EA043415B1 - HAPLOIDIZATION IN SORGHUM - Google Patents

Description

Field of technology to which the invention relates

The invention relates to the field of simplifying labor-intensive crossbreeding programs through molecular biological methods, marker technology and genetic engineering. In particular, sorghum plants are provided that are capable of inducing haploidy through modifications in the genome with respect to, preferably, pollen-specific expressed patatin phospholipase, through which haploid progeny is produced, and capable of producing inbred lines for hybrid crossing in a short time through chromosome doubling. In particular, sorghum plants are provided that have mutations in patatin phospholipase, methods for producing and identifying these mutations or the mutated plant, and a corresponding nucleic acid molecule that encodes the mutated patatin phospholipase, and vectors and host cells, in particular plant cells. , containing a nucleic acid molecule, and plants generated from such plant cells, capable of producing haploidy and their progeny, crossbred products, inbred lines and their respective plant parts and products.

Based on the knowledge of sorghum, the present invention provides methods for producing and identifying haploid inducers of transgenic and non-transgenic plants and corresponding plants that have acquired the property of haploid induction, or in which the induction efficiency has been increased. In addition, the present invention also includes seeds or progeny, organs, plant parts, tissues or plant cells of the present invention and their use.

Prerequisites for creating the invention

Sorghum is a relatively new crop in Germany, but due to its capacity for mass growth, drought tolerance and high efficiency of water and nutrient use, it is the focus of research and practice aimed at finding new high-yielding crops for substrate production, in particular related to biogas production, as well as for use as feed, food and ethanol production.

The main goals of crossing sorghum are adaptation to the climate of Central Europe, that is, increasing cold resistance, increasing yield, stability, combined with the good development of young plants and the development of resistance to diseases and pests.

Sorghum can be improved through crossbreeding, as is possible in a self-pollinating crop. Population improvement can be achieved through a designed crossbreeding scheme through targeted selection of the best plants for seed production for the next crop year. In addition, hybrid crossing has created new options for improving varieties. However, the process of hybrid crossing is based on several generations of continuous generation of homozygous paternal and maternal lines, which in turn makes the process of hybrid crossing time-consuming and expensive, despite good results.

Therefore, the object of the present invention was to propose an effective crossbreeding system for sorghum.

Summary of the invention

The present invention relates to the field of simplifying labor-intensive programs for crossing, marker technology and genetic engineering. The present invention provides sorghum plants capable of inducing haploidy through modifications in the genome in relation to pollen-specific expressed patatin phospholipase, through which haploid progeny is produced, and which can be obtained for hybrid crossing in a short time by doubling chromosomes in inbred lines, that is, in homozygous paternal and maternal lines. In addition, the results obtained can be used to produce inducers of haploids in transgenic and non-transgenic plants or to increase the efficiency of plant induction.

Therefore, the present invention relates to the embodiments listed in the following paragraphs [1] to [29] and which are illustrated by examples.

[1] A sorghum plant capable of inducing haploidy, characterized in that the plant has at least one modification related to endogenous patatin phospholipase, which is, in a preferred embodiment of the present invention, pollen-specific expressed.

[2] The plant according to paragraph [1], characterized in that the patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 1 or 2, or is encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions, or that contains the amino acid sequence shown in SEQ ID NO: 3, or a homologous amino acid sequence.

[3] The plant according to [1] or [2], wherein the modification of patatin phospholipase is the reason why it is suitable as a haploid inducer.

- 1 043415

[4] A plant according to one of points [1]-[3], characterized in that at least one modification

a) represent at least one mutation in the endogenous gene encoding patatin phospholipase as defined in paragraph [2], in a preferred embodiment of the present invention, a substitution resulting in at least one amino acid exchange or the generation of a stop codon ;

b) are at least one expression cassette insert that contains a promoter operably linked to (i) a nucleic acid molecule encoding a dsRNA, wherein the dsRNA contains at least 19 nucleotides that is complementary to a partial sequence of the nucleotide sequence , defined in paragraph [2]; or (ii) a nucleic acid molecule encoding patatin phospholipase as defined in paragraph [2], or with a functional part of patatin phospholipase, wherein the encoded patatin phospholipase or functional part thereof has at least one mutation leading to, at least one amino acid exchange or generation of a stop codon; or

c) causes knockout of patatin phospholipase.

[5] A plant according to item [4], characterized in that at least one mutation results in an amino acid exchange

a) in the range of amino acid positions from 37 to 240 in accordance with SEQ ID NO: 3, in a preferred embodiment of the present invention, this range corresponding to the functional domain of patatin phospholipase; and/or

b) in the range of amino acid positions from 241 to 385 in accordance with SEQ ID NO: 3, or

c) to generate a stop codon in the range of amino acid positions from 241 to 385 in accordance with SEQ ID NO: 3.

[6] The plant of claim [4] or [5], characterized in that at least one mutation for amino acid metabolism occurs in the range of amino acid positions 40-93, 135-204 and/or 270-320 in accordance with SEQ ID NO: 3, in a preferred embodiment of the present invention, in the range of amino acid positions 53-85, 150-192 and/or 285-311 in accordance with SEQ ID NO: 3, in a more preferred embodiment of the present invention, in the range of amino acid positions 55-75, 157-167 and/or 285-298 in accordance with SEQ ID NO: 3, or stop codon generation occurs in the range of amino acid positions 322-402 in accordance with SEQ ID NO: 3, in a preferred embodiment of the present invention , in the range of amino acid positions 342392 in accordance with SEQ ID NO: 3, in a more preferred embodiment of the present invention, in the range of amino acid positions 362-382 in accordance with SEQ ID NO: 3.

[7] The plant according to one of claims [4]-[6], characterized in that at least one mutation results in an amino acid exchange at amino acid position 59, 162 and/or 291 in accordance with SEQ ID NO: 3 and /or to the stop codon at amino acid position 372 in accordance with SEQ ID NO: 3.

[8] The plant according to one of paragraphs [1]-[7], characterized in that the modified patatin phospholipase (i) contains an amino acid sequence in accordance with SEQ ID NO: 3 or a homologous amino acid sequence in which at least one exchange of amino acids, wherein arginine (R) at position 59, valine (V) at position 162 and/or serine (S) at position 291 in accordance with SEQ ID NO: 3 is replaced by another amino acid, in a preferred embodiment of the present invention, glutamine (Q) at position 59, isoleucine (I) at position 162 and/or leucine (L) at position 291;

(ii) is encoded by a nucleotide sequence that contains the coding sequence of a DNA sequence in accordance with SEQ ID NO: 1 (derived from the corresponding cDNA in accordance with SEQ ID NO: 2) or a DNA sequence that is at least 80% identical to SEQ ID NO: 1, in which there is at least one nucleotide exchange resulting in an amino acid exchange, wherein at least one nucleotide is exchanged at nucleotide positions 421-423, 815-817, 1420-1422 and/or 1663 -1665 according to SEQ ID NO: 1 (corresponding to nucleotide positions 175-177, 484-486, 871-873 and/or 1114-1116 according to SEQ ID NO: 2);

(iii) contains an amino acid sequence according to SEQ ID NO: 6, 9 or 12; or (iv) is encoded by a nucleotide sequence that contains the coding sequence of a DNA sequence in accordance with SEQ ID NO: 4 (derived from the corresponding cDNA in accordance with SEQ ID NO: 5), SEQ ID NO: 7 (derived from the corresponding cDNA in accordance with SEQ ID NO: 8), SEQ ID NO: 10 (derived from the corresponding cDNA in accordance with SEQ ID NO: 11), SEQ ID NO: 13 (derived from the corresponding cDNA in accordance with SEQ ID NO: 14).

[9] A plant according to one of paragraphs [1]-[8], characterized in that the plant is homozygous or heterozygous for at least one mutation, in a preferred embodiment of the present invention, characterized in that the plant is homozygous for, at least one mutation.

- 2 043415

[10] A nucleic acid molecule encoding one of the identified patatin phospholipases, mutated in points [4]-[8].

[11] The nucleic acid molecule of claim [10], characterized in that its presence in the plant, in a preferred embodiment of the present invention, in the absence of wild-type patatin phospholipase, results in the plant being capable of inducing haploidy.

[12] A nucleic acid molecule comprising a length of at least 15, 16, 17, 18, 19 or 20, in a preferred embodiment of the present invention at least 21, 22, 23, 24 or 25, more in a preferred embodiment of the present invention, at least 30, 35, 40, 45 or 50 and in a most preferred embodiment of the present invention, at least 100, 200, 300, 500 or 1000 nucleotides that specifically hybridizes to the nucleotide sequence as defined in one of [5] to [8], and contains one of the mutations or a pair of nucleic acid molecules suitable for a range containing at least one of the mutations for polymerase chain reaction (PCR) amplification, in in a preferred embodiment of the present invention, in the form of an oligonucleotide, in a preferred embodiment of the present invention, having a length of a maximum of 50 nucleotides, which, in a preferred embodiment of the present invention, has one of the nucleotide sequences in accordance with SEQ ID NO: 28-30, 32 -34, 36-38 or 40-42.

[13] A vector, in a preferred embodiment of the present invention, a plant vector containing a nucleic acid molecule according to one of paragraphs [10]-[12] or an expression cassette as defined in paragraph [4].

[14] The host cell, in a preferred embodiment of the present invention, a plant cell containing a nucleic acid molecule according to one of paragraphs [10]-[12], an expression cassette as defined in paragraph [4], a vector according to paragraph [13], or a nucleic acid molecule according to one of paragraphs [10]-[12] as a transgene, optionally regulated by a heterologous promoter, in a preferred embodiment of the present invention, a pollen-specific promoter.

