One-step in vivo doubled haploid induction in plants

Abstract

The present invention relates to a method for generating a doubled haploid inducer plant (DHI), the method comprising providing a first plant which is a haploid inducer (HI) plant, wherein the haploid inducer plant is a maternal haploid inducer or paternal haploid inducer; modifying at least an endogenous gene in the haploid inducer plant that is involved in one or more pathways related a to a process which results in chromosome doubling of haploid cells, thereby obtaining a doubled haploid inducer plant.

Description

[0001] Dusseldorf, 20 January 2026 Our reference: KD 41971

[0002] KWS SAAT SE & Co. KGaA

[0003] One-step in vivo doubled haploid induction in plants

[0004] REFERENCE TO SEQUENCE LISTING

[0005] Pursuant to the EFS-web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an xml 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference, for the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

[0006] FIELD OF THE INVENTION

[0007] The present application relates to methods in vivo doubled haploid induction in plants.

[0008] BACKGROUND

[0009] Plant breeding depends on the development of pure-breeding lines (so-called inbred lines). Inbred line development is the most time-consuming part of plant breeding. Traditional methods for developing inbred typically require 6 to 10 generations of recurrent self-pollination and selection to reach sufficient homozygosity (>98%).In contrast, doubled haploid (DH) systems can create completely homozygous and homogeneous lines within two generations. DH technology dramatically speeds up breeding line development in plants. Since DH plants are derived from recombinant haploid gametes and are recombinant inbred after doubling, DH technology does not only provide diverse inbred pools which are readily available for breeding selection, but also offers a vital approach for rapid generation of homozygous mapping populations and recessive mutants. The latter is especially important for gene discovery and trait development.

[0010] DH technology currently includes two approaches - in vitro culture of haploid gametes (i.e., microspore, anther, or ovule culture) and in vivo (also known as in planta) crossing of a haploid inducer line with a source germplasm.

[0011] In vitro DH technology involves gamete cell culture and regeneration, which it is highly species- and genotype-dependent, and successful only in limited species and genotypes. In vivo DH technology is relatively fast, simple, less genotype-dependent, less gameto-clonal variation, and hence deemed superior to the in vitro approach.

[0012] In vivo haploid production involves four steps: (1) haploid induction via the crossing of a haploid inducer line with source germplasms; (2) haploid seed selection; (3) genome doubling by chemical treatment of the haploid seedlings with e.g. colchicine; and (4) doubled haploid seed production by self-pollination.

[0013] In vivo DH technology has become a routine application in many crops, like e.g., maize. It has dramatically reduced the “time to market” and allowed rapid breeding development of plant varieties comprising traits of interest.

[0014] During the past two decades, substantial progress has been achieved in in vivo DH technology. Several maize maternal haploid inducers with satisfactory haploid induction rates (HIR) (about 10% in average) have been developed. Maize has a spontaneous haploid rate of 0.1%.

[0015] Some early haploid inducers, like Stock 6, show haploid induction rates of up to 3%. (Trentin et al (2020), Coe (1959)). Some more recently developed proprietary lines are reported to have haploid induction rates of over 10% (Trentin et al (2020)).Single fertilization and chromosome elimination are the two mechanisms proposed for in vivo haploid induction.

[0016] In single fertilization, plant egg cells fail to fuse with one of the two sperms, such that fertilization occurs only to the central cell. The resulting seed is composed of a triploid endosperm and a haploid embryo, which stems from embryogenesis of the pseudo-fertilized egg-

[0017] In the case of chromosome elimination, an egg cell is fertilized, but the chromosomes which stem from the inducer are eliminated during subsequent cell divisions.

[0018] There are two types of in vivo haploid induction which depend on the origin of the genome in the resulting haploid progeny after crossing. One type of haploid induction is gynogenesis, which is defined as the ability to induce a haploid embryo deriving exclusively from the female source parent (also called “maternal haploid induction”). In this type, the haploid inducer is called “maternal haploid inducer”, and is used as the male pollen donor, whereas the source germplasm serves as the female pollen recipient. Pollen from maternal haploid inducers triggers the development of the egg cell from the source germplasm into an embryo but makes no genetic contribution to the embryo. The resulting haploids receive both the cytoplasmic and nuclear genome from the female parent. This phenomenon may make maternal haploid induction more appealing for maize breeding programs. Maternal haploid inducers are mostly derivatives of Stock 6.

[0019] The other type of haploid induction is androgenesis, in which the embryo contains only the paternal germplasm genome (also called “paternal haploid induction”). In maize plants, androgenesis occurs mostly when a derivative of an igl / igl mutant is used as the female parent. Haploid induction gene IG1 (Indeterminate Gametophyte 7) is a cell proliferation suppressor and required to limit cell divisions. Loss-of-function igl mutation leads to prolonged cell proliferation in leaves, and about 3% paternal haploid induction rate (HIR) in maize (Evans (2007); Kindiger et al (1993).

[0020] It was shown that the two quantitative trait loci (QTL) qhirl and qhir8 are essential for the maternal haploid induction in maize (Prigge et al. (2012); Liu, C. et al. (2015). A frameshift mutation (4-bp insertion) in the last exon of gene GRMZM2G471240 [encoding a pollen -specific phospholipase, named MATRILINEAL (MTL), ZEA MAYS PHOSPHOLIPASE Al (ZmPLAl), or NOT LIKE DAD (NLD)] is found to be responsible up to 2 / 3 of the maternal haploid induction capability observed in Stock 6 and the derivatives. ZmPLAl is located within QTL qhirl. The haploid induction rate (HIR) in the zmplal mutants is 2-3% (Kelliher et al. (2017); Liu et al. (2017); Gilles et al (2017).

[0021] A sperm-specific DOMAIN OF UNKNOWN FUNCTION 679 membrane protein ZmDMP was identified from the haploid induction mapping locus qhir8. A single-base substitution mutation in ZmDMP was considered to contribute to the high haploid induction rate (HIR) observed for CAU5. The haploid induction rate (HIR) in the zmdmp single mutants is only 0.1-0.3%, and it can be increased to 6-10% in the zmplal / zmdmp double mutants (Liu et al (2015); Zhong et al (2019)).

[0022] Two Arabidopsis DMP proteins of AtDMP8 and AtDMP9, the orthologs of ZmDMP in Arabidopsis, were shown to facilitate gamete fusion and double fertilization. Knockout dmp8 / dmp9 double mutant resulted in 40% single fertilization of the central cell in Arabidopsis. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Loss-of-function mutations of AtDMp8 and AtDMP9 induce maternal haploids in a rate of 2-3% in Arabidopsis (Takahashi et al. (2018); Cyprys et al. (2019).

[0023] Following haploid induction, doubling of the haploid genome is indispensable for fertility recovery and final establishment of a DH line. Spontaneous doubling occurs rarely (less than in 1% of cases), and artificial doubling treatment is likely required for most plant double haploid systems. Up to date, the artificial doubling is built on colchicine-based chemical treatments of the haploid seedlings in following generation. Colchicine and colchicine-like chemicals bind to soluble tubulin to form tubulin-colchicine complexes, which prevent the elongation of the microtubules and impair mitotic spindle formation. Colchicine and colchicine-like chemicals thus inhibit sister chromatid segregation in metaphase and lead to chromosome doubling, typically with a doubling rate of 10%-30%.

[0024] Since colchicine and colchicine-like chemicals are toxic, proper protection equipment and procedure are needed when handling (the haploid seedlings need to be handled and treated manually and individually). Following the treatment, the treated seedlings need to be transplanted into soil.As a result chemically induced haploid doubling is a labor-intensive, hazardous, timeconsuming, and costly approach. Moreover, the efficacy of chemical doubling differs dramatically among different source germplasm pools and seed batches, which makes the estimation for the amounts of starting haploid seeds difficulty. To ensure meeting the targeted number of doubled haploid line production, an exceed number of haploid seeds is typically used (e.g., 10-fold over the targeted number). Furthermore, the treatment usually results in chimeric plants (Do) comprising of undoubled haploid and doubled dihaploid cells. Being haploid at early developmental stages, the chimeric Do plants are indistinguishable from the haploid plants, and all treated haploid plants are generally kept until mature. All these factors increase the cost for doubled haploid line production.

[0025] Taken together, genome doubling based on current approaches represents a major bottleneck for doubled haploid technology in plant breeding.

[0026] It is hence one object of the present invention to provide a method which facilitates and / or accelerates the production of doubled haploid inducer lines.

[0027] It is one other object of the present invention to provide a method which reduces the costs and / or labour involved with the production of doubled haploid inducer lines.

[0028] It is one other object of the present invention to provide a method which reduces the toxic burden involved with the production of doubled haploid inducer lines.

