# US 2017/0280646 A1 · Generation of haploid plants

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- Number: US 2017/0280646 A1
- Document ID: US20170280646A1
- Status: Published application
- Kind: A1
- Application number: 15/507,538
- Filed: 2015-08-28
- Published: 2017-10-05
- Earliest claimed: 2014-08-28
- Assignees: Kws Saat AG
- Applicants: Kws Saat SE
- 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/US20170280646A1

## 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 centromer histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence comprises a mutation causing in the CATD domain an alteration of the amino acid sequence of the CENH3 protein, wherein said alteration confers the biological activity of a haploid inducer.

### Claim 2 (depends on 1)

The plant according to claim 1, wherein the mutation causes an alternation in a loop1 corresponding to nucleotides from position 340 to position 378 set forth in SEQ ID No. 37 of the CENH3 protein derived from Arabidopsis thaliana set forth in SEQ ID No. 38 and being positioned within the CATD domain, wherein the alteration confers the biological activity of a haploid inducer, or the mutation causes an alternation in an α2-helix corresponding to nucleotides from position 379 to position 465 set forth in SEQ ID No. 37 of the CENH3 protein derived from Arabidopsis thaliana set forth in SEQ ID No. 38 and being positioned within the CATD domain, wherein the alteration confers the biological activity of a haploid inducer.

### Claim 3 (depends on 1)

The plant according to claim 1, wherein the mutation causes one or more of the following substitutions or deletions of a specified amino acid as defined:
Amino acid(s)
Position within the loop1
1
T, S or A
2
H, Q, N, A, Y, F, G, D or E
3
M, Q, I, F, Y, A, E, N, R, L, H or G
4
L, F, V, I or Y
5
A, T, S, C or M
6
P, N, D, R, A, T, F, R, H, S or K
7
X
8
Q, Y, D, K, R, E, G, S, P, H, N or A
9
I, V or P
10
N, G, T, E, or S
11
R or P
12
W or Y
13
T, Q or S
Position within the α2-helix
1
A, P, V or L
2
E, D, Q, H or L
3
A
4
L or V
5
V, L, M, I, R, Y or T
6
S or A
7
I or L
8
Q
9
E
10
A or S
11
A or T
12
E
13
D, N, F, I or Y
14
Y, F or H
15
L, I or V
16
V or I
17
G, R, E, H, N, T, E, D or Q
18
L, M or I
19
F, M or L
20
S, E, D or G
21
D, M, V, N, E, A, R or K
22
S, G, A or T
23
M, W, N or H
24
L or H
25
C or L
26
A or T
27
L or I
28
H
29
A or S

### Claim 4 (depends on 1)

The plant according to claim 1, wherein the mutation causes a substitution or deletion of a specified amino acid of SEQ ID No. 1 or 49.

### Claim 5 (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 6 (depends on 1)

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

### Claim 7 (depends on 6)

The plant according to claim 6, wherein the nucleotide sequence comprising the mutation is a transgene and at least one endogenous gene encoding a CENH3 protein is inactivated or knocked out.

### Claim 8 (depends on 1)

The plant according to claim 1, wherein the amino acid asparagine at position 2 of SEQ ID No. 49 is substituted, or the amino acid alanine at position 95 of SEQ ID No. 55 is substituted, or the amino acid proline at position 6 of SEQ ID No. 49 is substituted, or the amino acid proline at position 121 of SEQ ID No. 52 is substituted, or the amino acid tryptophan at position 12 of SEQ ID No. 49 is substituted, or the amino acid tryptophan at position 127 of SEQ ID No. 52 is substituted, or the amino acid alanine at position 1 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 107 of SEQ ID No. 58 is substituted, or the amino acid leucine at position 4 of SEQ ID No. 1 is substituted, or the amino acid leucine at position 132 of SEQ ID No. 52 or position 92 of SEQ ID No. 34 or position 130 of SEQ ID No. 38 or position 106 of SEQ ID No. 61 is substituted, or the amino acid leucine at position 7 of SEQ ID No. 1 is substituted, or the amino acid leucine at position 109 of SEQ ID No. 61 is substituted, or the amino acid glutamine at position 8 of SEQ ID No. 1 is substituted, or the amino acid glutamine at position 114 of SEQ ID No. 58 or position 110 of SEQ ID No. 61 is substituted, or the amino acid alanine at position 10 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 138 of SEQ ID No. 52 is substituted, or the amino acid cysteine at position 25 of SEQ ID No. 1 is substituted, or the amino acid cysteine at position 153 of SEQ ID No. 52 is substituted, or the amino acid alanine at position 26 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 154 of SEQ ID No. 52 is substituted.

### Claim 9 (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 of the CENH3 protein and said alteration confers the biological activity of a haploid inducer.

### Claim 10 (depends on 1)

A haploid plant obtainable by crossing a plant according to claim 1 with a plant expressing wildtype CENH3 protein.

### Claim 11 (depends on 10)

A double haploid plant obtainable by converting the haploid plant according to claim 10 into a double haploid plant.

### Claim 12 (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 13 (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 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

A method of facilitating a cytoplasm exchange, comprising the steps of:
x) crossing a plant according to claims 1 to 8 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 15 (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 possessing an alteration of the amino acid sequence in the CATD domain of CENH3.

### Claim 16

A nucleotide sequence encoding at least the CATD domain of a CENH3 protein comprising a mutation causing in the CATD domain an alteration of the amino acid sequence of the CENH3 protein.

### Claim 17 (depends on 16)

A vector comprising the nucleotide sequence of claim 16.

### Claim 18 (depends on 16)

A plant cell or a host cell comprising the nucleotide sequence of claim 16, or a vector comprising said nucleotide sequence, as a transgene.

### Claim 19 (depends on 16)

A method of generating a transgenic plant comprising the steps of:
yy) transforming a plant cell with the nucleotide sequence of claim 16, 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 20 (depends on 9)

The part of the plant according to claim 9, wherein the part is a shoot vegetative organ, root, flower or floral organ, seed, fruit, ovule, embryo, plant tissue or cell.