WO2018015956A1

WO2018015956A1 - Compositions and methods for generating a haploid of a target plant

Info

Publication number: WO2018015956A1
Application number: PCT/IL2017/050816
Authority: WO
Inventors: Kevin A. COOK; Yaniv LERENTHAL; Lilah Rothem
Original assignee: Kaiima Bio Agritech Ltd.
Priority date: 2016-07-21
Filing date: 2017-07-19
Publication date: 2018-01-25

Abstract

A haploid inducer plant line genetically modified with a nucleic acid molecule encoding a DNA editing agent is provided. Also provided is a method of genetically modifying a haploid inducer, the method comprising genetically modifying the haploid inducer plant with a nucleic acid molecule encoding a DNA editing agent, thereby genetically modifying the haploid inducer. Also provided are methods of using such haploid plants in breeding.

Description

COMPOSITIONS AND METHODS FOR GENERATING A HAPLOID OF A

TARGET PLANT

FIELD AND BACKGROUND OF THE INVENTION

Genome editing tools are becoming widely available to make various types of nucleotide sequence or other modifications to specific targeted sites within genomes. This work is typically done in transformation systems (callus, immature embryos, protoplasts etc.) from which plants containing targeted genetic modifications can be regenerated. The transformation process and timeline is rate limiting for broad application in breeding programs. Transformation systems utilize specific germplasm (e.g., in maize) selected for compatibility with and efficiency in the transformation system. These germplasms are usually not elite, i.e. the finished transformation product will not be directly utilized in a commercial pipeline because the transformation germplasm is not competitive in that pipeline. Seed companies often desire to broadly employ valuable modification(s) in their germplasm pipelines. Tools that create novel genomic variation utilizing genome editing tools also require a method for rapid implementation across multiple genetic backgrounds to be impactful. Producing a gene edited site requires the genome editing tools to be expressed for only a short time to induce the genomic event. Once the genome is edited, the tool (e.g.,- CRISPR, TALEN, T-GEE, meganuclease, zinc finger nuclease, etc.) is no longer needed, it must be removed from the plant (through breeding methods or other) in order to develop a product that can be assessed for commercialization by regulatory agencies and to prevent additional genomic events from occurring (off targets).

Introduction of the trait into a breeding pipeline is dependent upon the creation of a single plant carrying the new DNA sequence(s) followed by seed increase and backcrossing to commercially relevant inbred lines (or other pipeline germplasms). The backcrossing process produces new inbred lines equivalent to the starting commercially relevant inbreds, but containing the newly added or modified gene(s) or other DNA sequence(s). This process is particularly common in species that are difficult to transform efficiently, such as maize, and in germplasms of other species which is difficult to transform efficiently. The line/variety containing modified sequences could also be directly utilized as a parent in breeding populations targeting the development of novel new inbred lines or varieties through concurrent selection for the target DNA sequence(s) and desired genetic, performance and phenotypic characteristics in the progeny. This application, too, is limited by transformation germplasm which is noncompetitive in the target market of the crop. Either way, the process is slow and costly. SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present disclosure there is provided a haploid inducer plant line genetically modified with a nucleic acid molecule encoding a DNA editing agent. According to some embodiments of the disclosure, the nucleic acid molecule encoding the DNA editing agent is integrated in the chromosomal genome of the inducer plant. According to some embodiments of the disclosure, the DNA editing agent is expressed in the inducer plant in an inducible manner or developmentally regulated manner. According to some embodiments of the disclosure, the inducer plant is an inducer line. According to certain embodiments of the disclosure, the DNA editing agent is expressed in the target plant in an inducible or developmentally regulated manner.

According to an aspect of some embodiments of the present disclosure there is provided a method of genetically modifying a haploid inducer, the method comprising genetically modifying the haploid inducer plant with a nucleic acid molecule encoding a DNA editing agent, thereby genetically modifying the haploid inducer. According to some embodiments of the disclosure, the method further comprises recovering the haploid inducer plant containing the nucleic acid molecule encoding the DNA editing agent.

According to an aspect of some embodiments of the present disclosure there is provided a method of generating a haploid of a target plant, the method comprising crossing a haploid inducer plant genetically modified with a nucleic acid molecule encoding a DNA editing agent with a target plant of interest, thereby generating a haploid plant. According to some embodiments of the disclosure, the methods can further comprise recovering a haploid progeny of the target plant following the crossing. According to some embodiments of the disclosure, the methods can further comprise selecting for the haploid plant following the crossing of the inducer plant comprising the nucleic acid molecule encoding the DNA editing agent with the target plant of interest. According to some embodiments of the disclosure, the haploid plant is a haploid plant having a DNA editing event in its genome. According to some embodiments of the disclosure, the methods can further comprise selecting for the haploid plant having a DNA editing event in its genome following the crossing of the inducer plant comprising the nucleic acid molecule encoding the DNA editing agent with the target plant of interest. According to some embodiments of the disclosure, the selection can comprise a selection for a biochemical, phenotypic, or genomic sequence modification that results from the gene editing event. According to some embodiments of the disclosure, the methods can further comprise genomically multiplying chromosomes of the selected haploid plant having a DNA editing event by treatment with a chromosome doubling agent and recovering a double haploid or polyhaploid target plant following the treating that has the gene editing event. According to some embodiments of the disclosure, the methods can further comprise selfing or crossing the double haploid or polyhaploid target plant that has the DNA editing event. According to some embodiments of the disclosure, the genetically modified haploid inducer is used as a pollen donor in the cross to provide a maternal haploid. According to some embodiments of the disclosure, the genetically modified haploid inducer is used as a pollen recipient in the cross to provide a paternal haploid.

According to an aspect of some embodiments of the present disclosure there is provided a method of genomically multiplying chromosomes of a target plant having a genetic modification (e.g. , gene editing event) of interest, the method comprising treating the haploid target plant generated according to any of the aforementioned or other methods described herein to a chromosome doubling agent, thereby generating a double haploid or polyhaploid target plant having the genetic modification (e.g., gene editing event) of interest. According to some embodiments of the disclosure, the method further comprises recovering the double haploid or polyhaploid target plant following the treating.

According to an aspect of some embodiments of the present disclosure there is provided a method of breeding, the method comprising: (i) crossing the double haploid or polyhaploid target plant having the genetic modification (e.g. , gene editing event) of interest generated according to any of the aforementioned or other methods described herein with a plant of interest or (ii) selfing the double haploid or polyhaploid target plant having the genetic modification (e.g. , gene editing event) of interest generated according to any of the aforementioned or other methods described herein. In certain embodiments of the disclosure, the method can further comprise recovering seed or progeny plants having the DNA editing event from the cross or self.

According to an aspect of some embodiments of the present disclosure there is provided a cell of the genetically modified inducer or genetically edited plant described herein. In certain embodiments, the genetically edited cell is a haploid cell, a doubled haploid cell, or a polyhaploid cell. In certain embodiments, the genetically edited cell is a haploid cell, a doubled haploid cell, or a polyhaploid cell made by the methods provided herein.

According to an aspect of some embodiments of the present disclosure there is provided a seed or other propagule of the genetically modified inducer or genetically edited plant described herein. In certain embodiments, the genetically edited seed or other propagule is a haploid, a doubled haploid, or a polyhaploid seed or other propagule. In certain embodiments, the genetically edited seed or other propagule is a haploid, a doubled haploid, or a polyhaploid seed or other propagule made by the methods provided herein.

According to an aspect of some embodiments of the present disclosure there is provided a pollen of the genetically modified inducer or genetically edited plant described herein.

According to an aspect of some embodiments of the present disclosure, the nucleic acid molecule encoding the DNA editing agent is integrated in the genome of any one of the aforementioned or other cells, seeds, propagules, or pollens described herein.

According to some embodiments of the disclosure, the haploid plant is a maternal haploid. According to some embodiments of the disclosure, the haploid plant having the DNA editing event is a maternal haploid.

According to some embodiments of the disclosure, the genetically modifying comprises transforming the inducer plant or plant line with the nucleic acid molecule encoding the DNA editing agent.

According to some embodiments of the disclosure, the genetically modifying comprises crossing a parental inducer plant or plant line with a plant comprising the nucleic acid molecule encoding the DNA editing agent and selecting a progeny inducer plant comprising the nucleic acid molecule encoding the DNA editing agent.

According to some embodiments of the disclosure, the inducer plant is of a different species of the target plant.

According to some embodiments of the disclosure, the target plant is an inbred line.

According to some embodiments of the disclosure, the selecting is performed using a marker.

According to some embodiments of the disclosure, the method further comprises validating the presence of a DNA editing event induced by the DNA editing agent in the haploid plant, target plant or progeny thereof.

According to some embodiments of the disclosure, the method further comprises validating the absence of the nucleic acid molecule encoding the DNA editing agent in the target plant or progeny thereof.

According to some embodiments of the disclosure, the DNA editing agent is directed to a target sequence of interest.

According to some embodiments of the disclosure, the nucleic acid molecule comprises a gene or an expression cassette.

According to some embodiments of the disclosure, the DNA editing agent is directed to a plurality of target sequences of interest.

According to some embodiments of the disclosure, the DNA editing agent is directed to an endogenous sequence in a target plant.

According to some embodiments of the disclosure, the DNA editing agent is directed to an exogenous sequence in a target plant.

According to some embodiments of the disclosure, the DNA editing agent does not induce an editing event in the inducer plant.

According to some embodiments of the disclosure, the DNA editing agent is directed to a sequence selected from the group consisting of coding sequence, splice junction, miR binding sequence and a regulatory sequence.

According to some embodiments of the disclosure, the endogenous sequence or plurality of sequences comprises a genomic repeat sequence. According to some embodiments of the disclosure, a DNA editing event induced by the DNA editing agent is selected from the group consisting of a deletion, insertion, insertion-deletion (Indel), inversion and substitution.

According to some embodiments of the disclosure, a DNA editing event induced by the DNA editing agent comprises a sub-chromosomal structural variation.

According to some embodiments of the disclosure, the DNA editing agent is expressed under a pollen specific promoter.

According to some embodiments of the disclosure, the DNA editing agent is selected from the group consisting of meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments provided herein, examples of methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the disclosure are, by way of example, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the disclosure. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the disclosure can be practiced.

In the drawings:

Figure 1 is a schematic illustration of a double gRNA expressing plasmid targeting sites flanking the EPSPS genome locus.

Figure 2 is a schematic illustration of a control vector used for protocol setup:

BAR-intron selection marker followed by GFP under regulation of maize ubiquitin promoter. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present disclosure, in some embodiments thereof, relates to compositions and methods for generating a gene edited haploid of a target plant.

Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The disclosure is capable of other embodiments or of being practiced or carried out in various ways.

The doubled haploid (DH) technology was a major breakthrough in the development of new cultivars either as final elite lines or as parental lines. Using DH production systems, homozygosity is achieved in one generation, eliminating the need for several generations of self-pollination. The time saving is substantial, particularly in biennial crops and in crops with a long juvenile period. For self incompatible species, dioecious species and species that suffer from inbreeding depression due to self- pollination, haploidy can be the only practical way to develop inbred lines.

However, there is still a need to efficiently introduce genomic diversity for the generation of novel cultivars. While transformation of crop plants such as maize has become accessible, it remains difficult and time consuming. Even with optimized material, initial transformation attempts can lead to failure.

In certain embodiments, a haploid inducer plant expressing one or more DNA editing agent(s) is crossed to a target plant to introduce a genomic variation of interest in the target genetic background during the process of haploid generation. Introducing the DNA editing agent into the inducer plant does not leave any traces of the editing agent(s) in the target plant since the haploid generation process results in the elimination of haploid inducer plant chromosomes during haploid production. Introducing the nucleic acids encoding the DNA editing agent(s) into the inducer plant also does not efficiently transmit chromosomally integrated nucleic acids encoding the editing agent(s) to the haploid target plant since the haploid generation process results in the elimination of inducer plant chromosomes during haploid plant production.

Thus, the plants and methods provided herein combine the creation of the desired genomic variation in a target genetic background without going through a costly transformation process each time anew. In certain embodiments, the methods provided herein are applied to fixed inbred lines. In certain embodiments, the methods can be used to create a new inbred line.

Thus, according to an aspect of the disclosure there is provided a method of generating a haploid of a target plant, the method comprising crossing a haploid inducer plant comprising a DNA editing agent with a target plant of interest, thereby generating a haploid plant.

As used herein "haploid" refers to a plant (sporophyte) that contains a gametic chromosome number (n).

Haploids are smaller and typically exhibit a lower vigor compared to the donor plants and are sterile due to the inability of their chromosomes to pair during meiosis. In order to propagate them through seed and to include them in breeding programs, their fertility has to be restored with spontaneous or induced chromosome doubling (as further discussed hereinbelow). The genomically multiplied double haploids (DHs) are homozygous at all loci and can represent a new variety (self-pollinated crops) or parental inbred line for the production of hybrid varieties (cross-pollinated crops). In fact, cross pollinated species often express a high degree of inbreeding depression. For these species, the induction process per se can serve not only as a fast method for the production of homozygous lines but also as a selection tool for the elimination of genotypes expressing strong inbreeding depression.

According to some embodiments, the haploid is from diploid plants or from polyploid plants. In the latter case of polyploid plants, haploids from polyploid species have more than one set of chromosomes and are polyhaploids; for example dihaploids (2n=2x) from tetrahaploid potato (2n=4x), trihaploids (2n=3x) hexahaploid (2n=6x) etc. Dihaploids and trihaploids are not homozygous like doubled haploids, because they contain more than one set of chromosomes. They cannot be used as true-breeding lines but they enable the breeding of polyploid species at the diploid level and crossings with related cultivated or wild diploid species carrying genes of interest.

According to some embodiments, the haploid is a maternal haploid. In such a case the inducer line/donor is male and its chromosomes are eliminated during fertilization and/or early embryo development of the haploidization process. The resultant haploid seed consists of the maternal haploid genotype. According to some embodiments of the disclosure the haploid is a paternal haploid. In such a case the inducer line/donor is female and its chromosomes are eliminated during haploidization process and the haploid is constituted of the parental gamete's haploid genotype.

