Methods for enhancing genomic engineering efficiency

Abstract

The present document relates to methods and materials for genome engineering in eukaryotic cells, and in particular to methods of increasing the efficiency of genome engineering (i.e. Transformation or genome editing) by co-delivering one or more chemicals such as a protein deacetylase inhibitor, a plant hormone and / or a regeneration enhancing gene with a genome engineering component.

Description

[0001] This application is a divisional application of the invention patent application with the application date of June 14, 2019, application number 201980053117.7, and invention name “Method for enhancing the efficiency of genome engineering”. Technical Field

[0002] This document relates to methods and materials for genome engineering in eukaryotic cells, and in particular to methods for increasing the efficiency of genome engineering (i.e., transformation or genome editing) by co-delivering one or more chemicals (such as epigenetic regulatory chemicals, plant hormones and / or regeneration enhancing genes) with genome engineering components. Background of the Invention

[0004] Traditional breeding has produced domesticated plants and animals, while modern biotechnology (especially genome engineering) is expanding breeding capabilities and achieving improvements that cannot be achieved with traditional hybridization of close species alone. Using biotechnology, a variety of traits such as high yield, herbicide tolerance and insect resistance have been introduced into crops, which has significantly improved global agriculture and food security. However, biotechnology has raised biosafety and environmental issues due to the presence of foreign DNA in the products.

[0005] By isolating the integrated DNA, genome editing technology can be used to generate site-specific modifications of the target genome without the presence of foreign DNA in the final plant. In addition, through transient expression, genome editing only involves transient editing activity to produce site-specific modifications without DNA integration at any point in the process. Genome-edited plants, particularly those derived from transient activity, are significantly different from conventional genome-modified plants and may not be regulated as genetically modified (GM) plants. Genome editing technology (especially through transient editing methods) provides highly accurate, safe and powerful plant breeding and development tools in agriculture.

[0006] However, genome engineering based on transient activity faces more challenges. Compared with stable transformation, transient engineering usually leads to fewer modified cells and when there is no integrated selectable marker, it is highly challenging to identify engineered cells and achieve homogeneous (homogenous) modification in regenerated plants. These challenges hinder the routine implementation of transient gene editing as a breeding tool for improved plants. Therefore, new methods and materials that enhance genome engineering efficiency are highly needed. Summary of the invention

[0007] In the present invention, it was surprisingly found that the efficiency of genome engineering in plant cells can be improved by co-delivering the genome engineering components with a second compound into the cells, wherein the second compound is selected from epigenetic regulatory chemicals such as protein deacetylase inhibitors or DNA methyltransferase inhibitors, particularly histone deacetylase inhibitors (HDACI, such as trichostatin A (TSA)), plant hormones (such as auxins, cytokinins) and / or proteins that cause improved plant regeneration from somatic tissue, callus or embryonic tissue. In addition, the co-delivery of the promoting chemical with the genome engineering components provides a growth benefit (particularly for transformed cells) and is therefore used as a positive selection strategy for recovering transformed cells.

[0008] Therefore, a first aspect of the present invention is a method for genetic modification in plant cells comprising

[0009] (a) The following are co-introduced into plant cells

[0010] (i) Genome engineering components and

[0011] (ii) a second compound comprising

[0012] (ii.1) epigenetic regulatory chemicals or their active derivatives, in particular DNA methyltransferase inhibitors or protein deacetylase inhibitors, preferably histone deacetylase inhibitors (HDACi), and / or

[0013] (ii.2) plant hormones or their active derivatives, and / or

[0014] (ii.3) a protein which leads to improved plant regeneration from somatic cells, callus cells or embryonic cells, or an expression cassette comprising a nucleic acid encoding said protein, and

[0015] (b) cultivating the plant cell under conditions that allow genetic modification of the genome of the plant cell by the activity of the genome engineering component in the presence of a second compound,

[0016] Preferably, the genome engineered component (i) and / or the second compound (ii) are transiently active and / or transiently present in the plant cell.

[0017] It has been found that all three types of compounds (ii.1), (ii.2) and (ii.3) are independently capable of increasing the efficiency of genetic modification of plant cells affected by genome engineering component (i). Compounds (ii.1), (ii.2) and (ii.3) can be used alone or in combination of two or more compounds or compound types, such as two or more compounds of type (ii.1) or one compound of type (ii.1) and one compound of type (ii.2), etc. When referring to all three compound types (ii.1), (ii.2) and (ii.3), the term "compound (ii)" is used synonymously.

[0018] The method of genetic modification in plant cells may comprise a further step c), which obtains and / or selects genetically modified plant cells or plants or parts thereof, which comprise genetically modified plant cells or are derived from genetically modified plant cells and which comprise a genetic modification of the genome in at least one cell. The selection may be carried out by means of detecting the genetic modification, for example by means based on PCR methods, or by means of phenotypic characteristics such as herbicide resistance, color or fluorescent markers, morphological characteristics such as plant height, etc. Such phenotypic characteristics may be conferred by exogenous (marker) genes, which are stably integrated into the genome of the plant cell.

[0019] In a particularly preferred embodiment, the above method does not comprise a selection process based on an exogenous selectable marker gene stably integrated into the genome of the plant cell.

[0020] In another particularly preferred embodiment, co-introduction causes one or more proteins or an expression cassette comprising a nucleic acid encoding the one or more proteins that improve plant regeneration from somatic cells, callus cells or embryonic cells. "One or more" can be at least one, at least two, at least three, at least four or can be one, two, three, four or more. Preferably, the one or more proteins have an additive effect or even a synergistic effect with respect to improved plant regeneration.

[0021] Suitable plant cells

[0022] The plant cells used in the present invention can be parts of or derived from any type of plant material, preferably buds, hypocotyls, cotyledons, stems, leaves, petioles, roots, embryos, callus, flowers, gametophytes or parts thereof. Isolated plant cells as well as plant materials, i.e. whole plants or parts of plants containing plant cells, can be used.

[0023] A part or parts of a plant may be attached to or separated from the entire intact plant. Such plant parts include, but are not limited to, organs, tissues and cells of the plant, and preferably seeds.

