# US 2008/0189803 A1 · Universal chloroplast integration and expression vectors, transformed plants and products thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2008/0189803 A1
- Document ID: US20080189803A1
- Status: Published application
- Kind: A1
- Application number: 11/379,833
- Filed: 2006-04-24
- Published: 2008-08-07
- Earliest claimed: 1996-01-25
- Assignees: Auburn University
- Inventors: Henry Daniell
- Web page: https://patentgrep.com/patent/US20080189803A1

## CPC

- C12N 15/8214: {Plastid transformation}
- C12N 15/8275: {Glyphosate}
- C12N 15/8278: {Sulfonylurea}
- C12N 15/8286: {for insect resistance}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention provides universal chloroplast integration and expression vectors which are competent to stably transform and integrate genes of interest into chloroplast genome of multiple species of plants. Transformed plants and their progeny are provided. Monocotyledonous and dicotyledonous plants are transformed which have never been transformed heretofore. Plants transformed with a synthetic gene express valuable biodegradable protein-based polymers (PBPs). Transformed plants produce high value molecules. Resistance is provided to agricultural crops against the major classes of chemical herbicides. Herbicide resistance is used as a lethal selectable marker for chloroplast transformation. The transformed plants are capable of expressing in addition to the targeted trait, a desirable, secondary non-targeted trait. Insect resistance is provided to transformed plants, both against insects that are susceptible to Bt toxins and against insects that have developed resistance to Bt toxins.

## Claims

### Claim 1

A stably transformed herbicide resistant target plant species or the progeny thereof, which comprises chloroplast stably transformed with a universal integration and expression vector competent for stable transformation of the chloroplast of different plants species which comprises an expression cassette which comprises a heterologous protein of interest expressed by a heterologous DNA sequence in the chloroplast genome of the target plant species, a selected phenotype other than tolerance to said herbicide, and flanking each side of the expression cassette, flanking DNA sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous protein into the chloroplast genome of the target plant was facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome.

### Claim 2 (depends on 1)

The herbicide resistant target plant of claim 1 in which the protein of interest is a mutant form of an enzyme which has decreased affinity for the herbicide than does the naturally occurring enzyme.

### Claim 3 (depends on 1)

The herbicide resistant target plant of claim 1 wherein the herbicide is glyphosate.

### Claim 4 (depends on 3)

The herbicide resistant target plant of claim 3 wherein the enzyme is EPSP synthase.

### Claim 5 (depends on 2)

The herbicide resistant target plant of claim 2 wherein the herbicide is selected from at least one of the following types: PSI, PSII, APP, auxin analog, mitotic, tertiary amino methyl phosphoric acids type and ALS inhibiting types.

### Claim 6 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is the PSI type selected from paraquat and diquat.

### Claim 7 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is selected from atrazine, dinoseb, lenacil and metribuzine.

### Claim 8 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is of the APP type selected from cyclohexanedione, haloxyfop, clethodim and phenoxaprop, and the lower alkyl-substituted compound thereof.

### Claim 9 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is an auxin analog selected from MCPA and 2,4-D.

### Claim 10 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is a mitotic type herbicide, which is dinitroahiline.

### Claim 11 (depends on 2)

The herbicide resistant target plant of claim 2 wherein the herbicide is a tertiary amino methyl phosphoric acid type, which is glyphosate.

### Claim 12 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is an ALS inhibiting type selected from sulfonylureas and imidazolines.

### Claim 13 (depends on 5)

The herbicide resistant target plant of claim 5 wherein the herbicide is selected from bromoxynil, methyl sulfuron, chlorsulfuron, phosphinothricin and imazapyr.

### Claim 14 (depends on 1)

The herbicide resistant target plant of claim 1 wherein the expressed phenotype is antibiotic resistance.

### Claim 15 (depends on 1)

The herbicide resistant plant of claim 1 wherein the selectable phenotype is encoded by the hygromycin gene that confers herbicide resistance.