As used herein, the terms "recover", "recovered", "recovering" and any other conjugates thereof, when used in the context of genetically modified inducer plants or haploid, doubled haploid, or polyhaploid plants having a DNA editing event, refer to methods and/or resultant haploid, doubled haploid, or polyhaploid plants, cells, tissues, seeds or other propagules thereof having a desired feature (e.g., a genetic modification or DNA editing event) are identified and physically isolated from other haploid, doubled haploid, or polyhaploid plants, cells, tissues, seeds or other propagules that lack the desired feature. In certain embodiments, such recovered haploid, doubled haploid, or polyhaploid plants, cells, tissues, seeds or other propagules thereof having the desired feature can be used in subsequent breeding steps (e.g., crossing or selfing) to introduce the desired feature into other genetic backgrounds or to expand the population having the desired feature.

As used herein, the terms "select", "selected", "selection", "selecting" and any other conjugates thereof, when used in the context of plant breeding and/or plant transformation, refer to methods and/or resultant plants, cells, tissues, seeds or other propagules thereof having a desired feature are identified and physically isolated from other plants, cells, tissues, seeds or other propagules that lack the desired feature. In certain embodiments, such selected plants, cells, tissues, seeds or other propagules thereof having the desired feature can be used in subsequent breeding steps (e.g., crossing or selfing) to introduce the desired feature into other genetic backgrounds or to expand the population having the desired feature. The desired features) include biochemical, phenotypic, and genotypic characteristics.

As used herein "a target plant" or "target genetic background" refers to a plant into which a genomic variation (e.g., a gene editing event) is introduced (by the genome editing agent). Typically, the target plant is a crop plant.

According to some embodiments, the target plant is an inbred crop plant line.

The term '"plant" as used herein encompasses whole plants, a grafted plant, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues and organs. The plant can be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, seed and microspores.

According to a specific embodiment the plant part (e.g., of the inducer plant) is pollen.

According to a specific embodiment the plant part is seed (e.g., of the inducer plant or target plant or progeny thereof).

Plants that are particularly useful in the methods provided herein include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canadensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees. Alternatively algae and other non-Viridiplantae can be used for the methods provided herein.

According to a specific embodiment, the target plant is selected from the group consisting of maize, wheat, soybean, rice, cotton and rapeseed.

According to a specific embodiment, the target plant is a cereal plant.

According to a specific embodiment, the target plant is a maize plant.

The inducer line is utilized as the parent and crossed to a target plant, which can be homogeneous (e.g., inbred or Fl hybrid) or heterogeneous (e.g., F2, F3, F4, etc.).

Certain embodiments involve crossing the target plant with a haploid inducer.

A "haploid inducer" refers to a plant that when crossed with a target plant elicits a haploid progeny in frequency (also referred to as "induction rate"), which is higher than that is naturally occurring in a population of a given crop.

The rate of induction is defined as the number of seeds with haploid embryos divided by all seeds investigated. These rates range from 2 % to 25 %.

While haploidization may occur spontaneously, in most crop species the rate is low (~0.1%-1%). (Chase SS, Genetics. 1949 Can;34(3):328-32).

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-25 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-20 %. According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-15 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-12 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 3-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 4-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 5-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 6-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 7-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 8-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 9-10 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-9 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-8 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-7 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-6 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-5 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 2-4 %. According to a specific embodiment, the rate of induction of haploids by the inducer plant is 3-8 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 3-7 %.

According to a specific embodiment, the rate of induction of haploids by the inducer plant is 3-6 %.

For maternal haploids, the inducer plant is typically selected producing enough pollen and has to have acceptable agronomic characteristics to facilitate maintenance.

According to a specific embodiment, the inducer plant is an inducer plant line. According to a specific embodiment, the inducer plant/line comprises (e.g., genetically modified with) a haploid marker for the identification of the haploid plants produced within the progeny population. For example, the inducer can comprise a dominant anthocyanin marker genes allowing haploids to be identified at different stages (dry seeds, seedlings and mature plants). Haploid markers can be introduced into the inducer line by methods known in the art e.g., genetic modification, crossing or combinations of same. Haploid markers (e.g., Rl-nj, B l or Pll) are further described hereinbelow.

According to a specific embodiment the inducer plant is of the same species as the target plant.

According to a specific embodiment, the inducer plant is of a different species as the target plant (also referred to herein as "interspecies cross"), yet it is able to cross with the target plant (either naturally or artificially facilitated), as described below to induce a haploid progeny.

Haploid induction is a technique which is well known in the art. To date there are published protocols for over 250 plant species belonging to almost all families of the plant kingdom (reviewed in Maluszynski, M.; Kasha, K.J. & Szarejko, I. (2003). Published doubled haploid protocols in plant species. In: Doubled Haploid Production in Crop Plants: A Manual, Maluszynski, M., Kasha, K.J., Forster, B.P. & Szarejko, I., pp. 309-335, Kluwer Academic Publishers, ISBN 1-4020-1544-5, Dordrecht Maluszynski, M.; Kasha, K.J.; Forster, B.P. & Szarejko, I. (Eds.). (2003.) Doubled Haploid Production in Crop Plants: A Manual, Kluwer Academic Publishers, ISBN 1- 4020-1544-5, Dordrecht, each of which is hereby incorporated by reference in its entirety).

Following are non-limiting examples for inducing plants, lines and protocols. Additional embodiments are described in the Examples section which follows or in the art such as provided herein.

Inducer line

Exemplified herein with respect to maize, yet this description is by no way meant to be limiting. Other examples are provided hereinbelow. Maternal haploid induction in maize (Zea mays L.) is a result of crossing within one species with selected inducing genotypes (line, single cross or population). It results in a majority of regular hybrid (diploid) embryos and a smaller proportion of haploid maternal embryos with normal triploid endosperms. The first recognized inducer line is the genetic strain Stock 6, with a haploid induction rate of up to 2.3 % (Coe, E. H. Am. Nat. 3, 381-382 (1959).), which has been subsequently improved by breeding and selection for improved rates. Today, modern haploid inducing lines display high induction rates of 8 to 12% (Geiger & Gordillo, 2009; Iowa State University Doubled Haploid Facility website, 2016) or higher, normal germination rate and lead to viable haploid seedlings. Haploid embryos can be selected early in the breeding process, based on morphological and physiological markers as further described hereinbelow.

Another inducer system is based on modification of centromere structure (Ravi and Chan 2010Nature 464:615-618, reviewed in Tel et al. Turk J. Agric. For. 38: 1-6, US Patent No. 9,215,849, each of which is incorporated by reference in its entirety). CENH3 is the centromere- specific variant of HISTONE3 (H3) and is required for kinetochore nucleation and spindle attachment in mitosis and meiosis. Haploid inducer lines can be engineered into crops by complementing CENH3^~~ with a tail-altered version of CENH3 (CENH3-tailswap - e.g., A CENH3 hyper-variable tail sequence is replaced with the maize H3 tail sequence) in which the N-terminal tail was swapped with the shorter H3 tail, Nature. 2010 Mar 25; 464(7288):615-8, Front Plant Sci. 2016; 7: 414, Front Plant Sci. 2016; 7: 357).

Centromeres constructed with tail-altered CENH3 proteins function normally until they are forced to compete with wild-type centromeres for centromere loading with kinetochore components in the hybrid zygote and early embryo. This causes reduced spindle attachment of the inducer genome, leading to elimination of those chromosomes via fragmentation, and micronuclei formation during mitosis. Haploid inducer lines using centromere engineering are available for Banana, Barley, Brachypodium, Cassava, Cotton, Rice, Soybean, Sugarbeet, Switchgrass, Tobacco as described in Tek 2014, supra.

Irradiated pollen

Irradiated pollen is another embodiment for inducing the formation of maternal haploids using intra-specific pollination. Embryo development is stimulated by pollen germination on the stigma and growth of the pollen tube within the style, although irradiated pollen is unable to fertilize the egg cell. It has been used successfully in several species including, but not limited to, apple, blackberry, carnation, cucumber, European plum, kiwifruit, mandarin, melon, onion, pear, petunia, rose, Nicotiana, squash, sunflower, sweet cherry and watermelon, as reviewed in Murovec 2012 (Haploids and Doubled Haploids in Plant Breeding, Plant Breeding, Dr. Ibrokhim Abdurakhmonov (Ed.), ISBN: 978-953-307-932-5, InTech, Available on the World Wide Web internet site intechopen(dot)com/books/plant-breeding/haploids-and- doubled-haploids-in-plant-breeding, U.S. Pat. Number 8,969,658, each of which is hereby incorporated by reference in its entirety).

In maize breeding, haploid induction systems are typically based upon the characteristics of the Stock 6 population. The R-navajo (Rl-nj) gene, which causes anthocyanin expression, is generally used as the haploid marker to identify putative haploid kernels. Inbred or population inducer lines with improved agronomic and haploid induction characteristics have been developed and are in use by public and private breeding programs. Some percentage of the seed harvested from a cross between the inducer line and another plant population (inbred, Fl, F2, F3, open pollinated, or other heterogeneous population) will have a maternal haploid genotype. Putative haploid seed are identified by selecting seed with a purple endosperm (indicating fertilization by a plant containing Rl-nj) and a colorless endosperm (indicating that the chromosome containing Rl-nj is not transferred into the developing embryo). Other selection systems are contemplated and some of them are described hereinbelow. The inducer line is typically selected from pre-existing ones or developed according to the intended use. Non-limiting examples of inducer lines include, but are not limited to, B0223B and B2923B in onion (B. Bohanec et al, J. Amer. Soc. Hort. Sci. July 2003 128:571- 574), other examples for maize: KMS (Korichnevy Marker Saratovsky) and ZMS, both derived from Stock 6; (2) WS 14, developed from a cross between lines W23ig and Stock 6 ; (3) KEMS (Krasnador Embryo Marker Synthetic), derived from a cross ; (4); MHI (Moldovian Haploid Inducer), derived from a cross KMS x ZMS ; (5) RWS (Russian inducer KEMS + WS 14), descendant of the cross KEMS x WS 14 ; (6) UH400, developed at University of Hohenheim from KEMS ; (7) PK6 (Barret et ah, 2008); (8) HZI1, derived from Stock 6 ; (9) CAUHOI, derived at China Agricultural University from a cross between Stock 6 and Beijing High Oil Population, and (10) PHI (Procera Haploid Inducer), derived from a cross between MHI and Stock 6.

Examples of haploid inducers especially useful under a given abiotic stress are described for maize hereinbelow. This description is not aimed to be limiting for maize.

Temperate haploid inducers: A number of haploid inducer lines with high haploid induction rates (HIR) and for commercial use have been derived from Stock 6 as the founder; these include: KMS and ZMS both derived from Stock 6; WS 14, developed from a cross between lines W23ig and Stock 6; KEMS derived from a cross; MHI derived from a cross KMS x ZMS; RWS (Russian inducer KEMS + WS 14), descendant of the cross KEMS x WS 14; UH400, developed at University of Hohenheim from KEMS; PK6 ; HZI1, derived from Stock 6; CAUHOI, derived at China Agricultural University from a cross between Stock 6 and Beijing High Oil Population; and PHI (Procera Haploid Inducer), derived from a cross between MHI and Stock 6.

Tropicalized haploid inducers: Since 2007, CIMMYT Global Maize Program has been intensively engaged in optimization of the DH technology especially for the tropical/subtropical maize growing environments, in partnership with the University of Hohenheim, Germany. Tropically adapted inducer lines (TAILs; with 8 - 10% HIR) have been developed through this collaboration (Reviewed by Murovec 2012, supra).

Another inducer system is the indeterminate gametophyte system, which has been used to produce haploids in maize, as described in Kindiger and Hamann 1993 Crop Sci. 33:342-344, which is hereby incorporated by reference in its entirety. Haploid induction by interspecies crossing

It has been shown that interspecies crossing of specific plant species can be used as a method for haploid induction in crop plants (e.g., wheat flowers fertilized by maize pollen (Zhang W. at al. Botanical Studies An International Journal 2014 55:26)). Other examples of intercross able to induce haploidy is described in Murovec J. and Bohanec B. "Plant Breeding", book edited by Ibrokhim Y. Abdurakhmonov, ISBN 978-953-307- 932-5, Published: January 11, 2012 chapter 5 p.87-106.

It should be noted that in interspecific haploidization, there is no need for a pollen specific or inducible promoter (for expressing the DNA editing agent described below) to prevent damage to the haploid inducer pollen donor as specific targeting sequences (e.g., gRNAs) can be designed not to target any site on the inducer and be specific to the target genetic background.

According to a specific embodiment, the use of species-specific promoters -for expressing the DNA editing agent only in the target plant are also contemplated.

Wide crossing between species has been shown to be a very effective method for haploid induction and has been used successfully in several cultivated species. It exploits haploidy from the female gametic line and involves both inter- specific and inter- generic pollinations. The fertilization of polar nuclei and production of functional endosperm can trigger the parthenogenetic development of haploid embryos, which mature normally and are propagated through seeds (e.g., potato). In other cases, fertilization of ovules is followed by paternal chromosome elimination in hybrid embryos. The endosperms are absent or poorly developed, so embryo rescue and further in vitro culture of embryos are contemplated in such cases (e.g., barley). Non-limiting examples are provided below.

In barley, for instance, haploid production is the result of wide hybridization between cultivated barley (Hordeum vulgare, 2n=2x=14) as the female and wild H. bulbosum (2n=2x=14) as the male. After fertilization, a hybrid embryo containing the chromosomes of both parents is produced. During early embryogenesis, chromosomes of the wild relative are preferentially eliminated from the cells of developing embryo, leading to the formation of a haploid embryo, which is due to the failure of endosperm development. A haploid embryo is later extracted and grown in vitro. The 'bulbosum' method is the first haploid induction method to produce large numbers of haploids across most genotypes and quickly entered into breeding programs.

Pollination with maize pollen can also be used for the production of haploid barley plants.

Paternal chromosome elimination has also been observed after interspecific crosses between wheat (Triticum aestivum) and maize. After pollination, a hybrid embryo between wheat and maize develops but, in the further process, the maize chromosomes are eliminated so that haploid wheat plantlets can be obtained. Such haploid wheat embryos usually cannot develop further when left on the plant, because the endosperm fails to develop in such seeds. By applying growth regulator 2,4- dichlorophenoxyacetic acid in planta, embryo growth is maintained to the stage suitable for embryo isolation and further in vitro culture.