[0024] The present invention is applicable to any plant species, whether monocots or dicots. Preferably, the plant that can be subjected to the methods and uses of the present invention is a plant selected from the following genera: Hordeum, Sorghum, Saccharum, Zea, Setaria, Oryza, Triticum, Secale, Triticale, Malus, Brachypodium, Aegilops, Daucus, Beta, Eucalyptus, Nicotiana, Solanum, Coffea, Vitis, Erythrante, Genlisea, Cucumis, Marus, Arabidopsis rabidopsis, Crucihimalaya, Cardamine, Lepidium, Capsella, Olmarabidopsis, Arabias, Brassica, Eruca, Raphanus, Citrus, Jatropha, Populus, Medicago, Cicer, Cajanus, Phaseolus, Glycine, Gossypium, Astragalus, Lotus, Torenia, Allium, or Helianthus. More preferably, the plant is a plant selected from the group consisting of: barley (Hordeum vulgare), bulbous barley (Hordeum bulbusom), bicolor sorghum (Sorghum bicolor), sugarcane (Saccharumofficinarium), Zea spp. including corn (Zea mays).), millet (Setariaitalica), small-grain rice (Oryza minuta), rice (Oryza sativa), Australian wild rice (Oryza australiensis), tall wild rice (Oryza alta), wheat (Triticum aestivum), durum wheat (Triticum durum), rye (Secale cereale), triticale (Triticale), apple (Malusdomestica), Brachypodium distachyon, seashore barley (Hordeum marinum), tauschii (Aegilops tauschii), Daucus glochidiatus, Beta spp. including Beta vulgaris), Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotianatomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Coffeacanephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii), Cardamine nexuosa, Lepidium virginicum, Capsella bursapastoris, Olmarabidopsis pumila, Arabis hirsute, Brassicanapus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicariasubsp.Sativa), Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Jerusalem artichoke, tuberosus and / or leek (Allium tuberosum). Particularly preferred are sugar beet (Betavulgaris), corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), sunflower (Helianthus annuus), potato (Solanum tuberosum), bicolor sorghum (Sorghum bicolor), turnip (Brassica rapa), rapeseed (Brassica napus), mustard (Brassicajuncacea), cabbage (Brassica oleracea), radish (Raphanus sativus), rice (Oryza sativa), soybean (Glycine max) and / or cotton (Gossypium sp.). .

[0025] Genome engineering components

[0026] The term "genome engineering" as used herein refers to the methodology of genetic modification in plants, i.e., the methodology for modifying the genome of a plant. Preferably, the term refers to a) transformation of a plant or plant cell, preferably a stable transformation and b) genome editing of a plant or plant cell. Genome engineering can be performed in isolated plant cells or plant tissues, preferably in cell culture or in intact plants, i.e., it can be performed in vitro or in vivo.

[0027] The genome engineering components may be introduced as proteins and / or nucleic acids encoding the genome engineering components, in particular as DNA such as plasmid DNA, RNA, mRNA or RNP.

[0028] Genome engineering can be used to prepare transgenic plant materials. The term "transgenic" used according to the disclosure herein refers to a plant, plant cell, tissue, organ or material comprising a gene or genetic construct, which comprises a "transgene" that has been transferred from another organism to a plant, plant cell, tissue organ or material by natural means or by means of transformation technology. The term "transgenic" comprises a nucleic acid sequence (including DNA or RNA) or an amino acid sequence, or a combination or mixture thereof. Therefore, the term "transgenic" is not limited to sequences that are usually identified as "genes", i.e., sequences that encode proteins. It can also refer to, for example, DNA or RNA sequences that do not encode proteins. Therefore, the term "transgenic" generally means that the corresponding nucleic acid or amino acid sequence is not naturally present in the corresponding target cell (including plants, plant cells, tissues, organs or materials). Therefore, the term "transgenic" or "transgenic" used herein refers to such a nucleic acid sequence or amino acid sequence, which is obtained or synthesized from the genome of an organism, and then introduced into another organism in a transient or stable manner by artificial techniques such as molecular biology, genetics, etc. As used herein, "plant material" refers to any material that can be obtained from a plant during any developmental stage. Plant material can be obtained in planta or from in vitro cultures of plants or their plant tissues or organs. Thus, the term encompasses plant cells, tissues and organs and developing plant structures and subcellular components, such as nucleic acids, polypeptides and all chemical plant substances or metabolites that can be found in plant cells or compartments and / or can be produced by plants or can be obtained from extracts of any plant cell, tissue or plant at any stage of development. The term also encompasses derivatives of plant material (e.g., protoplasts) derived from at least one plant cell comprised by the plant material. Thus, the term also encompasses meristem cells or meristems of plants.

[0029] For the plant cells to be modified, although transformation methods based on biological methods, such as Agrobacterium transformation or viral vector-mediated plant transformation and transformation methods based on physical delivery methods, such as particle bombardment or microinjection have developed into prominent technologies for introducing genetic material into plant cells or tissues of interest. Helenius et al., ("Gene delivery into intact plants using the HeliosTM gene Gun", Plant Molecular Biology Reporter, 2000, 18 (3): 287-288) disclose a particle bombardment as a physical method for introducing material into plant cells. At present, there are therefore a variety of plant transformation methods to introduce genetic material in the form of genetic constructs into plant cells of interest, including biological and physical means known to those skilled in the field of plant biotechnology and which can be used to introduce at least one gene encoding at least one wall-related kinase into at least one cell of at least one of a plant cell, tissue, organ or intact plant. It is worth noting that the delivery methods for transformation and transfection can be applied simultaneously to the introduction of the tools of the present invention. A common biological means is transformation with Agrobacterium, which has been used for decades for a variety of different plant materials. Viral vector-mediated plant transformation represents another strategy for introducing genetic material into cells of interest. The physical means found to be applied in plant biology is particle bombardment, also known as biolistic transfection or microparticle-mediated gene transfer, which refers to a physical delivery method in which coated microparticles or nanoparticles containing nucleic acids or genetic constructs of interest are transferred to target cells or tissues. Physical introduction means are suitable for introducing nucleic acids, i.e., RNA and / or DNA, and proteins. Similarly, there are specific transformation or transfection methods for specifically introducing nucleic acids or amino acid constructs of interest into plant cells, including electroporation, microinjection, nanoparticles, and cell penetrating peptides (CPP). In addition, there are chemical-based transfection methods to introduce genetic constructs and / or nucleic acids and / or proteins, particularly including transfection with calcium phosphate, transfection using liposomes (e.g., cationic liposomes), or transfection using cationic polymers (including DEAD-dextran or polyethyleneimine) or combinations thereof. The above delivery techniques, alone or in combination, can be used in vivo (including plant in situ) or in vitro methods.