[15] A method for producing a plant capable of inducing haploidy or having an increased rate of induction relative to the wild type, comprising the following steps:

(a) (i) mutagenesis of plant cells and then regeneration of plants from plant cells subjected to mutagenesis, or plant mutagenesis;

(ii) identifying the plant from step (i) having at least one mutation in the endogenous DNA sequence that matches and/or that results in at least one of the mutations identified in paragraphs [4]-[ 8], wherein aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position 203 of the amino acid sequence according to SEQ ID NO: 3 is replaced by another amino acid, in a preferred embodiment of the present invention, asparagine (N) at position 75, arginine (R) at position 79 and/or leucine (L) at position 203, or their corresponding amino acids, and capable of inducing the production of haploid progeny at an increased rate compared to a plant not subjected to mutagenesis; or (b) (i) introducing a nucleic acid molecule having at least one mutation corresponding to and/or leading to at least one of the mutations identified in paragraphs [4] to [8], when aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position 203 of the amino acid sequence according to SEQ ID NO: 3 is replaced by another amino acid, in a preferred embodiment of the present invention, asparagine ( N) at position 75, arginine (R) at position 79 and/or leucine (L) at position 203, or their corresponding amino acids, in plant cells, or introducing the expression cassette defined in paragraph [4] into plant cells; and (ii) regenerating a plant, eg a transgenic plant, from the plant cells of step (i).

[16] The method of claim [15], wherein the endogenous DNA sequence or nucleic acid molecule is a coding nucleotide sequence that contains the amino acid sequences shown in SEQ ID NO: 3 for sorghum (Sorghum bicolor), SEQ ID NO: 18 for sunflower (Helianthus annuus), SEQ ID NO: 21 for barley (Hordeum vulgare) or in SEQ ID NO: 24 or 27 for Beta vulgaris (for example, for sugar beets).

[17] The method according to paragraph [16], wherein the introduction of a nucleic acid molecule can be carried out, for example, through the transformation of Agrobacterium, homologous recombination, for example, through the CRISPR/Cas or CRISPR/Cpf1 systems and a template for repair, and it contains mutagenesis of chemical substances and physical mutagenesis, TILLING, targeted mutagenesis, for example, using zinc finger nucleases, TALE nucleases (TALE - transcription activator-like effector), meganucleases and the CRISPR/Cas or CRISPR/Cpf1 systems.

[18] A plant containing a plant cell according to item [14], and/or obtained by a method according to one of items [15]-[17], in a preferred embodiment of the present invention, wherein the plant is sorghum, sunflower, rye, wheat, potatoes, barley or sugar -

- 3 043415 new beets.

[19] An organ, part of a plant, tissue or cell of a plant according to one of paragraphs [1]-[9] or [18], or seeds or progeny of a plant according to one of paragraphs [1]-[9] or [18], in this case, the seed or offspring have a mutation defined in paragraphs [4]-[8], and/or a nucleic acid molecule according to one of paragraphs [10]-[12], an expression cassette according to paragraph [4] or a vector according to paragraph [ 13].

[20] A method for producing a haploid plant, comprising the following steps:

(a) crossing a plant according to one of paragraphs [1] to [9] or [18] with a plant of the same genus, in a preferred embodiment of the present invention, the same species, (b) selecting a pollinated haploid seed or embryo, and (c ) production of a haploid plant from the seed or embryo from step (b).

[21] A haploid plant, haploid pollinated seed or embryo can be obtained by the method according to paragraph [20].

[22] An organ, part of a plant, tissue, cell, seed or progeny of a plant under paragraph [21].

[23] A method for producing a diploid plant, comprising the following steps:

(a) producing a haploid plant by the method according to paragraph [20];

(b) doubling the haploid number of chromosomes in at least one cell of the haploid plant, and (c) regenerating a diploid plant from the cell of step (b).

[24] A diploid plant can be obtained by the method according to paragraph [23].

[25] A method for producing hybrid plants, comprising the following steps:

(a) crossing the plant of [24] with a second plant of the same genus, in a preferred embodiment of the present invention, the same species, (b) selecting hybrid plants based on the desired trait.

[26] A hybrid plant that can be obtained by the method according to paragraph [25].

[27] A method for identifying a plant according to one of paragraphs [1]-[9] or [18] by detecting a mutation in the patatin phospholipase gene, for example, a mutation defined in paragraphs [4]-[9], or a marker allele that linked to the mutation, in a preferred embodiment of the present invention, using the nucleic acid of [12] as a molecular marker.

[28] Use of a nucleic acid as defined in [10] or [11], an expression cassette as defined in [4], or a vector as defined in [13] in a plant to impart haploid inducer properties or to increase the rate of induction for producing a plant or transgenic plant capable of inducing haploidy.

[29] Use of a plant according to one of paragraphs [1]-[9] or [18] to produce a haploid pollinated seed or embryo, or a haploid plant.

[30] Use of nucleic acid according to paragraph [12] as a molecular marker for detecting mutations in the patatin phospholipase gene.

First, some of the terms used in the invention are explained in more detail as follows:

The expression conferring the property of a haploid inducer or conferring the property of a haploid inducer, or capable of inducing haploidy means expression comparable to that in a plant, which is carried out using a nucleic acid according to the present invention or through modification of the genome, in particular through a mutation of patatin phospholipase, altered thereby so that it is capable of producing pollinated seeds or embryos having a simple (haploid) set of chromosomes, as a result of crossing with a plant of the same genus, in a preferred embodiment of the present invention, the same species, which does not have the property of a haploid inducer. The haploid inducer property, represented as the absolute rate of haploid induction, means that at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 , 0.9 or 1%, in a preferred embodiment of the present invention, at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5%, in a more preferred embodiment of the present invention, at least 6, 7, 8, 9, 10, 11, 12 13, 14 or 15%, or in an even more preferred embodiment of the present invention, at least 20, 25, 30, 35, 40 , 45 or 50% of pollinated seeds or embryos have a haploid set of chromosomes.

The term functional nucleotide sequence fragment means a region of nucleotide sequence that has functionality identical to or comparable to the overall nucleotide sequence from which the functional fragment is derived. As such, the functional fragment may have a nucleotide sequence identical or homologous to the overall nucleotide sequence by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, 97, 98, or 99% in length. In addition, the term functional fragment of a nucleotide sequence can also mean a region of a nucleotide sequence that changes the functionality of the entire nucleotide sequence, for example, during post-transcriptional or transcriptional gene silencing. As such, a functional nucleotide sequence fragment may contain at least 14, 15, 16, 17, 18, 19,

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20, 21, 22, 23, 24 or 25, in a preferred embodiment of the present invention, at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120 or 140, more preferably in an embodiment of the present invention, at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of total nucleotide sequence.

The term functional part of a protein means a region of a protein or a region of amino acid sequence that encodes a protein, and this region may implement functionality identical to or comparable to the functionality of a general protein in a plant cell. The functional portion of the protein has a length that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, 97, 98, or 99% identical or similar to the amino acid sequence, taking into account conservative and semi-conservative amino acid metabolisms, like the protein from which this functional part is derived.

The term haploid inducer also means an inducer of haploids under in vivo conditions.

The term heterologous means that the introduced polynucleotide, for example, comes from the same cell or organism having a different genetic background of the same species or a different species, or it is homologous to a prokaryotic or eukaryotic host cell, but is then located in a different genetic environment and thus thus, it is different from any corresponding naturally occurring polynucleotide. A heterologous polynucleotide may be present in addition to the corresponding endogenous gene.

The term hybridization or hybridization refers to the process in which a single-stranded nucleic acid molecule attaches to a practically complementary nucleic acid chain, that is, it enters into base pairing with it. Standard hybridization methods are described, for example, in Sambrook et al., Molecular Cloning: Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. In a preferred embodiment of the present invention , it is meant that at least 80 or 85%, in a preferred embodiment of the present invention, at least 90, 91, 92, 93, 94 or 95%, in a particularly preferred embodiment of the present invention, at least 96, 97, 98, or 99% of the bases of a nucleic acid molecule undergo base pairing with a substantially complementary nucleic acid strand. The possibility of such an attachment depends on the severity of the hybridization conditions. The term stringency refers to the hybridization conditions. High rigidity is specified when base pairing is difficult, and low rigidity is specified when base pairing is facilitated. The stringency of hybridization depends, for example, on salt concentration or ionic strength and temperature. Typically, hardness can be increased by increasing the temperature and/or decreasing the salt content. The term stringent hybridization conditions refers to those conditions under which hybridization occurs predominantly only between homologous nucleic acid molecules and homologs. The term hybridization conditions refers not only to the conditions prevailing during the actual addition of the nucleic acids, but also to the conditions prevailing during subsequent washing steps. Stringent hybridization conditions are, for example, conditions under which only those nucleic acid molecules that have at least 80, at least 85, at least 90, or at least 95% sequence identity are hybridized. Stringent hybridization conditions include, for example, hybridization in 4× sodium chloride citrate (SSC) at 65°C followed by multiple washes in 0.1× SSC at 65°C for a total of approximately 1 hour. As used herein, the term stringent hybridization conditions may also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% bovine serum albumin (BSA) for 16 hours followed by washing twice in 2x SSC and 0.1% SDS at 68°C. In a preferred embodiment of the present invention, hybridization occurs under harsh conditions.

The expression increasing the induction efficiency of a haploid inducer or similar expressions means that the rate of haploid induction in a plant having the property of a haploid inducer is increased. Thus, the number of pollinated seeds that have a haploid set of chromosomes and crossing a haploid inducer with a plant of the same genus, in a preferred embodiment of the present invention, the same species that does not have the property of a haploid inducer, increased by at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1%, in a preferred embodiment of the present invention by at least 1. 5, 2, 2.5, 3, 3.5, 4, 4.5 or 5%, and in the most preferred embodiment of the present invention at least 6, 7, 8, 9, 10, 15, 20 , 30 or 50% higher than the number of pollinated seeds achieved without the use of nucleic acid or without modification of the genome, in particular without mutation of patatin phospholipase in the context of the present invention, that is, the rate of haploid induction can be increased by at least 0 .1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1%, in a preferred embodiment of the present invention by at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5%, and in the most preferred embodiment of the present invention at least 6, 7, 8, 9, 10, 15, 20, 30 or 50% relative

- 5 043415 corresponding to the previously achieved rate of haploid induction.

The term complementary nucleotide sequence to a nucleic acid in the form of double-stranded DNA means that the second strand of DNA, complementary to the first strand of DNA, has nucleotides according to base-pairing rules that correspond to the bases of the first strand, corresponding to the Watson-Crick rules.

