17, wherein the screenable marker is β-glucuronidase (GUS), luciferase or green fluorescent protein (GFP).

### Claim 19 (depends on 13)

The method of claim 13, wherein the expression vector comprises a gene editing system.

### Claim 20

(canceled)

### Claim 21 (depends on 19)

The method of claim 19, wherein the gene editing system comprises TALENs, zinc-finger nucleases, or a CRISPR/Cas9 system.

### Claim at position 22

22-30. (canceled)