[0030] As used herein, the term "genome editing" refers to the strategies and techniques for targeting, specific modification of any genetic information or genome of plant cells. Therefore, these terms include both gene editing and the editing of regions other than the gene coding region of the genome, such as intron sequences, non-coding RNA, miRNA, regulatory element sequences (such as promoters, terminators, transcription activator binding sites, cis or trans-acting elements). Additionally, the term may include base editing for targeted substitution of a single nuclear base. It may further include the editing of the nuclear genome and other genetic information (i.e., mitochondrial genome or chloroplast genome and miRNA, pre-mRNA or mRNA) of plant cells. In addition, "genome editing" may include epigenetic editing or engineering that may cause heritable changes in gene expression, i.e. targeted modification, such as DNA methylation or histone modification (such as histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination).

[0031] According to a preferred embodiment of the present invention, the genome engineering component comprises

[0032] a) a double strand DNA break (DSB) inducing enzyme or a nucleic acid encoding the same, which preferably recognizes a predetermined site in the genome of the cell, and optionally comprises a repair nucleic acid molecule, or

[0033] b) a single strand DNA or RNA break (SSB) inducing enzyme or a nucleic acid encoding the same, which preferably recognizes a predetermined site in the genome of said cell, and optionally comprises a repair nucleic acid molecule, or

[0034] c) a base editor enzyme, optionally fused to a disarmed DSB-inducing enzyme or SSB-inducing enzyme, which preferably recognizes a predetermined site in the genome of said cell, or

[0035] d) an enzyme affecting DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination, which is preferably fused to a disarmed DSB-inducing enzyme or SSB-inducing enzyme, which preferably recognizes a predetermined site in the genome of the cell.

[0036] As used herein, a "double-stranded DNA break-inducing enzyme" or "DSBI enzyme" is an enzyme that can induce double-stranded DNA breaks at a specific nucleotide sequence (referred to as a "recognition site" or "predetermined site"). Thus, a "single-stranded DNA or RNA break-inducing enzyme" or "SSBI enzyme" is an enzyme that can induce single-stranded DNA or RNA breaks at a specific nucleotide sequence (referred to as a "recognition site" or "predetermined site").

[0037] In order to have a break at a predetermined target site, the enzyme preferably includes a binding / recognition domain and a cleavage domain. Specific enzymes that can induce double-stranded or single-stranded breaks are nucleases or nickases and variants thereof, including molecules that no longer contain nuclease or nickase functions but act as recognition molecules in combination with another enzyme. In recent years, many suitable nucleases have been developed, particularly customized endonucleases, including meganucleases, zinc finger nucleases, TALE nucleases, Argonaute nucleases (derived from, for example, Natronobacterium gregoryi) and CRISPR nucleases (including, for example, Cas9, Cpf1, CasX or CasY nucleases as part of a clustered regularly interspaced short palindromic repeat (CRISPR) system). Therefore, in a preferred aspect of the present invention, the genome engineering component comprises a DSB or SSB inducing enzyme or a variant thereof, which is selected from CRISPR / Cas endonuclease, preferably CRISPR / Cas9 endonuclease or CRISPR / Cpf1 endonuclease, zinc finger nuclease (ZFN), homing endonuclease, large range nuclease and TAL effector nuclease.

[0038] Rare-cleaving endonucleases are DSBI / SSBI enzymes with a recognition site of preferably about 14 to 70 continuous nucleotides, and therefore have a very low cutting frequency even in larger genomes (such as most plant genomes). Homing endonucleases, also known as meganucleases, constitute a family of such rare-cutting endonucleases. They can be encoded by introns, independent genes or intervening sequences, and present surprising structural and functional properties that distinguish them from more classical restriction enzymes, usually from bacterial restriction modification type II systems. Their recognition sites have a general asymmetry, which is contrary to the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns or inteins have been shown to promote their respective genetic elements to home to alleles without introns or intein sites. By making site-specific double-strand breaks in intronless or inteinless alleles, these nucleases generate recombinogenic ends that participate in the gene conversion process that replicates the coding sequence and results in the insertion of introns or intervening sequences at the DNA level. A list of other rare-cutting meganucleases and their respective recognition sites is provided in Table I (pages 17 to 20) of WO03 / 004659, which is incorporated herein by reference.

[0039] In addition, there are methods for designing customized rare-cutting endonucleases that recognize essentially any target nucleotide sequence of choice. In short, a hybrid between a zinc finger domain designed to recognize a specific nucleotide sequence and a nonspecific DNA cleavage domain from a natural restriction enzyme such as Fokl can be used to prepare a chimeric restriction enzyme. Such methods are described, for example, in WO03 / 080809, WO94 / 18313 or WO95 / 09233 and Isalan et al., (2001), A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nature biotechnology, 19 (7): 656; Liu et al., (1997), Design of polydactyl zinc-finger proteins for unique addressing within complex genomes, Proceedings of the National Academy of Sciences, 94 (11): 5525-5530.).