Yet according to another example, the maize chromosome elimination system in wheat enables the production of large numbers of haploids from any genotype. Pollination with maize is also effective for inducing haploid embryos in several other cereals, such as barley, triticale (x Triticosecale), rye (Secale cereale) and oats (Avena sativa) (Wedzony, 2009, Progress in doubled haploid technology in higher plants. In: Advances in Haploid Production in Higher Plants, Touraev, A., Forster, B.P., & Jain, S.M., pp. 1-34, Springer Science + Business Media B.V, ISBN 978-1-4020-8853-7, which is hereby incorporated by reference in its entirety). Similar processes of paternal chromosome elimination can occur after the pollination of wheat with wild barley (H. bulbosum), sorghum (Sorghum bicolour L. Moench) and pearl millet (Pennisetum glaucum).

In contrast to maize and pearl millet pollination, pollination with H. bulbosum is strongly influenced by the maternal genotype. Haploid production in cultivated potato (Solanum tuberosum L. ssp. tuberosum, 2n=4x) can be achieved by inter-generic pollination with selected haploid inducer clones of S. phureja (2n=2x). The tetraploid female S. tuberosum produces an embryo sac containing one egg cell and two endosperm nuclei, all with the genetic constitution n=2x, while the diploid pollinator S. phurea produces two sperms of the genetic constitution n=x or 2x.

After pollination, dihaploid (2n=2x) embryos can develop from un-fertilized egg cells, which are supported by a 6x endosperm formed by the fusion of polar nuclei with both reduced sperm cells. The frequency of dihaploid seeds is low; they have to be selected from hybrid seeds containing 3x or 4x embryos developed from egg cells (n=2x) fertilized with haploid (n=x) or diploid (n=2x) sperm cells. (Maine, 2003). Dihaploid potatoes can be used for breeding purposes, including alien germplasm introgression or selection at the diploid level, but such plants are not homozygous.

An exemplary protocol for haploid induction using interspecies crossing is described in Bakos et al. 2005 Acta Biologica Cracoviensia 47/1: 167-171, which is hereby incorporated by reference in its entirety. Briefly, Haploid wheat plants are produced by a method of zygote rescue carried out after distant pollination. Wheat stigmas are pollinated with maize pollen or rice pollen and subsequently the activated egg cells from the elongated ovaries are rescued for in vitro plant development in single cell culture. As the control, 2-week-old embryos are also dissected and then cultured. Because the lack of a normal endosperm hampers embryo development even in the early stages, early zygote rescue (two days after distant pollination) can represent a more efficient way of producing double haploid (DH) plants in cultivars that are recalcitrant in androgenic cultures.

Another protocol for haploid induction using interspecies crossing is described in Kazuhiro et al. Plant Cell Reports 1989 8:263-266, which is hereby incorporated by reference in its entirety.

Yet another protocol for haploid induction using interspecies crossing is described in Zhang et al. 2014 Botanical Studies 2014 55:26, describing successful haploid generation of dwarf male sterile wheat using a corn inducer, which is hereby incorporated by reference in its entirety.

Thus, once a haploid inducer is in hand, the present teachings further contemplate a method of genetically modifying a haploid inducer. In certain embodiments, the methods comprise genetically modifying the haploid inducer plant with a nucleic acid molecule encoding a DNA editing agent(s).

In certain embodiments, genetically modifying is such that the nucleic acid molecule(s) encoding the DNA editing agent(s) is integrated in the genome of the inducer plant, such that upon completion of the haploid embryo developmental process (in vitro or in vivo), the DNA editing agent will have completed its role in modifying the genome of the embryo {e.g., introducing a DNA editing event in the genome of the embryo) and will have been completely eliminated from the haploid along with the entire genome of the inducer.

Thus the haploid inducer plant is introduced with a nucleic acid molecule encoding the DNA editing agent(s).

In certain embodiments, genetic modification of the inducer plant can be effected by directly genetically modifying. Accordingly, the inducer plant is directly transformed with one or more expression cassettes encoding one or more DNA editing agent(s) that provide for expression of the agent(s) in the inducer plant or for expression at least during the haploid induction process. Methods of introducing genetic material (e.g., expression constructs or genes encoding DNA editing agents) to plants are further described hereinbelow and in the Examples section which follows.

In other cases genetic modification of the inducer plant is effected by indirect genetic modification. In certain embodiments, the indirect genetic modification comprises crossing the inducer plant with a plant comprising the nucleic acid molecule(s) encoding the DNA editing agent(s) and selecting the inducer line comprising the expression cassette(s). When needed, pedigree breeding is practiced including backcrossing to the recurrent parent (i.e., the inducer plant).

At least one advantage of such an embodiment is the use of "easily" transformable germplasm such as B 104, or Hi II as pollen donors for target crop editing. (Examples of which can be found in: Production and identification of haploid dwarf male sterile wheat plants induced by corn inducer, Zhang et al. Botanical Studies 2014, 55:26, which is hereby incorporated by reference in its entirety).

As used herein "a DNA editing agent" refers to a single stranded or double stranded engineered DNA endonuclease and in certain embodiments ancillary agents (e.g., gRNA(s), donor DNA sequences) causing insertion, deletion, insertion-deletion, substitution, insertion, or any combination thereof in a genome of an organism.

Throughout the disclosure it is submitted that a "DNA editing agent" refers to one or more DNA editing agent and is also referred to as "DNA editing agent(s)".

According to a specific embodiment, the DNA editing agent is directed to a target sequence of interest.

According to a specific embodiment, the DNA editing agent is directed to a plurality of target sequences of interest (e.g., 2, 3 or more). According to a specific embodiment, the DNA editing agent can be directed to a plurality of target sequences of interest and can comprise a whole library inducing tens, hundreds or thousands of genetic alteration in the target genome.

According to a specific embodiment, the DNA editing agent modifies the target genome but not the inducer plant genome.

According to a specific embodiment, the DNA editing agent modifies the target sequence of interest (in the target genome) and is devoid of "off target" activity, i.e., does not modify other sequences in the target genome. According to other embodiments, the DNA editing agent modifies the target sequence of interest (in the target genome) and is significantly reduced in "off target" activity. Significant reductions in "off target" activity include reductions of off target modification rates to less than 2%, 1%, or 0.1%. Methods that can be used to assess off target modification rates include those of Haeussler et al. (Genome Biology, 2016, 17: 148).

This is especially true when the target genome and the inducer genome are not from the same species or are of the same species but not the same cultivars.

• According to a specific embodiment, the DNA editing agent comprises an "off target activity" on a non-essential gene in the target genome or inducer plant.

Non-essential refers to a gene that when modified by the DNA editing agent does not affect the phenotype of the target genome in an agriculturally valuable manner (e.g., biomass, vigor, yield, selection, biotic/abiotic stress tolerance and the like).

In certain embodiments the DNA editing agent can produce off-target effects that are beneficial (e.g. can be used to generate SVs).

According to a specific embodiment, the DNA editing agent is directed to an endogenous sequence in a target plant.

According to a specific embodiment, the DNA editing agent is directed to an exogenous sequence in a target plant (e.g., a transgene expressing an agriculturally valuable trait).

The DNA editing agent can be directed to any target sequence of interest. Examples include, but are not limited to, coding sequences, splice junctions, miR binding sequence, a regulatory sequence (e.g., promoter), a non-coding sequence (e.g., for tagging), a euchromatic sequence or a heterochromatic sequence. According to a specific embodiment, the endogenous sequence or plurality of sequences comprises a genomic repeat sequence. Accordingly, a single agent can cause a number of variations in the target genome.

For instance, in certain embodiments where a DNA editing tool such as CRISPR (as described hereinbelow) directed to a repeat sequence is used. The Zea_mays annotated repeats v3.0 from the http internet site "maize(dot)jcvi(dot)org" tool is employed.

The repeat sequences are mapped to the Zea_mays.AGPv3.22 genome.

The number of hits for each repeat is listed. Next the list is filtered for repeats with hits from all 12 chromosomes.

A proprietary script, based on the CasOT tool [Xiao A. et ah , CasOT: a genome- wide Cas9/gRNA off-target searching tool Bioinformatics. 2014 Jan 21.] is used to design gRNAs to the selected repeat sequences, and find their targets and off-targets.

For each selected gRNA, one target is chosen and a set of PCR primers is designed for that target, using Primer3 tool for target validation [Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Research 40(15):el l5] .

Alternatively or additionally, the DNA editing agent is directed to a gene cluster flanking region. In order to edit a sequence of interest, two gRNAs targeting the region flanking the sequence of interest can be used (instead of one targeting the gene itself). Using two gRNA's also opens the possibility to induce duplication or inversion of the region as well as deletion of the whole region. Using such a method allows deleting a cluster of genes using a single editing event, instead of editing each gene within the cluster individually.

DNA editing agent(s) is selected from the group of agents that can affect a genetic modification (e.g., DNA editing event) selected from the group consisting of a deletion, insertion, insertion-deletion (Indel), inversion, substitution and combinations thereof.

According to a specific embodiment, the DNA editing agent(s) can provide a genetic modification (e.g., DNA editing event) that upregulates expression of an expression product (i.e., RNA or protein) in the target plant. According to a specific embodiment, the DNA editing agent(s) can provide a genetic modification (e.g., DNA editing event) that downregulates expression of an expression product (i.e., RNA or protein) in the target plant.

The expression product can have any advantage (or disadvantage) to the target plant. Thus, it can be involved in abiotic or biotic stress tolerance, herbicide resistance (e.g., tolerance), crossability (e.g., male sterility), biomass, vigor, yield or any other trait that is agriculturally valuable (or invaluable).

Alternatively, the expression product can be of commercial value such as a pharmaceutical, cosmetic, health product, commodity, food and the like.

According to a specific embodiment, the genetic modification introduced by the DNA editing agent(s) doesn't affect expression of an expression product (i.e., RNA or protein) in the target plant. Such a setting can be used for tagging or for introduction of variant sequences with distinct enzymatic or regulatory activities.

According to a specific embodiment, a DNA editing event induced by the DNA editing agent comprises a sub-chromosomal structural variation.

As used herein a "sub-chromosomal structural variation", also referred to herein as SV, refers to a genomic variation that involves sub-chromosomal structural variation a segment of DNA (as opposed to a point mutation/SNP) e.g., above 100 bp long. According to a specific embodiment, the DNA segment is larger than 1 kb (e.g., 1Kb- 2Mb e.g., smaller than 10 Mb). The DNA segment can comprise a plurality of variations. According to a specific embodiment, the SV comprises a coding region, a non-coding region or a combination of same.

According to a specific embodiment, the structural variation is sub- chromosomal, namely, involves segments within a given chromosome and not a reciprocal chromosomal variation such as that occurring during translocations. Yet, it should be understood that a number of SVs can occur in different chromosomes and/or the same chromosome in different locations (e.g., QTLs).

According to a specific embodiment, the sub-chromosomal structural variation is sub-microscopic, i.e., not detected using a light/fluorescent microscope. Thus, according to a specific embodiment, the sub-chromosomal structural variation is not detectable using karyotypic analysis nor Giemse staining. According to a specific embodiment, the sub-chromosomal structural variation is smaller than a whole chromosome arm.

According to a specific embodiment, the sub-chromosomal structural variation is selected from the group consisting of presence-absence variation (PAV), insertions, deletions, insertions/deletions (InDels), inversions, translocations and combinations thereof.

As used herein the term "presence-absence variation" or "PAV" refers to sequences that are present in one genome and absent in another. PAVs can be considered to be extreme CNVs, where the sequence is completely missing from one or more individual. According to a specific embodiment, the DNA segment which creates the PAV is larger than 100b or larger than 1Kb (e.g., 100b-2Mb, lKb-2Mb e.g., smaller than 10 Mb).

As used herein the term "insertion/deletion" or "Indel" a difference in length between DNA sequences based on an insertion of DNA base(s) in the longer sequence or deletion of DNA base(s) in the shorter sequence. According to a specific embodiment, the DNA segment which creates the indel in the contest of sub- chromosomal structural variation is larger than 100b or larger than 1Kb (e.g., 100b- 2Mb, lKb-2Mb e.g., smaller than 10 Mb).

Methods of introducing nucleic acid alterations to a gene of interest are well known in the art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244: 1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed by publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

Following is a description of various non-limiting examples of methods and

DNA editing agents used to introduce nucleic acid alterations to a gene of interest and agents for implementing same that can be used according to specific embodiments of the present disclosure.

Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double- stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDS) and nonhomologous end-joining (NHEJF). NHEJF directly joins the DNA ends in a double- stranded break, while HDR utilizes a homologous donor sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a donor DNA repair template containing the desired sequence must be present during HDR.

Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and these sequences often will be found in many locations across the genome resulting in multiple cuts which are not limited to a desired location. To overcome this challenge and create site-specific single- or double- stranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system.

Meganucleases - Meganucleases are commonly grouped into four families: the

LAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location.

This can be exploited to make site-specific double-stranded breaks in genome editing. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence.

Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent No s. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double- stranded breaks (Christian et al, 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

Basically, ZFNs and TALENs restriction endo nuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the double- stranded break.

Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the Fokl domains heterodimerize to create a double-stranded break. Repair of these double- stranded breaks through the non-homologous end-joining (NHEJ) pathway often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site.

The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have been successfully generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the double-stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al, 2010; Urnov et al, 2005).

Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers are typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

T-GEE system (TargetGene's Genome Editing Engine) - A programmable nucleoprotein molecular complex containing a polypeptide moiety and a specificity conferring nucleic acid (SCNA) which assembles in-vivo, in a target cell, and is capable of interacting with the predetermined target nucleic acid sequence is provided. The programmable nucleoprotein molecular complex is capable of specifically modifying and/or editing a target site within the target nucleic acid sequence and/or modifying the function of the target nucleic acid sequence. Nucleoprotein composition comprises (a) polynucleotide molecule encoding a chimeric polypeptide and comprising (i) a functional domain capable of modifying the target site, and (ii) a linking domain that is capable of interacting with a specificity conferring nucleic acid, and (b) specificity conferring nucleic acid (SCNA) comprising (i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site, and (ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide. The composition enables modifying a predetermined nucleic acid sequence target precisely, reliably and cost-effectively with high specificity and binding capabilities of molecular complex to the target nucleic acid through base-pairing of specificity-conferring nucleic acid and a target nucleic acid. The composition is less genotoxic, modular in their assembly, utilize single platform without customization, practical for independent use outside of specialized core-facilities, and has shorter development time frame and reduced costs.

CRISPR-Cas system (also referred to herein as "CRISPR")- Many bacteria and archea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) nucleotide sequences that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to the DNA of specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR/Cas system of Streptococcus pyogenes have shown that three components form an RNA/protein complex and together are sufficient for sequence- specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821.).

