# US 11,895,960 B2 · Generation of haploid plants

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- Number: US 11,895,960 B2
- Document ID: US11895960B2
- Status: Granted
- Kind: B2
- Application number: 17/218,984
- Filed: 2021-03-31
- Published: 2021-08-26
- Granted: 2024-02-13
- Earliest claimed: 2014-08-28
- Assignees: KWS SAAT SE & Co. KGaA
- Applicants: KWS SAAT SE & Co. KGaA
- Inventors: Andreas Houben; Raheleh Karimi-Ashiyani; Takayoshi Ishii; Nils Stein; Jochen Kumlehm; Christof Bolduan; Frank Breuer; Monika Kloiber-Maitz; Markus Niessen; Milena Ouzunova; Britta Schulz; Silke Wieckhorst
- Web page: https://patentgrep.com/patent/US11895960B2

## CPC

- A01H 1/08: Methods for producing changes in chromosome number
- A01H 5/06: Roots
- A01H 5/10: Seeds
- A01H 6/20: Brassicaceae, e.g. canola, broccoli or rucola
- C07K 14/415: from plants
- C12N 15/8287: {for fertility modification, e.g. apomixis}
- C12N 15/8289: {Male sterility}
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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 present invention relates to non-transgenic and transgenic plants, preferably crop plants, comprising a mutation causing an alteration of the amino acid sequence in the CATD domain of the centromere histone H3 (CENH3), preferably within the loop1 or the α2-helix of the CATD domain, which have the biological activity of a haploid inducer. Further, the present invention provides methods of generating the plants of the present invention and haploid and double haploid plants obtainable by crossing the plants of the present invention with wildtype plants as well as methods of facilitating cytoplasm exchange.

## Claims

### Claim 1

A plant having biological activity of a haploid inducer and comprising a nucleotide sequence encoding a centromere histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence comprises a mutation encoding a polypeptide having one or more amino acid substitutions at positions 2, 6, and 12 of a loop1 consensus sequence, wherein said alteration confers the biological activity of a haploid inducer, wherein the plant is homozygous with respect to the mutation and is not able to express a native CENH3 protein,
wherein the loop1 consensus sequence comprises: T, S, or A at position 1: H, Q, N, A, Y, F, G, D or E at position 2; M, Q, I, F, Y, A, E, N, R, L, H or G at position 3; L, F, V, I or Y at position 4; A, T, S, C or M at position 5; P, N, D, R, A, T, F, R, H, S or K at position 6; Q, Y, D, K, R, E, G, S, P, H, N or A at position 8; I, V or P at position 9; N, G, T, E or S at position 10; R or P at position 11; W or Y at position 12; and T, Q or S at position 13, and
wherein the plant is selected from the group consisting of Sorghum bicolor, Saccharum officinarium, Zea mays, Arabidopsis thaliana, Brassica rapa, and Brassica juncea.

### Claim 2 (depends on 1)

The plant according to claim 1, wherein crossing between the plant and a wildtype plant or a plant expressing wildtype CENH3 protein yields at least 0.1% haploid progeny.

### Claim 3 (depends on 1)

The plant according to claim 1, wherein the nucleotide sequence comprising the mutation is an endogenous gene or a transgene.

### Claim 4 (depends on 3)

The plant according to claim 3, wherein the nucleotide sequence comprising the mutation is a transgene.

### Claim 5 (depends on 1)

The plant according to claim 1, wherein the one or more amino acid substitutions comprise:
a substitution for the amino acid H, Q, N, A, Y, F, G, D or E at position 2 of the loop1 consensus sequence;
a substitution for the amino acid P, N, D, R, A, T, F, R, H, S or K at position 6 of the loop1 consensus sequence; and
a substitution for the amino acid W or Y at position 12 of the loop1 consensus sequence.

### Claim 6 (depends on 1)

A part of the plant according to claim 1 comprising a mutation causing in the CATD domain an alteration of the amino acid sequence at positions 2, 6, and 12 of the loop1 sequence of the loop1 consensus sequence.

### Claim 7 (depends on 1)

A method of generating a haploid plant, comprising the steps of:
a) crossing a plant according to claim 1 to a plant expressing wildtype CENH3 protein, and
b) identifying the haploid progeny plant generated from the crossing step.

### Claim 8 (depends on 1)

A method of generating a double haploid plant, comprising the steps of:
a) crossing a plant according to claim 1 to a plant expressing wildtype CENH3 protein,
b) identifying a haploid progeny plant generated from the crossing step, and
c) converting the haploid progeny plant into a double haploid plant.

### Claim 9 (depends on 1)

A method of facilitating a cytoplasm exchange, comprising the steps of:
x) crossing a plant according to claim 1 as ovule parent with a plant expressing wildtype CENH3 protein as pollen parent, and
y) obtaining a haploid progeny plant comprising the chromosomes of the pollen parent and the cytoplasm of ovule parent.

### Claim 10 (depends on 1)

A method of generating a plant according to claim 1, comprising the steps of:
i) subjecting seeds of a plant to a sufficient amount of the mutagen ethylmethane sulfonate to obtain M1 plants,
ii) allowing sufficient production of fertile M2 plants,
iii) isolating genomic DNA of M2 plants and
iv) selecting individuals comprising said nucleotide sequence comprising said one or more mutations of the loop1 consensus sequence, wherein said substitution confers the biological activity of a haploid inducer, and wherein the plant is homozygous with respect to the mutation and is not able to express a native CENH3 protein.

### Claim 11

An isolated nucleotide sequence encoding a centromere histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence encodes a polypeptide having substitutions at one or more of positions 2, 6, and 12 of a loop1 consensus sequence of SEQ ID NO: 49,
wherein the substitution at position 2 is not H, Q, N, A, Y, F, G, D or E,
wherein the substitution at position 6 is not P, N, D, R, A, T, F, R, H, S or K,
wherein the substitution at position 12 is not W or Y, and
wherein the polypeptide has the biological activity of a haploid inducer.

### Claim 12 (depends on 11)

A vector comprising the nucleotide sequence of claim 11.

### Claim 13 (depends on 11)

A plant cell or a host cell comprising the nucleotide sequence of claim 11, or a vector comprising said nucleotide sequence, as a transgene.

### Claim 14 (depends on 11)

A method of generating a transgenic plant comprising the steps of:
yy) transforming a plant cell with the nucleotide sequence of claim 11, or a vector comprising said nucleotide sequence, and
zz) regenerating a plant having the biological activity of a haploid inducer from the plant cell.

### Claim 15 (depends on 6)

The part of the plant according to claim 6, wherein the part is a shoot vegetative organ, root, flower or floral organ, seed, fruit, ovule, embryo, plant tissue or cell.

### Claim 16 (depends on 1)

The plant according to claim 1, wherein the one or more mutations comprise:
a V at position 2 of the loop1 sequence of the loop1 consensus sequence.