[0029] It is one other object of the present invention to provide a method which increases the efficacy of the production of doubled haploid inducer lines.

[0030] These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.

[0031] BRIEF DESCRIPTION OF THE FIGURESFigure 1: shows 29-day-old maize Al 88 plants growing in 50-well tray in green house are ready for immature tassel harvesting.Figure 2: shows a Bright field image (A) or tDTomato fluorescence image of the immature tassels 18 hours after bombardment (B) from 29-day-old maize Al 88 plants.

[0032] Figure 3: shows a Bright field image (A) or tDTomato fluorescence image of the immature tassels 18 hours after bombardment (B) from 29-day-old maize Al 88 plants.

[0033] Figure 4: shows the callus development in a MRM3 medium for 7 days (A), shoot development in shooting medium (SM) for 10 days (B), and regenerated To plantlets 5 days in a rooting medium (RM) phytatray (C) from the bombarded Al 88 immature tassels.

[0034] Figure 5: shows the genome editing nuclease MAD7 expression construct GEMT510 map. tdTomato defines tdTomato report gene. MAD7 defines the maize codon optimized CDS of MAD7 nuclease (Inscripta). BdUBIlO defines the Brachypodium Ubiquitin 10 promoter. Tnos defines the nos terminator.

[0035] Figure 6: shows the genome editing nuclease LbCpfl RR version expression construct GEMT505 map. tdTomato defines tdTomato report gene. LbCpfl defines the maize codon optimized CDS of LbCpfl RR nuclease. BdUBIlO defines the Brachypodium Ubiquitin 10 promoter. Tnos defines the nos terminator.

[0036] Figure 7: shows the guide crRNA expression construct CTA001-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous Cyclin-Bl-1 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0037] Figure 8: shows the guide crRNA expression construct CTA005-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmCyclin-Bl-5 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0038] Figure 9: shows the guide crRNA expression construct CTA046-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous Zm Cyclin-B2-3 gene.ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0039] Figure 10: Guide crRNA expression construct CTA102-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmCDKBl-1 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0040] Figure 11: shows the guide crRNA expression construct CTA087-pA0-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmNC80 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0041] Figure 12: shows the guide crRNA expression construct CTA097 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmSPC25 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0042] Figure 13: shows the guide crRNA expression construct CTA079 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmKinesinl gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0043] Figure 14: Guide crRNA expression construct CTA069-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmKinesin2 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0044] Figure 15: Guide crRNA expression construct CTA142-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous Zmkrpl gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0045] Figure 16: shows the guide crRNA expression construct CTA183 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmMAP gene, encoding a 187kDa microtubule-associated protein. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.Figure 17: shows the guide crRNA expression construct CTA194 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmMAP6 gene, encoding a 65-kDa microtubule-associated protein 6. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0046] Figure 18: shows the guide crRNA expression construct CTA197-pA-01 map. crRNA-1 to crRNA-5 define the five protospacer targeting to the maize endogenous ZmAurora3 gene. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0047] Figure 19: shows the maize PLT5 expression construct pABM-BdEFl_ZmPLT5 map. ZmPLT5 is driven by the strong constitutive EFl promoter from Brachypodium (pBdEFl). Tnos defines the nos terminator.

[0048] Figure 20: shows the maize KWS RBP8 expression construct pABM-BdEFl_ RBP8 map. KWS RBP8 is driven by the strong constitutive EFl promoter from Brachypodium (pBdEFl). Tnos defines the nos terminator.

[0049] Figure 21: shows 22-day-old haploid inducer EMPC plants growing in 50-well tray in green house are ready for immature tassel harvesting.

[0050] Figure 22: shows a fresh isolate immature tassel from a 22-day-old haploid inducer EMPC plant is ready for bombardment.

[0051] Figure 23: shows a Bright field image (A) or tDTomato fluorescence image of the immature tassels 18 hours after bombardment (B) from 22-day-old EMPC plants.

[0052] Figure 24: shows the EMPC callus development in MRM3 for 10 days (A) and shoot development a SM medium for 10 days (B), and plantlet development in a rooting (RM) phytatray for 10 days (C).

[0053] Figure 25: shows a sequence alignment comparing the edited target gene from the edit of CTA005-T261 with the non-edited gene from wild-type (WT) EMPC. (A) shows the comparison for ZmCyclin Bl-1, and (B) for ZmCyclin B-l-5.Figure 26: shows a sequence alignment comparing the edited ZmCyclin-B2-3 gene from the edit of CTA013-T183 with the non-edited gene from wild-type (WT) EMPC.

[0054] Figure 27: shows a sequence alignment comparing the edited Zmkrpl gene from the edit of CTA069-T179 with the non-edited gene from wild-type (WT) EMPC.

[0055] Figure 28: shows a sequence alignment comparing the edited ZmCDKBl-1 gene from the edit of CTA102-T183 with the non-edited gene from wild-type (WT) EMPC.

[0056] Figure 29: shows a sequence alignment comparing the edited ZmSPC25 gene from the edit of CTA097-T256 with the non-edited gene from wild-type (WT) EMPC.

[0057] Figure 30: shows a sequence alignment comparing the edited ZmMAP gene from the edit of CTA182-T069 with the non-edited gene from wild-type (WT) EMPC.

[0058] Figure 31: shows a sequence alignment comparing the edited ZmMAP6 gene from the edit of CTA189-T085 with the non-edited gene from wild-type (WT) EMPC.

[0059] Figure 32: shows a sequence alignment comparing the edited ZmAurora3 gene from the edit of CTA197-T090 with the non-edited gene from wild-type (WT) EMPC.

[0060] Figure 33: shows a workflow for DH induction and identification.

[0061] Figure 34: shows a DH induction cross between the edited EMPC (pollen donor) and glossy Fl tester om greenhouse.

[0062] DETAILED DESCRIPTION

[0063] According to one aspect of the invention, a method for generating a doubled haploid inducer plant (DHI) is provided, the method comprising

[0064] a) providing a first plant which is a haploid inducer (HI) plant, wherein the haploid inducer plant is a maternal haploid inducer or paternal haploid inducer;b) modifying at least an endogenous gene in the haploid inducer plant that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells,

[0065] c) thereby obtaining a doubled haploid inducer (DHI) plant.

[0066] The doubled haploid inducer plant (DHI) is capable of inducing at least a doubled haploid progeny plat (DHP) by one-step crossing with a second plant.

[0067] According to an embodiment of such method according to the invention, the method further comprises

[0068] d) providing a second plant, wherein the second plant is, or comprises, a source germplasm e) crossing the doubled haploid inducer plant and the second plant;

[0069] f) obtaining progeny of said crossing

[0070] g) testing, in said progeny, whether it comprises a doubled haploid genome derived from the source germplasm, thereby

[0071] h) obtaining at least a doubled haploid plant (DHP).

[0072] In this context, it needs to be stated that the “crossing”, as used herein, does not necessarily imply “sexual crossing”, i.e., a process in which the whole genomes of the two parental plants are crossed and genetical information of the two parental plants is recombined in the offspring. In fact, when "crossing" a source germplasm with a DHI, the genomes are not crossed and the genome of the source germplasm and the genome of the source germplasm remains unchanged. This is reason why we use the DHI - it is not meant to be a trait supplier, but as a kind of catalyst to "help" the source germplasm to clone itself). As such, the “crossing”, as used herein, does not qualify as an essentially biological process.

[0073] The term “haploid inducer” as used herein, refers to a plant, either male or female, which, when crossed with a regular plant, yields progeny plants that are haploid, meaning that that they will only contain half of the parental plant genome (as e.g. opposed to the usual two copies, of the parent is diploid). Importantly, all their genetic material comes from the parent which is not the haploid inducer. Hence, if a plant egg is pollinated with an inducer pollen, such pollination triggers the development of the egg cell into an embryo containing only a haploid maternal genome. Naturally occurring haploid inducer lines have been discovered in Maize in the 1950s (Coe, 1959), yet have also been created inter alia in Arabidopsis thaliana and Brassica juncea.See, in this context, also patent applications W02016030019, WO2016075255, WO2016102665, W02018158301, WO2020157197, WO2021239986

[0074] As used herein, the term “doubled haploid inducer (DHI) plant” relates to a plant that is capable of inducing at least a doubled haploid progeny plat (DHP) by one-step crossing with a second plant.

[0075] The term “source germplasm” as used herein relates to a seed, plant, or plant part useful in crop breeding, characterized by carrying one or more genetic traits of interest.

[0076] Many haploid inducers are derived from the haploid inducer line Stock6 (see Kalinowska et al (2018), the content of which is incorporated herein by reference for enablement purposes).

