# US 9,518,257 B2 · Reverse breeding

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- Number: US 9,518,257 B2
- Document ID: US9518257B2
- Status: Granted
- Kind: B2
- Application number: 14/177,751
- Filed: 2014-02-11
- Published: 2014-09-11
- Granted: 2016-12-13
- Earliest claimed: 2001-08-23
- Assignees: Rijk Zwaan Zaadteelt En Zaadhandel B.V.
- Applicants: Rijk Zwaan Zaadteelt En Zaadhandel B.V.
- Inventors: Robert Helene Ghislain Dirks; Cornelis Maria Petrus Van Dun; Kornelius Reinink; Jacobus Petrus Cornelis De Wit
- Web page: https://patentgrep.com/patent/US9518257B2

## CPC

- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for efficiently producing homozygous organisms from a heterozygous non-human starting organism, comprising providing of a heterozygous starting organism; allowing the starting organism to produce haploid cells; creating homozygous organisms from the haploid cells thus obtained; and selecting the organisms having the desired set of chromosomes; wherein during production of the haploid cells no recombination occurs in order to obtain a limited number of genetically different haploid cells. Recombination can also be prevented or suppressed.

## Claims

### Claim 1

A method for producing homozygous plants from a heterozygous starting plant, comprising:
at least partially preventing or suppressing recombination by interfering with at least one target gene involved in double strand breaks, chromosome pairing and/or strand exchange while allowing the heterozygous starting plant to produce haploid cells, whereby a limited number of genetically different haploid cells are thus obtained;
creating homozygous plants from the haploid cells by forming doubled haploid cells; and selecting from the plants those having the desired set of chromosomes,
wherein the interfering with the at least one target gene includes destabilizing the at least one target gene mRNA or transcript;
wherein the at least one target gene mRNA or transcript is destabilized by nucleic acid molecule(s) complementary to the at least one target gene mRNA or transcript; and
wherein the nucleic acid molecule(s) complementary to the at least one target gene mRNA or transcript further comprises a co-suppressor molecule or a DNA oligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one target gene is selected from the group consisting of SPO11, RHD54/TID1, DMC1, SAE3, RED1, HOP1, HOP2, REC8, MER1, MRE2, ZIP1, ZIP2, MEI5, RAD51, RAD52, RAD54, RAD55, RAD57, RPA, SMC3, SCC1, MSH2, MSH3, MSH6, PMS1, SOLODANCERS, HIM6, CHK2, MER2, MEI4, REC102, REC104, REC114, MEK1/MRE4, RAD50, MRE11, XRS2, and their functional homologues.

### Claim 3 (depends on 1)

The method of claim 1, further comprising crossing the homozygous plants to produce a progeny plant.

### Claim 4 (depends on 1)

The method of claim 1, further including transferring cytoplasmic male sterility (CMS).

### Claim 5 (depends on 1)

The method of claim 1, including making parental lines to produce F1 hybrid seeds.