The term molecular marker is a nucleic acid that is polymorphic in a plant population and is used as a starting or targeting point. A marker for detecting a recombination event must be capable of tracking differences or polymorphisms within a plant population. Thus, such a marker is capable of detecting and distinguishing between a variety of allelic states (alleles). The term molecular marker also refers to nucleotide sequences that are complementary or at least substantially complementary or homologous to genomic sequences, such as nucleic acids, that are used as probes or primers. For markers, these differences can be detected at the DNA level and are, for example, differences in polynucleotide sequences such as SSR (simple sequence repeat), RFLP (restriction fragment length polymorphism), FLP (fragment length polymorphism) or SNP (single nucleotide polymorphism). Markers can be derived from genomic or expressed nucleic acids, such as spliced RNA, cDNA or EST, and they can also refer to nucleic acids that are used and considered suitable as a probe or primer pairs for amplifying a fragment of a sequence using methods based on PCR. Markers describing genetic polymorphism (between parts of a population) can be detected using well known methods in accordance with the prior art (Introduction to Genetic Analysis. 7th edition, Griffiths, Miller, Suzuki et al., 2000). These include, for example, DNA sequencing, PCR-based sequence-specific amplification, RFLP detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), detection of amplified plant genome variable sequences, 3SR (self-sustaining sequence replication) detection, SSR detection, SNP, RFLP or AFLP (amplified fragment length polymorphism). In addition, methods for detecting ESTs (expressed sequence markers) and SSR markers derived from EST and RAPD (random amplified polymorphic DNA) sequences are also known. Depending on the context, the term marker as used herein may also refer to a specific chromosomal position in the genome of a species in which a specific marker (eg, SNP) may be identified.

The term operably linked means linked into a common nucleic acid molecule in such a way that the combined elements are arranged and oriented towards each other in such a way that transcription of the nucleic acid molecule can occur. DNA operably linked to a promoter is regulated by that promoter at the transcriptional level.

The term plant in the context of the present invention may, unless otherwise indicated, refer to any species of dicotyledonous and monocotyledonous plants. Preference is given to plants grown in agriculture or horticulture, or for the production of bioenergy (bioethanol, biogas, etc.). These include, for example, Solarium tuberosum, Triticum aestivum, Triticum durum, Triticum spelta, Helianthus annuus, Secale cereale, Hordeum vulgare, Hordeum bulbosum, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncacea, Brassica nigra, Glycine max, Gossypium sp ., Sorghum bicolor, Sorghum sudanense, Sorghum bicolor x Sorghum sudanense, triticale, Saccharum officinarium, Setaria italica, Oryza sativa, Oryza minuta, Oryza australiensis, Oryza alta, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Daucus p usillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea, Musa sp., Avena sp., Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosiformis, Solanum lycopersicum, Coffea canephora, Vitis vinifera, Cucumis sativus, Morus notabilis, Crucihimalaya himalaica , Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa-pastoris, Olmarabidopsis pumila, Arabis hirsuta, Raphanus sativus, Eruca vesicaria sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa or Beta vulgaris.

The sorghum plant of the present invention is a plant of the genus Sorghum, in particular the species Sorghum bicolor, Sorghum sudanense, Sorghum bicolor x Sorghum sudanense, Sorghum x almum (Sorghum bicolor x Sorghum halepense), Sorghum arundinaceum, Sorghum x drummondii, Sorghum halepense and/or Sorghum propinquum, or their hybrids and all varieties derived from them.

The term plant organs means, for example, leaves, stem, stem, roots, vegetative buds, meristems, embryos, anthers, ovules, seeds or fruits, in particular grains. The term plant part or plant parts includes, but is not limited to, shoot axis or stem, leaves, flowers, inflorescences, roots, fruits and seeds, and pollen. The term plant parts also means a combination of several organs, such as a flower or a seed, or part of an organ, such as a cross section from the axis of a shoot. The term plant tissues are, for example, callus tissue, storage tissue, meristematic tissue, leaf tissue, shoot tissue, root tissue, tumor growth tissue

- 6 043415 niya or reproductive tissue, and developed tissue, ground tissue (the so-called parenchyma), xylem, supporting-trophic tissue and integumentary tissue (the so-called epidermis). However, fabric is not limited to this list. The term plant cells refers, for example, to isolated cells having a cell wall or its aggregates, or protoplasts.

The term promoter refers to the untranslated segment of DNA, usually located upstream of the coding region, which includes the RNA polymerase binding site and initiates DNA transcription. The promoter also contains other elements that act as a gene regulator of gene expression (eg, cis-regulatory elements). The term core or minimal promoter means a promoter that has at least the basic elements necessary for initiation of transcription (eg, TATA box and/or initiator).

In the context of the present invention, the term modification refers to a nucleotide sequence that affects the specificity and/or level of expression, for example, one in which the regulatory sequence mediates tissue specificity. Such a regulatory sequence may be located upstream or downstream of the transcription initiation point of a minimal promoter, for example, in a transcribed but untranslated leader sequence or in an intron.

The term transgenic plant refers to a plant in which at least one polynucleotide, in a preferred embodiment of the present invention, a heterologous polynucleotide, has been integrated into its genome. In a preferred embodiment of the present invention, the polynucleotide is stably integrated, meaning that the integrated polynucleotide is stably maintained in the plant, expressed and can be stably inherited by progeny. The stable introduction of a polynucleotide into the plant genome also includes integration into the plant genome of the previous parental generation, and the polynucleotide can be stably inherited in the future. The term heterologous means that the introduced polynucleotide, for example, comes from the same cell or organism having a different genetic background of the same species or a different species, or it is homologous to a prokaryotic or eukaryotic host cell, but is then located in a different genetic environment and thus thus, it is different from any corresponding naturally occurring polynucleotide. A heterologous polynucleotide may be present in addition to the corresponding endogenous gene.

The expression suitable for use as a haploid inducer or capable of inducing haploidy means that the plant is capable of producing pollinated seeds having a simple (haploid) set of chromosomes as a result of crossing with a plant of the same genus, in a preferred embodiment of the present invention, the same species that does not have the property of a haploid inducer. Using a haploid inducer, represented as the absolute rate of haploid induction, means that at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 , 0.9 or 1%, in a preferred embodiment of the present invention, at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5%, in a more preferred embodiment of the present invention, at least 6, 7, 8, 9, 10, 11, 12 13, 14 or 15%, or in an even more preferred embodiment of the present invention, at least 20, 25, 30, 35, 40 , 45 or 50% of pollinated seeds or embryos have a haploid set of chromosomes.

Circuits and embodiments of the present invention are described by way of example with reference to the accompanying figures and sequences:

Fig. 1. Sequence alignment (CLUSTAL O 1.2.4) of the amino acid sequences of wild-type patatin phospholipase from sorghum (Sorghum_PPL_AA, SEQ ID NO: 3), patatin phospholipase from sorghum with the exchange of the amino acid arginine (R) for glutamine (Q) at amino acid position 59 ( Sorghum_PPL_R59Q, SEQ ID NO: 6), patatin phospholipases from sorghum with the exchange of the amino acid valine (V) for isoleucine (I) at amino acid position 162 (Sorghum_PPL_V162I, SEQ ID NO: 9), patatin phospholipases from sorghum with the exchange of the amino acid serine ( S) to leucine (L) at amino acid position 291 (Sorghum_PPL_S291L, SEQ ID NO: 12) and patatin phospholipase from sorghum with the exchange of the amino acid glutamine (Q) for a stop codon at amino acid position 372 (Sorghum_PPL_Q372Stop, SEQ ID NO: 15) . Amino acid positions 59, 162 and 291 are highlighted in black.

Fig. 2. Sequence alignment (CLUSTAL O 1.2.4) of amino acid sequences of wild-type patatin phospholipase from sorghum (Sorghum bicolor_PPL_AA, SEQ ID NO: 3), patatin phospholipase from barley (Hordeum vulgare_PPL_AA, SEQ ID NO: 21), patatin phospholipase from sunflower (Helianthus annuus_PPL_AA, SEQ ID NO: 18), patatin phospholipase 1 from sugar beet (Beta vulgaris_PPL1_AA, SEQ ID NO: 24) and patatin phospholipase 2 from sugar beet (phospholipase Beta vulgaris 2_AA, SEQ ID NO: 27). The amino acid positions that constitute the functional phospholipase domain are shown in bold.

Detailed Description of Embodiments of the Invention

The present invention provides a plant of the genus Sorghum, hereinafter also referred to as sorghum plant, which is capable of inducing haploidy. Thanks to this property of haploid induction, the plant is capable of producing pollinated seeds or embryos that have a simple (haploid)

- 7 043415 set of chromosomes resulting from crossing with a plant of the same genus, in a preferred embodiment of the present invention, the same species, which does not have the property of a haploid inducer. Thanks to this system, the inventors were able to propose an effective system for crossing sorghum plants, since inoculation lines, that is, homozygous paternal and maternal lines for the hybrid cross, could be generated by doubling the chromosomes of the haploid progeny.

The sorghum plants of the present invention are characterized in that they have at least one modification that relates to patatin phospholipase, which either imparts haploid induction property, or also improves the naturally present haploid induction property, or increases the induction efficiency. Thus, the modification to patatin phospholipase makes the sorghum plant of the present invention suitable for use as a haploid inducer in crossbreeding programs.

The plants of the present invention are characterized in that they have an induction rate of, in a preferred embodiment of the present invention, at least 0.5% and are thereby distinguished from wild-type plants or inducers that are not haploid inducers.

In experiments carried out in the context of the present invention, it was found that patatin phospholipase in sorghum is pollen-specifically expressed and therefore presumably affects pollen tube growth or interactions between gametophytes. Thus, the sorghum plant of the present invention, in a preferred embodiment of the present invention, is characterized in that the patatin phospholipase is pollen-specifically expressed and/or affects pollen tube growth or interaction between gametophytes.