[0077] Reference is in this context made to published US patent application US2019106703 Al, which discloses methods for increasing the frequency of spontaneous haploid genome doubling in plants. The method comprises introducing to a plant a nucleic acid molecule that encodes a variant BUBR1 polypeptide operably linked to a promoter capable of driving expression in a plant. Said variant BUBR1 polypeptide confers increased spontaneous haploid genome doubling when crossed with a haploid inducer line compared to haploids derived from a control plant. The increased SHGD associated with a variant BUBR1 gene can be further introgressed into maize population by backcrosses and QTL-assisted selections. Furthermore, a modified plant with high frequency of spontaneous haploid genome doubling can be generated using a gene editing technology (e.g., ZFNs, TALENs, CRISPR / Cas9) to introduce one or more mutations into a BUBR1 endogenous nucleic acid sequence.

[0078] US2019106703A1 further discloses a method of producing doubled haploid plants at a high frequency without a chromosomal doubling agent. The method comprises crossing a plant produced by the method as described above with a second plant to produce an FI plant; (transgenic variant BUBR1 Fl), and pollinating said Fl plant with an inducer line to produce a population of haploid embryos. Said population of haploid embryos is then planted, so that the haploid embryos undergo spontaneous haploid genome doubling at a high frequency to produce a population of doubled haploid plants. As a key feature, said population of haploid embryos are not treated with a chromosomal doubling agent.The method as disclosed in US2019106703A1 is called “spontaneous haploid genome doubling (SHGD. This means that the first plant used is a modified hybrid (Fl) plant or a modified plant containing a mutation (=variant BUBR) that enables SHGD after haploid induction. The second plant is a Haploid Inducer (HI). As such, the doubling mutation needs to be built in breeding plant pools and will remain in the resulting new DH lines. Moreover, SHGD requires, in many cases, a full GMO approach.

[0079] In the one-step DH method according to the invention, on the contrary, a Doubled Haploid Inducer (DHI) line is generated by mutagenesis of one or more genes in a preexisting haploid inducer line (HI), so as to turn the haploid inducer (HI) into a Doubled Haploid Inducer (DHI). The Doubled Haploid Inducer line can induce haploid plants and double the haploid genome simultaneously or consequently when crossed e.g. with a source germplasm. The first plant in one-step DH is a Doubled Haploid Inducer (DHI) and the second plant is e.g. a WT hybrid source plant or source plant. A DHI can be used for crossing with all the source plants from different breeding pools, and the mutations in a DHI will be eliminated after haploid induction and leads to clean DH lines in one-step DH crossing.

[0080] Furthermore, as disclosed herein, the method according to the present invention does not necessarily require a GMO approach.

[0081] According to one embodiment of the method according to the invention, the haploid inducer plant is characterized by at least one mutation in a gene or quantitative trait locus selected from the group consisting of

[0082] • MATRILINEAL (MTL) / ZEA MAYS PHOSPHOLIPASE Al (ZmPLAl) / NOT LIKE DAD (NLD) (maternal haploid inducer)

[0083] • Indeterminate gametophyte (igl) gene (paternal haploid inducer)

[0084] • CENH3 (CENTROMERIC HIST0NE3) (maternal and paternal haploid inducer), and / or

[0085] • ZmDMP (DUF679 Domain Membrane Protein)

[0086] • ZmigN (patermal haploid inducer)

[0087] or orthologues thereof.As used herein, the term “orthologue” relates to a gene within a different species which performs the same or a similar function as the gene it is orthologue to. In some embodiments, the term “orthologue” relates to a gene "within a different species that shares a common ancestor and was separated by a speciation event.

[0088] As used herein, the term “quantitative trait locus” (QTL) relates to a gene locus that correlates with variation of a quantitative trait in the phenotype of a population of organisms. QTLs are mapped by identifying which molecular markers (such as SNPs or AFLPs) correlate with an observed trait. This is often an early step in identifying the actual genes that cause the trait variation. QTLs are typically associated with a specific phenotype or trait with continuous variance, rather than traits with discrete variance, such as height or skin color.

[0089] A frameshift mutation in a gene coding for a pollen-specific phospholipase, named MATRILINEAL (MTL) / ZEA MAYS PHOSPHOLIPASE Al (ZmPLAl) / NOT LIKE DAD (NLD), causes haploid induction in maternal inducers, providing a ~2% haploid induction rate (HIR) (see Kelliher et al (2017), Liu et al (2017) or Gilles et al (2017), the contents of which are incorporated herein by reference for enablement purposes).

[0090] The igl mutation was first observed as a spontaneous mutation in the inbred line Wisconsin-23 (W23) leading to about 3% haploid induction rates (HIR) of paternal haploids. The inducer containing igl is used as female parent. Paternal haploids thus contain the cytoplasm of the female inducer and the haploid genome of the pollen donor (see Evans (2007) or Kindiger et al (1993), the content of which is incorporated herein by reference for enablement purposes).

[0091] cenh3 null mutants expressing altered CENH3 proteins which are crossed to wild type, cause chromosomes from the mutant to be are eliminated, producing haploid progeny (see Wang et al. (2021) and Ravi and Chan (2010), the content of which is incorporated herein by reference for enablement purposes).

[0092] The mutation of a non-k / wA6-originating gene in qhir8. ZmDMP, enhances and triggers haploid induction. ZmDMP was verified by CRISPR-Cas9-mediated knockout experiments. A single-nucleotide change in ZmDMP leads to a 2-3 -fold increase in the HIR. ZmDMP knockout triggered haploid induction with a HIR of 0.1-0.3% and exhibited a greater ability to increasethe HIR by 5-6-fold in the presence of mtllzmplallnld (see Zhong et al (2019), the content of which is incorporated herein by reference for enablement purposes).

[0093] ZmDMP is a DUF679 Domain Membrane Protein in maize, which is encoded by maize GRMZM2G465053 gene. Specifically, ZmDMP is a sperm plasma membrane protein that facilitates the translocation of fusion factors to sperm membrane in response to signals from egg cells during fertilization. Loss-of-function mutation in ZmDMP induces maternal haploids with a haploid induction rate (HIR) of 0.1-0.3%, and the HIR is greatly increased by 5-6-fold when combined with a mutation from ZmMTL / ZmPLAl / ZmNLD gene (Zhong, et al, 2019).

[0094] Further gene mutations or quantitative trait loci which case haploid induction are e.g. described in Kalinowska et al (2018, the content of which is incorporated herein by reference for enablement purposes).

[0095] According to embodiments of such method according to the invention, the haploid inducer plant is a maize plant derived from the list consisting of Stock6, CAUB, CAU5, CAU079, CAUHOI, KMS, ZMS, RWS, RWP, RWK (RWK76), UH400, UH402, UH600, UH601, MHI, M741B, M741C, M741F, M741H, M741I, M741J, PHI- 1, PHI-2, PHI-3, PHI-4, PK6, TAILs, CIM2GTAILs, LI-ESALQ, FIGH1 and derivatives thereof or hybrids of these haploid inducers (e g., RWSxRWK76).

[0096] These inducers are maternal inducers and well described in the respective literature.

[0097] According to one embodiment of such method according to the invention, the haploid inducer plant is a maize plant derived from the igl mutants, igl-0

[0098] These inducers are paternal inducers and well described in the respective literature. Principles of maternal and paternal haploid inducers are for example discussed in Chaikam et al (2019), the content of which is incorporated herein by reference for enablement purposes.

[0099] According to embodiments of such method according to the invention, the endogenous gene that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells is a gene involved in at least one process which results inendopolyploidy, endoreduplication, polyploidization, and / or impaired chromosome segregation of cells.

[0100] As used herein, the term “endoreduplication” relates to replication of the nuclear genome in the absence of mitosis, which leads to elevated nuclear gene content and polyploidy. Endoreduplication is a special cell cycle, where chromosomes are replicated in S phase, but mitosis is entirely or partially skipped (endoreduplication, endoreplication, endocycle, or endocycling or endomitosis), or cytokinesis is avoided (cytokinesis failure). Endoreplication can be understood simply as a variant form of the mitotic cell cycle (G1-S-G2-M) in which mitosis is circumvented entirely, due to modulation of cyclin-dependent kinase (CDK) activity. Examples of endoreplication characterized in arthropod, mammalian, and plant species suggest that it is a universal developmental mechanism responsible for the differentiation and morphogenesis of cell types that fulfil an array of biological functions. While endoreplication is often limited to specific cell types in animals, it is considerably more widespread in plants, such that polyploidy can be detected in the majority of plant tissues.

[0101] As used herein, the term “impaired chromosome segregation” relates to chromosome segregation failure which results in doubling in chromosome content, producing polyploid cells or doubled haploid progeny cell when combined with haploid induction.