### Claim 16 (depends on 5)

The herbicide resistant target plant of claim 5 which is maize, rice, grass, rye, barley, oat, wheat, soybean, peanut, grape, potato, sweet potato, pea, canola, tobacco, tomato or cotton.

### Claim 17 (depends on 16)

The herbicide resistant target plant of claim 16 which is a homoplasmic plant.

### Claim 18

A process for conferring herbicide resistance to a target plant species which comprises introducing into the plant a universal integration and expression vector competent for stably transformed with a universal integration and expression vector competent for stably transforming the chloroplast of different plants species which comprises an expression cassette which comprises, operably joined, a heterologous DNA sequence coding for a protein of interest which confers resistance to a herbicide and control sequences positioned upstream from the 5′ and downstream from the 3′ ends of the coding sequence to provide expression of the coding sequence in the chloroplast of the target plant, a heterologous nucleotide sequence coding for a selectable phenotype other than for is tolerance to said herbicide, and flanking each side of the expression cassette, flanking sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle and the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous coding sequence into the chloroplast genome of the target plant is facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome and growing the transformed plant.

### Claim 19 (depends on 18)

The process of claim 18 wherein the DNA sequence codes for a mutant form of an enzyme which has decreased affinity for the herbicide than does the naturally occurring enzyme.

### Claim 20 (depends on 19)

The process of claim 19 wherein the enzyme is EPSP synthase and the herbicide is glyphosate.

### Claim 21 (depends on 20)

The process of claim 20 wherein DNA sequence is the EPSP synthase gene which is a mutant EPSP synthase gene.

### Claim 22 (depends on 18)

The process of claim 18 which comprise selecting the viable, transformed target plants from a medium which is lethal to non-transformed plants.

### Claim 23 (depends on 22)

The process of claim 22 wherein the viable transformed target plants are homoplasmic plants.

### Claim 24 (depends on 22)

The process of claim 22 wherein the viable transformed target plants are heteroplasmic plants.

### Claim 25 (depends on 18)

The process of claim 18 wherein the herbicide resistant target plant is tobacco, the nucleotide sequence codes for a selectable phenotype which is lethal to tobacco or for a visual trait that permits to select the transformed tobacco plants from the non-transformed plants.

### Claim 26 (depends on 25)

The process of claim 25 wherein the lethal selectable phenotype is hygromycin to which tobacco is not naturally resistant.

### Claim 27 (depends on 25)

The process of claim 25 wherein the visual trait is the expression of a color.

### Claim 28 (depends on 18)

The process of claim 18 for stably transforming the phenotype of a target plant species, whereby the grown transformed plant expresses the selectable phenotype and another trait in addition to the expression of that phenotype.

### Claim 29 (depends on 28)

The process of claim 28 wherein the selected phenotype is conferred by expression of the hygromycin 3-phosphotransferase gene and the additional trait that is conferred is resistance to the herbicide glyphosate.

### Claim 30

A process of determining chloroplast transformation and expression of a target trait on the basis of a target plant species acquisition of resistance to a selected herbicide due to the transformation of the plant species which comprises introducing into the plant a universal integration and expression vector competent for stably transforming the chloroplast of different plant species which comprises an expression cassette which comprises, operably joined, a heterologous DNA sequence coding for the desired target trait and control sequences positioned upstream from the 5′ and downstream from the 3′ ends of the coding sequence to provide expression of the coding sequence in the chloroplast of a target plant, a heterologous nucleotide sequence coding for a selectable phenotype and flanking each side of the expression cassette, flanking DNA sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle and the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous coding sequence into the chloroplast genome of the target plant is facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome, exposing the plants into which the vector has been introduced to a lethal concentration of the herbicide and selecting the plants which do not die from exposure thereto, thereby having selected the transformed plants which express the desired target trait.

### Claim 31 (depends on 30)

The process of claim 30 wherein the selected herbicide is selected from at least one of the following types: PSI, PSII, APP, auxin analog, mitotic, tertiary amino methyl phosphoric acids and ALS inhibiting types.