[0040] Another example of a specially designed endonuclease includes TALE nucleases (TALENs), which are based on transcription activator-like effectors (TALEs) from the genus Xanthomonas bacteria fused to the catalytic domain of the nuclease (e.g., FokI or a variant thereof). The DNA binding specificity of these TALEs is defined by repeated variable di-residues (RVDs) of 34 / 35 amino acid repeat units arranged in tandem, so that one RVD specifically recognizes one nucleotide in the target DNA. Repeat units can be assembled to recognize essentially any target sequence, and repeat units can be fused to the catalytic domain of the nuclease to produce sequence-specific endonucleases (see, e.g., Boch et al., (2009), Breaking the code of DNA binding specificity of TAL-type III effectors, Science, 326 (5959), 509-1512; Moscou & Bogdanove (2009). A simple cipher governs DNA recognition by TAL effectors, Science, 326 (5959), 1501-1501; and WO2010 / 079430, WO2011 / 072246, WO2011 / 154393, WO2011 / 146121, WO2012 / 001527, WO2012 / 093833, WO2012 / 104729, WO2012 / 138927, WO2012 / 138939). WO2012 / 138927 also describes monomeric (compact) TALENs and TALEs with various catalytic domains and combinations thereof.

[0041] Recently, a new type of customizable endonuclease system has been described; the so-called CRISPR / Cas system. The CRISPR system in its natural environment describes a molecular complex comprising at least one small and separate non-coding RNA combined with a Cas nuclease or another CRISPR nuclease such as a Cpf1 nuclease (Zetsche et al., "Cpf1Is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System", Cell, 163, pp. 1-13, October 2015) that can produce specific DNA double-strand breaks. Currently, CRISPR systems are divided into two categories, including five types of CRISPR systems, such as type II systems with Cas9 as effectors, and type V systems with Cpf1 as effector molecules (Makarova et al., Nature Rev. Microbiol., 2015). In artificial CRISPR systems, synthetic non-coding RNAs and CRISPR nucleases and / or optionally modified CRISPR nucleases (modified to act as nickases or lack any nuclease function) can be used in combination with at least one synthetic or artificial guide RNA or gRNA that combines the functions of crRNA and / or tracrRNA (Makarova et al., 2015, supra). CRISPR / Cas-mediated immune responses in natural systems require CRISPR-RNA (crRNA), in which the maturation of the guide RNA that controls the specific activation of CRISPR nucleases varies significantly between the various CRISPR systems characterized to date. First, the invading DNA (also called the spacer) is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. The type II CRISPR system encodes the Cas9 nuclease as a key enzyme in the interference step, and the system contains both crRNA and trans-activating RNA (tracrRNA) as a guide motif. These hybridize and form a double-stranded (ds) RNA region, which is recognized by RNAseIII and can be cut to form a mature crRNA. These in turn then associate with the Cas molecule in order to guide the nuclease specifically to the target nucleic acid region. Recombinant gRNA molecules can contain both variable DNA recognition regions and Cas interaction regions, and can therefore be designed independently of the specific target nucleic acid and the desired Cas nuclease. As another safety mechanism, PAM (motif adjacent to the pre-spacer region) must be present in the target nucleic acid region; these are DNA sequences directly linked to the DNA recognized from the Cas9 / RNA complex.The PAM sequence for Cas9 from Streptococcus pyogenes has been described as "NGG" or "NAG" (standard IUPAC nucleotide code) (Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science 2012, 337: 816-821). The PAM sequence for Cas9 from Staphylococcus aureus is "NNGRRT" or "NNGRR (N)". Other variant CRISPR / Cas9 systems are known. Thus, Neisseria meningitidis Cas9 cuts at the PAM sequence NNNNGATT. Streptococcus thermophilus Cas9 cuts at the PAM sequence NNAGAAW. Recently, another PAM motif NNNNRYAC has been described for the CRISPR system of Campylobacter (WO2016 / 021973A1). For the Cpf1 nuclease, it has been described that the Cpf1-crRNA complex without tracrRNA effectively recognizes and cuts the target DNA via a short T-rich PAM, in contrast to the G-rich PAM usually recognized by the Cas9 system (Zetsche et al., supra). In addition, by using modified CRISPR polypeptides, specific single-strand breaks can be obtained. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific DNA double-strand breaks by means of double DNA nicking. In addition, by using two gRNAs, the specificity of DNA binding can be optimized, and thus DNA cutting can be optimized. Meanwhile, other CRISPR effectors originally described for bacteria, such as CasX and CasY effectors are available and represent additional effectors that can be used for genome engineering purposes (Burstein et al., “New CRISPR-Cas systems from uncultivated microbes”, Nature, 2017, 542, 237-241).

[0042] The cleavage site of the DSBI / SSBI enzyme is related to the exact position of the induced break on the DNA or RNA. The cleavage site may or may not be included in the recognition site of the DSBI / SSBI enzyme (may overlap with the recognition site of the DSBI / SSBI enzyme or may not overlap with the recognition site of the DSBI / SSBI enzyme), and it is therefore said that the cleavage site of the DSBI / SSBI enzyme is located at or near its recognition site. The recognition site of the DSBI / SSBI enzyme, sometimes also referred to as the binding site, is a nucleotide sequence that is recognized by the DSBI / SSBI enzyme (specificity) and determines its binding specificity. For example, a TALEN or ZNF monomer has a recognition site determined by its RVD repeats or ZF repeats, respectively, and its cleavage site is determined by its nuclease domain (e.g., FokI) and is generally located outside the recognition site. In the case of a dimeric TALEN or ZFN, the cleavage site is located between the two recognition / binding sites of the corresponding monomer, and the intervening DNA or RNA region where the cleavage occurs is called the spacer region.

[0043] Those skilled in the art will be able to select DSBI / SSBI enzymes that recognize a specific recognition site and induce DSB or SSB at a cleavage site at or near a preselected / predetermined site, or engineer such DSBI / SSBI enzymes. Alternatively, a DSBI / SSBI enzyme recognition site can be introduced into the target genome using any conventional transformation method or by hybridization with an organism having a DSBI / SSBI enzyme recognition site in its genome, and then any desired nucleic acid can be introduced at or near the cleavage site of the DSBI / SSBI enzyme.