[0102] As used herein, the term “nuclear fusion“ relates to the fusion of nuclear membranes of two progeny nuclei to form a single nucleus composed of mixing of chromosomes from the two nuclei. Nuclear fusion results in doubling in chromosome content, producing polyploid cells or doubled haploid progeny cell when combined with haploid induction.

[0103] According to one embodiment of such method according to the invention, the endogenous gene that is involved in endoreduplication is involved in endomitosis, endocycle and cytokinesis.

[0104] As used herein, the term “endomitosis” relates to a mitotic process in which chromosome condensation occurs in the absence of spindle and nuclear membrane breakdown. By doubling the chromatids without subsequent cell and nuclear division, polyploid cells are formed.

[0105] As used herein, the term “endocycle” also referred to as endocycling, endoreduplication, or endoreplication, relates to replication of the nuclear genome in the complete absence of mitosis,which leads to elevated nuclear gene content and polyploidy. Endocycle can be understood simply as a variant form of the mitotic cell cycle (G1-S-G2-M) in which mitosis is circumvented entirely, due to modulation of cyclin-dependent kinase (CDK) activity.

[0106] As used herein, the term “cytokinesis” relates to the physical process of cell division, which divides the cytoplasm of a parental cell into two daughter cells. Cytokinesis failure relates to a dysfunction of cytokinesis. Cytokinesis is Cytokinesis failure leads to two nuclei or multiple micronuclei carrying a doubled genomic content in total within a single cell.

[0107] According to embodiments of such method according to the invention, the endogenous gene that is modified belongs to at least one group selected from the group consisting of

[0108] • Cyclins

[0109] • Cyclins dependent kinases (CDK)

[0110] • CDK-like protein kinases

[0111] • mitotic arrest deficient protein (MAD)

[0112] • MAD-like

[0113] • Kinase

[0114] • Microtube-associated protein (MAP)

[0115] • structural maintenance of chromosomes (SMC)

[0116] • Kinesins, and / or

[0117] • Cohesion subunit genes

[0118] According to embodiments of such method according to the invention, the endogenous gene that is modified is at least one gene shown in Table 1.

[0119] According to embodiments of such method according to the invention, the endogenous gene that is modified is at least selected from the group consisting of ZmCyclin Bl-1 (SEQ ID NO: 8), ZmCyclin Bl -5 (SEQ ID NO: 15), ZmCyclin B2-3 (SEQ ID NO: 9), Zmkrpl (SEQ ID NO: 57), ZmCDKBl-1 (SEQ ID NO: 12), ZmSPC25 (SEQ ID NO: 35), ZmAurora 3 (SEQ ID NO: 115), ZmKinesinl (SEQ ID NO: 116), ZmKinesin2 (SEQ ID NO: 117), ZmMAP (SEQ ID NO: 118) or ZmMAP6 (SEQ ID NO: 119).

[0120] According to embodiments of such method according to the invention, the gene modification is introduced by means of at least one of• an SDN-1 based intervention

[0121] • an SDN-derived gene editing system

[0122] • RNA interference or RNA-mediated gene silencing

[0123] • application of a chemical mutagen, and / or

[0124] • application of radiation.

[0125] For a general overview of genome editing techniques in plants see Kawall (2019), the content of which is incorporated herein by reference for enablement purposes.

[0126] In SDN-1 based interventions, mutations consisting of changes in a few base pairs, short deletions or insertions (indels) are generated by means of a site directed nuclease (SDN) in a predefined region in the genome as a result of an error-prone gene repair mechanisms of the cell (NHEJ). The repair mechanism does not require exogeneous delivered DNA.

[0127] Application of a chemical mutagen oftentimes involves to use of a mutagen like e.g. ethyl methanesulfonate (EMS). Application of a radiation oftentimes involves the use UV radiation or X rays.

[0128] The latter is preferably used in the so-called TILLING method (Targeting Induced Local Lesions in Genomes), which allows directed identification of mutations in a specific gene. The method combines a standard and efficient technique of mutagenesis, such as radiation or chemical mutagenesis, with a sensitive DNA screening-technique that identifies single base mutations in a target gene. The TILLING method relies on the formation of DNA heteroduplexes that are formed when multiple alleles are amplified by PCR and are then heated and slowly cooled. A “bubble” forms at the mismatch of the two DNA strands, which is then cleaved by a single stranded nuclease. The products are then separated by size on several different platforms. Mismatches may be due to induced mutation, heterozygosity within an individual, or natural variation between individuals. Since the advent of NGS sequencing technologies, TILLING-by-sequencing has been developed based on sequencing of target genes amplified from multidimensionally pooled templates to identify possible singlenucleotide changes.

[0129] Also, there are several sources for single strand nucleases that can be used in TILLLING. The first widely used enzyme was mung bean nuclease, but this nuclease has been shown to havehigh non-specific activity, and only works at low pH, which can degrade PCR products and dye-labelled primers. The original source for single strand nuclease was from CEL1, or CJE (celery juice extract), but other products have entered the market including Frontier Genomics’ SNiPerase enzymes, which have been optimized for use on platforms that use labelled and unlabelled PCR products. Transgenomic isolated the single strand nuclease protein and sells it as a recombinant form. The advantage of the recombinant form is that unlike the enzyme mixtures, it does not contain non-specific nuclease activity, which can degrade the dyes on the PCR primers. Further enabling disclosure of the TILLING method can be found in McCallum et al (2000), the content of which is incorporated herein by reference for enablement purposes.

[0130] EcoTILLING (Nieto et al (2007), the content of which is incorporated herein by reference for enablement purposes) is a method that uses TILLING techniques to look for natural mutations in individuals, usually for population genetics analysis. DEcoTILLING (Garvin et al, 2007, the content of which is incorporated herein by reference for enablement purposes) is a modification of TILLING and EcoTILLING which uses an inexpensive method to identify fragments. Since the advent of NGS sequencing technologies, TILLING-by-sequencing (Tsai et al (2011), the content of which is incorporated herein by reference for enablement purposes) has been developed based on Illumina sequencing of target genes amplified from multidimensionally pooled templates to identify possible single-nucleotide changes.

[0131] RNA interference or RNA-mediated gene silencing techniques are well established in plants. Principles and techniques are for example disclosed in Hung and Slotkin (2021), the content of which is incorporated herein by reference for enablement purposes.

[0132] According to embodiments of such method according to the invention,

[0133] a. the SDN-1 based intervention employs at least one enzyme or system selected from the group consisting of

[0134] • Meganucleases,

[0135] • Zinc-Finger Nucleases,

[0136] • TALENs and / or

[0137] • CRISPR endonucleases,

[0138] and / or

[0139] b. the SDN-derived gene editing is at least one selected from the group consisting of• base editing,

[0140] • prime editing,

[0141] • CRISPR activation (CRISPRa), and / or

[0142] • CRISPR interference (CRISPRi).

[0143] According to embodiments of such method according to the invention, the CRISPR endonuclease is selected from the group consisting of Cas9 nuclease, Cfpl / Casl2a nuclease, MAD7 nuclease, dCas9-FokI, dCpfl-Fokl, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FENl-FokI, and Mega-TALs, a nickase Cas9 (nCas9), chimeric dCas9 non-Foklnuclease and dCpfl non-Fokl nuclease.

[0144] Prime editing is a genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired endonuclease (e.g., a CRISPR Cas9 endonuclease) fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. Technical details of prime editing are for example disclosed in Anzalone et al (2012) as well as in WO2021072328A1, the contents of which are incorporated herein by reference for enablement purposes.

[0145] Base editing is a genome editing method that directly generates precise point mutations in genomic DNA or in cellular RNA without directly generating DSBs, requiring a DNA donor template or relying on cellular HDR. Since base editors do not normally create DSBs, they minimize the formation of DSB-associated byproducts. Instead, DNA base editors (BEs) comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme that operates on single-stranded DNA (ssDNA) but not double-stranded DNA (dsDNA). Upon binding to its target locus in DNA, base pairing between the guide RNA and target DNA strand leads to displacement of a small segment of single-stranded DNA in an “R-loop”. DNA bases within this single-stranded DNA bubble are modified by the deaminase enzyme. To improve efficiency in eukaryotic cells, the catalytically disabled nuclease alsogenerates a nick in the non-edited DNA strand, inducing cells to repair the non-edited strand using the edited strand as a template.

[0146] Two classes of DNA base editor have been described: cytosine base editors (CBEs) convert a OG base pair into a T»A base pair, and adenine base editors (ABEs) convert an A»T base pair to a G»C base pair. Collectively, CBEs and ABEs can mediate all four possible transition mutations (C to T, Ato G, T to C, and Gto A). Technical details of base editing are for example disclosed in Rees and Liu (2018) as well as inW02020181195Al, the contents of which are incorporated herein by reference for enablement purposes.