In a preferred embodiment of the present invention, patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a nucleotide sequence that is at least 80, in a preferred embodiment of the present invention, 85, in a more preferred embodiment embodiment of the present invention, 90, in an even more preferred embodiment of the present invention, 95, and in the most preferred embodiment of the present invention, 99% identical to SEQ ID NO: 1 or 2, or encoded by a nucleotide sequence that has a sequence complementary nucleotide sequence in accordance with SEQ ID NO: 1 or 2 under stringent conditions, or contains the amino acid sequence shown in SEQ ID NO: 3, or a homologous amino acid sequence. As mentioned above, the sorghum plant of the present invention contains plants of any species of the genus Sorghum, in particular the species Sorghum bicolor, Sorghum sudanense and Sorghum bicolorx Sorghum sudanense or hybrids thereof and all varieties derived therefrom. Accordingly, it is reasonable to assume that during the process of speciation and crossing of varieties, the nucleotide sequence and, accordingly, the amino acid sequence of patatin phospholipase changed. In this context, the term homologous means that the genes in question (from two different plant species or cultivars) have essentially the same function and common ancestor, and therefore usually exhibit substantial identity in their nucleic acid or encoded amino acid sequence, accordingly, in a preferred embodiment of the present invention by at least 80%.

In the context of the present invention, the term homolog refers to a protein that has the same phylogenetic origin, the term analog refers to a protein that performs the same function but has a different phylogenetic origin, and the term ortholog refers to a protein from a plant of a different species that performs the same function. , and the term paralogue refers to a protein that was created through duplication within a species, and this copy either retains the same protein function, it changes the gene expression profile, but not the function, it changes the function of the protein, or the function of the ancestral gene divided between both copies.

An encoded protein (or amino acid sequence) is essentially a homologue in the context of the present invention when it performs the same function, regardless of whether it has the same or different phylogenetic origins, or is derived from the same or different kind. The homolog is also capable of complementing the haploid inducing property, that is, by modifying the gene product encoded by the homologue of the plant from which the gene originates, imparting the haploid inducing property or increasing the induction efficiency of the haploid inducer. Accordingly, a homologue corresponding to patatin phospholipase encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or containing an amino acid sequence in accordance with SEQ ID NO: 3, can, in a preferred embodiment of the present invention, be characterized as having the ability complement the haploid inducer property that is observed in the sorghum plant of the present invention. Additionally or alternatively, the patatin phospholipase homolog may, in a preferred embodiment of the present invention, be characterized by mediating the haploid inducer property or increasing the induction efficiency of the haploid inducer by mo

- 8 043415 modification of the gene product encoded by the homologue.

Those skilled in the art will know appropriate methods and techniques for genetic complementation in sorghum, for example, from Lee et al., J. Genet. 94 (2015), 445-452, in which the brown midrib phenotype of sorghum, which is caused by a point mutation in the bmr-6 gene, was complemented by introduction of the wild-type bmr-6 gene.

As shown in Example 2, the amino acid sequence of the amino acid sequence of patatin phospholipase from sorghum (SEQ ID NO: 3) results in a haploid induction rate in sorghum that is partially greater than 1.5%. It can be speculated that a further increase in the rate of induction may result from additional amino acid exchanges, which leads to further modification of the patatin phospholipase coding sequence. Thus, sorghum plants containing at least one modification or mutation related to patatin phospholipase are included in the present invention.

In the context of the present invention, the above-mentioned modifications which relate to patatin phospholipase, in a preferred embodiment of the present invention, are characterized by the fact that they are mutations that lead to at least one amino acid exchange or to the production of a stop codon in the endogenous sequence DNA encoding patatin phospholipase.

Mutation means a modification at the DNA level, that is, a change in genetics and/or epigenetics. For example, a change in genetics may involve an exchange of at least one nucleotide base in an endogenous DNA sequence or in a regulatory sequence of an endogenous DNA sequence. If such a nucleotide base exchange occurs, for example, in a promoter, then this can lead to a change in the activity of the promoter, since, for example, cis-regulatory elements are modified in such a way that the affinity of the transcription factor for the mutated cis-regulatory element, compared with the wild-type promoter, changes such that the activity of a promoter with a mutated cis-regulatory element is increased or decreased depending on whether the transcription factor is a repressor or an inducer, or whether the transcription factor's affinity for the mutated cis-regulatory element is increased or decreased. If such a nucleotide base exchange occurs, for example, in the coding region of an endogenous DNA sequence, it may result in an exchange of amino acids in the encoded protein, which may cause a change in the activity or stability of the protein compared to the wild-type protein. Another example of a change in genetics is the deletion of nucleotides in a regulatory sequence and/or endogenous DNA sequence and the addition of nucleotides in a regulatory sequence and/or endogenous DNA sequence. Changes in epigenetics can occur, for example, as a result of changes in DNA methylation profiles.

Methods for mutagenizing DNA sequences are described in more detail in the context of a method for producing a plant haploid inducer.

Since the described mutations lead to a change or shortening of the amino acid sequence of patatin phospholipase, it can be assumed that the mutations change the activity or stability of patatin phospholipase, encoded by the endogenous DNA sequence in the sorghum plant, compared to the wild type plant, that is, they increase or decrease.

Generation of stop codons in functional domains of a protein usually results in loss of protein function, whereas generation of a stop codon after a functional domain could also result in increased activity or stabilization of the protein. In the case of mutations, which, in a preferred embodiment of the present invention, are amino acid exchanges, an increase in protein activity may occur, for example through sequence optimization or, equally, through inhibition or loss of activity. Mutations in the promoter region can also lead to changes in gene expression.

Therefore, according to the present invention, the described mutation alters the biological activity of patatin phospholipase such that its original function in pollen is no longer performed to the same extent as in wild-type sorghum.

This can be achieved, on the one hand, through gene overexpression associated with increased protein quantity and activity, for example through mutations in the promoter region, through increased protein stability, for example through protein truncation through the generation of a stop codon or through the formation of a more active form of patatin -phospholipases, for example, through amino acid exchanges in the functional domain, for example, which could lead to faster pollen tube growth, uncoupling of generative cell transport in the pollen tube as it grows, or disrupted gametophyte interactions with subsequent incomplete pollination followed by chromosome removal. On the other hand, overexpression of the gene could also inhibit, prevent or reduce the proper formation, folding and/or stability of patatin phospholipase. However, reduced production of functional patatin phospholipase could also occur, or production of functional patatin phospholipase could be absent, for example through mutations in the promoter region to produce less

- 9 043415 active, inactive or unstable form of patatin phospholipase, for example, through the generation of a stop codon or through amino acid exchanges in the functional domain, which in turn could lead to improper pollination. In addition, the localization of patatin phospholipase could be altered by the described mutations so that, for example, it is no longer pollen-specifically expressed.

As a result, the present invention also includes sorghum plants having at least one expression cassette insert that is a nucleic acid molecule encoding patatin phospholipase which, by virtue of the nucleic acid sequence of SEQ ID NO: 1 or 2, or by a nucleotide sequence that is at least 80, in a preferred embodiment of the present invention, 85, in a more preferred embodiment of the present invention, 90, in an even more preferred embodiment of the present invention, 95, and in the most preferred embodiment embodiment of the present invention, is 99% identical to SEQ ID NO: 1 or 2, or encoding a functional portion of the patatin phospholipase, or containing a nucleic acid molecule having a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence in accordance with SEQ ID No : 1 or 2 under stringent conditions, or which contains a nucleic acid molecule encoding patatin phospholipase having an amino acid sequence in accordance with SEQ ID No: 3 or a homologous amino acid sequence or a functional portion thereof, and operably linked to a promoter, in a preferred embodiment of the present invention, with a pollen-specific promoter, wherein the encoded patatin phospholipase or a functional portion thereof has at least one mutation resulting in at least one amino acid exchange or the generation of a stop codon, and thus a patatin phospholipase is provided , encoding overexpression of a gene encoding a plant or part thereof compared to a wild-type plant or corresponding part thereof.

In addition, the present invention also includes sorghum plants in which the expression of patatin phospholipase is partially or completely inhibited, or a reduced amount of patatin phospholipase protein is present, or functional patatin phospholipase is not formed. Thus, the present invention also includes sorghum plants in which the expression of said patatin phospholipase is partially or completely inhibited by RNA interference (Fier et al., Nature 391 (1998), 806-811). Accordingly, the plant of the present invention is also characterized in that it has at least one expression cassette insert containing a promoter and, optionally, a terminator that is operably linked to a dsRNA-encoding nucleic acid molecule that contains at least 19 or 20, in a preferred embodiment of the present invention at least 21, 22, 23, 24 or 25, in a more preferred embodiment of the present invention at least 30, 35, 40, 45 or 50 and in the most preferred embodiment in an embodiment of the present invention, at least 100, 200, 300, or 500 nucleotides complementary to a partial sequence of a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a partial sequence of a nucleotide sequence that is at least 80 in in a preferred embodiment of the present invention, 85, in a more preferred embodiment of the present invention, 90, in an even more preferred embodiment of the present invention, 95, and in the most preferred embodiment of the present invention, 99% identical to a partial sequence of the nucleotide sequence according to with SEQ ID NO: 1 or 2.

In addition, the present invention also contains sorghum plants in which patatin phospholipase is knocked out through mutation.

Suitable promoters used in expression cassettes may be promoters that are constitutively induced (eg the 35S promoter from Cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812), suitable promoters are promoters that which exhibit plant development-specific (eg, flower-specific promoters) or tissue-specific promoters, in particular promoters that exhibit pollen-specific activity (examples: Chen et al., Molecular Biology Reports 37 (2010), 737- 744, Zhao et al., Planta 224 (2006), 405-412 or Twell et al., Genes & Development 5 (1991), 496-507) Suitable promoters may also be synthetic or chimeric promoters that do not occur naturally, which consist of several elements and contain a minimal promoter, and above the minimal promoter they have at least one cis-regulatory element that serves as a binding site for specific transcription factors. Chimeric promoters can be designed to the desired specificity and induced or repressed by various factors. Examples of such promoters can be found in the work of Gurr and Rushton (TRENDS in Biotechnology 23 (2005), 275-282) or in the work of Venter (Trends in Plant Science 12 (2007), 118-1249). A suitable terminator is, for example, the nopaline synthase terminator (Depicker et al., Journal of Molecular and Applied Genetics 126 (1982), 561-573). Promoters and other transcriptional regulatory elements are well known and available to those skilled in the art; see for example WO 00/75359 on p.