[0044] There are two main and unique ways to repair breaks - homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires the presence of a homologous sequence (e.g., a "donor") as a template to guide the cell repair process, and the results of the repair are error-free and predictable. When the template (or "donor") sequence for homologous recombination is missing, cells usually attempt to repair the break by a non-homologous end joining (NHEJ) process.

[0045] In the particularly preferred aspect of this embodiment, the repair nucleic acid molecule is additionally imported into the plant cell.As used herein, "repair nucleic acid molecule" is a single-stranded or double-stranded DNA molecule or RNA molecule, which is used as a template for modifying the genomic DNA or RNA at or near the preselected site of the cleavage site.As used herein, "template for modifying genomic DNA" refers to homologous recombination between the flanking region and the corresponding homologous region in the flanking target genome at the preselected site, optionally combined with non-homologous end connection (NHEJ) at one end of the two ends of the repair nucleic acid molecule (for example, in the case of only one flanking region), and the repair nucleic acid molecule is replicated or integrated at a predetermined site.Integration by homologous recombination will allow the repair nucleic acid molecule to be accurately connected to the target genome until the nucleotide level, and NHEJ can cause the small insertion / deletion of the junction between the repair nucleic acid molecule and the genomic DNA.

[0046] As used herein, "modification of a genome" means that the genome has been altered at least one nucleotide. This can occur by inserting a transgene, preferably an expression cassette comprising a transgene of interest, substituting at least one nucleotide and / or deleting at least one nucleotide and / or inserting at least one nucleotide, as long as it results in a total change of at least one nucleotide compared to the nucleotide sequence of the preselected genomic target site before modification, thereby allowing identification of the modification, for example by techniques such as sequencing or PCR analysis, etc., well known to those skilled in the art.

[0047] As used herein, "preselected site", "predetermined site" or "predefined site" refers to a specific nucleotide sequence in a genome (e.g., a nuclear genome or a chloroplast genome) at which one or more nucleotides are expected to be inserted, substituted and / or deleted. This can be, for example, an endogenous locus or a specific nucleotide sequence in or connected to a previously introduced foreign DNA, RNA or transgene. The preselected site can be a specific nucleotide position at which (after) an insertion of one or more nucleotides is intended to be made. The preselected site can also include a sequence of one or more nucleotides to be exchanged (substituted) or deleted.

[0048] As used in the context of this application, the term "about" refers to + / -10% of the value, preferably + / -5% of the value. For example, about 100 nucleotides (nt) should be understood as a value between 90nt and 110nt, preferably between 95nt and 105nt.

[0049] As used herein, "flanking region" is a region of a repair nucleic acid molecule having a nucleotide sequence homologous to the nucleotide sequence of the DNA region located at the flank of the preselected site (i.e., upstream or downstream). Obviously, the length of the flanking region and the sequence identity percentage should be selected so that homologous recombination can be carried out between the flanking region and their corresponding DNA regions upstream or downstream of the preselected site. One or more DNA regions located at the flank of the preselected site that have homology with one or more flanking DNA regions of the repair nucleic acid molecule are also referred to as one or more homologous regions in the genomic DNA.

[0050] In order to have enough recombinant homology, the length of the flanking DNA region of the repair nucleic acid molecule can be different, and the length should be at least about 10nt, about 15nt, about 20nt, about 25nt, about 30nt, about 40nt or about 50nt. However, the flanking region can be as long as possible (for example, up to about 100-150kb, such as complete bacterial artificial chromosome (BAC)). Preferably, the flanking region will be about 50nt to about 2000nt, such as about 100nt, 200nt, 500nt or 1000nt. In addition, the region positioned at the DNA flank of interest need not be identical with the homology region (the DNA region positioned at the flanking preselected site), and can have the sequence identity of about 80% to about 100% with the DNA region positioned at the flanking preselected site, preferably have the sequence identity of about 95% to about 100%. The longer the flanking region, the less stringent the requirement to homology. Furthermore, in order to achieve exchange of the target DNA sequence at the preselected site without changing the DNA sequence of the adjacent DNA sequences, the flanking DNA sequence should preferably be identical to the upstream and downstream DNA regions flanking the preselected site.

[0051] As used herein, "upstream" refers to a position on a nucleic acid molecule that is closer to the 5' end of the nucleic acid molecule. Similarly, the term "downstream" refers to a position on a nucleic acid molecule that is closer to the 3' end of the nucleic acid molecule. For the avoidance of doubt, nucleic acid molecules and their sequences are usually expressed in their 5' to 3' direction (from left to right).

[0052] In order to modify the target sequence at the preselected site, the flanking region must be selected so that the 3' end of the upstream flanking region and / or the 5' end of the downstream flanking region are aligned with the end of the predefined site. In this way, the 3' end of the upstream flanking region determines the 5' end of the predefined site, while the 5' end of the downstream flanking region determines the 3' end of the predefined site.

[0053] As used herein, the preselected site is located outside or away from the cleavage (and / or recognition) site, which means that the site (preselected site) intended for genome modification does not contain a cleavage site and / or a recognition site for a DSBI / SSBI enzyme, i.e., the preselected site does not overlap with the cleavage (and / or recognition) site. Therefore, in this regard, outside / away refers to upstream or downstream of the cleavage (and / or recognition) site.

[0054] As used herein, "base editor" refers to a protein or fragment thereof having the same catalytic activity as the protein derived therefrom, which has the ability to mediate targeted base modification when provided alone or in a molecular complex (referred to herein as a base editing complex), that is, if the base conversion does not cause a silent mutation but causes a conversion of an amino acid encoded by a codon containing a position to be converted by the base editor to be used, the conversion of the base of interest leads to a point mutation of interest, which in turn can lead to a targeted mutation. Preferably, at least one base editor according to the present invention is temporarily or permanently connected to at least one site-specific DSBI / SSBI enzyme complex or at least one modified site-specific DSBI / SSBI enzyme complex or optionally to a component of the at least one site-specific DSBI / SSBI enzyme complex. The connection can be covalent and / or non-covalent.