[0147] CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). The CRISPR effector is guided to the target by a complementary guide RNA. However, CRISPR activation systems are fused to transcriptional activators to increase expression of genes of interest. Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest. The most commonly used effector is based on Cas9 but other effectors like Casl2a (Type V) have been used as well.

[0148] Typically, CRISPRa uses an enzymatically dysfunctional nuclease mutant, like e.g. Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9. Therein, endonuclease activity is removed through point mutations in its endonuclease domains. Cas9 has 2 endonuclease domains called the RuvC and HNH domains. The point mutations D10A and H840A change 2 important residues for endonuclease activity that ultimately result in its deactivation.

[0149] Although dCas9 lacks endonuclease activity, it is still capable of binding to its guide RNA and the DNA strand that is being targeted because such binding is managed by other domains. This alone is often enough to attenuate if not outright block transcription of the targeted gene if the gRNA positions dCas9 in a way that prevents transcriptional factors and RNA polymerase from accessing the DNA. However, this ability to bind DNA can also be exploited for activation since dCas9 has modifiable regions, typically the N and C terminus of the protein, that can be used to attach transcriptional activators. Technical details of CRISPRa are for example disclosed in Perez-Pinera et al. (2013) the content of which is incorporated herein by reference for enablement purposes.CRISPR interference (CRISPRi) can sterically repress transcription by blocking either transcriptional initiation or elongation. This is accomplished by designing sgRNA complementary to the promoter or the exonic sequences. The level of transcriptional repression with a target within the coding sequence is strand-specific. Depending on the nature of the CRISPR effector, either the template or non-template strand leads to stronger repression. For dCas9 (based on a Type-2 CRISPR system), repression is stronger when the guide RNA is complementary to the non-template strand. It has been suggested that this is due to the activity of helicase, which unwinds the RNA:DNA heteroduplex ahead of RNA pol II when the sgRNA is complementary to the template strand. Unlike transcription elongation block, silencing is independent of the targeted DNA strand when targeting the transcriptional start site. In prokaryotes, this steric inhibition can repress transcription of the target gene by almost 99.9%; in archaea, more than 90% repression was achieved; in human cells, up to 90% repression was observed.

[0150] Technical details of CRISPRi are for example disclosed in Qi et al (2013), the content of which is incorporated herein by reference for enablement purposes.

[0151] According to embodiments of such method according to the invention, the gene modification is a loss-of-function mutation in a gene, or in a regulatory element thereof.

[0152] According to embodiments of such method according to the invention, the gene modification is a gain-of-function mutation in a gene, or in a regulatory element thereof.

[0153] According to embodiments of such method according to the invention, the method comprises a step of identifying one or more gene modifications conferred to the haploid inducer plant.

[0154] According to embodiments of such method according to the invention, said identification comprises TILLING screening.

[0155] According to another aspect of the invention, a doubled haploid inducer (DHI) plant is provided, which plant has been produced according to the method of claim 1 or a doubled haploid plant (DHP), which plant has been produced according to the method as described above.According to another aspect of the invention, a doubled haploid inducer (DHI) plant is provided, which plant has one or more features that qualify it as a haploid inducer (HI) plant, which plant has furthermore a modification in at least an endogenous gene that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells,

[0156] With regard to the feature “endogenous gene that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells” the disclosure and examples set forth hereinabove shall apply mutatis mutandis, to avoid lengthy repetitions.

[0157] With regard to the feature “one or more features that qualify it as a haploid inducer (HI) plant”, the disclosure set forth hereinabove describing by which gene mutations HI plants can be characterized set forth hereinabove, and examples for such HI plants set forth hereinabove, shall apply mutatis mutandis, to avoid lengthy repetitions.

[0158] According to another aspect of the invention, a method for producing doubled haploid plants (DHP) is provided, which method comprises the use of a doubled haploid inducer (DHI) plant according to the above description, and a second plant, wherein the second plant is a source germplasm.

[0159] According to one embodiment, such method comprises at least one feature selected from the group consisting of

[0160] a) providing such doubled haploid inducer (DHI) line

[0161] b) providing a second plant, wherein the second plant is a source germplasm

[0162] c) crossing the doubled haploid inducer line plant and the second plant

[0163] d) obtaining progeny of said crossing

[0164] g) testing said progeny for haploidy or doubled haploidy; and

[0165] e) selecting at least one doubled haploid progeny; and

[0166] f) establishing at least one doubled haploid line by self-pollination

[0167] In this context, it needs to be stated that the “crossing”, as used herein, does not necessarily imply “sexual crossing”, i.e., a process in which the whole genomes of the two parental plants are crossed and genetical information of the two parental plants is recombined in the offspring.In fact, when "crossing" a source germplasm with a DHI, the genomes are not crossed, and the genome of the source germplasm and the genome of the source germplasm remains unchanged. This is reason why we use the DHI - it is not meant to be a trait supplier, but as a kind of catalyst to "help" the source germplasm to clone itself). As such, the “crossing”, as used herein, does not qualify as an essentially biological process.

[0168] According to one embodiment, the method further comprises testing a progeny of that method for haploidy or doubled haploidy is carried out using a selection marker.

[0169] Such selection marker can e.g. be taken from the list comprising

[0170] • blue color marker from Rl-nj (Navajo) gene expression in seeds,

[0171] • red color from PH gene expression in roots,

[0172] • purple color from Bl & PH gene expressions in stem and sheaths, and / or

[0173] • oil content from the expressions of multiple genes of led, DGAT1-2, 0BAP1, WRI1 in seeds.

[0174] These markers are well described in the respective literature, as for example in Trentin et al (2020), the content of which is incorporated herein by reference for enablement purposes.

[0175] According to one embodiment of such method according to the invention, the selection of at least one doubled haploid progeny is carried out using cell flow cytometry and marker analysis

[0176] According to embodiments of such method according to the invention, the second plant is a pollen donor or pollen recipient.

[0177] According to one embodiment of such method according to the invention, the second plant is of the same species as the first plant.

[0178] According to one embodiment of such method according to the invention, the second plant is of a different species than the first plant.

[0179] According to embodiments of such method according to the invention, the first plant and / or the second plant is a monocot or a dicot, optionally selected from the group consisting of maize,wheat, rice, rye, barley, oats, triticale, sorghum, pearl millet, teosinte, sugar cane, onion, garlic, sugar beets, sunflower, oil see rape, tomato, potato, soybean, pea, pepper, and / or melon.

[0180] According to another aspect of the invention, a doubled haploid plant produced with the method according to the above description is provided.

[0181] EXAMPLES

[0182] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration(s) and description(s) are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0183] Example 1. Generating target mutation via transient genome editing (GE) SDN-1 in maize A188

[0184] Maize immature inflorescence preparation

[0185] The immature inflorescences from maize Al 88 at the developmental stages of late V6 to late V7 are used. It most likely takes 25-32 days (most likely 28 days) to reach these stages after seed planting. The developmental stages of immature inflorescences are determined using a Zeiss stereo microscope. The immature inflorescence is manually isolated under aseptic conditions.

[0186] Genome editing SDN-1 via transient transformation of particle bombardment

[0187] The fresh isolated immature inflorescences were placed onto an osmatic medium plate (e.g N6 0SM medium) for 4 hours. Particle bombardment is conducted using a Bio-Rad PDS-1000 / He particle gun. The bombardment conditions were: 28-30 mm / Hg vacuum, 650-1,550 psi helium pressure. Per bombardment, 100-1000 ng each of CRISPR constructs (Cas nuclease,guide RNA expression cassettes), and at least one regeneration booster construct (e.g., RBP2) are co-coated onto 10-300 pg of 0.4 or 0.6 pm gold particles using calcium-spermidine method. Per sample plate 3-4 bombardments are performed. The bombarded immature inflorescences were kept on the osmotic medium plate for another 16-20 hours after the bombardment. The fluorescence report gene, tDTomato was used for monitoring biolistic transformation and the gene expression.

[0188] Plant regeneration

[0189] 16-20 hours after the bombardment the Al 88 immature inflorescence was subject to regeneration, comprising the steps of:

[0190] a) Embryogenic callus induction: cut the bombarded immature inflorescences into a segment of 2-5 mm in length, with a sharp blade, and place onto a callus induction medium (e.g., N6_5Ag) in petri dish plate (25 x 100 mm) with the bombarded surface up. Seal the plate with surgical tape and culture at 27°C, dark, for about 1 week.

[0191] b) Embryogenic callus development: transfer the segments from callus induction onto a fresh MRM3 medium, and incubate the plate at 27 °C, dark for another 2 weeks for embryogenic callus development.