- 10 043415

23, line 5 - page 24, line 17.

As shown in Example 2, the exchange of the amino acid arginine for glutamine at amino acid position 59 in accordance with the amino acid sequence of patatin phospholipase shown in SEQ ID NO: 6, or the exchange of the amino acid valine for isoleucine at amino acid position 162 in accordance with the amino acid sequence of patatin phospholipase , shown in SEQ ID NO: 9, results in a haploid induction rate in sorghum greater than 0.5%. From fig. Figure 2 shows that these amino acid positions lie in the functional domain of patatin phospholipase, which corresponds to the range of amino acid positions from 37 to 240.

Thus, the present invention provides sorghum plants having at least one mutation resulting in an amino acid exchange in the range of amino acid positions 37 to 240 in accordance with SEQ ID NO: 3, corresponding, in a preferred embodiment of the present invention, to a functional domain patatin phospholipases. In a preferred embodiment of the present invention, at least one mutation in the range of amino acid positions 40-93 or 135-204, in a preferred embodiment of the present invention, in the range of amino acid positions 53-85 or 150-192, in a more preferred embodiment of the present invention of the invention, in the range of amino acid positions 55-75 or 157-167 leads to amino acid exchange.

In addition, the exchange of the amino acid serine for leucine at amino acid position 291 according to the amino acid sequence of patatin phospholipase shown in SEQ ID NO: 12 in sorghum plants results in a haploid induction rate greater than 1.5%. Additionally, a sorghum haploid inducer could be generated by a mutation in the nucleotide sequence of SEQ ID NO: 1 that results in a stop codon at amino acid position 372 of SEQ ID NO: 3. This mutation replaces the amino acid glutamine (Q) at position 372 there is a stop codon.

From fig. 2 shows that these positions lie outside the functional domain of patatin phospholipase.

Thus, the present invention also provides sorghum plants having at least one mutation resulting in an amino acid exchange in the range of amino acid positions 241 to 385 in accordance with SEQ ID NO: 3, corresponding, in a preferred embodiment of the present invention, to the region , located outside the functional domain of patatin phospholipase, while the mutation can also lead to a stop codon and, consequently, to a shortening of patatin phospholipase. In a preferred embodiment of the present invention, at least one mutation in the range of amino acid positions from 270 to 320, in a preferred embodiment of the present invention, in the range of amino acid positions from 285 to 311, in a more preferred embodiment of the present invention, in the range of amino acid positions 285 to 298, results in an amino acid exchange and/or a stop codon in the range of amino acid positions 322-402, in a preferred embodiment of the present invention, in the range of amino acid positions 342-392, in a more preferred embodiment of the present invention, in the range of positions amino acids 362-382.

In a particularly preferred embodiment of the present invention, the plant of the present invention is characterized in that at least one mutation results in an amino acid exchange at amino acid position 59, 162 and/or 291, and/or a stop codon at amino acid position 372 in according to SEQ ID: 3.

Accordingly, in one embodiment of the present invention, the modified patatin phospholipase contains an amino acid sequence in accordance with SEQ ID NO: 3, in which there is at least one amino acid exchange, with arginine (R) at position 59 , valine (V) at position 162 and/or serine (S) at position 291 is replaced by another amino acid, in a preferred embodiment of the present invention, glutamine (Q) at position 59, isoleucine (I) at position 162 and/or leucine (L ) at position 291 or encoded by a nucleotide sequence in accordance with SEQ ID NO: 1, in which there is at least one nucleotide exchange leading to an amino acid exchange and/or a stop codon, wherein at least one nucleotide exchanges at positions 421-423, 815-817, 1420-1422 and/or 1663-1665 according to SEQ ID NO: 1 (corresponding to nucleotide positions 175-177, 484-486, 871-873 and/or 1114-1116 according to SEQ ID NO: 2).

Plants, like eukaryotes, have at least two copies of their genetic information per cell. Each gene is usually represented by two alleles, which may be identical in the homozygous state or different in the heterozygous state. The phenotype of the plant of the present invention is caused by at least one mutation in patatin phospholipase, wherein the plant of the present invention is homozygous or heterozygous, in a preferred embodiment of the present invention, homozygous for the mutated patatin phospholipase.

In another embodiment of the present invention, a nucleic acid molecule is provided that contains previously defined specific mutations resulting in amino acid exchanges R59Q, V162I, S291L and/or Q372stop based on the amino acid sequence in accordance with

- 11 043415

SEQ ID NO: 3. In this case, the nucleic acid molecule of the present invention is characterized in that its presence in a plant results in the plant being able to induce haploidy, or in that the induction efficiency of a plant already capable of inducing haploid is improved. In a preferred embodiment of the present invention, the presence of a nucleic acid molecule of the present invention in the absence of wild-type patatin phospholipase in the plant results in the plant being able to induce haploidy or improving the induction efficiency of a plant already capable of inducing haploidy.

The nucleic acid molecule of the present invention can be used as a transgene to impart haploid inducer properties to a plant or to improve the induction efficiency of a haploid inducer. In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention is an isolated nucleic acid molecule that has been isolated from its natural or original environment, that is, outside its genetic context. A nucleic acid molecule can be double-stranded or single-stranded, linear or circular. This can be genomic DNA, synthetic DNA, cDNA, or a type of RNA, for example, siRNA or miRNA, in which the nucleotide base uracil is included in the RNA instead of the nucleotide base thymine.

In a preferred embodiment of the present invention, the nucleic acid of the present invention, or the RNA encoded by the nucleic acid, or the protein or polypeptide encoded by the nucleic acid, affects the growth of a pollen tube in a plant, the interaction between gametophytes, or pollination itself.

Hybridization DNA probes derived from a modified patatin phospholipase sequence, that is, containing any of the above-described mutations, can be used to identify plants of the present invention, that is, to detect mutations in the patatin phospholipase gene. To achieve specific hybridization, such probes must be specific and at least 15 nucleotides in length, in a preferred embodiment of the present invention at least 20 nucleotides. A detailed guide to nucleic acid hybridization can be found in Thiesen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part 1, Chapter 2, Review of Hybridization Principles and Strategy for Nucleic Acid Assays, Elsevier, New York (1993); and in, Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley Interscience, New York (1995). The probes can also be used to amplify a variety of modified patatin phospholipase sequences that have received at least one of the previously described mutations through the known polymerase chain reaction (PCR) process.

Thus, a nucleic acid molecule comprising a length of at least 15, 16, 17, 18, 19 or 20, in a preferred embodiment of the present invention at least 21, 22, 23, 24 or 25, more in a preferred embodiment of the present invention, at least 30, 35, 40, 45 or 50 and in the most preferred embodiment of the present invention, at least 100, 200, 300, 500 or 1000 nucleotides are the object of the present invention, with herein, said nucleic acid molecule specifically hybridizes to a previously described nucleotide sequence containing a modified patatin phospholipase gene, and contains one of the mutations or a pair of suitable nucleic acid molecules in the region containing at least one of the mutations, for amplification in the polymerase chain reaction ( PCR), in a preferred embodiment of the present invention, in the form of an oligonucleotide, in a preferred embodiment of the present invention, with a maximum length of 50 nucleotides. A preferred embodiment of the nucleic acid molecule of the present invention is described in paragraph [12].

Another object of the present invention are vectors containing a nucleic acid molecule of the present invention or the above-mentioned expression cassette. The vector of the present invention may contain a mutant patatin phospholipase gene having the above-mentioned nucleotide sequence characteristics operably linked to a heterologous promoter, or it may contain a mutant patatin phospholipase gene together with its natural promoter.

Another vector may contain a wild-type patatin phospholipase gene operably linked to a heterologous promoter. In addition, the vector may contain a recombinant DNA molecule that has a nucleotide sequence that encodes double-stranded RNA and therefore results in expression of the patatin phospholipase gene once expressed in a plant cell.

In addition, the vector may contain a previously described nucleic acid molecule that specifically binds to the mutated patatin phospholipase nucleotide sequence.

The described vector may be a plasmid, cosmid, bacteriophage or expression vector, transformation vector, bifunctional vector or cloning vector, it may be double-stranded or single-stranded, linear or circular, or it may transform a prokaryotic or eukaryotic host either by integration into its genome, or extra

- 12 043415 chromosomal. In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention is operably linked in an expression vector having at least one regulatory sequence that allows transcription and, optionally, expression in a prokaryotic or eukaryotic host cell; see, for example, Sambrook et al., Molecular Cloning: Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 and International Application WO 00/75359 at page 21 , line 20 to page 22, line 32. The regulatory sequence, in a preferred embodiment of the present invention, DNA, may be homologous or heterologous to the nucleic acid of the present invention. In a preferred embodiment of the present invention, these regulatory sequences are promoters or terminators, in particular a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal. In addition, vectors typically contain indicator/reporter genes or resistance genes to detect the transfer of the desired vector or DNA molecule/nucleic acid molecule and to select individuals containing them, since direct detection of gene expression is quite difficult. In a preferred embodiment of the present invention, the vector is a plant vector.

In addition to the above-described vectors, the present invention also provides a method comprising introducing the described vector into a host cell. The vector can be introduced, for example, by conjugation, mobilization, biolistic transformation, Agrobacterium-mediated transformation, transfection, transduction, vacuum infiltration or electroporation. Such methods and methods for producing the described vectors are known to one skilled in the art (Sambrook et al., 2001).

In another aspect, the present invention relates to host cells containing the described vectors, nucleic acid molecules or expression cassettes. The host cell in the context of the present invention may be a prokaryotic (eg bacterial) or eukaryotic cell (eg plant cell or yeast cell). In a preferred embodiment of the present invention, the host cell is an Agrobacterium, for example, Agrobacterium tumefaciens or Agrobacterium rhizogenes, or a plant cell containing the nucleic acid of the present invention, the described vector or expression cassette. One skilled in the art knows numerous methods, such as conjugation or electroporation, by which he can introduce a nucleic acid of the present invention, a vector of the present invention, or an expression cassette into Agrobacterium, and methods, such as various transformation methods (biolistic transformation, transformation mediated by Agrobacterium) by which it can introduce a nucleic acid of the present invention, a vector or an expression cassette of the present invention into a plant cell (Sambrook et al., 2001).