[0055] Any base editor or site-specific DSBI / SSBI enzyme complex disclosed herein, or a catalytically active fragment thereof, or any component of a base editor complex or any component of a site-specific DSBI / SSBI enzyme complex can be introduced into a cell as a nucleic acid fragment representing or encoding a DNA, RNA or protein effector, or it can be introduced as DNA, RNA and / or protein or any combination thereof.

[0056] Base editors are proteins or fragments thereof that have the ability to mediate targeted base modifications (i.e., conversion of bases of interest resulting in point mutations of interest). Preferably, in the context of the present invention, at least one base editor is temporarily or permanently fused to at least one DSBI / SSBI enzyme, or optionally fused to a component of at least one DSBI / SSBI. Fusion can be covalent and / or non-covalent. Multiple publications have shown targeted base conversions (mainly from cytosine (C) to thymine (T)) using CRISPR / Cas9 nickases or non-functional nucleases connected to cytidine deaminase domains (apolipoprotein B mRNA editing catalytic polypeptide (APOBEC1) (e.g., APOBEC from rats)). The deamination of cytosine (C) is catalyzed by cytosine deaminase, and uracil (U) with base pairing properties of thymine (T) is produced. Most known cytidine deaminases act on RNA, and a few examples of known acceptance of DNA require single-stranded (ss) DNA. Studies on dCas9-target DNA complexes reveal that after forming the Cas9-guide RNA-DNA "R-loop" complex, at least nine nucleotides (nt) of the replaced DNA strand are unpaired (Jore et al., Nat. Struct. Mol. Biol., 18, 529-536 (2011)). Indeed, in the structure of the Cas9 R-loop complex, the first 11 nt of the pre-spacer sequence on the replaced DNA strand are disordered, indicating that their movement is not highly restricted. It has also been speculated that the mutations in the cytosine of the non-template strand induced by the Cas9 nickase may be caused by the accessibility of the cellular cytosine deaminase to them. It is reasonable to believe that a subset of this ssDNA fragment in the R-loop can be used as an effective substrate for the cytidine deaminase connected by dCas9- to achieve direct programmable conversion of C to U in DNA (Komor et al., supra). Recently, Goudelli et al. ((2017), Programmable base editing of A·T to G·C ingenomic DNA without DNA cleavage, Nature, 551(7681), 464) described an adenine base editor (ABE) that mediates A·T to G·C conversion in genomic DNA.

[0057] Enzymes and histone modifying enzymes that affect DNA methylation have been identified in the art. Histone post-translational modification plays an important role in regulating chromatin structure and gene expression. For example, in Sterner DE, Berger SL (June 2000): "Acetylation of histones and transcription-related factors", Microbiol.Mol.Biol.Rev.64 (2): 435–59, described enzymes for histone acetylation. Enzymes that affect histone methylation are described in Zhang Y., Reinberg D (2001): "Transcription regulation by histonemethylation: interplay between different covalent modifications of the corehistone tails", Genes Dev.15 (18): 2343–60. Ubiquitination of histones is described in Shilatifard A (2006): "Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression", Annu.Rev.Biochem.75: 243–69. Enzymes for histone phosphorylation are described in Nowak SJ., Corces VG. (April 2004): "Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation", Trends genet. 20(4): 214–20. Enzymes for histone SUMOylation are described in Nathan D., Ingvarsdottir K., Sterner DE. et al., (April 2006): "Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications", Genes Dev. 20(8): 966–76.Enzymes for histone ribosylation are described in Hassa PO., Haenni SS., Elser M, Hottiger MO. (September 2006): "Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going?", Microbiol. Mol. Biol. Rev. 70(3): 789–829. Histone citrullination is catalyzed by an enzyme, for example, called peptidyl arginine deiminase 4 (PAD4, also known as PADI4), which converts both histone arginine (Arg) and monomethylarginine residues to citrulline.

[0058] Enzymes affecting DNA methylation and histone modifying enzymes may be fused to disarming DSB or SSB inducing enzymes, which preferentially recognize predetermined sites in the genome of the cell.

[0059] Epigenetic regulatory chemicals

[0060] As used herein, "epigenetic regulatory chemical" refers to any chemical involved in regulating the epigenetic state of plant cells (e.g., DNA methylation, protein methylation, particularly histone methylation and acetylation, particularly histone acetylation). According to a first embodiment of the present invention, epigenetic regulatory chemical, such as protein deacetylase inhibitor (ii.1) is co-introduced with genome engineering components. Preferred epigenetic regulatory chemical used according to the present invention is a histone deacetylase inhibitor (HDACI) such as trichostatin A (TSA) or a DNA methyltransferase inhibitor. As used herein, "histone deacetylase inhibitor (HDACI)" refers to any substance that inhibits histone deacetylase activity, and "DNA methyltransferase inhibitor" refers to any substance that inhibits DNA methyltransferase activity.

[0061] It is hypothesized that the co-delivered epigenetic regulatory chemicals (ii.1) (particularly HDACI) relax the chromatin structure of the plant, promoting DNA accessibility to genome engineering components in the bombarded cells, and thus subsequently promoting genome engineering (i.e., transformation and genome editing) efficiency. The reason for this hypothesis is that the basic structural and functional unit of genetic material is the nucleosome, in which negatively charged DNA is wrapped around positively charged histone octamers and associated linker histones. The nucleosomal unit is further folded and stacked into chromatin (Andrews, AJ, and Luger, K. (2011). Nucleosome structure (s) and stability: Variations on a theme. Annu. Rev. Biophys. 40: 99–117). DNA accessibility depends largely on the compactness of nucleosomes and chromatin. Chromatin remodeling enzymes can dynamically modify the lysine or other amino acids of histones, which leads to changes in their charge and interactions with DNA and other proteins, and causes chromatin folding or unfolding (Bannister AJ., Kouzarides T. (2011) Regulation of chromatin by histone modifications. Cell Res 21: 381–95). Acetylation and deacetylation of lysine residues in histones are often involved in the reversible regulation of chromatin structure in eukaryotes by adding or removing acetyl groups, and mediate chromatin accessibility and gene expression regulation. Histone deacetylase (HDAC) is an enzyme that removes acetyl groups from lysine residues located at the N-terminal tail of histones, which makes histones more positively charged and thus allows histones to wrap DNA more tightly. Inhibiting HDAC may help chromatin unfolding and make DNA more accessible.