[0192] c) Shoot development: separate embryogenic calluses into small pieces of 2-5 mm in diameter and transfer the calluses onto a Shooting medium plate (25x 100mm). Seal the plate with surgical tape and culture at 25°C, weak light, for ~3-5 days (20-100 pmol m-2 s— 1) and then full light (>100 pmol m-2 s— 1) for another 7 ~ 14 days.

[0193] d) Root and plantlet development: transfer the developing shoots onto a rooting medium in a phytotray and culture the shoots in the full light chamber at 25 °C. The regenerated TO plants are ready for sampling and moving to soil in 5-7 days.

[0194] Molecular screening for SDN-1 events in the regenerated To plants

[0195] A 5-10 mm leaf tip from each of the leaves of a To plant are collected for DNA extraction. Genome editing SDN-1 in the regenerated To plants are screened by TaqMan real-time PCR (qPCR), marker capillary electrophoresis analysis, and TaqMan Digital Droplet PCR. Site-specific modification is further conformed by next generation sequencing (NGS) or Sanger sequencing.

[0196] Grow the edited To plants and Ti seed production

[0197] After the molecular screening and confirmation, the selected To edits are transferred to soil, and grown in a growth chamber or greenhouse under the suitable growth conditions. To plants are phenotypically analyzed and grown for Ti seed production by self-cross or back-crossing to maize WT Al 88.

[0198] Media

[0199] 5Ag medium: N6, 1.0 mg / L of 2, 4-D, 100 mg / L of Caseine, 2.9 g / L of L-proline, 20 g / L sucrose, 5g / L of glucose, 5 mg / L of AgNCL, 3 g / L of Gelzn, pH 5.8

[0200] N6OSM medium: N6, 100 mg / L of Caseine, 0.7 g / L of L-proline, 0.2 M Mannitol (36.4 g / L), 0.2 M sorbitol (36.4 g / L), 20 g / L sucrose, 15 g / L of Bacto-agar, pH 5.8

[0201] MRM3: LS, 0.7g / L of Proline, 0.5 mg / L of dicamba, 5% PEG 4000, 3% of sucrose, 4.0 g / L of Gelzan, pH 5.8

[0202] Shooting medium: MS, 100 mg / L of myoinositol, 3% sucrose, 5mg / L Zeatin, 3 g / L of Gelzan, pH 5.8

[0203] Rooting medium: MS, 100 mg / L of myoinositol, 2% sucrose, 0. 5 mg / L of IBA, 3g / L of Gelzan, pH 5.8

[0204] Example 2: Genome doubling via endoreduplication induction or chromosome segregation inhibition

[0205] This experiment identified genes involved in one or more pathways related to a process which results in endopolyploidy or a process resulting in polyploidization of cells (e.g. gametes and zygote cells) and result in doubling of haploid genomes when mutated in a haploid inducer line and crossed with a source germplasm.

[0206] Endoreduplication is a special cell cycle, where chromosomes are replicated in S phase, but mitosis is entirely or partially skipped (endoreduplication, endoreplication, endocycle, orendocycling), or cytokinesis is avoided (cytokinesis failure). The induction of endoreduplication thus leads to genome doubling. Cyclin B (CYCB) is a mitotic cyclin, called Mitosis Promoting Factor (MPF). CYCB and CYCB dependent kinase (CDKB) activities are required for the G2 / M transition, and they also inhibit G1 entry. Lack of CYCB and CDKB / CYCB activities may result in endoreduplication due to cell cycle transition directly from G2 to Gl. Anaphase-promoting complex / cyclosome (APC / C) is an E3 ubiquitin ligase, a key player in cell cycle regulation, and involved in the degradation of CYCB and other important proteins (e.g. DNA replication inhibitors and anaphase inhibitors, etc.). APC / C is required for CYCB degradation. For example, Arabidopsis GIGAS CELLI (GIG 1 1 OMISSION OF SECOND DIVISION 1 (0SD1) gene encodes a plant-specific inhibitor of APC / C. The loss-of-function mutation of this gene triggers ectopic endoreduplication and caused gigas cells with doubled chromosomes (4n) in cotyledon (Iwata, et. al., 2011).

[0207] If any errors occur during mitotic chromosome segregation, the replicated sister chromatids may not separate, and the chromosomes may either be doubled or eliminated (haploid induction) in the daughter cells. Therefore, modifications in the genes involved in chromosome segregation may trigger chromosome doubling and / or haploid induction. Thousands of genes involve in chromosome segregation at many steps, e.g. chromatin condensation, kinetochore and spindle assemble, kinetochore-spindle attachment, and chromosome migration, etc. A maize mutant elongate 1 (the causing gene has not been identified yet) has elongated chromosomes (defect in chromatin condensation) in meiosis and leads to gametes with doubled chromosomes.

[0208] The candidate genes with expression in reproductive cells, e.g. gametes, zygote and following cells, were identified. Examples of genes involved in one or more pathways related to a process which results in chromosome doubling of haploid cells, and that were modified by means of SDN-1 based intervention, are listed in Table 1. The function domains / motifs of each candidate were analyzed in silico or manually. The desirable modifications were introduced via SDN-1 mutagenesis in haploid inducer lines. A rapid and transgene-free genome editing regeneration system with immature inflorescence (disclosed in WO2021170785, the content of which is incorporated herein by reference for enablement purposes) was used to generate desirable modifications directly in a haploid inducer, e.g. a maternal haploid inducer line (e.g. AX6008, RWPc, EMPc or 5F279 HIR1-HIR8) or paternal haploid inducer line (e.g. Ig- Alvey), based on their expression profiles. The modifications include:1. Dominant negative modifications of a negative regulator via genome editing SDN-1 in the preferred function domain.

[0209] 2. Gain-of-function modifications of a positive regulators via genome editing SDN-1 in its promoter, 5'UTR, or upstream open reading frames (uORF) within the 5’ untranslated region (5’UTR) (uORF typically inhibits downstream expression of the primary ORFs).

[0210] In this process, a haploid inducer was reformed into a doubled haploid inducer (DHI) if the modification(s) could trigger doubling of haploid genome. The dominant modifications in a DHI were expressed in and / or delivered to the zygote cell after in vivo crossing, where trigger chromosome doubling, and were eliminated in the zygote and following cells during haploid induction.

[0211] The edited haploid inducer plants were evaluated for DH induction capability via crossing with a source germplasm as pollen donors(matemal) or recipients (paternal). The resulting haploid or doubled haploid kernels were selected based on the haploid selection markers and sown directly into soil in green houses, growth chambers, or in the nursery field. Haploid progeny seedlings, which are short and small with narrow upward leaves, were discarded. The doubled haploid plants were identified by visual phenotypes (e.g. normal stature, size, fertile) and confirmed by cell flow cytometry and marker analysis. The DH plants could be pre-selected for the desired trait(s) and multiplied by selfing.

[0212] The resulting DH inducers and DH lines can be directly used as breeding materials in genome editing friendly regions, while the complete non-GM doubling trait can be developed by TILLING.

[0213] Maize immature inflorescence preparation

[0214] The immature inflorescences from maize haploid inducer plants (e.g. AX6008, RWPc, EMPc) at late V6 to early V7 stages are used. It most likely takes 22-32 days to reach these stages after seed planting. Immature inflorescence from maize plants at the suitable development stage is harvested by retrieving the stem segment between the base (near to soil) and the youngest leaf collar. The leaf sheathes wrapping the stem segment are manually removed one by one and sterilized by surface spray with 70% ethanol in a laminar hood. Finally, the immatureinflorescences are isolated from the stem segment under aseptic conditions. The developmental stages of immature inflorescences are determined using a Zeiss stereo microscope.

[0215] Multiplex genome editing SDN-1 via transient co-bombardment

[0216] The fresh isolated immature inflorescences are placed onto an osmatic medium plate (e.g. N6 0SM medium) for 4 hours. Particle bombardment is conducted using a Bio-Rad PDS-1000 / He particle gun. The bombardment conditions are: 28-30 mm / Hg vacuum, 1,100 psi helium pressure. Per bombardment, 100-1000 ng each of CRISPR constructs (Cas nuclease, multiplex crRNA guide RNA constructs), and at least one regeneration booster construct (e.g. RBP2, see e.g. WO2019238909A1) are co-coated onto 100 pg of 0.6 pm gold particles using calcium-spermidine method. Four bombardments per sample plate are performed. The bombarded immature inflorescences are kept on the osmotic medium plate for another 16-20 hours after the bombardment. The fluorescence report gene, tDTomato is used for monitoring biolistic transformation and the gene expression.