In a more preferred embodiment, the present invention relates to a transgenic plant cell containing a nucleic acid molecule of the present invention as a transgene, a vector of the present invention or an expression cassette, and a transgenic plant or part thereof that contains the transgenic plant cell. Such a transgenic plant cell or plant is, for example, a plant cell or plant that, in a preferred embodiment of the present invention, is stably transformed with a nucleic acid molecule of the present invention, a vector of the present invention, or an expression cassette. The transgenic plant of the present invention, in a preferred embodiment of the present invention, is suitable for use as a haploid inducer. In a preferred embodiment of the transgenic plant cell of the present invention, the nucleic acid molecule is operably linked to at least one regulatory sequence that allows transcription and, if necessary, expression in the plant cell. The regulatory sequence, in a preferred embodiment of the present invention DNA, may be homologous or heterologous to the nucleic acid of the present invention. The overall design of the nucleic acid molecule of the present invention and the regulatory sequence(s) subsequently constitutes a transgene. The plant part may be a pollinated or unpollinated seed, embryo, pollen, tissue, organ or plant cell, wherein the pollinated or unpollinated seed, embryo or pollen is produced on a transgenic plant and in its genome, the nucleic acid of the present invention is integrated as a transgene , vector or expression cassette. In addition, the present invention also includes progeny of a transgenic plant into whose genome the nucleic acid of the present invention is integrated as a transgene, vector or expression cassette, and which is suitable for use as a haploid inducer.

In another aspect, the present invention relates to a method for producing a plant suitable for use as a haploid inducer. The method may contain the following steps:

(a) mutagenesis of plant cells and then regeneration of plants from plant cells, subjected

- 13 043415 plant mutagenesis, or mutagenesis of plants; and (b) identifying the plant from step (a) having at least one mutation in the endogenous DNA sequence encoding the above-described patatin phospholipase, resulting in at least one amino acid exchange or generation of a stop codon, in in a preferred embodiment of the present invention to at least one of the described amino acid exchanges S291L, R59Q, V162I and Q372stop in accordance with the amino acid sequence of SEQ ID NO: 3 or leads to at least one amino acid exchange in the plant patatin phospholipase sequences corresponding exchange of amino acids S291L, R59Q, V162I and Q372stop in accordance with the amino acid sequence of SEQ ID NO: 3, and which is capable of inducing the production of haploid progeny at a higher rate compared to a plant not subjected to mutagenesis. In this case, at least one mutation results in imparting haploid inducer properties to the identified plant or increasing the induction efficiency of the haploid inducer.

In addition, further studies indicate that there are additional potential mutagenic sites that are suitable for imparting haploid inducing properties to plants or increasing their induction efficiency. These mutations result in the exchange of aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position 203 of the amino acid sequence in accordance with SEQ ID NO: 3 for another amino acid, in a preferred embodiment of the present invention, to asparagine (N) at position 75, arginine (R) at position 79 and/or leucine (L) at position 203 or to such amino acid exchanges that correspond to the amino acid exchange occurring in plant patatin phospholipases.

The endogenous DNA sequence from step (b) of the above method may encode essentially any plant patatin phospholipase. In a preferred embodiment of the present invention, the endogenous DNA sequence encodes patatin phospholipase from sorghum (SEQ ID NO: 3), sunflower (SEQ ID NO: 18), barley (SEQ ID NO: 21) or sugar beet, wherein sugar beet has two putative patatin phospholipases (SEQ ID NOs: 24 and 27). The plant patatin phospholipases mentioned in step (b), in a preferred embodiment of the present invention, are plant patatin phospholipases from sunflower (SEQ ID NO: 18), barley (SEQ ID NO: 21) or sugar beet, wherein sugar beet has two putative patatin phospholipases (SEQ ID NO: 24 and 27).

A person skilled in the art knows how a mutation can be achieved in the context of the present invention through the mutagenization process in step (a) of the method for producing a plant that is suitable for use as a haploid inducer. As used herein, mutagenesis includes both conventional mutagenesis and site-directed mutagenesis or genome editing. Modification at the DNA level is not intentionally induced by conventional mutagenesis. A plant cell or plant is subjected to mutagenic conditions, such as TILLING, through irradiation with ultraviolet light or through the use of chemicals (Till et al., BMC Plant Biology 4 (2004), 12). Another method of random mutagenesis is transposon mutagenesis. Site-directed mutagenesis allows modification to be introduced at the DNA level, targeting predefined DNA sites. For example, TALEN methods (WO 2010/079430, WO 2011/072246), meganucleases (Silva et al., Current Gene Therapy 11 (2011), 11), homing endonucleases (Chevalier, Molecular Cell 10 (2002), 895 -905), zinc finger nucleases (Lloyd et al., Proceedings of the National Academy of Sciences of the United States of America 102 (2005), 2232-237 or the CRISPR/Cas system (Guy et al., Trends in Biotechnology 31 (2013), 397-405).

Plant identification in step (b) can be accomplished, for example, by means of molecular markers or probes. DNA probes are, for example, primers or pairs of primers that can be used in a PCR reaction. For example, TILLING mutants (TILLING - Targeting Induced Local Lesions in Genomes - reverse genetics method) can be detected or identified by sequencing the target gene in the TILLING population or by other methods that detect erroneous base pairing in DNA, such as melting temperature assays or the use of mismatch-specific nucleases. The present invention also includes a primer/pairs of primers that can be used for this purpose, for example primers for patatin phospholipase.

In addition, mutants generated by transposons can be detected using transposon-specific primers and target gene-specific primers in PCRs of the entire population and in PCR products subjected to subsequent sequencing. The present invention also contains such primers. Those skilled in the art are aware of other methods and methods that they can use to identify the plant in step (b). The present invention also relates to molecular markers that detect the presence or absence of a mutation in an endogenous DNA sequence or in a regulatory sequence of an endogenous DNA sequence. For example, such markers are based on SNP (Single Nucleotide Polymorphism

- 14 043415 single nucleotide polymorphism), and they are mutation specific (examples: KASPar or TaqMan markers).

Identification of the plant in step (b) can also be carried out by testing the efficiency of induction, as described in Example 1. Thus, the present invention also relates to a method for identifying the plant of the present invention by detecting a mutation in the patatin phospholipase gene or by detecting a marker allele , which is connected to the mutation, in a preferred embodiment of the present invention, using the molecular markers described above.

An example of a plant produced and identified in this manner is the sorghum plant of the present invention.

The present invention also relates to a plant that can be produced or produced by the above method, or to a part of such a plant, wherein the part of the plant can be a pollinated or unpollinated seed, embryo, pollen, tissue, organ or plant cell, wherein pollinated or the unpollinated seed, embryo or pollen is produced on a transgenic plant and has at least one mutation in its genome. In addition, the present invention also includes a progeny plant that has at least one mutation and is suitable for use as a haploid inducer. Essentially, this method can be applied to any plant containing patatin phospholipase and thus can be given haploid inducing properties. In a preferred embodiment of the present invention, the plant is sorghum, sunflower, barley, sugar beet, rye, wheat or potato.

In another aspect, the present invention relates to a method for producing a transgenic plant suitable for use as a haploid inducer. The method may contain the following steps:

(a) introducing a nucleic acid molecule of the present invention that encodes a patatin phospholipase as described above and has at least one mutation resulting in at least one of the described amino acid exchanges or the generation of a stop codon, in a preferred embodiment of the present invention, resulting in at least one amino acid exchange selected from S291L, R59Q, V162I and Q372stop in accordance with the amino acid sequence of SEQ ID No. 3, introduction of a nucleic acid molecule having at least one mutation resulting in at least one amino acid exchange in the plant patatin phospholipase sequences corresponding to the amino acid exchanges S291L, R59Q, V162I and Q372stop in accordance with the amino acid sequence SEQ ID No. 3, the introduction of a previously described expression cassette containing either a nucleic acid molecule encoding a modified plant patatin phospholipase of the present invention and operably linked to a promoter, or containing a promoter and, if necessary, a terminator operably linked to a nucleic acid molecule encoding a dsRNA containing at least 19 nucleotides of a nucleotide sequence according to SEQ ID NO: 1 or 2, or having a nucleotide sequence that is at least 80% identical to a nucleotide sequence according to SEQ ID NO: 1 or 2, or introduction the vector of the present invention into a plant cell; and (b) regenerating the transgenic plants from the plant cells of step (a).

The method of producing a transgenic plant suitable for use as a haploid inducer also includes providing at least two of the above-described nucleic acids, as well as, by selecting various embodiments of the nucleic acid of the present invention and, if necessary, at least one vector, and transforming plant cells by introducing at least two nucleic acids. In an alternative or additional embodiment of the present invention, in addition to the nucleic acid of the present invention, at least one nucleic acid known to be suitable for generating a haploid inducer (e.g., subjected to manipulation of the cenh3 gene (Ravi and Chan, Nature 464 (2010), 615-618, EP 2989889 A1; EP 3037540 A1, WO 2016/138021 A1).

The nucleic acid molecule from step (a) of the method for producing a plant suitable for use as a haploid inducer may encode patatin phospholipase from any plant. In a preferred embodiment of the present invention, the endogenous DNA sequence encodes patatin phospholipase from sorghum (SEQ ID NO: 3), sunflower (SEQ ID NO: 18), barley (SEQ ID NO: 21) or sugar beet, wherein sugar beet has two putative patatin phospholipases (SEQ ID NOs: 24 and 27). The plant patatin phospholipases mentioned in the same step (b) are, in a preferred embodiment of the present invention, patatin phospholipases from sunflower (SEQ ID NO: 18), barley (SEQ ID NO: 21) or sugar beet, wherein sugar beets have two putative patatin phospholipases (SEQ ID NOs: 24 and 27).