[0062] Chromatin remodeling and other epigenetic modifications undoubtedly play an important role in regulating cell totipotency and regeneration (Zhang, H., and Ogas, J. (2009). An epigenetic perspective on developmental regulation of seed genes. Mol. Plant 2: 610–627). It has been shown that inhibition of histone deacetylase (HDAC) activity is associated with plant regeneration and microspore embryogenesis (Miguel, C., and L. Marum, 2011. Anepigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J. Exp. Bot. 62: 3713–3725., Li Hui et al. (2014) The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte PLANTCELL, 26: 195–209). Inhibition of HDAC activity or downstream HDAC-mediated pathways plays a major role in initiating stress-induced haploid embryogenesis. One such HDACi is trichostatin A (TSA). TSA has been shown to induce a large number of embryogenic cell proliferations in the male gametophytes of Brassica napus. TSA treatment results in a high frequency of sporophytic cell divisions in cultured microspores and pollen.

[0063] This document describes methods to improve genome engineering efficiency in the presence of one or more epigenetic regulatory chemicals such as protein deacetylase inhibitors, particularly HDACi. Such HDACi can be trichostatin A (TSA), N-hydroxy-7-(4-dimethylaminobenzoyl)-aminoheptylamide (M344), suberoylanilide hydroxamic acid (SAHA) or other. These HDACIs are selected from chemicals based on hydroxamic acid (HA), and their target is zinc-dependent HDAC.

[0064] Plant hormones

[0065] According to a second embodiment of the present invention, one or more plant hormones (ii.2), such as auxins and cytokinins, such as 2,4-D, 6-benzylaminopurine (6-BA) and zeatin, are delivered together with the genome engineering component (i). As used herein, "plant hormone" refers to any naturally occurring or synthetic material and chemical substance that promotes plant cell division and / or plant morphogenesis.

[0066] Plant somatic cells can resume cell division and regenerate into whole plants in vitro through somatic embryogenesis or organogenesis, which mainly depends on plant hormones such as auxin and cytokinin. In the present invention, it is found that plant hormones promote cell proliferation, increase the sensitivity of plant cells to genome engineering, and thus improve the efficiency of genome engineering (i.e., transformation and genome editing).

[0067] One of the auxins is 2,4-dichlorophenoxyacetic acid (2,4-D), which is almost essential for somatic embryogenesis and cell regeneration in monocots such as corn and wheat. At the same time, cytokinins such as 6-benzylaminopurine (6-BA) or zeatin are essential for plant organogenesis and the initiation and development of shoot meristems. This document describes a method for improving genome engineering efficiency by delivering one or more plant hormones (2,4-D, 6-BA, zeatin, etc.) together with genome engineering components.

[0068] Regeneration enhancing gene

[0069] According to a third embodiment of the present invention, an expression cassette (ii.3) of a protein or a nucleic acid encoding said protein that causes improved plant regeneration from somatic cells, callus cells or embryonic cells is co-introduced with the genome engineering component (i). This type of compound (ii.3) is also referred to herein as a "regeneration enhancing gene".

[0070] It is believed that the reproducibility of the transformed cells is lower than that of wild-type cells. Due to the presence of foreign DNA in the cells, the transformed cells are susceptible to programmed cell death. The stress caused by delivery (such as bombardment) may also cause cell death. Therefore, promoting cell division is essential for the regeneration of modified cells. In addition, genome engineering efficiency is controlled to a great extent by the host cell state. The cells (like those in plant meristems) that experience rapid cell division are the most suitable receptors for genome engineering. Promoting cell division will likely improve DNA integration or modification in DNA replication and fission processes, and thus improve genome engineering efficiency.

[0071] Enhancer genes were selected based on their roles in promoting cell division and plant morphogenesis. Each candidate gene was cloned and driven by a strong constitutive promoter and evaluated by transient expression in maize cells without selection. Examples of booster genes are PLT5 (PLETHORA5; SEQ ID NOs: 1, 2, 13 and 14), PLT7 (PLETHORA7; SEQ ID NOs: 3, 4, 15 and 16) and RKD genes (RKD2: SEQ ID NOs: 5, 6, 29, 30, 31 and 32; RKD4: SEQ ID NOs: 11, 12, 17, 18, 27 and 28; e.g., Waki, T., Hiki, T., Watanabe, R., Hashimoto, T., & Nakajima, K. (2011). The Arabidopsis RWP-RK protein RKD4 triggers gene expression and pattern formation in early embryogenesis. Current Biology, 21(15), 1277-1281)

[0072] PLT (PLETHORA), also known as AIL (AINTEGUMENT-LIKE) gene, is a member of the transcriptional regulator AP2 family. Transcription factor AP2 family members play an important role in plant cell proliferation and embryogenesis (El Ouakfaoui, S., Schnell, J., Abdeen, A., Colville, A., Labbé, H., Han, S., Baum, B., Laberge, S., Miki, B (2010) Control of somatic embryogenesis and embryo development by AP2 transcription factors. PLANT MOLECULAR BIOLOGY 74 (4-5): 313-326.). The PLT gene is mainly expressed in the developmental tissues of buds and roots, and is necessary for stem cell homeostasis, cell division and regeneration, and organ primordium patterning.

[0073] The PLT family contains the AP2 subbranch with six members. Four PLT members, PLT1 / AIL3 (SEQ ID NOs: 19 and 20), PLT2 / AIL4 (SEQ ID NOs: 21 and 22), PLT3 / AIL6 (SEQ ID NOs: 9, 10, 23 and 24) and BBM / PLT4 / AIL2 (SEQ ID NOs: 7, 8, 25 and 26) are partially overlappingly expressed in the root apical meristem (RAM) and are required for the expression of QC (quiescent center) markers in the correct position within the stem cell niche. These genes play redundant roles in maintaining cell division and preventing cell differentiation in the root apical meristem.