[0217] Plant regeneration

[0218] 16-20 hours after the bombardment the immature inflorescence is subjected to regeneration, which comprises the steps of:

[0219] a) Embry ogenic callus induction: bombarded immature inflorescence is cut into a segment of 1-3 mm in length, with a sharp blade, and place onto a callus induction medium (e.g., N6_5Ag) in petri dish plate (25 x 100 mm) with the bombarded surface up. Plate is sealed with surgical tape and. Segment is cultured at 27°C, dark, for about 1 week.

[0220] b) Embryogenic callus development: Segments from callus induction are transferred onto a fresh MRM3 medium and incubated at 27 °C. Segments are kept in the dark for another 2 weeks for embryogenic callus development.

[0221] c) Shoot development: Embryogenic calluses are separated into small pieces of 1 - 3 mm in diameter and transferred onto a shooting medium plate (25x 100mm). Plate is sealed with surgical tape and cultured at 25°C, weak light, for ~3-5 days (20-50 pmol m2s ') and then full light (>50 pmol m2s ') for another 7 ~ 14 days.d) Root and plantlet development: Developing shoots are transferred onto a rooting medium in a phytotray and cultured in the full light chamber at 25 °C. The regenerated TO plants are ready for sampling and moving to soil in 5-7 days.

[0222] Screen for genome editing events in the regenerated To plants

[0223] After one week in a rooting medium, the regenerated plantlets are ready for leaf sampling for molecular analysis. A 5-10 mm leaf tip from each of the leaves of a To event are collected for DNA extraction. The edited genomes in the regenerated To plants derived from the bombarded inflorescence cells are screened by TaqMan real-time PCR (qPCR), marker capillary electrophoresis analysis, or TaqMan Digital Droplet PCR. Site-specific modification is further conformed by next generation sequencing (NGS) or Sanger sequencing.

[0224] Growing and phenotyping the edited To haploid inducer plants

[0225] The edited To haploid inducer plants are transferred to soil and grown in a growth chamber or greenhouse under the suitable growth conditions. To plants are phenotypically analyzed via visual observation along the plant growth. The size of stomata guard cells in abaxial leaf surface is examined under a microscope. The Gametophyte development is monitored from VT stage (lowest branch of the inflorescence is visible). Pollen amount and size are observed with a dissection microscope, while pollen viability is evaluated by pollen germination in a pollen germination medium. Kernel abortion is examined 10-14 days after pollination. Kernel count, embryo abortion, and seed weight are measured when seeds are fully mature.

[0226] DH induction via in vivo crossing

[0227] The edited To haploid inducer plants are used as the pollen donors (maternal) or recipients to cross with a source germplasm. The resulting ears are matured in greenhouse and seeds are harvested.

[0228] Table 1 shows genes involved in one or more pathways related to a process which results in chromosome doubling of haploid cells, and that were modified by means of SDN-1 based intervention and their cDNA / CDS sequences as well as their amino acid sequences (AA). Note that the cDNA / CDS sequences do not necessarily start with an ATG start codon, but may comprise untranslated 5’ and 3’ sequencesTable 1: selected genes that are involved in one or more pathways related to a process which results in chromosome doubling of haploid cells. The Gene IDs have been taken from the database iMaizeGDB https: / / www.maizegdb.org / )

[0229]

[0230]

[0231] Example 3: Developing maternal doubled haploid (DH) inducer via genome editing mutagenesis on maize maternal haploid inducer EMPC

[0232] The cDNA and protein sequences for the haploid doubling gene are provided in Table 2. Figures 5 and 6 present two CRISPR Cas nuclease expression constructs. Guide crRNA sequences targeting the specified gene are detailed in Table 3, while the multiplex crRNA constructs are illustrated in Figures 7 to 18. Additionally, two regeneration boosters (shown in Figures 19 to 29) were co-delivered with the genome editing components into EMPC immature tassels.

[0233] For details on target mutagenesis by transient genome editing (GE) SDN-1 in EMPC — including bombardment, regeneration, and molecular screening — refer to Example 1, Example 2, and Figures 21-24. Editing sequence results are in Figures 25-32. Edited TO plants were grown for T1 seed production in a greenhouse. The DH induction and identification workflow appears in Figure 33: edited T1 plants served as pollen donors crossed with glossy Fl plants(Figure 34). Haploid and doubled haploid selection was based on Rl-nj expression and glossy phenotyping, with further confirmation by ploidy and marker chip analyses. Table 2 summarizes DH induction data, while table 3 shows the resulting Doubled Haploid Induction Rate (DHIR)

[0234] Table 2: An SDN-1 mutation in the target gene induces haploid genome doubling

[0235]

[0236] Table 3: Doubled Haploid Induction Rate (DHIR) reached over 2% via maternal DH induction.

[0237]

[0238]

[0239] Table 4: List of multiplex guide RNA sequences for a specific target gene

[0240]

[0241] References

[0242] Comai, L.; Young, K.; Till, B. J.; Reynolds, S. H.; Greene, E. A.; Codomo, C. A.; Enns, L. C.; Johnson, J. E.; Burtner, C.; Odden, A. R.; Henikoff, S. (2004). "Efficient discovery of DNA polymorphisms in natural populations by Ecotilling". The Plant Journal. 37 (5): 778-786.

[0243] Cyprys, Philipp & Lindemeier, Maria & Sprunck, Stefanie. (2019). Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins. Nature Plants. 5. 253.

[0244] 10.1038 / s41477-019-0382-3.

[0245] Garvin, M. R.; Gharrett, A. J. (2007). "DEco-TILLING: An inexpensive method for single nucleotide polymorphism discovery that reduces ascertainment bias". Molecular Ecology Notes. 7 (5): 735-746.

[0246] Coe EH (1959) A line of maize with high haploid frequency. Am Nat 93:381-382.Takahashi, T., Mori, T., Ueda, K., Yamada, L., Nagahara, S., Higashiyama, T., Sawada, H., and Igawa, T. (2018). The male gamete membrane protein DMP9 / DAU2 is required for double fertilization in flowering plants. Development 145 (23).

[0247] Nieto, C.; Piron, F.; Dalmais, M.; Marco, C. F.; Moriones, E.; Gomez-Guillamon, M. L.; Truniger, V.; Gomez, P.; Garcia-Mas, J.; Aranda, M. A.; Bendahmane, A. (2007). "EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility". BMC Plant Biology. 7: 34.

[0248] Helen Tsai, Tyson Howell, Rebecca Nitcher, Victor Missirian, Brian Watson, Kathie J. Ngo, Meric Lieberman, Joseph Fass, Cristobal Uauy, Robert K. Tran, Asif Ali Khan, Vladimir Filkov, Thomas H. Tai, Jorge Dubcovsky, Luca Comai, Discovery of Rare Mutations in Populations: TILLING by Sequencing, Plant Physiology, Volume 156, Issue 3, July 2011, Pages 1257-1268.

[0249] Kalinowska K, Chamas S, Unkel K, Demidov D, Lermontova I, Dresselhaus T, Kumlehn J, Dunemann F, Houben A. State-of-the-art and novel developments of in vivo haploid technologies. Theor Appl Genet. 2019 Mar;132(3):593-605.

[0250] Kelliher, T.; Starr, D.; Richbourg, L.; Chintamanani, S.; Delzer, B.; Nuccio, M.L.; Green, J.; Chen, Z.;McCuiston, J.; Wang, W.; et al. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 2017, 542, 105-109.

[0251] Liu, C.; Li, X.; Meng, D.; Zhong, Y.; Chen, C.; Dong, X.; Xu, X.; Chen, B.; Li, W.; Li, L.; et al. A 4-bp Insertion at ZmPLAl Encoding a Putative Phospholipase A Generates Haploid Induction in Maize. Mol. Plant 2017, 10, 520-522.

[0252] Gilles, L.M.; Khaled, A.; Laaire, J.; Chaignon, S.; Gendrot, G.; Laplaige, J.; Berges, H.; Beydon, G.; Bayle, V.; Barret, P.; et al. Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize. EMBO J. 2017, 36, 707-717.

[0253] Evans, M.M.S. The indeterminate gametophytel Gene of Maize Encodes a LOB Domain Protein Required for Embryo Sac and Leaf Development. Plant Cell Online 2007, 19, 46-62.Kindiger, B.; Hamann, S. Generation of Haploids in Maize: A Modification of the Indeterminate Gametophyte (ig) System. Crop Sci. 1993, 33, 342-344.

[0254] Wang N, Gent JI, Dawe RK. Haploid induction by a maize cenh3 null mutant. Sci Adv. 2021 Jan 20;7(4): eabe2299.

[0255] Ravi, M.; Chan, S.W.L. Haploid plants produced by centromere-mediated genome elimination. Nature 2010, 464, 615-618.

[0256] Zhong, Y.; Liu, C.; Qi, X.; Jiao, Y.; Wang, D.; Wang, Y.; Liu, Z.; Chen, C.; Chen, B.; Tian, X.; et al. Mutation of ZmDMP enhances haploid induction in maize. Nat. Plants 2019, 5, 575-580.