It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double- stranded brakes in a variety of different species (Cho et al, 2013; Cong et al, 2013; DiCarlo et al, 2013; Hwang et al, 2013a,b; Jinek et al, 2013; Mali et al, 2013).

The CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas 9.

The gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Just as with ZFNs and TALENs, the double- stranded breaks produced by CRISPR/Cas can undergo homologous recombination or NHEJ and are susceptible to specific sequence modification during DNA repair.

The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA. A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs. This creates a system that can be readily modified to target modifications at different genomic sites and/or to target different modifications at the same site. Additionally, protocols have been established which enable simultaneous targeting of multiple genes. The majority of cells carrying the mutation present biallelic mutations in the targeted genes.

However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.

Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called 'nickases'. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single- strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system. A double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.

Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

There are a number of publically available tools available to help choose and/or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.

Non-limiting examples of a gRNA that can be used in the present disclosure include those described in the Example section which follows.

In order to use the CRISPR system, both gRNA and a CAS endonuclease (e.g.

Cas9) should be expressed in a target cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px330 plasmid from Addgene (75 Sidney St, Suite 550A · Cambridge, MA 02139). Use of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology and a Cas endonuclease for modifying plant genomes are also at least disclosed by Svitashev et al, 2015, Plant Physiology, 169 (2): 931-945; Kumar and Jain, 2015, J Exp Bot 66: 47-57; and in U.S. Patent Application Publication No. 20150082478, which is specifically incorporated herein by reference in its entirety. CAS endonucleases that can be used to effect DNA editing with gRNA include, but are not limited to, Cas9, Cpfl (Zetsche et al., 2015, Cell. 163(3):759-71), C2cl, C2c2, and C2c3 (Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97).

"Hit and run" or "in-out" - involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive/negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences. The "double-replacement" or "tag and exchange" strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive/negative selectable cassette near the location where the mutation is to be introduced. After electroporation and positive selection, homologously targeted clones are identified. Next, a second targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

Site-Specific Recombinases - The Cre recombinase derived from the PI bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed "Lox" and "FRT", respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site- specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.

Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue- specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT "scar" of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.

Thus, Cre/Lox and Flp/FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.

According to a specific embodiment, the DNA editing agent is expressed in the inducer plant in an inducible manner.

For example, the DNA editing agent can be configured having a pollen inducible promoter (e.g., Zml3 and ZM58).

Examples of inducible promoters and developmentally regulated promoters include, but are not limited to, heat-inducible promoter in wheat: the barley Hvhspl7 gene promoter is induced by heatshock (Freeman J. et al., plant Biotechnology J. (2011) 9, pp. 788-796). Pathogen infection and chemical elicitors such as benzothiadiazole are used to induce PR-la promoter and other is the maize In2-2 (Inducible gene 2-2) promoter, which is induced by benzenesulfonamide safeners (herbicide tolerance increasing agrochemicals of plants) (Shah et al., (2015) American-Eurasian J. Agric. & Enviroon. Sci., 15(4) pp 664-675).

Embryo promoters: promoters for globulin- 1 and globulin-2 expressed highly in the maize embryo (Streatfield S.J et al., GM Crops (2010)). Maize defensin-like protein genes Defl and Def2 functions as an embryo -specific asymmetric bidirectional promoter (Liu X. et al., (2016) J. of Experimental Botany Advance).

Following are some non-limiting examples of configurations of DNA editing agents that can be employed according to some embodiments of the disclosure. While these examples focus on the CRISPR system it is envisaged for any DNA editing agent, such as described hereinabove.

Thus, the DNA editing agent (e.g., CRISPR) can be designed to target a specific locus inducing a double strand break that is repaired by NHEJ and in some cases cause sequence deletion for a small number of nucleotides causing a frameshift mutation and a premature stop codon or other functional deactivation of the gene. In this case, a single gRNA is selected targeting for example the coding region of a gene, a splice site (causing mis splicing event) or a regulatory sequence such as a critical promoter binding motif or an miRNA binding sequence that modifies expression of the gene. The sequence is designed to have the lowest number of "of targets" possible by using tools such as CRISPR-plant (available on the World Wide Web internet site "genome.arizona.edu/crispr/" or in the internet site CRISPR-P "cbi.hzau.edu.cn/crispr/".

The DNA editing agent(s) can target one or more genes of interest. The double strand breaks induced on both sides of the genomic locus can cause a deletion of the whole region or in other cases different types of structural variations such as an inversion or duplication. To induce a targeted structural variation, two gRNA's are cloned on one CRISPR constructs having a U6 specific promoter (atU6 for dicots and osU6 for monocots) gRNA designed is done using CasOT (CRISPR/Cas system (Cas9/gRNA) Off-Targeter) (available on the World Wide Web "eendb(dot)zfgenetics(dot)org/casot/"). Sequence search can be restricted to length (17- 20nt not including PAM sequence), PAM sequence (NGG) and number of mutations allowed in sequence (seed or other).

The DNA editing agent can be designed to target repetitive genomic sequence such as transposable elements causing genome wide double strand breaks that are repaired by HR or NHEJ and can result in de novo structural variation. The structural variation induced by these gRNA's can range from small to large deletions, inversions, duplications and translocations (inter and intra chromosomal). For the above described strategy one or more gRNA are designed on a single construct consisting of a Cas9 nuclease (or a cas9 variant endonuclease able to induce a double strand break in a gRNA directed manner) under a constitutive (eg. CMV 35S promoter) or a signal induced (e.g., safener inducible maize glutathione-S-transferase (GST-II-27) promoter, (Front Plant Sci. 2014; 5: 379.). (The GST-II-27 gene has been shown previously (International Application Number WO 90/08826) to be induced by certain chemical compounds, known as "herbicide safeners", which can be applied, as a spray, for example, to growing plants.)

In order to prevent haploid inducer/donor line from undergoing genome editing, several strategies can be taken including the above mentioned inducible promoter or other examples. A second strategy can include establishment of a pollen specific promoter (eg. Zmgl3 or Lat56 US patent no.5412085 A) regulated "universal" Cas9 trangenic HI line and hybridize with a U6:gRNA transgene prior to haploid induction. This strategy prolongs the procedure by a season but can reduce HI genomic instabilitywhen using a targeting strategy that introduces DNA breaks at many locations, such the targeting of repetitive elements in the creation of a random/mutator CRISPR gRNA inducer line). This is of significance when an inducer capable of creating significant genomic variation is introduced contemplated.

Figure 1 provides an example of a double gRNA expressing plasmid targeting sites flanking the EPSPS genome locus (Figure 2 being a control thereof).

Methods of transforming DNA editing agents into plants are well known in the art. Some are described infra.

Constructs useful in the methods according to some embodiments of the disclosure can be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs can be inserted into vectors, which can be commercially available vectors, suitable for transforming into plants and suitable for expression of the gene or expression cassette encoding a gene editing agent in transformed cells, plants, tissues, and/or developmental stages.

In certain embodiments, the expression cassette encoding a gene editing agent can comprise a promoter that is operably linked to a sequence encoding a gene editing agent, which is in turn operably linked to a sequence encoding a polyadenylation site. The genetic construct can be an expression vector wherein said nucleic acid molecule is operably linked to one or more regulatory sequences allowing expression in the plant cells.

In a particular embodiment of some embodiments of the disclosure the regulatory sequence is a plant-expressible promoter.

As used herein the phrase "plant-expressible" refers to a promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, including a monocotyledonous or dicotyledonous plant cell, tissue, or organ. Non-limiting examples of promoters useful for certain embodiments of the methods provided herein are presented in Table 1 and 2, below. Table 1

Non-limiting examples of constitutive promoters for use in the performance

of some embodiments of the disclosure

Nucleic acid sequences of the polypeptides of some embodiments of the disclosure can be optimized for plant expression. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.

The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).

In this method, the standard deviation of codon usage, a measure of codon usage bias, can be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU = n = 1 N [ ( Xn - Yn ) / Yn ] 2 / N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

Plant cells can be transformed stably with the nucleic acid constructs in some embodiments. In stable transformation, the nucleic acid molecule of some embodiments of the disclosure is integrated into the plant genome and as such it represents a stable and inherited trait.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al, Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant

Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923- 926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. In certain embodiment, the transformed plant can be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant with the addition of the introduced nucleotide sequence or gene. Regeneration of plantlets by micropropagation can be utilized to produce homogeneous plants following transformation

Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the tissue containing the nucleic acid molecule encoding the DNA editing agent. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants, include but are not limited to, the speed of plant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.

Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression of non- viral exogenous nucleic acid sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al, Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231: 1294- 1297; and Takamatsu et al. FEBS Letters (1990) 269:73-76.

When a DNA virus is utilized, suitable modifications can be made to the virus itself. Alternatively, the virus DNA can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. A bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsulate the viral DNA. If an RNA virus is utilized, the virus is generally cloned as a cDNA and inserted into a plasmid. The DNA of the plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsulate the viral RNA.

Construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous nucleic acid sequences is demonstrated by the above references as well as in U.S. Pat. No. 5,316,931. Provided herein are embodiments where the non- viral exogenous sequences used in such vectors comprise sequences encoding one or more DNA editing agents.

In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, including, but not limited to, the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted. Alternatively, the coat protein gene can be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced. The recombinant plant viral nucleic acid can contain one or more additional non-native subgenomic promoters.

Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) nucleic acid sequences can be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non- native coat protein coding sequence.

In a third embodiment, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences can be inserted adjacent the non-native subgenomic plant viral promoters such that said sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vectors are encapsulated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired protein. In addition to the above, the nucleic acid molecule of some embodiments of the disclosure can also be introduced into a plastid genome thereby enabling plastid expression.

A technique for introducing exogenous nucleic acid molecules to the genome of plastids is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of plastids per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the plastids. The exogenous nucleic acid is selected such that it is integratable into the plastid's genome via homologous recombination which is readily effected by enzymes inherent to the plastid.

To this end, the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the plastid's genome. In addition, the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the plastid genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference in their entireties.

Thus, the present teachings provide for a haploid inducer plant genetically modified with a nucleic acid molecule encoding the DNA editing agent(s).

Once such a plant is in hand it can in certain embodiments be crossed with a target plant of interest, thereby generating a haploid plant. Crossing is effected using methods which are well known in the art including, but not limited to, emasculation of the pollen recipient followed by pollen transfer from the pollen donor. In certain embodiments, marker-assisted breeding can be used although phenotypic selection can also be used.

Identification ofhaploids:

Several direct and indirect approaches are available for determining the ploidy level of regenerated plants. Indirect approaches are based on comparisons between regenerated and donor plants in terms of plant morphology (plant height, leaf dimensions and flower morphology), plant vigor and fertility, number of chloroplasts and their size in stomatal guard cells.

Direct methods for ploidy determination are more robust and reliable and include conventional cytological techniques, such as counting the chromosome number in root tip cells and measurement of DNA content using flow cytometry. The latter provides a rapid and simple option for large-scale ploidy determination as early as in the in vitro culturing phase. It also enables detection of mixoploid regenerants (having cells with different ploidy) and the determination of their proportion.

A fast and reliable haploid identification method is needed for large scale production of haploids. Morphological markers expressed at the embryo, seed or early seedling stages can be used. In maize, the most common haploid identification marker is the Rl-nj 'red crown' kernel trait, which causes deep pigmentation of the aleurone layer in the crown region (endosperm) and scutellum (embryo tissue). In a haploid inducing cross, the marker should be homozygous recessive in the female parent and homozygous dominant in the pollinator inducer line. After pollination, kernels with a red aleurone crown (resulting from regular fertilization of polar nuclei) containing a nonpigmented scutellum are visually selected from the hybrid kernel of regular fertilization with both aleurone and scutellum pigmented.

Alternatively, the use of haploid inducers with anthocyanin marker genes B l (Boosterl) and Pll (Purplel) that result in sunlight-independent purple pigmentation in the plant tissue (coleoptile and root) is found suitable for cases where haploid sorting is not possible at dry seed stage. In this case, a pigmented coleoptile or root in the early developmental stage indicates diploid state, while the nonpigmented seedlings could be designated as haploids.

Alternatively, expression of GFP protein or utilization of a high oil inducer line is contemplated. In the case of the latter, seed oil content is impacted by the genotype of the male parent. A high oil male parent is therefore able to cause expression of a high oil percentage in progeny seeds had it successfully integrates into the embryonic cells upon fertilization. Selection for normal oil content in the progeny seed is therefore a tool for identifying those individual seed which are fertilized but in which the high oil genome is eliminated from the embryonic tissue or is not integrated in the first place. A similar approach is used in potato, in which selection is based on a

homozygous dominant color marker gene carried by the pollinator line.

Weichnag et al. (Mol. Breeding 2016 36:5) describes a green-fluorescent protein haploid inducer line for haploid screens and is hereby incorporated by reference in its entirety.

Haploid identification in maize based on oil content is described in Melchinger et al. 2013 Scientific Reports 3:2129 and is hereby incorporated by reference in its entirety.

Once the haploid is identified it can be subjected to validation for the presence of a DNA editing event induced by the DNA editing agent in the haploid plant.

Additionally or alternatively, absence of the DNA editing agent in the haploid is validated.

In certain embodiments, haploid plants having a DNA editing event can be selected. Such selections can include assaying a cell, a tissue, or any portion of a haploid plant or propagule thereof for the presence of the DNA editing event followed by isolation of those haploid plants or propagules thereof that have the DNA editing event. Assays that can be used to select haploids having one or more DNA editing event(s) include assays that identify biochemical features, phenotypic features, or genomic sequence alterations that result from the DNA editing event(s). In certain embodiments, the biochemical features, phenotypic features, or genomic sequence alterations that result from the DNA editing event(s) in the haploid plant or propagule thereof are identified by comparing assay results obtained from a candidate haploid plant that has been subjected to the DNA editing agent to a control plant or propagule that has not been treated with a DNA editing agent. Control plants and propagules include inbred diploid target plants and target haploid plants that have not been treated with the DNA editing agent. In certain embodiments, the biochemical feature that is selected is an enzymatic activity or compositional feature. Such compositional features that can be selected include various quality traits {e.g., protein, oil, starch, or other nutrient content or profile). Phenotypic features that can be selected include yield, stature {e.g., increased or decreased as desired), root mass, abiotic or biotic stress tolerance, herbicide tolerance, and the like. In certain embodiments, the assayed genomic sequence modification is selected from the group consisting of a sub- chromosomal structural variation, presence-absence variation, deletion, insertion, insertion-deletion (Indel), inversion, substitution, and combinations thereof.

