# US 2019/0376072 A1 · Methods and Vectors for Producing Transgenic Plants

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- Number: US 2019/0376072 A1
- Document ID: US20190376072A1
- Status: Published application
- Kind: A1
- Application number: 16/550,636
- Filed: 2019-08-26
- Published: 2019-12-12
- Earliest claimed: 2008-07-16
- Assignees: Monsanto Technology LLC
- Applicants: Monsanto Technology LLC
- Inventors: Larry A. GILBERTSON; Susan J. JOHNSON
- Web page: https://patentgrep.com/patent/US20190376072A1

## CPC

- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8209: {Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers}
- C12N 15/821: {Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8291: {Hormone-influenced development}
- C12N 15/8297: {Gibberellins; GA3}
- C12N 15/8202: {by biological means, e.g. cell mediated or natural vector}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods of, and compositions for, assembling one or more transcription units in a genome without a linked selectable marker or other unwanted transcription unit are provided. Also provided methods of, and compositions for, assembling one or more transcription units in a genome with a reduced frequency of vector backbone.

## Claims

### Claim at position 1

1-27. (canceled)

### Claim 28

A recombinant nucleic acid molecule comprising:
a first DNA segment comprising a first transcription unit flanked on both sides and adjacent to a first and a second shorter transcribable nucleic acid sequence in opposite orientation to each other and located between a first left T-DNA border region and a first right T-DNA border region;
a second DNA segment comprising a second transcription unit flanked on both sides and adjacent to a third and a fourth shorter transcribable nucleic acid sequence in opposite orientation to each other and located between a second left T-DNA border region and a second right T-DNA border region, wherein said third shorter transcribable nucleic acid sequence is operably linked to a plant-expressible promoter, wherein a fifth shorter transcribable nucleic acid sequence is located on the opposite side of, and adjacent to, said second right T-DNA border region and in opposite orientation to said fourth shorter transcribable nucleic acid sequence; and
wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are homologous to a portion of said second transcription unit; and
a third transcription unit comprising a non-lethal negative selectable marker gene located between said first DNA segment and said second DNA segment;
wherein said T-DNA border regions are capable of enabling the transfer of said segments into a plant genome.

### Claim 29 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are each at least 75 contiguous nucleotides in length, and share greater than 90% sequence identity to each other and to a portion of said second transcription unit.

### Claim 30 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are each at least 21 contiguous nucleotides in length, and share greater than 90% sequence identity to each other and to a portion of said second transcription unit.

### Claim 31 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein said first and second DNA segments are not identical.

### Claim 32 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.

### Claim 33 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein the transcription of said second and fourth shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit.

### Claim 34 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein the transcription of said first and third shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit.

### Claim 35 (depends on 28)

The nucleic acid molecule of claim 28, wherein the transcription of said fourth and fifth shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit.

### Claim 36 (depends on 28)

The nucleic acid molecule of claim 28, wherein said first and second shorter transcribable nucleic acid sequences are homologous to a portion of a sequence of interest within said first transcription unit.

### Claim 37 (depends on 28)

The recombinant nucleic acid molecule of claim 28, wherein said first transcription unit comprises a sequence of interest and said second transcription unit comprises a selectable marker gene.

### Claim 38 (depends on 37)

The nucleic acid molecule of claim 37, wherein said sequence of interest comprises a nucleic acid sequence that causes the targeted expression or over-expression of an exogenous nucleic acid sequence.

### Claim 39 (depends on 37)

The nucleic acid molecule of claim 37, wherein said sequence of interest comprises a protein coding sequence.

### Claim 40 (depends on 37)

The nucleic acid molecule of claim 37, wherein said sequence of interest causes the inhibition of expression of an endogenous gene via gene silencing.

### Claim 41 (depends on 37)

A method of selecting for unlinked first and second DNA segments in a plant cell, comprising:
a. transforming one or more plant cells with the recombinant nucleic acid molecule of claim 37;
b. growing said transformed one or more plant cells;
c. selecting from said transformed one or more plant cells a plant cell comprising said first DNA segment and said second DNA segment and lacking said third transcription unit; wherein said transforming is mediated through a bacterial cell selected from the group consisting of Agrobacterium spp., Rhizobium spp., Sinorhizobium spp., Mesorhizobium spp., Phyllobacterium spp., Ochrobactrum spp., and Bradyrhizobium spp.

### Claim 42 (depends on 41)

The method of claim 41, wherein said selecting further comprises selecting for said second DNA segment using said selectable marker gene.

### Claim 43 (depends on 41)

The method of claim 41, wherein said plant cell is a sugarcane, wheat, corn, soybean, cotton, or canola plant cell.

### Claim 45 (depends on 41)

The method of claim 41, wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.

### Claim 46 (depends on 41)

The method of claim 41, further comprising regenerating a plant from said transformed one or more plant cells.

### Claim 47 (depends on 41)

The method of claim 41, wherein said first transcription unit provides a desirable trait selected from the group consisting of disease resistance, insect resistance, pest tolerance, herbicide tolerance, yield improvement, nutritional enhancement, and stress tolerance.