Introduction of the nucleic acid molecule or vector from step (a) occurs by transformation, in a preferred embodiment of the present invention, by stabilization

- 15 043415 new transformation of plant cells. The vector can be introduced, for example, by conjugation, mobilization, biolistic transformation, Agrobacterium-mediated transformation, transfection, transduction, vacuum infiltration or electroporation. Such methods are known to one skilled in the art (Sambrook et al., 2001). In addition, the nucleic acid molecule can also be introduced into the plant genome through homologous recombination, for example, through the CRISPR/Cas system or the CRISPR/Cpf1 system and repair template.

The present invention also relates to a transgenic plant that can be produced or produced by this method, or to a part of such a plant, wherein the plant part can be a pollinated or unpollinated seed, embryo, pollen, tissue, organ or plant cell, wherein, a pollinated or unpollinated seed, embryo or pollen is produced on a transgenic plant, and into the genome of which the introduced nucleic acid is integrated as a transgene or vector. Moreover, the present invention also includes progeny of a transgenic plant which has an introduced nucleic acid as a transgene and is suitable for use as a haploid inducer. Essentially, this method can be applied to any plant containing patatin phospholipase and thus can be given haploid inducing properties. In a preferred embodiment of the present invention, the plant is sorghum, sunflower, barley, sugar beet, rye, wheat or potato.

In another aspect, the present invention relates to a method for producing a haploid plant, comprising the following steps:

(a) crossing a non-transgenic or transgenic induced plant of the present invention, which is suitable for use as a haploid inducer, with a plant of the same genus, in a preferred embodiment of the present invention, the same species, (b) selecting a pollinated haploid seed or embryo, and (c) producing a haploid plant from the seed or embryo from step (b).

In a preferred embodiment of the present invention, a plant suitable for use as a haploid inducer is used as a parent form and crossed with a maternal form of the same genus, in a preferred embodiment of the present invention, the same species. A plant suitable for use as a haploid inducer can also be used as a maternal form for crossing with a paternal form of the same genus, in a preferred embodiment of the present invention, the same species. Both crossing partners in step (a), that is, the maternal form and the paternal form, may also be the same individual. The crossing stage then represents self-pollination.

Selection of the haploid pollinated seed or embryo may comprise the step of detecting haploidy and separating the haploid pollinated seed or embryo from the polyploid pollinated seeds or embryos. Detection of haploidy in a pollinated sperm or embryo may be phenotypic or genotypic, for example by offering an inducer with an embryo-specific dominant marker that is visible in all diploid progeny but not in induced haploid progeny. Additionally, ploidy status can be determined by flow cytometry. In addition, the pattern of complete homozygosity of molecular markers indicates haploid plants. The separation may be automated, for example based on data obtained from haploidy detection.

The present invention also relates to a haploid pollinated seed or embryo that is obtained by crossing in step (a) of the method for producing a haploid plant, and to a haploid plant that can be produced or produced by this method, or to a part of this plant, wherein a part a plant may be a seed, embryo, tissue, organ or plant cell. In addition, the present invention also includes plant progeny.

In addition, the present invention also includes a double haploid (diploid) plant or part thereof, wherein the double haploid (diploid) plant or part thereof was produced by doubling the chromosomes of the haploid plant or part thereof. These double haploid (diploid) plants can be produced by the following method:

(a) producing a haploid plant by the method of the present invention;

(b) doubling the haploid set of chromosomes in at least one cell of the haploid plant, and (c) regenerating a diploid plant from the cell of step (b).

In the method for producing double haploid plants of the present invention, the haploid plants from step (a) are treated with the cell division inhibitor colchicine. This leads to the doubling of chromosomes. One skilled in the art is aware of this process and it is described, for example, in Seguy-Cimarro and Nuez, Cytogenetic and Genome Research 120 (2008), 358-369.

General methods for producing haploid and double haploid plants are known to those skilled in the art, for example from the work of Dwivedi et al., Biotechnol. Adv. 33 (2015), 812-29 and from Murovets and Boganets, Biochemistry, Genetics and Molecular Biology Plant Crossing (2012), eds. Abdurakhmonov, Chapter 5, and they can be applied to the present invention.

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Another embodiment of the present invention comprises a method for producing hybrid plants through the following steps:

(a) crossing a double haploid (diploid) plant of the present invention with a second plant of the same genus, in a preferred embodiment of the present invention, the same species, (b) selecting hybrid plants for the desired trait.

Double haploid plants are homozygous, and the known effect of heterosis occurs when two homozygous plants are crossed, resulting in particularly pronounced efficiency of hybrid plants. Accordingly, hybrid plants obtained by this method are also the subject of the present invention.

In another aspect, the present invention relates to the use of a nucleic acid of the present invention, a vector of the present invention or an expression cassette as described above in a plant to impart haploid inducer properties or to improve the induction efficiency of a haploid inducer, or to the use of a nucleic acid of the present invention, a vector of the present the invention or a previously described expression cassette for producing a plant or transgenic plant suitable for use as a haploid inducer. Moreover, the present invention also includes the use of a plant according to the above invention, which is suitable for use as a haploid inducer for producing a haploid pollinated seed or embryo, or a haploid plant. The above explanations of the objects and methods of the present invention also apply to the mentioned use cases.

In experiments with sorghum plants of the present invention, a gene, in particular the patatin phospholipase gene, could be identified having at least one mutation that would be suitable for conferring haploidy inducer properties on the plant and an induction efficiency of at least from 0.4 to 1.5% or more, whereby for the first time an efficient and therefore economically applicable system for producing haploid and double haploid sorghum plants for hybrid crossing could be provided. The method of the present invention for producing such haploid inducers and identifying patatin phospholipases in future crops can also be transferred to this system.

The following examples demonstrate the present invention, however they do not limit the scope of the present invention. Unless otherwise noted, standard molecular biology techniques were used, see, for example, (Sambrook et al., Molecular Cloning: Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), Fritsch et al., Cold Spring Harbor Laboratory Press: 1989; Mayer et al., Immunochemical Methods in Cellular and Molecular Biology, ed., Academic Press, London, 1987) and Weir et al., Handbook of Experimental Immunology, Volumes I-FV, Blackwell, ed., 1986).

Examples

1. Identification of patatin phospholipase as a target for imparting haploid induction properties to sorghum.

In the TILLING population, a sorghum plant was identified that was capable of inducing haploidy with an efficiency of approximately 1%. Particular attention is paid to various genes as potential targets in the search for the genetic basis that confers this trait on the identified sorghum plant. The genes were selected based on the fact that they are predominantly expressed in the reproductive organs of the plant and somehow play a role in pollination, because chromosome elimination can occur through defective or incomplete pollination, for example, in the absence or incorrect transport of generative cells into female ovules or by influencing the energy metabolism of pollen, which leads to a haploid set of chromosomes.

According to preliminary studies in the context of the present invention, a mutated gene was subsequently identified that could be identified as patatin phospholipase in sorghum through the bioinformatic methods BLASTP and Synteny Study (Altschul et al., Nucleic Acid Res. 25 (1997) , 3389-3402), the nucleotide and amino acid sequences of which are illustrated in SEQ ID NO: 1 or SEQ ID NO: 3, and were identified as pollen-specific expressed patatin phospholipase by RNA sequencing (PHKSeq). The mutated gene had a point mutation in the nucleotide sequence of patatin phospholipase (see SEQ ID NO: 10), which caused the amino acid substitution of serine to leucine at position 291 (see SEQ ID NO: 12). A point mutation in the nucleotide sequence of patatin phospholipase was identified using the PCR method with primers in accordance with SEQ ID Nos.: 44 and 45.

To verify that the mutated gene is the cause of the observed haploid induction, a locus containing the gene was introduced into the genetic background of the non-inducer through crossbreeding and biotechnological methods. This allowed the haploid inducer property to be introduced into a non-inducer with an average efficiency of 1.5% when the mutation was homozygous and 1.2% when the mutation was heterozygous. During verification, the mutant gene could be followed by PCR with primers according to SEQ ID Nos.: 36-38. The induction efficiency of a potential inducer was achieved by

- 17 043415 pollination of sorghum plants by mutant sorghum plants (containing a mutated gene). In this case, the wild-type sorghum plants used were genetically distinct from several markers of the potential inducer lineage. These markers were used to identify homozygous plants, which were subsequently tested for haploidy by flow cytometry.

2. Creation in vivo of other inducers of sorghum haploids.

After verifying, through the previously described experiment, that patatin phospholipase in sorghum is a suitable target for conferring haploid inducing properties on sorghum, additional mutations were introduced into the endogenous wild-type gene. A mutation was introduced that resulted in the exchange of the amino acid glutamine (Q) for a stop codon at position 372. This resulted in a shortening of the patatin phospholipase protein and gave sorghum plants containing this mutation the property of haploid induction with an average efficiency of 1 to 3%. As shown in FIG. 2, the previously described mutations S291L and Q372stop are outside the functional domain of patatin phospholipase. Consequently, in the next experiment, mutations were introduced into the patatin phospholipase gene, which, on the one hand, caused the amino acid exchange R59Q and, on the other hand, the amino acid exchange V162I. These two mutations are within the functional domain and cause sorghum to become a haploid inducer, with initial experiments showing induction rates in excess of approximately 0.5%. Sequence alignments of the four mutants with the wild-type protein are shown in Fig. 1.

It is expected that additional or different mutations, or a combination of multiple mutations in patatin phospholipase will lead to an increase or further increase in the rate of induction, so further experiments can be conducted to screen TILLING populations to identify a plant with increased induction efficiency.

In addition, mutations can also be introduced into patatin phospholipase not only through TILLING or other mutagenesis techniques in various sorghum species and sorghum-derived species, but, for example, additional haploid inducers can be produced through transgenic expression of patatin phospholipase. For this purpose, the corresponding genes, including their promoters, from sorghum inducer lines having the S291L, R59Q, V162I and/or Q372stop mutations in patatin phospholipase according to SEQ ID NO: 3 must be cloned. These genes can be cloned into a suitable transformation vector and transformed into the desired plant.

The above-described induction property of sorghum plants can be attributed to the illustrated mutations in patatin phospholipase, which usually lead to changes in their biological activity. However, biological activity can be altered not only by introducing the described mutations, but also by numerous additional genetic engineering techniques.