[0257] Prigge V, Xu X, Li L, Babu R, Chen S, Atlin GN, Melchinger AE. New insights into the genetics of in vivo induction of maternal haploids, the backbone of doubled haploid technology in maize. Genetics. 2012 Feb;190(2):781-93.

[0258] Uliana Trentin H, Frei UK, Lubberstedt T. Breeding Maize Maternal Haploid Inducers. Plants (Basel). 2020 May 12;9(5):614.

[0259] Chaikam, V., Molenaar, W., Melchinger, A.E. et al. Doubled haploid technology for line development in maize: technical advances and prospects. Theor Appl Genet 132, 3227-3243 (2019). https: / / doi.org / 10.1007 / s00122-019-03433-x S.: 23.

[0260] Kawall K. New Possibilities on the Horizon: Genome Editing Makes the Whole Genome Accessible for Changes. Front Plant Sci. 2019 Apr 24; 10:525. doi: 10.3389 / fpls.2019.00525. PMID: 31068963; PMCID: PMC6491833.

[0261] McCallum, CM; Comai, L; Greene, EA; Henikoff, S (Apr 2000). "Targeted screening for induced mutations". Nat Biotechnol. 18 (4): 455-7. doi:10.1038 / 74542. PMID 10748531. S2CID 12063367. S.: 26.

[0262] Yu-Hung Hung, R Keith Slotkin, The initiation of RNA interference (RNAi) in plants, Current Opinion in Plant Biology, Volume 61, 2021, 102014, ISSN 1369-5266 S.: 26.Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R. (21 October 2019). "Search-and-replace genome editing without double-strand breaks or donor DNA". Nature. 576 (7785): 149-157.

[0263] Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-1. Erratum in: Nat Rev Genet. 2018 Oct 19;: PMID: 30323312; PMCID: PMC6535181. S.: 27.

[0264] Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, et al. (October 2013). "RNA-guided gene activation by CRISPR-Cas9-based transcription factors". Nature Methods. 10 (10): 973-6. doi:10.1038 / nmeth.2600. PMC 3911785. PMID 23892895. S.: 28.

[0265] Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (February 2013). "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression". Cell. 152 (5): 1173-1183. doi: 10.1016 / j cell.2013.02.022. PMC 3664290. PMID 23452860 S.: 29.

[0266] Rustgi S, Naveed S, Windham J, Zhang H, Demirer GS. Plant biomacromolecule delivery methods in the 21st century. Front Genome Ed. 2022 Oct 14;4: 1011934. doi: 10.3389 / fgeed.2022.1011934. PMID: 36311974; PMCID: PMC9614364. S.: 31.

[0267] SEQUENCES

[0268] The sequences disclosed in the enclosed WIPO ST26 compatible electronic sequence listing provided with this application form part of the disclosure of the present application.

[0269] In some cases, signal peptides may be encompassed in the reproduced sequences. In such case the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php7SignalP

Claims

What is claimed is:

1. A method for generating a doubled haploid inducer plant (DHI), the method comprising a) providing a first plant which is a haploid inducer (HI) plant, wherein the haploid inducer plant is a maternal haploid inducer or paternal haploid inducer;b) modifying at least an endogenous gene in the haploid inducer plant that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells,c) thereby obtaining a doubled haploid inducer (DHI) plant.

2. The method according to claim 1, which further comprisesd) providing a second plant, wherein the second plant is, or comprises, a source germplasm e) crossing the doubled haploid inducer plant and the second plant;f) obtaining progeny of said crossingg) testing, in said progeny, whether it comprises a doubled haploid genome derived from the source germplasm, therebyh) obtaining at least a doubled haploid plant (DHP).

3. The method according to any one of the aforementioned claims, wherein the haploid inducer plant is characterized by at least one mutation in a gene or quantitative trait locus selected from the group consisting of• MATRILINEAL (MTL) / ZEA MAYS PHOSPHOLIPASE Al (ZmPLAl) / NOT LIKE DAD (NLD) (maternal haploid inducer)• Indeterminate gametophyte (igl) gene (paternal haploid inducer)• CENH3 (CENTROMERIC HIST0NE3) (maternal and paternal haploid inducer), and / or• ZmDMP (DUF679 Domain Membrane Protein)• ZmigN (patermal haploid inducer)or orthologues thereof.

384. The method according to any one the aforementioned claims, wherein the haploid inducer plant is a maize plant derived from the list consisting of Stock6, CAUB, CAU5, CAU079, CAUHOI, KMS, ZMS, RWS, RWP, RWK (RWK76), UH400, UH402, UH600, UH601, MHI, M741B, M741C, M741F, M741H, M741I, M741J, PHI- 1, PHI-2, PHI-3, PHI-4, PK6, TAILs, CIM2GTAILs, LI-ESALQ, FIGH1 and derivatives thereof or hybrids of these haploid inducers (e.g., RWSxRWK76).

5. The method according to any one the aforementioned claims, wherein the haploid inducer plant is a maize plant derived from the igl mutants, igl-O.

6. The method according to any one of the aforementioned claims, wherein the endogenous gene that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells is a gene involved in at least one process which results in endopolyploidy, endoreduplication, polyploidization, and / or impaired chromosome segregation of cells.

7. The method according to claim 6, wherein the endogenous gene that is involved in endoreduplication is involved in endomitosis, endocycle and cytokinesis.

8. The method according to any one of the aforementioned claims, wherein the endogenous gene that is modified belongs to at least one group selected from the group consisting of • Cyclins• Cyclins dependent kinases (CDK)• CDK-like protein kinases• mitotic arrest deficient protein (MAD)• MAD-like• Kinase• Microtube-associated protein (MAP)• structural maintenance of chromosomes (SMC)• Kinesins, and / or• Cohesion subunit genes.

9. The method according to any one of the aforementioned claims, wherein the endogenous gene that is modified is at least selected from the group consisting of39ZmCyclin Bl-1 (SEQ ID NO: 8), ZmCyclin Bl -5 (SEQ ID NO: 15), ZmCyclin B2-3 (SEQ ID NO: 9), Zmkrpl (SEQ ID NO: 57), ZmCDKBl-1 (SEQ ID NO: 12), ZmSPC25 (SEQ ID NO: 35), ZmAurora 3 (SEQ ID NO: 115), ZmKinesinl (SEQ ID NO: 116), ZmKinesin2 (SEQ ID NO: 117), ZmMAP (SEQ ID NO: 118) or ZmMAP6 (SEQ ID NO: 119).

10. The method according to any one of the aforementioned claims, wherein the endogenous gene that is modified is at least one gene shown in Table 1.

11. The method according to any one of the aforementioned claims, wherein the gene modification is introduced by means of at least one of• an SDN-1 based intervention• an SDN-derived gene editing system• RNA interference or RNA-mediated gene silencing• application of a chemical mutagen, and / or• application of radiation.

12. The method according to any one of claims 1 - 11, wherein the gene modification is a loss- of-function mutation in a gene, or in a regulatory element thereof.

13. The method according to any one of claims 1 - 11 wherein the gene modification is a gain- of-function mutation in a gene, or in a regulatory element thereof.

14. The method according to claim 13, which further comprises a step of identifying one or more gene modifications conferred to the haploid inducer plant, optionally wherein said identification comprises TILLING screening.

15. A doubled haploid inducer (DHI) plant, which plant has been produced according to the method of claim 1 or a doubled haploid plant (DHP), which plant has been produced according to the method of claim 2.

16. A doubled haploid inducer (DHI) plant, which plant has one or more features that qualify it as a haploid inducer (HI) plant,40which plant has furthermore a modification in at least an endogenous gene that is involved in one or more pathways related to a process which results in chromosome doubling of haploid cells.

17. A method for producing doubled haploid plants (DHP), which method comprises the use of a doubled haploid inducer (DHI) plant according to any one of claims 15 and / or 16, and a second plant, wherein the second plant is a source germplasm.

18. The method according to claim 17, which method further comprises testing a progeny of that method for haploidy or doubled haploidy is carried out using a selection marker.

19. The method according to claim 18, wherein the selection of at least one doubled haploid progeny is carried out using cell flow cytometry and marker analysis.

20. The method according to any one of the aforementioned, claims wherein the first plant and / or the second plant is a monocot or a dicot, optionally selected from the group consisting of maize, wheat, rice, rye, barley, oats, triticale, sorghum, pearl millet, teosinte, sugar cane, onion, garlic, sugar beets, sunflower, oil see rape, tomato, potato, soybean, pea, pepper, and / or melon.

21. A doubled haploid plant produced with the method according to any one of claims 17 -20.

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