In certain embodiments, a fertile plant having the DNA editing event is obtained from the haploid plant for use as a commercial product or for further development such as by breeding. In certain embodiments, the haploid carrying the DNA editing event can be subjected to a chromosome doubling agent, thereby generating a fertile double haploid or polyhaploid target plant, dependent on the type of genome having the genetic modification or gene editing event of interest.

In certain embodiments, the selected putative haploid seed is germinated and treated to induce genome duplication and grown in a growth chamber, greenhouse, and/or field environment. Double haploid or polyhaploid progeny plants or propagules can then obtained from the treated haploid plant.

Again, once multiplied, a molecular test that selects for occurrence of the desired genomic event (e.g., DNA editing event) and confirms that no nucleic acid molecules encoding the genome editing agent has been transferred to the diploid or polyploid or polyhaploid target plant can be performed. It should be noted that spontaneously doubled haploids can also occur, thus negating the need for chromosome doubling.

In certain embodiments, doubled haploid or polyhaploid plants having a DNA editing event can be selected. Such selections can include assaying a cell, a tissue, or any portion of a doubled haploid or polyhaploid plant or propagule thereof for the presence of the gene editing event followed by isolation of those doubled haploid or polyhaploid plant plants or propagules thereof that have the gene editing event. Assays that can be used to select doubled haploid or polyhaploid plant having one or more DNA editing event(s) include assays that identify biochemical features, phenotypic features, or genomic sequence alterations that result from the DNA editing event(s). Assays that identify biochemical features, phenotypic features, or genomic sequence alterations described above in reference to selections of haploid plants or propagules thereof having one or more DNA editing event(s) can also be applied to doubled haploid or polyhaploid plants having one or more DNA editing event(s).

Artificial chromosome doubling or polyploidization (dependent on the source material) can be induced using methods which are well known in the art. Typically, a G2/M cycle inhibitor is used in such protocols. According to a specific embodiment, the G2/M cycle inhibitor comprises a microtubule polymerization inhibitor.

Examples of microtubule cycle inhibitors include, but are not limited to oryzalin, colchicine, colcemid, trifluralin, benzimidazole carbamates (e.g. nocodazole, oncodazole, mebendazole, R 17934, MBC), o-isopropyl N-phenyl carbamate, chloroisopropyl N-phenyl carbamate, amiprophos-methyl, taxol, vinblastine, griseofulvin, caffeine, bis-ANS, maytansine, vinbalstine, vinblastine sulphate and podophyllo toxin .

Microtubule polymerization inhibitor can be applied at various stages of androgenesis, such as being incorporated into microspore pretreatment media.

Colchicine application on anther culture medium, for instance, shows a significant increase in embryo formation and green plant regeneration in wheat.

Alternatively, duplication treatments are applied after regeneration at either embryo, shoot or plantlet level. Similarly, treatments of gynogenically derived embryos with colchicine have also been found to be appropriate. Alternatively, treatment of plants at later developmental stages has the advantage that only already tested haploid regenerants are treated either in vitro (for instance at the shoot culture stage) or in vivo following acclimatization.

Alternatively, treatment with nitrogen oxide (N₂0), which is developed for maize seedlings is also contemplated.

Chemical treatment might be avoided by using in vitro adventitious somatic regeneration, which itself frequently leads to increased ploidy. Such an approach was found efficient in onion.

Also provided is a method of breeding, the method comprising crossing the double haploid or polyhaploid target plant having the genetic modification (e.g., DNA editing event) of interest generated according to the method described above with a plant of interest.

A typical breeding protocol involves crossing and backcrossing.

The term "backcrossing" as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental plants. The parental plant which contributes the genetic event for the desired characteristic is termed the non-recurrent or donor parent. This terminology refers to the fact that the non-recurrent parent is used one time in the backcross protocol and therefore does not recur. The parental plant to which the gene from the non-recurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.

In a typical backcross protocol, a plant from the original varieties of interest

(recurrent parent) is crossed to a plant selected from second varieties (non-recurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a plant is obtained wherein essentially all or most of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the non-recurrent parent.

Thus, near-isogenic lines (NIL) can be created by many backcrosses to produce an array of individuals that are nearly identical in genetic composition except for the trait or genomic region under investigation.

Backcrossing methods can be used with the plants provided herein to improve or introduce a characteristic into the parent lines. Marker assisted breeding (selection) as described above can be used in this method.

Also provided is a method of breeding, the method comprising selfing (i.e., self pollination) the double haploid or polyhaploid target plant having the genetic modification (e.g., DNA editing event) of interest. Progeny of such selfing can be used in a variety of subsequent selections or breeding steps. In certain embodiments, the progeny of the self are used in selections or evaluations for biochemical or phenotypic features of interest. In certain embodiments, progeny of the self can be used to bulk up (i.e., increase) the double haploid or polyhaploid seed. In certain embodiments, progeny of the self can be used to as a pollen recipient or donor in a cross with a genetically distinct population of pollen donor or recipient plants, respectively, to produce hybrid seed.

In certain embodiments, presence of the genetic editing event and/or absence of the DNA editing agent can be validated in the dihaploid/haploid target plant or progeny thereof.

In certain embodiments, progeny of doubled haploid or polyhaploid plants having the DNA editing event are selected. Assays that can be used to select doubled haploid or polyhaploid plant having one or more DNA editing event(s) include assays that identify biochemical features, phenotypic features, or genomic sequence alterations that result from the DNA editing event(s). Examples of such assays that identify biochemical features, phenotypic features, or genomic sequence alterations described above in reference to selections of haploid plants or propagules thereof having one or more DNA editing event(s) can also be applied to progeny of doubled haploid or polyhaploid plants having one or more DNA editing event(s).

Methods for detecting sequence alterations are well known in the art and include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis. Various methods used for detection of single nucleotide polymorphisms (SNPs) can also be used.

It will be appreciated that dependent on the type of alteration, biochemical or phenotypic assays can also be used to determine the presence of the editing event.

According to certain embodiments there is provided a gene-edited plantobtainable by the methods described herein.

According to certain embodiments there is provided a cell of the gene edited plant.

According to certain embodiments there is provided a seed, pollen or other propagule of the gene edited plant.

According to certain embodiments there is provided a non-regenerable processed plant product obtained from the gene edited plant, wherein the product comprises a detectable amount of gene-edited genomic DNA.

According to certain embodiments the product is a meal.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure can include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a gene editing agent" or "at least one gene editing agent" can include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format. For example, SEQ ID NO: XXX is expressed in a DNA sequence format (e.g. , reciting T for thymine), but it can refer to either a DNA sequence that corresponds to an XXX nucleic acid sequence, or the RNA sequence of an RNA molecule nucleic acid sequence. Similarly, though some sequences are expressed in a RNA sequence format (e.g. , reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Maize haploid inducer line based deployment of a single locus targeting gRNA based CRISPR system

Workflow overview

Plasmid design and construction;

Transformation into agrobacteria EHA105;

Agro based transformation into a haploid inducer (HI) line;

Selection of positive expressing HI donor line;

Pollination of target line;

Selection of haploid seed by a selection marker;

Diploidization of a haploid seed;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector. gRNA design and plasmid preparation:

For proof of concept purposes sequences are chosen according to the published literature (Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System. J Genet Genomics. 2016 Jan 20;43(l):37-43. Feng C. et al.). gRNA targeting GRMZM2G332562 (Hsg3) (accgataagcacagcagctgtgg, SEQ ID NO: 1), GRMZM2G080129 (Hsg4) (gatgtcttcatcatggatccagg, SEQ ID NO: 2), GRMZM2G170586 (Hsg6) (acgagagctgcaggcggccatgg, SEQ ID NO: 3), GRMZM2G438243 (Hsgl2) (cggcgtggcgccggagctcacgg, SEQ ID NO: 4) (Feng C. et. al. J Genet Genomics. 2016 Jan 20;43(l):37-43) are cloned into a B330 plasmid back bone having a Bar-intron selection marker under regulation of 2X 35S promoter sequences, a plant codon optimized Cas9 intron coding sequence under regulation of zmUbi promoter and a single gRNA expression cassette (Cong et al. Science. 2013 Feb 15;339(6121):819-23.) regulated by OsU6 promoter sequence. Plasmid transformation into agrobacterium

EHA105 agrobacterium is transformed by heat shock and grown on LB agar plates supplemented with 100 μg/ml spectinomycin for 2-3 days at 28 °C until colonies appearing positive colonies are selected and are frozen in -80 °C as glycerol stocks for further use. Transformed haploid inducer line establishment

A haploid inducer line [eg. Stock 6 (Coe, 1959), KMS and ZMS (Tyrnov and Zavalishina 1984), WS 14 (Lashermes and Beckert 1988), KEMS (Sarkar et al. 1994), MHI and M741H (Eder and Chalyk 2002), RWS (Rober et al. 2005), UH400 (Chang and Coe 2009), PK6 (Barret et al. 2008), HZI1 (Zhang et al. 2008), CAUHOI (Chen and Song 2003) and PHI (Rotarenco et al. 2010)1 is transformed according to the following protocol which is an adaptation from Frame B. et al (2002) Agrobacterium-mediated transformation of maize embryos is effected using a standard binary vector system. Plant Physiology 129: 13-22 and V. Sidorov and D. Duncan (2009), Agrobacterium- mediated maize transformation: immature embryos versus callus. In M. Paul Scott (ed.) Methods in Molecular Biology: Transgenic maize, vol. 526. pp47-58.

Agrobacterium Preparation

Plasmid transformed Agrobacterium are streaked out from glycerol stock on to YEP solid medium (5 g/L yeast extract, 10 g/L peptone, 5 g/L NaC12, 15 g/L Bacto- agar) supplemented with 100 μg/ml spectinomycin (S0692 sigma-aldrich, st. Louis MU) in a 100 x 15 mm petri dish to generate single colonies. Plates are sealed with Parafilm M and placed upside down in a 28 °C incubator for 3 days in the dark. Two days before inoculating calli, one colony of the Agrobacterium is picked from the plate and inoculated in 25 ml of liquid LB supplemented with 100 μg/ml spectinomycin in a 250 ml flask. The flask is placed on a shaker at 150 rpm in the dark and incubates in 26°C overnight. Next day a 1:5 dilution of the Agrobacterium culture in LB liquid media is made and incubated for additional 6 hours in the dark at 26 °C. Following incubation, the Agrobacterium culture is divided into 50 ml tubes, centrifuged at 3500 rpm for 15 minutes and resuspended in 10 ml of an infection medium containing freshly added AS ]N6 salts and vitamins, 1.5 mg/L 2,4-D, 0.7 g/L L-proline, 68.4 g/L sucrose and 36 g/L glucose, pH5.2), which is filter sterilized and stored at 4°C. Filtered sterilized acetosyringone (to a final concentration 100 μΜ)]. O.D. 660 is checked and adjusted to 0.2 with the infection medium. Thereafter, the resuspension flask is placed on a shaker at 150 rpm in the dark and incubated overnight at 26 °C. The next morning, agrobacterium cells are centrifuged and resuspended in 6-10 ml of the infection medium (freshly added with AS). OD660 is checked and adjusted to 0.5.

Inoculation and co-cultivation:

The HI list is: BHI 305 (IA SPI5), BHI 307, BHI 306 and BHI201 (BHI 102; IA-MAI) from Iowa State University (ISU).

10-12 days following fertilization ears are harvested, decontaminated in bleach for 30 min and washed in sterilized water. Embryos are grown for seven days post subculturing, on an infection medium approximately. 10 ml of established callus pieces are collected in 50 ml sterile centrifuge tubes and supplemented with an Agrobacterium solution sufficient to cover the tissue for 30 minutes at room temperature after which the Agrobacterium suspension is removed with a narrow bore 5 ml pipette. The inoculated cali are then dumped onto a sterile whatman filter paper (GE healthcare) to blot dry and transferred to a sterile 8.5 cm filter paper in a 100 x 25 mm Petri dishes and the dishes are placed in a 24 °C growth chamber for 2 days in the dark. Alternatively a non-desiccation cysteine treatment is done: the inoculated calli are dumped onto a filter paper to blot dry and transferred to a solid co-cultivation medium (N6 salts and vitamins, 1.5 mg/L 2,4-D, 0.7 g/L L-proline, 30 g/L sucrose, 3.0 g/L gelrite, pH 5.8. Post autoclaving add filter sterilized AgN03 to a final concentration of 0.85 mg/L, acetosyringone to a final concentration of 100 μΜ, and cysteine to a final concentration of 300 mg/L) and incubated in the dark at 20 °C for three days. Resting and Selection:

Following co-cultivation, calli are carefully transferred to a fresh non-selection Resting Medium [N6 salts and vitamins, 1.5 mg/L 2,4-D, 0.7 g/L L-proline, 30 g/L sucrose. 0.5 mg/L MES, 3 g/L gelrite, pH 5.8. Post autoclaving (when media is cooled) add filter sterilized carbenicillin to a final concentration of 200 mg/L, and AgN0₃ to a final concentration of 0.85 mg/L] for 7 days incubation at 28°C in the dark. Rested calli are then transferred to a selection Medium I [N6 salts and vitamins, 1.5 mg/L 2,4-D, 0.7 g/L L-proline, 30 g/L sucrose. 0.5 mg/L MES, 3 g/L gelrite, pH 5.8. Post autoclaving (when media is cooled) add filter sterilized Bialaphos to a final concentration of 1.5 mg/L, carbenicillin to a final concentration of 200 mg/L, and AgN0₃ to a final concentration of 0.85 mg/L] for two weeks incubation at 28°C in the dark and then transferred carefully to a Selection Medium II [N6 salts and vitamins, 1.5 mg/L 2,4-D, 0.7 g/L L-proline, 30 g/L sucrose. 0.5 mg/L MES, 3 g/L gelrite, pH 5.8. Post autoclaving (when media is cooled) add filter sterilized Bialaphos to a final concentration of 3.0 mg/L, carbenicillin to a final concentration of 200 mg/L, and AgN0₃ to a final concentration of 0.85 mg/L] for an additional selective incubation at 28°C in the dark. Two weeks later, the medium is replaced with new selection medium II plates for an additional two weeks incubation at 28 °C. Several weeks post-infection the transgenic callus is visible as rapidly growing healthy tissue. Regeneration and plant development

After selection, surviving healthy tissues are placed on regeneration medium I (MS salts and vitamins, 5.0 mg/L BAP, 0.25 mg/L 2,4-D, 30 g/L sucrose, 3g/L gelrite, and pH 5.8. Post autoclaving add (when media is cooled) add filter sterilized Bialaphos to a final concentration of 3.0 mg/L (glufosinate can also be substituted for the Bialaphos in this medium as well) carbenicillin to a final concentration of 200 mg/L) and incubate in low light for two week at 16 hour photoperiod. Germinating shoots are transferred to Regeneration Medium II (MS salts and vitamins, 100 mg/L myo-inositol, 30 g/L sucrose, 3 g/L gelrite, pH 5.8 ) and incubated at 16 hours photoperiod at 80 μΕιη^" V¹ until shoots are 3-5 mm in length. Once grown, shoots are transferred to a Shoot Elongation medium (SH salts and vitamins, 100 mg/L myo-inositol, 30 g/L sucrose, 2.5 g/L gelrite, pH 5.8) and incubated at 80 μΕι ^"28 until they reached the top of the tube. Healthy shoots are then transferred to the greenhouse for hardening and replanting to complete plant development.