For example, a wild-type patatin phospholipase gene together with a suitable promoter can be cloned into a transformation vector and transformed into the desired plant, resulting in overexpression of patatin phospholipase. In addition, the activity of patatin phospholipase can be reduced by RNA interference. To do this, for example, it is necessary to produce hairpin constructs, which are then cloned into a suitable transformation vector and transformed into the desired plant, including a suitable promoter and terminator that allow transcription of the hairpin construct to occur before or during pollen production. In an alternative embodiment of the present invention, knockout mutants can be identified that further reduce activity.

3. Creation of new haploid inducers under in vivo conditions.

The first studies in the context of the present invention suggest that it is possible to provide certain other crops with the property of haploid induction or improvement of this property by modifications of patatin phospholipase, in particular by means of the corresponding mutations for the sorghum plant described above or in accordance with the method of the invention described above. These are target genes encoding putative patatin phospholipases having one of the following amino acid sequences in sunflower (SEQ ID NO: 18), barley (SEQ ID NO: 21) or sugar beet, wherein sugar beet has two potential phospholipases (SEQ ID NO: 24 and 27). Sequence alignments of protein sequences from sorghum, sunflower, barley and sugar beet are illustrated in FIG. 2, which further characterizes (in bold) the putative functional domain. Expression of these genes in pollen can be accomplished, for example, by pollen PHKSeq.

In accordance with the above-described method of the present invention, modifications related to patatin phospholipase can now be introduced, by selection, into sunflower, barley (and other cereal crops such as wheat, rye and oats) and sugar beets. For example, by aligning the sequences in FIG. 2, amino acids corresponding to and exchanging amino acids at positions 291, 59, 162 and 372 can be identified in accordance with SEQ ID NO: 3. Accordingly, sunflower, barley or cereal plants and sugar beet plants are also the object of the present invention, which are capable of inducing haploidy and character

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Claims (17)

characterized in that they have at least one modification, one endogenous, in a preferred embodiment of the present invention, pollen-specific expressed patatin phospholipase, respectively related to one of the embodiments of the present invention, characterized in the above paragraphs [] or in the claims , in particular for sorghum plants, and/or for plants obtained by the method according to the above-described invention or described in the claims. There are already first indications that amino acid exchanges at position 75 (aspartic acid (D) is replaced by asparagine (N) (D75N)), at position 79 (glycine (G) is replaced by arginine (R) (G79R)) and/or position 203 (proline (P) is replaced by leucine (L) (P203L)) of the amino acid sequence according to SEQ ID NO: 3 can also confer haploid induction in both sorghum and other crops. CLAIM 1. A sorghum plant that is capable of inducing haploidy, characterized in that it has at least one modification related to an endogenous gene encoding patatin phospholipase, wherein patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2 , or a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 1 or 2, or encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions , or which contains the amino acid sequence shown in SEQ ID NO: 3, or a homologous amino acid sequence, wherein at least one modification is at least one mutation in an endogenous gene encoding patatin phospholipase and at least At least one amino acid change mutation occurs in the range of amino acid positions 53-85, 150-192 and/or 285-311 according to SEQ ID NO: 3. 2. The plant according to claim 1, characterized in that at least one mutation results in an amino acid change at amino acid position 59, 162 and/or 291 in accordance with SEQ ID NO: 3. 3. A plant according to claim 1 or 2, characterized in that the modified patatin phospholipase (i) contains an amino acid sequence in accordance with SEQ ID NO: 3 or a homologous amino acid sequence in which at least one amino acid substitution is present, with wherein arginine (R) at position 59, valine (V) at position 162 and/or serine (S) at position 291 in accordance with SEQ ID NO: 3 is replaced by another amino acid, preferably glutamine (Q) at position 59, isoleucine (I ) at position 162 and/or leucine (L) at position 291; (ii) is encoded by a nucleotide sequence containing the coding sequence of a DNA sequence in accordance with SEQ ID NO: 1 or a DNA sequence that is at least 80% identical to SEQ ID NO: 1 in which at least one substitution is present nucleotides resulting in an amino acid substitution, wherein at least one nucleotide is replaced at positions 421-423, 815-817 and/or 1420-1422 in accordance with SEQ ID NO: 1; (iii) contains an amino acid sequence according to SEQ ID NO: 6, 9 or 12; or (iv) is encoded by a nucleotide sequence comprising the coding sequence of a DNA sequence in accordance with SEQ ID NO: 4, SEQ ID No: 7 or SEQ ID NO: 10. 4. A nucleic acid molecule that encodes a mutated patatin phospholipase according to any one of claims 1 to 3 and its presence in the plant, in the absence of wild type patatin phospholipase, results in the plant being able to induce haploidy. 5. An oligonucleotide of at least 15, 16, 17, 18, 19 or 20 nucleotides in length that specifically hybridizes to the nucleotide sequence defined in any one of claims 1 to 3 and contains one of the mutations. 6. The oligonucleotide according to claim 5, wherein the oligonucleotide has one of the following nucleotide sequences according to SEQ ID No. : 28-30, 32-34 or 36-38. 7. A plant cell containing a nucleic acid molecule according to claim 4 or an oligonucleotide according to any claim 5, 6 or a vector containing a nucleic acid molecule according to claim 4 or an oligonucleotide according to any claim 5, 6. 8. A plant cell according to claim 7, in which the nucleic acid molecule according to claim 4 is a transgene. 9. The plant cell according to claim 8, in which the transgene is regulated by a heterologous promoter. 10. A method for producing a plant capable of inducing haploidy or having an increased rate of induction relative to the wild type, containing the following steps: (a) (i) mutagenesis of plant cells and then regeneration of plants from plant cells subjected to mutagenesis, or plant mutagenesis; (ii) identifying the plant from step (i) having in the endogenous DNA sequence at least one mutation, which is at least one mutation in the endogenous gene encoding patatin phospholipase, and at least one mutation causes replacement of ami - 19 043415 no acids in the range of amino acid positions 53-85, 150-192 and/or 285-311 in accordance with SEQ ID NO: 3, where patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or the sequence nucleotides that is at least 80% identical to SEQ ID NO: 1 or 2, or is encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions, or that contains an amino acid sequence shown in SEQ ID NO: 3, or a homologous amino acid sequence, and/or which results in the replacement of aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position 203 of the amino acid sequence in according to SEQ ID NO: 3, a different amino acid, and wherein the plant is capable of inducing haploid progeny at an increased rate compared to a plant not subjected to mutagenesis; or (b) (i) introduction of a nucleic acid molecule having at least one mutation of step (a) (ii), and/or which results in the replacement of aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position 203 of the amino acid sequence according to SEQ ID No. 3 replaced by another amino acid at a specified position in the plant cell, and (ii) regenerating the plant from the plant cell (i). 11. The method according to claim 10, wherein in step (a) (ii) or (b) (i) aspartic acid (D) at position 75, glycine (G) at position 79 and/or proline (P) at position The 203 amino acid sequence of SEQ ID NO: 3 is replaced by asparagine (N) at position 75, arginine (R) at position 79, and/or leucine (L) at position 203. 12. The method of claim 10 or 11, wherein the endogenous DNA sequence or nucleic acid molecule is a coding nucleotide sequence that contains the amino acid sequences shown in SEQ ID NO: 3 for sorghum (Sorghum bicolor), SEQ ID NO: 18 for sunflower (Helianthus annuus), SEQ ID NO: 21 for barley (Hordeum vulgare) or SEQ ID NO: 24 or 27 for Beta vulgaris. 13. A plant containing a plant cell according to any item 7-9 and/or obtained by a method according to any item 10-12. 14. The plant of claim 13 is sorghum, sunflower, rye, wheat, potato, barley or sugar beet. 15. A plant organ according to one of claims 1-3, 13 or 14, characterized in that the organ has at least one mutation in the endogenous gene encoding patatin phospholipase, and at least one mutation causes an amino acid substitution in the range of amino acid positions 53-85, 150-192 and/or 285-311 in accordance with SEQ ID NO: 3, where patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a nucleotide sequence of at least at least 80% identical to SEQ ID NO: 1 or 2, or encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2, under stringent conditions, or which contains the amino acid sequence of SEQ ID NO : 3, or a homologous amino acid sequence, and/or a nucleic acid molecule according to claim 4, an oligonucleotide according to any claims 5, 6, or a vector containing a nucleic acid molecule according to claim 4, or an oligonucleotide according to any claims 5, 6. 16. A plant part according to one of claims 1-3, 13 or 14, characterized in that the plant part has at least one mutation in the endogenous gene encoding patatin phospholipase, and at least one mutation causes a replacement amino acids in the range of amino acid positions 53-85, 150-192 and/or 285-311 in accordance with SEQ ID NO: 3, where patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a nucleotide sequence according to at least 80% identical to SEQ ID NO: 1 or 2, or encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2, under stringent conditions, or which contains the amino acid sequence of SEQ ID NO: 3, or a homologous amino acid sequence and/or nucleic acid molecule according to claim 4, an oligonucleotide according to any claims 5, 6, or a vector containing a nucleic acid molecule according to claim 4, or an oligonucleotide according to any claims 5, 6. 17. Plant tissue according to one of claims 1-3, 13 or 14, characterized in that the tissue has at least one mutation in the endogenous gene encoding patatin phospholipase, and at least one mutation causes the replacement of amino acids in range of amino acid positions 53-85, 150-192 and/or 285-311 in accordance with SEQ ID NO: 3, where patatin phospholipase is encoded by a nucleotide sequence in accordance with SEQ ID NO: 1 or 2, or a nucleotide sequence of at least 80% identical to SEQ ID NO: 1 or 2, or encoded by a nucleotide sequence that hybridizes to a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2, under stringent conditions, or that contains the amino acid sequence of SEQ ID NO: 3 , or a homologous amino acid sequence and/or nucleic acid molecule according to claim 4, an oligonucleotide according to any claims 5, 6, or a vector containing a nucleic acid molecule according to claim 4, or an oligonucleotide according to any claims 5, 6. -