# US 10,865,418 B2 · Use of multiple transformation enhancer sequences to improve plant transformation efficiency

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- Number: US 10,865,418 B2
- Document ID: US10865418B2
- Status: Granted
- Kind: B2
- Application number: 16/035,556
- Filed: 2018-07-13
- Published: 2019-01-03
- Granted: 2020-12-15
- Earliest claimed: 2006-07-19
- Assignees: Monsanto Technology LLC
- Applicants: Monsanto Technology LLC
- Inventors: Larry A. Gilbertson; Xudong Ye
- Web page: https://patentgrep.com/patent/US10865418B2

## CPC

- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 1/205: Bacterial isolates
- C12N 15/8202: {by biological means, e.g. cell mediated or natural vector}
- C12R 2001/41: Rhizobium
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

The present invention relates to methods and compositions for improving the efficiency of Agrobacterium- and Rhizobium-mediated plant cell transformation by use of additional transformation enhancer sequences, such as overdrive or TSS sequences, operably linked to a T-DNA border sequence on a recombinant construct that comprises T-DNA.

## Claims

### Claim 1

A method of increasing the efficiency of bacterially-mediated plant transformation, comprising the steps of:
a) introducing at least one additional transformation enhancer sequence into a plant transformation vector; and
b) transforming a plant cell with the vector by bacterially-mediated transformation, wherein the bacterium is competent for the transformation of the plant cell;
wherein the vector comprises at least one T-DNA border sequence and a transformation enhancer sequence operably linked to the T-DNA border sequence;
and wherein the transformation enhancer sequence comprises two or more copies of an overdrive (OD) sequence from Agrobacterium tumefaciens.

### Claim 2 (depends on 1)

The method of claim 1, wherein the overdrive sequence comprises the consensus sequence of SEQ ID NO:20 or SEQ ID NO:19.

### Claim 3 (depends on 2)

The method of claim 2, wherein the overdrive sequence comprises:
a) SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13; or
b) a sequence complementary to the full length of any of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13.

### Claim 4 (depends on 1)

The method of claim 1, wherein the transformation enhancer sequence is located proximal to a T-DNA right border (RB) sequence.

### Claim 5 (depends on 4)

The method of claim 4, wherein the overdrive sequence is from a nopaline or octopine Ti plasmid of A. tumefaciens.

### Claim 6 (depends on 1)

The method of claim 1, wherein the bacterially-mediated transformation is Agrobacterium-mediated transformation.

### Claim 7 (depends on 1)

The method of claim 1, wherein the bacterially-mediated transformation is Rhizobia-mediated transformation.

### Claim 8 (depends on 7)

The method of claim 7, wherein the Rhizobia-mediated transformation is Agrobacterium-, Rhizobium-, Sinorhizobium-, Mesorhizobium-, or Bradyrhizobium-mediated transformation.

### Claim 9 (depends on 1)

The method of claim 1, wherein the plant cell is from a plant selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato and watermelon.

### Claim 10 (depends on 1)

The method of claim 1, wherein the overdrive sequence comprises SEQ ID NO:19 or SEQ ID NO:20; or a sequence complementary to the full length of SEQ ID NO:19 or SEQ ID NO:20;
or wherein the transformation enhancer sequence comprises SEQ ID NO:10 or a sequence complementary to the full length of SEQ ID NO:10.

### Claim 11 (depends on 1)

The method of claim 1, wherein the plant cell is a corn or soybean cell.

### Claim 12 (depends on 1)

The method of claim 1, wherein the transformation enhancer sequence comprises from 2 to about 18 copies of said overdrive sequence.

### Claim 13 (depends on 1)

The method of claim 1, further comprising the step of:
c) regenerating a transgenic plant from said plant cell.

### Claim 14

A recombinant DNA construct comprising a T-DNA border sequence, said T-DNA sequence operably linked to a transformation enhancer sequence that comprises two or more copies of an overdrive (OD) sequence selected from the group consisting of:
a) SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:19, or SEQ ID NO:20;
b) the sequences complementary to the full length of any of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:19, or SEQ ID NO:20; and
c) combinations thereof.

### Claim 15 (depends on 14)

The construct of claim 14, wherein the enhancer sequence comprises at least about four copies of said overdrive sequence.

### Claim 16 (depends on 14)

The construct of claim 14, wherein the border sequence is a right border (RB) sequence.

### Claim 17 (depends on 14)

The construct of claim 14, wherein the border sequence is a left border (LB) sequence.

### Claim 18 (depends on 14)

The construct of claim 14, wherein the transformation enhancer sequence comprises SEQ ID NO:10 or a sequence complementary to the full length of SEQ ID NO:10.

### Claim 19 (depends on 16)

The construct of claim 16, wherein the RB sequence is from a nopaline Ti plasmid.

### Claim 20 (depends on 16)

The construct of claim 16, wherein the RB sequence is from an octopine Ti plasmid.

### Claim 21 (depends on 14)

A transgenic cell transformed with the construct of claim 14.

### Claim 22 (depends on 21)

The cell of claim 21, defined as a plant or bacterial cell.

### Claim 23 (depends on 22)

The cell of claim 22, wherein the bacterial cell is an Agrobacterium cell.

### Claim 24 (depends on 22)

The cell of claim 22, wherein the bacterial cell is a Rhizobium cell.

### Claim 25 (depends on 22)

The cell of claim 22, wherein the plant cell is from a plant selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato, and watermelon.

### Claim 26 (depends on 14)

A transgenic plant transformed with the construct of claim 14.

### Claim 27 (depends on 26)

The transgenic plant of claim 26, defined as selected from the group consisting of soybean, corn, cotton, canola, rice, wheat, alfalfa, common bean, peanut, tobacco, sunflower, barley, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, sorghum, spinach, squash, sugarbeet, tomato, and watermelon.