Selection of positive transformed plants is done at the DNA level to validate that the plasmid sequence is stably integrated and in parallel on RNA samples to validate expression by PCR using the following primers; RNA vs. DNA. Bar_FWl ctcgtcgctgaggtggatg (SEQ ID NO: 5) Bar_REl gagaagtcgagctgccagaa (SEQ ID NO: 6) (amplicon size on DNA: 518bp and on cDNA: 329bp ) Cas9_CDS_FWl aagcagcgtaccttcgacaa (SEQ ID NO: 7) Cas9_CDS_REl ccgctgatctcgacagagtc (SEQ ID NO: 8) (amplicon size on DNA and cDNA: 545bp ) Cas9_Vec_FWl cgcagacgggatcgatctag (SEQ ID NO: 9) Cas9_Vec_REl aagagaagggcgccaatcaa (SEQ ID NO: 10) (amplicon size on DNA: 512bp and on cDNA: N/A).

DNA is extracted from the leaf tissue using crude DNA extraction protocol described in (Paris M and Carter M Plant Mol Bio Rep Dec. 2000, Voll8, Issue 4, pp 357-360). A small leaf tissue is placed in 40 μΐ_^ of ddH20 and crushed with forceps until the solution becomes green. Thereafter, 40 μΐ extraction solution (500 mM NAOH in ddH20) are added and mixed. A 5ul aliquot of the mix is then added in 40 μΐ of Neutralizing solution (80mM Tris, ImM EDTA) and further diluted by adding 120μ1 ddH₂0. after extraction Ιμΐ of the samples is used for PCR as template.

RNA is extracted from the leaf tissue using RNA Mini Kit (Plant) and according to manufacturer's protocol (Real Biotech Corporation). Following RNA extraction and quantification, cDNA is synthesized by Verso cDNA Synthesis Kit (Thermo Scientific) according to manufacturer's protocol: RNA template (^g) is added into an enzyme mix containing cDNA synthesis buffer, dNTP Mix RNA Primer (random hexamer), RT Enhancer and Verso Enzyme Mix and added with ddH₂0 to complete volume of 20ul. The reaction is then incubated at 42 °C for 30 min on a thermocycler followed by a 2 min. cycle at 95 °C for inactivation. 1 μΐ of the DNA template is used in the PCR reaction to compare with the results obtained from the genomic DNA sample.

Establishment of CRISPR donor line can also be accomplished through transformation of an easily transformable maize line (eg. B 104) following the agrobacterium transformation protocol described above and backcrossing to the inducer line and selecting for the transgene and the required color selection marker (eg Rl-nj) as described in Yu, W. & Birchler, J.A. Mol Breeding (2016) 36: 5. After 3 generations of backcrossing to the inducer line (BC3), progeny will optimally have -87.5 % of the genetic material homozygous from the recurrent backcrossing haploid inducer parent. Several BC3F1 clones are then selected and self- pollinated to achieve homozygousity of the CRISPR transgene. Selection for homozygousity is done by relative copy number variation based on melt data as described in (Zhou LI et al. Clin Chem. 2015 May;61(5):724-33.) using primers targeting the bar selection marker Bar cDNA_FW_l ACAAGCAGAAGAACGGCATC (SEQ ID NO: 11) and Bar cDNA_RE_l AGTTGCACGCTTCCATCTTC (SEQ ID NO: 12) and using the following PCR primers (867F TTTGGGCCTCCTCTGATCTTTC, SEQ ID NO: 13; 867R TTTCTTTGGCAGCGAAACCG, SEQ ID NO: 14) are used as reference for the copy number evaluation.

BC3F2 progeny will be then tested for haploid induction efficiency before continuing with molecular selection for the CRISPR edited genomic event. Deployment of CRISPR donor pollen material onto a target line:

Induction of haploidy/CRISPR editing on target is carried out in the field or greenhouse. Plants of the transformed haploid inducer and of the target germplasm are grown under conditions that allow plant growth, pollination, and maturity of seeds. The planting dates of inducer and target germplasm should be adjusted to synchronize pollen shedding of the inducer with silking of the source germplasm. Tassels of all source germplasm plants should be removed to avoid pollen contamination during pollination with the inducer. Before silk emergence, the target germplasm ear shoots are covered with shoot bags to avoid uncontrolled pollinations. During anthesis, the tassels of the inducer plants are covered before shedding with pollination bags to collect pollen. Pollen-filled bags collected from the inducer tassels are used to pollinate ears of target plants by covering the silk with the filled pollen bag and fastening with a stapler. After grains have reached physiological maturity, cross -pollinated ears of target germplasm are harvested, subjected to postharvest insecticide treatment, dried down to storage moisture content, and shelled for selection. For large-scale in vivo haploid/CRISPR induction, the plants are grown, inducers and target germplasm in alternate rows in an isolated block (the number of rows of inducer and source germplasm depends on the inducer's pollen shedding ability). When the tassel emerges on the target plants, the plants are detasseled such hat these are pollinated by wind with pollen of the inducer/donor pollen. This eliminates manual pollinations (Prigge V and Melchinger AE.Methods Mol Biol. 2012;877: 161-72.). Selection of haploid seed

Selection is done using the Rl-nj marker system which is carried in this case by the inducers. Seeds are selected according to the following criteria, haploid seeds with unpigmented (haploid) embryo and purple-colored (triploid) endosperm, compared to normal Fl seeds which have a purple-colored (diploid) embryo and a purple-colored (triploid) endosperm and completely unpigmented seeds that can originate from a random outcross.

Chromosome Doubling

For chromosome doubling, selected haploid seeds are germinated in a labeled germination tray with a wet filter-paper inlay under controlled conditions in darkness with an adequate moisture supply. When coleoptiles of the seedlings are about 2 cm long, seedlings are prepared for colchicine treatment by taking each seedling individually from the tray and cutting off a few millimeters of the tip of its coleoptile with a scalpel or razor blade. After cutting, the seedling are placed into a mesh bag and sunk in a colchicine treatment container. The container is then filled with the colchicine solution (0.06 % Colchicine, 0.5 % DMSO in deionized water) until all seedlings are well covered for 8 h at room temperature. After the treatment, the seedlings are rinsed with tap water three times to remove residual colchicines and planted in pots for about 10 days so that they recover from the colchicine treatment and grow to the three or four- leaf stage during this period. Conditions are maintained favorable for seedling growth. Putative diploid plants are grown and self pollinated and selected for the genomic CRISPR edited event by PCR.

PCR for selection of CRISPR dependent event

Leaf tissue from putative dihaploid plants is collected and DNA is extracted as described above (Paris M and Carter M Plant Mol Bio Rep Dec. 2000, Voll8, Issue 4, pp 357-360). The samples are run by PCR using the following primers H3F gtgcttgccaatttcgactc (SEQ ID NO: 15) and H3R attcgtggtactgctgctca (SEQ ID NO: 16) for Hsg3 gRNA; H4F ggagcggttatgccccaaag (SEQ ID NO: 17), H4R gcaagttcgcgggcaagatc (SEQ ID NO: 18) for Hsg4 gRNA; H6F gccgcgtccctttgtttga (SEQ ID NO: 19) and H6R gggcaaataatggagggctg (SEQ ID NO: 20) for Hsg6 gRNA and H12F cgcgtggaagagggagaaag (SEQ ID NO: 21) and H12F ccaaccagcaaatgcacaac (SEQ ID NO: 22) for Hsgl2 (Feng C. et. al, J Genet Genomics. 2016 Jan 20;43(l):37-43). Following amplification, induced indels are detected by Surveyor® Mutation Detection Kit (IDT- Integrated DNA Technologies, Inc.) (Methods Mol Biol. 2010; (649):247- 56.) by multiplexing PCR products prepared from samples and a wild type reference and treating the mix heteroduplex/homoduplex with Surveyor Nuclease. After the enzymatic reaction, the samples are analyzed by agarose gel to compare cut amplicon (positive - having mismatched sequence) to an uncut reference.

In parallel PCR is conducted to verify CRISPR plasmid is not present in the positive plants using primers Bar_FWl ctcgtcgctgaggtggatg (SEQ ID NO: 5) Bar_REl gagaagtcgagctgccagaa (SEQ ID NO: 6) (amplicon size on DNA: 518bp) Cas9_CDS_FWl aagcagcgtaccttcgacaa (SEQ ID NO: 7) Cas9_CDS_REl ccgctgatctcgacagagtc (SEQ ID NO: 8 (amplicon size on DNA) Cas9_Vec_FWl cgcagacgggatcgatctag (SEQ ID NO: 9) Cas9_Vec_REl aagagaagggcgccaatcaa (SEQ ID NO: 10) (amplicon size on DNA: 512bp) as previously described. EXAMPLE 2

Maize CENH3-tailswap complemented haploid inducer line based deployment of a single locus targeting gRNA based CRISPR system

Workflow overview

Establishment of a CENH3 complemented inducer line by expressing CenH3- tailswap followed by knockout of endogenous CENH3;

Plasmid design and construction;

Transformation into agrobacteria EHA105;

Agro based transformation into a CENH3 Haploid Inducer line;

Selection of positive expressing HI donor line; Pollination of target line/use of target to pollinate donor line (use as maternal or paternal haploid inducer); Selection of haploid seeds/plants by selection marker/phenotype;

Diploidization of haploid seed;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector.

A detailed protocol is found in "Generation of haploid plants" WO 2014110274, and US 9,215,849 which are both hereby incorporated by reference in its entirety.

The protocol is similar to Example 1 with differences in the starting haploid inducer/donor material which can be potentially any given maize line (eg. B 104) as described in (Kelliher T et al.Front Plant Sci. 2016; 7: 414.)

EXAMPLE 3

Interspecies crossing - Wheat (or Barley, Rye) - Maize intercross delivery of editing system via haploid embryo

Workflow overview

Plasmid design and construction;

Transformation into agrobacteria EHA105;

Agrobacterium based transformation into a maize line;

Selection of positive expressing maize pollen donor line;

Pollination of target wheat line;

Embryo rescue of haploid embryos - optional GFP or other selection method to identify haploid embryo;

Diploidization of Haploid embryos;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector.

CRISPR gRNA sequence to target wheat inositol oxygenase (inox) is taken from (G3 (Bethesda). 2013 Dec; 3(12): 2233-2238.)

ta_inox 1 AGACGTACGAGTTTGTGCAGCGG (SEQ ID NO: 23) ta_inox 2 CAAGACGGAGATGAGCATCTGGG (SEQ ID NO: 24). Selection primers for validating the editing event by Surveyor® Mutation Detection Kit (IDT- Integrated DNA Technologies, Inc.) or the T7 nuclease assay using the following primers: ta_inox_for CAGGGACTACGACGCGGAG (SEQ ID NO: 25) and ta_inox_rev GGATGGCCTCGGCGGTTTG (SEQ ID NO: 26).

Establishment of maize pollen CRISPR stably transformed donor material is done according to the agrobacterium transformation protocol described above (Example 1) with the change of the germplasm used is not a haploid inducer line but a transformable maize line (eg. A188, B 104). Validation of transformation event and expression is performed also as described in Example 1.

The established maize pollen donor and the target wheat germplasm (eg. 'Chinese Spring') are planted in a timely manner to synchronize fresh pollen shedding of the maize donor/inducer with flower development of the wheat germplasm (protocol in Pak. J. Bot, 38(2): 393-406, 2006). This protocol claims to have 100% efficiency in haploid induction. Basic protocol from Hereditas Volume 116, Issue Supplement si, pages 117-120, June 1992.

At ear emergence, wheat tillers with spikes approaching the conventional emasculation stage (3-5 days prior to anthesis) are cut off at the base of the growing plant and cultured in a flask with tap water. Spikes are then either manually emasculated by opening the lemma and palea and removing the anthers or alternatively, treated with hot-water (43 °C for 3 minutes) to induce emasculation. After pollination with fresh maize donor pollen on the emasculated spikes the tillers are cultured for 4 days in a solution containing 40 g/1 sucrose, 8 ml/1 sulfurous acid (6% S02) and 100 mg/1 2,4-D. After 4 days, the tillers are transferred to a solution containing only sucrose and sulfurous acid until ready for embryo rescue after about 10 to 12 days i.e., 15-17 days after pollination. The procedures up to this stage are conducted under glasshouse- controlled regimes as described earlier. 15-17 days after pollination, immature embryos are aseptically excised from all seed set on the wheat spikes, and transferred onto half- strength Murashige & Skoog (MS) culture medium supplemented with 20 g/1 sucrose and 6 g/1 agarose. Selection of only those enlarged seed of a spike that have an embryo is done using the 'inverted light technique' (Bains et. al. Plant Breeding 117, 191— 192 (1998)) by placing a light source (60 W bulb) above the seeds which makes the embryos within them visible when viewed from below. The rescued embryos are incubated at 25 °C, 12-hr day length and approximately 5000 lux light intensity. Haploid seedlings regenerated from embryos duplication are achieved by treating the seedlings with colchicine solution [0.05% colchicine, 2% dimethyl sulfoxide (DMSO), 15 drops/1 Tween-20], incubating for 12-15 h in darkness at room temperature, after which the seedling are rinsed with water, and transferred to soil. Plants are then grown to maturity and validated by PCR for gene editing event employing the Surveyor® Mutation Detection Kit (IDT- Integrated DNA Technologies, Inc.) and using primers flanking the editing event: ta_inox_for CAGGGACTACGACGCGGAG (SEQ ID NO: 23) and ta_inox_rev GGATGGCCTCGGCGGTTTG (SEQ ID NO: 24). Positive plants are self pollinated and propagated.

