Methods for selecting plants after genome editing

Patent metadata

Field	Value
Publication number	US-10487336-B2
Application number	US-201515303707-A
Country	US
Kind code	B2
Filing date	May 7, 2015
Priority date	May 9, 2014
Publication date	Nov 26, 2019
Grant date	Nov 26, 2019

Abstract

The disclosure provides methods for selecting modified plants with a mutation in a target gene and plants produced by the methods. Specifically, the disclosure provides methods comprising introducing a recombinant expression cassette encoding a genome editing protein into meristematic or germline cells of a parent plant, wherein the genome editing protein specifically recognizes a target gene; crossing or selfing the parent plant, thereby producing a plurality of progeny seeds; and selecting progeny plants grown from the progeny seeds that express a phenotype that can be selected at the intact plant level.

First claim

What is claimed is:

1. A method for selecting modified plants with a mutation in a target gene wherein the method does not use tissue culture, the method comprising
a) introducing a first recombinant expression cassette encoding a first genome editing protein into meristematic or germline cells of a parent plant, wherein the first genome editing protein specifically recognizes the target gene;
b) introducing a second recombinant expression cassette encoding a second genome editing protein into meristematic or germline cells of the parent plant, wherein the second genome editing protein specifically recognizes a gene controlling seed germination, wherein the gene is LsNCED4;
c) after steps (a) and (b), crossing or selfing the parent plant, thereby producing a plurality of progeny seed;
d) selecting progeny plants grown from the progeny seed that are capable of germinating under heat conditions that inhibit germination of seed which lack a mutation in the gene controlling seed germination;
e) identifying the progeny plants selected in step (d) that comprise a mutation in the target gene, thereby selecting modified plants with a mutation in the target gene.

2. The method of claim 1 , wherein the meristematic cells are in an apical meristem.

3. The method of claim 1 , wherein the step of selecting progeny plants is carried out using first generation progeny.

4. The method of claim 1 , wherein the first or second genome editing protein is a zinc finger nuclease.

5. The method of claim 1 , wherein the first or second genome editing protein is a TALEN.

6. The method of claim 1 , wherein the first or second genome editing protein is a Cas-9 nuclease guided by a guide RNA (gRNA).

7. The method of claims 1 , wherein the first or second genome editing protein introduces a single strand nick or a double strand break in the target gene.

8. The method of claim 1 , wherein the first genome editing protein is used to introduce a desired nucleotide sequence into the target gene by homologous recombination.

9. The method of claim 1 , wherein the first or second recombinant expression cassette is introduced into the cells using Agrobacterium.

10. The method of claim 1 , wherein the first or second recombinant expression cassette is introduced into the cells using particle bombardment.

11. The method of claim 1 , wherein the first or second recombinant expression cassette is introduced into the cells using microinjection.

12. The method of claim 1 , wherein the first or the second recombinant expression cassette is transiently expressed in the cells.

13. The method of claim 1 , wherein the first and second expression cassettes are introduced into the plant in planta.

14. The method of claim 1 , wherein the mutation in the target gene confers resistance to a plant pathogen.

15. The method of claim 1 , wherein the mutation in the target gene confers resistance to stress conditions.

16. The method of claim 1 , wherein the mutation in the target gene increases yield.

17. The method of claim 1 , wherein the mutation in the target gene changes plant development, plant color, or sensitivity to a chemical.

Assignees

• Univ California

Inventors

• Michelmore Richard

Classifications

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title