Example 4

Arabidopsis CENH3-tailswap complemented inducer line CRISPR

deployment system of a single gene

According to "Generation of haploid plants and improved plant breeding" US 20140090099 and US 9,215,849, which are hereby incorporated by reference in their r entireties.

Workflow overview

Similar to that described in example 2 with the following exceptions:

Establish a CENH3 inducer line on a selected crop (eg. Arabidopsis thaliana tomato, strawberry and chickpea ) transformed with the target CRISPR;

Cross inducer with desired target to induce haploids;

Select for haploid and duplicate genome to restore diploidy;

Select for the desired CRISPR edited event carrying individual.

CENH3 protocol for Arabidopsis thaliana

Based on a protocol described by Ravi Maruthachalam and Simon Chan 1. (Ravi, M. and Chan, S.W.L. Nature, 464, 615-618 (2010).

Haploid inducer

The haploid inducer is a cenh3-l null mutant in the Col-0 ecotype that is complemented by a CENH3 transgene called GFP-tailswap (cenh3-l is embryo lethal). It has a vegetative phenotype that varies in intensity depending on growth conditions. Rosette leaves are slightly curled, and it also has shorter internodes after bolting. The mutant is mostly male sterile, but has reasonable female fertility (about 60-70 % of wild type in our hands). It can be maintained as a homozygote because it yields a few hundred seed per plant if treated carefully. Later siliques (seed capsule) of 2 fused carpels with the length being more than three times the width )have higher fertility. Importantly, a majority (90%) of the offspring are diploids similar to the parent plant.

Low frequency of aneuploid offspring are observed, which can be easily distinguished from diploid siblings by their distinctive phenotypes. Seed collected from cenh3-l/+ GFP-tailswap/GFP-tailswap plants are available from ABRC and from NASC (stock number CS66982). A quarter of the population have the desired cenh3-l GFP-tailswap.

Genotyping the cenh3-l mutation and the GFP-tailswap transgene

Cenh3-1 is a point mutation in the CENH3 gene (also known as HTR12). The mutation is G161A relative to ATG = +1. The mutation can be identified using the following dCAPS primers:

Primer 1: GGTGCGATTTCTCCAGCAGTAAAAATC (SEQ ID NO: 27), Primer 2: CTGAGAAGATGAAGCACCGGCGATAT (SEQ ID NO: 28), total amplicon length: 215bp, dCAPs restriction polymorphism with EcoRV cutting only the WT allele to 191 bp and 24bp fragment which is identified by running the digested PCR products in a 2.5 % gel. GFP-tailswap is a Hyg marked transgene on chromosome 1 (identified by TAIL PCR) that complements the embryo-lethal phenotype of cenh3-l. It has the native CENH3 promoter and terminator, a N-terminal GFP tag, the N-terminal tail domain of histone H3.3, the C-terminal histone fold domain of CENH3.

The cenh3-tailswap is genotyped with the following primers: Primer 1 for wild type and T-DNA : CACATACTCGCTACTGGTCAGAGAATC (SEQ ID NO: 29), Primer 2 for wild type only: CTGAAGCTGAACCTTCGTCTCG (SEQ ID NO: 30), Primer 3 for the T-DNA: AATCCAGATCCCCCGAATTA (SEQ ID NO: 31), WT (Primer 2 + Primer 1): 518 bp TDNA (Primer 3 + Primer 1) = ~450bp.

Establishing an Arabidopsis CENH3 CRISPR stable donor:

Once established as a CENH3-tailswap complemented inducer the line is transformed with a CAS9/gRNA of choice. As an example gRNA targeting 3 genes are chosen (described in Xing HL et al., BMC Plant Biology201414:327) TRY gRNA: AGAGGAGATAGAGAGATATTGG (SEQ ID NO: 32); CPC gRNA: GAGGAGATAGAGAGATATTGG (SEQ ID NO: 33); ETC2 gRNA: GAAGTGAGTAGCATCGAATGGG (SEQ ID NO: 34).

Transformation of Arabidopsis plants are performed on by floral-dip protocol

(Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J 16:735-43).

Arabidopsis plants are grown until they are flowering. Growing is done under long days in pots in soil covered with cheesecloth. In order to encourage proliferation of many secondary bolts first bolts clipping is recommended. Plants are ready in roughly 4-6 days after clipping. Optimally plants that have many immature flower clusters and not many fertilized siliques are used.

Agrobacterium tumefaciens strain LBA4404 carrying the crispR expression plasmid is prepared by growing a large liquid culture at 28 °C in LB supplemented with antibiotics (eg spectinomycin 10(Vg/ml) to select for the binary plasmid. For optimal transformation mid-log culture is harvested, centrifuged 3500RPM for 15min and resuspend to OD600 = -0.8 in 5% Sucrose solution. Before dipping, Silwet L-77 is added to a concentration of 0.05% (500 ul/L) and mixed well. If there are problems with L-77 toxicity, a concentration of 0.02% or as low as 0.005% is used. Above-ground parts of plants are then in Agrobacterium solution for 2 to 3 seconds, with gentle agitation. After dipping plants are placed under a cover for 16 to 24 hours to maintain high humidity and should not be exposed to excessive sunlight. Following initial incubation plants are watered and grown normally until seeds mature and dried after which they are harvested.

Selection for transformants is done using antibiotic or herbicide selectable marker (eg kanamycin). For example, vapor-phase sterilize and plate 40 mg = 2000 seed (resuspended in 4 ml 0.1% agarose) on 0.5X MS/0.8% tissue culture Agar plates with 50 ug/ml Kanamycin, cold treat for 2 days, and grow under continuous light (50- 100 μΕϊηβίεϊηβ) for 7-10 days.

After selection putative transformants are transplanted in soil to grow, and validate by PCR on DNA and RNA level for expressing clones utilizing NPTII targeting primers NptII_FWl CGTGAAGACTGACCTCTCCG (SEQ ID NO: 35) and NptII_REl GGTCATCCTGGTCCACAAGG (SEQ ID NO: 36) with expected amplicon length of 518bp on the genomic template and 329bp on the RNA/cDNA template, Cas9 primers described on example can also be utilized for this purpose with no difference in amplicon size Cas9_CDS_FWl aagcagcgtaccttcgacaa (SEQ ID NO: 7) Cas9_CDS_REl ccgctgatctcgacagagtc (SEQ ID NO: 8) (amplicon size on DNA) Cas9_Vec_FWl cgcagacgggatcgatctag (SEQ ID NO: 9) Cas9_Vec_REl aagagaagggcgccaatcaa (SEQ ID NO: 10) (amplicon size on DNA: 512bp) as previously described.

Crossing the haploid inducer donor with a target line

Haploid Arabidopsis are produced by crossing the haploid inducer to a selected target line. In the fertilized zygote, chromosomes from the mutant parent are eliminated at a high frequency, resulting in a substantial fraction of haploid plants in the Fl that carry only chromosomes from the wild type parent. The haploid inducer is used as a male or a female parent in a cross. If used as a female parent in a cross, then entire female genome is eliminated in the resultant zygote and the haploids are purely paternal in origin. On the other hand, if it is used as a male parent, the paternal genome is eliminated following fertilization and thus the haploids are maternal in origin.

Although it is possible to generate either maternal or paternal haploids, the frequency of haploids in the Fl is higher when the haploid inducer plant is used as the female parent. As the mutant is mostly male sterile, no emasculation is done before pollination. There are a lot of aborted seed (-80 %) if the haploid inducer is fertilized by wild types. Note that the procedure above yield haploid plants in which the cytoplasm is derived from Col-0. Crossing the haploid inducer as the male is also possible as some flowers have reasonable amounts of viable pollen. To identify the fertile flowers a dissecting scope is used for the crosses to turn up the magnification to the point where pollen being deposited on the stigma is visible, using >10 haploid inducer flowers per target plant stigma can get 20-50 seeds per cross.

Selecting haploids in the Fl of a cross to the haploid inducer.

Among viable plants in the Fl four types of plants are detectible, diploid hybrids, aneuploidy hybrids (with >10 chromosomes), self-fertilized plants (rare, these can occur if emasculation is not done) and haploids. When the haploid inducer is the male in a cross, we observe mostly diploid hybrids, -5% haploids and a very low frequency of aneuploids. On the other hand, if the haploid inducer is used a female parent in a cross, 25-50% haploids can be obtained, 25-50% are aneuploids and 25% are diploid hybrids.

To increase germination seeds are plated on Murashige and Skoog medium (MS4.33g (Sigma M5519), 20 g sucrose, Bacto agar 7g, adjusted the pH to 5.7 with 2 N KOH per 1 L final volume H20.) plates, Late germinating seeds can be more likely to be haploid, so all the seed that germinate are transfered to soil. Haploid Arabidopsis are readily distinguished irrespective of ecotype. Rosette leaves are somewhat smaller than diploid, especially early in development. Leaves are also narrower than diploids. Haploids are vigorous, and these differences may diminish later on. The most obvious phenotypes are seen after bolting: flowers are very small (following the general pattern tetraploid > triploid > diploid > haploid), and the plants are sterile.

Aneuploids in Arabidopsis generally have distinctive developmental phenotypes that are more severe than haploids. In a large population very similar phenotypes represent the 5 trisomies are visible - a pale green phenotype, a brassinosteroid-like phenotype, a very sick dark green phenotype e.t.c. Haploids are more like diploids than all of the aneuploids in terms of vegetative phenotype.

In the absence of a phenotypic marker (e.g. recessive marker from wild type parent), the easiest way to pick out the desired haploids is to wait until bolting and pick the sterile individuals. Diploid and aneuploid hybrids are fertile.

Doubling haploids to create diploids.

Haploid plants are treated with colchicine before or after bolting (before is better) by preparing a solution of 0.25% colchicine, 0.2% Silwet and applying 20μί on the meristem. The meristem will appear to die, and the plant may become very sick. After it recovers, you will find fertile inflorescences that yield diploid seeds.

We routinely collect >50-500 diploid seeds from each haploid plant. As haploids produce very little pollen, it is easy for them to outcross to other plants in the room. To avoid this, it is recommend growing haploids apart from fertile diploid Arabidopsis thaliana.

Following genome doubling a selecting of the desire genome editing event is done using for example primers described in Xing HL et al., BMC Plant Biology201414:327 for the gRNA's mentioned above and applying the Surveyor® Mutation Detection Kit (IDT- Integrated DNA Technologies, Inc.) described in previous examples. For the CPC target region: CPC-IDF GGTCTAACTTACCGAGCTGTCAATG (SEQ ID NO: 37) and CPC-IDR CAAAATAGTAATTCAAGGACAGGTACAT (SEQ ID NO: 38) ; For the ETC2 target region: ETC2-IDF CAGTAGTTATGGATAATACCAACCGTCT (SEQ ID NO: 39) and ETC2-IDR ATCAGCTTTGATTTGTTACTCTCGCCAT (SEQ ID NO: 40) and For the TRY target region: TRY-IDF ATGTACAGACTTGTCGGTGATAGGT (SEQ ID NO: 41) and

TRY-IDR GTCTCATGGATTCGTTGTATAGCGT (SEQ ID NO: 42)

References for cenh3 system in Arabidopsis:

1. Ravi, M. and Chan, S.W.L. Haploid plants produced by centromere- mediated genome elimination Nature, 464, 615-618 (2010)

2. Ravi, M., Kwong, P.N., Menorca, R.M.G., Valencia, J.T., Ramahi, J.S., Stewart, J.L., Tran, R.K., Sundaresan, V., Comai, L. and Chan, S.W.L.

3. The rapidly evolving centromere- specific histone has stringent functional requirements in Arabidopsis thaliana Genetics, 186, 461-471 (2010)

Example 5

Targeting repetitive elements using CRISPR based induction system for delivery by a haploid inducer donor line in maize

Workflow overview:

Plasmid design and construction for a repeat sequence targeting gRNA/s;

Transformation into agrobacteria EHA105;

Agro based transformation into a haploid inducer (HI) line;

Selection of positive expressing HI donor line;

Pollination of target line;

Selection of haploid seed by a selection marker;

Diploidization of a haploid seed;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector Workflow in this example is similar to described in example 1 utilizing a CRISPR system gRNA targeting a repeat sequence which targets multiple locations in the genome and can potentially induce "random" structural variations (eg large deletions, inversions, insertions and translocations) as well as small localized indels.

Selection of maize repeat sequence targeting gRNA's is done by running gRNA design tool on sequences downloaded from the maize repeat sequences database (www.maize(dot)jcvi(dot)org/repeat_d(dot)shtml) followed by filtering for number of potential targets for each sequence. gRNA design for random targets

In order to find gRNAs which appear multiple times in the genome, we took the Zea_mays annotated repeats v3.0 from www.maize(dot)jcvi(dot)org/ or from plant repeat database www.plantrepeats(dot)plantbiology(dot)msu(dot)edu/downloads(dot)html repeat regions are mapped the to the Zea_mays.AGPv3.22 genome. The mapped results are summarized to get the number of hits for each repeat and filtered for repeats having with hits on all 10 chromosomes.

We use an in-house script, based on the CasOT tool (Xiao et al. Bioinformatics. 2014 Jan 21.) to design gRNAs to the selected repeat sequences, and find their targets and off-targets. We used the following settings: Maximum 2 mismatches in the seed, maximum 2 mismatches in the non-seed, PAM = NGG, require a G at the 5' position, length of the protospacer is 17-20bp (not including the PAM).

(CasOT parameters: -m=target -s=2 -n=2 -p=A -r=yes -1=17-20)

Next, we use additional in-house scripts to find how many targets/off-targets overlap genes (using gene annotation Zea_mays.AGPv3.22) and to filter the gRNAs found. The basic criteria for gRNA selection is 50-200 total targets (including off- targets) and 0-2 target genes (preferably 0 to avoid knocking out specific genes). For each selected gRNA, one target is chosen and primers were designed using Primer3 tool for its flanking region to enable basic validation of crispR activity using Surveyor® Mutation Detection Kit.

Exemplary repeat sequence targeting multiple loci in the maize genome that was designed: 8917_1509f GACGACTCTATTACAAGAAGGGG (SEQ ID NO: 43) with primers used for basic evaluation 8917_1509f_10_L CCTTGGAAGAGTTCTCTCCTTGAC (SEQ ID NO: 44) and 8917_1509f_10_R GTAGTGTCTTGGCAGATGTCGTAG (SEQ ID NO: 45)

To avoid potential genome instability caused by the CRISPR system on the haploid inducer donor line the binary vector is designed so the Cas9 expression cassette is regulated by an inducible promoter eg. GST-27 promoter induced by safener application (US Patent No. US5965387).

Alternatively the gRNA and the Cas9 expression cassettes are cloned on two separate expression vectors, the gRNA under U6 constitutive promoter and the Cas9 under pollen specific promoter regulation (eg. Zmgl3 or Lat56 US Patent No. 5412085), and transformed individually to an HI line. Prior to pollination of target lines the HI donors are hybridized to get a single pollen donor having both components of the crispR system expressed together in the pollen limiting the DNA damage induction to the target embryo. This system may increase haploid induction efficiency in comparison to an irradiated pollen system

After haploid induction, selection and genome duplication are performed as described previously in example 1. PCR will is performed to evaluate crispR activity on a selected target representing crispR genome editing potential by the Surveyor® Mutation Detection Kit assay or other mismatch detection assay using flanking region primers for example: 8917_1509f_10_L CCTTGGAAGAGTTCTCTCCTTGAC (SEQ ID NO: 44) and 8917_1509f_10_R GTAGTGTCTTGGCAGATGTCGTAG (SEQ ID NO: 45)

To complete variation induction evaluation plants are planted in the field and selected by phenotype.

Example 6

Targeting repetitive elements using CRISPR system for delivery by a haploid inducer donor line in Arabidopsis (CENH3-tailswap complemented HI line) Workflow overview

Establishment of a selected CENH3 mutant/CENH3-tailswap complemented line in Arabidopsis.

Plasmid design and construction for a repeat sequence targeting gRNA/s;

Transformation into agrobacteria LB4404;

Agrobacterium based transformation into a CENH3 haploid inducer (HI) line;

Selection of positive expressing HI donor line;

Pollination of target line/use of target to pollinate donor line (use as maternal or paternal haploid inducer);

Selection of haploid seeds/plants by selection marker/phenotype;;

Diploidization of a haploid seed;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector

Repeat sequence design is done based on the described in example 5. Exemplary gRNA sequences that are used : 1457_27f GGGATCCGGTGCATTAGTGCTGG (SEQ ID NO: 46) and 1739_296f GGGTTTAGAAAGTACGATTAGGG (SEQ ID NO: 47), and Plasmid construction can utilize an inducible promoter to regulate Cas9 expression and (Borghi, L. Inducible gene expression systems for plants. Methods in molecular biology. 655, (2010), 65-75.).

Following plasmid construction and transformation into LB4404 strain of agrobacterium the transformed bacteria is used to transform a pre-established CENH3 Haploid inducer line described in example 4 using the procedure described by Clough SJ and Bent AF, 1998. Plant J 16:735-43 and also described in detail in example 4.

After selection putative transformants are transplanted in soil to grow, PCR validation on DNA and RNA level for expressing clones is performed utilizing NPTII and CAS 9 targeting primers described in example 4. Haploid induction process using the HI line as paternal or maternal donor, selection of haploids and induced diploidization is also done as described below on example 4.

Selection of CrispR active clones is done using the Surveyor® Mutation Detection Kit assay utilizing primers targeting an amplicon flanking a selected target site: for gRNA 1457_27f: 1457_27fL GAATTCCTCGTGTTGCATCC (SEQ ID NO: 48) and 1457_27fR AAACAGAGGGATGCAACACG (SEQ ID NO: 49) and for gRNA 1739_296f: 1739_296fLCCTTCACCCCCTTATATTCACC (SEQ ID NO: 50) and 1739_296f_l_2_R GCCACAACTATTGAACGAAGC (SEQ ID NO: 51).

Once validated for activity, the plants are grown to evaluate phenotypical characteristics and selected accordingly.

Example 7

Targeting repetitive elements using CRISPR based induction system for delivery by a maize haploid inducer donor line hybridized by wheat or barley

Workflow overview

Plasmid design and construction for a repeat sequence targeting gRNA/s;

Transformation into agrobacteria EHA105;

Agrobacterium based transformation into a maize line;

Selection of positive expressing maize pollen donor line;

Pollination of target wheat line;

Diploidization of Haploid embryos;

Molecular selection of CRISPR edited event containing plant;

Validation of absence of CRISPR expressing vector.

Repeat sequence design is done based on the described in example 5. Using the wheat genome draft rWGSC CSS 1.0 with design strategy similar to described in example 6. Selected repeat sequence targeting gRNA which were chosen as an example are: 885_127f GGCCCAATAATAAGGTGATGTGG (SEQ ID NO: 52) and 717_143f GCATCTCGGAGTTGGAATATAGG (SEQ ID NO: 53). CrispR expression binary vector is constructed similar to what is described in example 1 with an optional Cas9 promoter selected from an either constitutive expression (eg. zmUbi promoter) inducible (eg GST-27 promoter) pollen specific (Zmgl3 or Lat56 from maize) or wheat specific.

Wheat haploid induction is done using stably transformed maize pollen, embryo rescued, selected and duplicated according to the procedures described on example 2.

Resulting dihaploid wheat plants are then evaluated by genome editing evaluation assay (eg. Surveyor kit or T7 nuclease mutation assay) for a representative crispR edited site using the following flanking region targeting primers: for gRNA 885_127f primers are used 127f_L TTACAAGAGCTCGTGCATGC (SEQ ID NO: 54) and 127f_R CATGGGTGCAAGTTATCGAC (SEQ ID NO: 55); for gRNA 717_143f primers 143f_L ATGATCGGTGAGCCAATGAG (SEQ ID NO: 56) and 143f_R CCAGTGTGATGCCCAATATG (SEQ ID NO: 57) are used.

Although the preceding Examples serve to illustrate specific embodiments of this disclosure, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting. REFERENCES

(other references are cited in the document)

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20. Coe, E. H. A line of maize with high haploid frequency. Am. Nat. 3, 381-382 (1959).

21. Paris M, Carter M: Cereal DNA: a rapid high-throughput extraction method for marker assisted selection. Plant Molecular Biology Reporter. 2000, 18: 357- 360.

22. Prigge V, Melchinger AE. Production of haploids and doubled haploids in maize. Methods Mol Biol. 2012;877: 161-72.

Claims

WHAT IS CLAIMED IS:

1. A haploid inducer plant line genetically modified with a nucleic acid molecule encoding a DNA editing agent.

2. A method of genetically modifying a haploid inducer, the method comprising genetically modifying said haploid inducer plant with a nucleic acid molecule encoding a DNA editing agent, thereby genetically modifying the haploid inducer.

3. A method of generating a haploid of a target plant, the method comprising crossing a haploid inducer plant genetically modified with a nucleic acid molecule encoding a DNA editing agent with a target plant of interest, thereby generating a haploid plant.

4. A method of genomically multiplying chromosomes of a target plant having a DNA editing event of interest, the method comprising treating said haploid target plant generated according to said method of claim 3 to a chromosome doubling agent, thereby generating a double haploid or polyhaploid target plant having said DNA editing event of interest.

5. A method of breeding, the method comprising crossing said double haploid or polyhaploid target plant having said DNA editing event of interest generated according to the method of claim 4 with a plant of interest or selfing said double haploid or polyhaploid target plant having said DNA editing event generated according to the method of claim 4.

6. A cell of the plant of claim 1.

7. A seed or other propagule of the plant of claim 1.

8. A pollen of the plant of claim 1.

9. The method of any one of claims 3-4, wherein said plant is a maternal haploid.

10. The method of claim 2, further comprising recovering said haploid inducer plant containing said nucleic acid molecule encoding said DNA editing agent.

11. The method of claim 3, further comprising recovering a haploid progeny of said target plant following said crossing.

12. The method of claim 11, wherein the recovered haploid progeny plant is a haploid progeny plant having the DNA editing event.

13. The method of claim 4 further comprising recovering said double haploid or polyhaploid target plant following said treating.

14. The method of claim 13, wherein the recovered doubled haploid or polyhaploid progeny plant having the DNA editing event.

15. The plant, method, cell, seed, pollen or ovule of any one of claims 1-9, wherein said nucleic acid molecule encoding said DNA editing agent is integrated in said genome of said inducer plant.

16. The plant, method, cell, seed, pollen or ovule of any one of claims 1-15, wherein said DNA editing agent is expressed in said inducer plant in an inducible manner or developmentally regulated manner.

17. The plant, method, cell, seed, pollen or ovule of any one of claims 1-3, 4- 16, wherein said inducer plant is an inducer line.

18. The method of any one of claims 2, 3, 4-5, 9-17, wherein said genetically modifying comprises transforming said inducer plant or plant line with said nucleic acid molecule encoding said DNA editing agent.

19. The method of any one of claims 2, 3, 4-5, 9-16, wherein said genetically modifying comprises crossing said inducer plant or plant line with a plant comprising said nucleic acid molecule encoding said DNA editing agent and selecting a genetically modified inducer plant comprising said nucleic acid molecule encoding said DNA editing agent.

20. The method of any one of claims 3-5, 9-19, wherein said inducer plant is of a different species of said target plant.

21. The method of any one of claims 3-5, 9-19, wherein said target plant is an inbred line.

22. The method of any one of claims 3-5, 15-20, further comprising selecting for said haploid plant following said crossing said inducer plant comprising said nucleic acid molecule encoding said DNA editing agent with said target plant of interest.

23. The method of claim 22, wherein said selecting is performed using a marker.

24. The method of any one of claims 3-5, 15-23, further comprising validating presence of a DNA editing event induced by said DNA editing agent in said haploid plant, target plant or progeny thereof.

25. The method of any one of claims 3-5, 15-24, further comprising validating absence of said nucleic acid molecule encoding said DNA editing agent in said target plant or progeny thereof.

26. The plant, method, cell, seed, pollen or ovule of any one of claims 1-25, wherein said DNA editing agent is directed to a target sequence of interest.

27. The plant, method, cell, seed, pollen or ovule of any one of claims 1-24, wherein said nucleic acid molecule comprises a gene or an expression cassette.

28. The plant, method, cell, seed, pollen or ovule of any one of claims 1-25, wherein said DNA editing agent is directed to a plurality of target sequences of interest.

29. The plant, method, cell, seed, pollen or ovule of any one of claims 1-28, wherein said DNA editing agent is directed to an endogenous sequence in a target plant.

30. The plant, method, cell, seed, pollen or ovule of any one of claims 1-25, wherein said DNA editing agent is directed to an exogenous sequence in a target plant.

31. The plant, method, cell, seed, pollen or ovule of any one of claims 1-30, wherein said DNA editing agent does not induce an editing event in said inducer plant.

32. The plant, method, cell, seed, pollen or ovule of any one of claims 1-31, wherein said DNA editing agent is directed to a sequence selected from the group consisting of coding sequence, splice junction, miR binding sequence and a regulatory sequence.

33. The plant, method, cell, seed, pollen or ovule of any one of claims 28-29, wherein said endogenous sequence or plurality of sequences comprises a genomic repeat sequence.

34. The plant, method, cell, seed, pollen or ovule of any one of claims 1-33, wherein a DNA editing event induced by said DNA editing agent is selected from the group consisting of a deletion, insertion, insertion-deletion (Indel), inversion and substitution.

35. The plant, method, cell, seed, pollen or ovule of any one of claims 1-33, wherein a DNA editing event induced by said DNA editing agent comprises a sub- chromosomal structural variation.

36. The plant, method, cell, seed, pollen or ovule of any one of claims 1-33, wherein said DNA editing agent is expressed under the control a pollen specific promoter.

37. The plant, method, cell, seed, pollen or ovule of any one of claims 1-36, wherein said DNA editing agent is selected from the group consisting of meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR.

38. The cell, seed, propagule, or pollen of any one of claims 6, 7, or 8, wherein the nucleic acid molecule encoding the DNA editing agent is integrated into the genome of the cell, seed, propagule, or pollen.

39. A haploid inducer plant line genetically modified with a nucleic acid molecule encoding a DNA editing agent directed to a genomic repeat sequence endogenous to a target plant of interest.

40. A method of genetically modifying a haploid inducer, the method comprising genetically modifying said haploid inducer plant with a nucleic acid molecule encoding a DNA editing agent directed to a genomic repeat sequence endogenous to a target plant of interest, thereby genetically modifying the haploid inducer.

41. A method of generating a haploid of a target plant, the method comprising crossing a haploid inducer plant genetically modified with a nucleic acid molecule encoding a DNA editing agent with a target plant of interest, thereby generating a haploid plant, wherein said DNA editing agent is directed to a genomic repeat sequence endogenous to said target plant of interest.

42. A method of genomically multiplying chromosomes of a target plant having a DNA editing event of interest, the method comprising treating said haploid target plant generated according to said method of claim 41 to a chromosome doubling agent, thereby generating a double haploid or polyhaploid target plant having said DNA editing event of interest.

43. A method of breeding, the method comprising crossing said double haploid or polyhaploid target plant having said DNA editing event of interest generated according to the method of claim 42 with a plant of interest or selfing said double haploid or polyhaploid target plant having said DNA editing event generated according to the method of claim 42.

44. A cell of the plant of claim 39.

45. A seed or other propagule of the plant of claim 39.

46. A pollen of the plant of claim 39.

47. The method of any one of claims 41-42, wherein said plant is a maternal haploid.

48. The method of any one of claims 2-5, 9-37, 41-43, further comprising phenotypic selection of a desired feature resulting from a DNA editing event by said DNA editing agent.

49. The method of claim 48, wherein said phenotypic selection is following said treatment with said chromosome doubling agent.

50. The method of claim 48 or 49, wherein said phenotypic selection is following crossing or selfing.

51. The method of claim 48, wherein said feature is selected from the group consisting of yield, stature, root mass, abiotic stress tolerance, biotic stress tolerance and herbicide tolerance.

52. The method of any one of claims 40-43 and 47-51 further comprising identifying sequence alterations of said DNA editing event.

53. A gene-edited plant obtainable by the method of any one of claims 41-43 and 47-52.

54. The method of any one of claims 40-41, wherein said DNA editing agent causes structural variations.