[0074] Three PLT genes, PLT3 / AIL6, PLT5 / AIL5, and PLT7 / AIL7, are expressed in the shoot apical meristem (SAM), where they play redundant roles in the positioning and outgrowth of lateral organs. PLT3, PLT5, and PLT7 regulate de novo shoot regeneration in Arabidopsis by controlling two distinct developmental events. PLT3, PLT5, and PLT7 are required to maintain high levels of PIN1 expression at the periphery of the meristem and regulate localized auxin production in the central region of the SAM, which underlies the phyllotactic transition. Cumulative loss of function of these three genes results in an intermediate cell mass, the callus, that fails to form shoot progenitors, whereas induction of PLT5 or PLT7 allows shoot regeneration in a hormone-independent manner. PLT3, PLT5, and PLT7 regulate and require the shoot-promoting factor CUP-SHAPEDCOTYLEDON2 (CUC2) to complete the shoot formation program. PLT3, PLT5 and PLT7 are also expressed in lateral root founder cells, where they redundantly activate the expression of PLT1 and PLT2 and subsequently regulate lateral root formation.

[0075] According to the present invention, a protein causing improved plant regeneration from somatic cells, callus cells or embryonic cells preferably comprises an amino acid sequence selected from the group consisting of

[0076] a) a sequence as shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31,

[0077] b) a sequence that is at least 60% identical to the sequence of (a),

[0078] c) a sequence encoded by a nucleic acid sequence as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32, and

[0079] d) a sequence encoded by a nucleic acid sequence that is at least 60% identical to the nucleic acid sequence of (c).

[0080] e) A sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions to a sequence complementary to the nucleic acid sequence defined in c).

[0081] For the amino acid sequence of (b) or the nucleic acid sequence of (d) above, the sequence identity over the entire sequence length is preferably at least 70%, at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99%.

[0082] For purposes of the present invention, " sequence identity " (expressed as percentage) of two related nucleotides or amino acid sequences refers to the number of positions in two optimally aligned sequences with identical residues (x100) divided by the number of positions compared. A gap, i.e., a position in the alignment of a residue that exists in a sequence and does not exist in another sequence, is considered to be a position with non-identical residues. The comparison of two sequences is carried out by Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The above computer-assisted sequence alignment can be easily carried out using standard software programs such as the NEEDLE program implemented in European Molecular Biology Open Software Suite (EMBOSS) as version 6.3.1.2 (Trends in genetics 16 (6), 276 (2000)) (its default parameters are for example for protein matrix=EBLOSUM62, gapopen=10.0 and gapextend=0.5).

[0083] As used herein, the term "hybridization" refers to the formation of a hybrid between two nucleic acid molecules by base pairing of complementary nucleotides. The term "hybridization under stringent conditions" refers to hybridization under specific conditions. An example of such conditions includes conditions under which substantially complementary chains (i.e., chains consisting of nucleotide sequences having at least 80% complementarity) hybridize with a given chain, while lower complementary chains do not hybridize. Alternatively, these conditions refer to specific hybridization conditions of sodium salt concentration, temperature, and washing conditions. For example, highly stringent conditions are included in 42°C, 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate, 5x Denhardt solution, 10x dextran sulfate, 20mg / ml sheared salmon sperm DNA incubated and washed in 0.2x SSC (SSC stands for 0.15M sodium chloride and 0.015M trisodium citrate buffer) at about 65°C. Alternatively, highly stringent conditions may mean hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDDS, 1 mM EDTA and 1% BSA for 16 hours, and washing twice with 2×SSC and 0.1% SDDS at 68° C. Further alternatively, highly stringent hybridization conditions are, for example, hybridization at 65° C. in 4×SSC, and then washing multiple times at 65° C. in 0.1×SSC for a total of about 1 hour, or hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours, and then washing twice with 2×SSC and 0.1% SDS at 68° C.

[0084] Co-import

[0085] The method of the invention for genetic modification in plant cells is characterized in that the genome engineering component (i) is co-introduced into a plant cell together with at least one of the compounds (ii.1), (ii.2) and (ii.3).

[0086] As used herein, "co-delivery" or "co-delivery" and "co-introduction" or "co-introduction" are used interchangeably. For the purposes of the present invention, "co-introduction" refers to a process in which at least two different components are delivered into the same plant cell at the same time. Thus, the genome engineered component (i) is introduced into the same plant cell together with compounds (ii.1), (ii.2) and / or (ii.3). Preferably, both types of components / compounds are introduced via a common construct.

[0087] Co-introduction into plant cells can be carried out by particle bombardment, microinjection, Agrobacterium-mediated transformation, electroporation, agroinfiltration or vacuum infiltration. According to the present invention, methods based on physical delivery (such as particle bombardment, microinjection, electroporation, nanoparticles and cell penetrating peptides (CPP)) are particularly preferred for co-introduction of component (i) and compound (ii). Co-introduction by particle bombardment is particularly preferred.

[0088] As used herein, the term "particle bombardment", also known as "biolistics transfection" or "microparticle-mediated gene transfer", refers to a physical delivery method for transferring coated microparticles or nanoparticles containing a construct of interest into target cells or tissues. For use in the present invention, the construct of interest comprises the genome engineering component (i) and at least one of compounds (ii.1), (ii.2) and (ii.3). The micro- or nanoparticles act as projectiles and are fired at the target structure of interest at high pressure using a suitable device (commonly referred to as a gene gun). Transformation by particle bombardment uses metal microprojectiles coated with the construct of interest, which are then fired at the target cell using a device called a "gene gun" (Sandford et al., 1987) at a high speed (~1500 km / h) fast enough to penetrate the target tissue cell wall, but not harsh enough to cause cell death. For protoplasts, whose cell wall is completely removed, the conditions are logically different. The precipitated construct on at least one microprojectile is released into the cell after bombardment. Acceleration of the microprojectile is achieved by high voltage discharge or compressed gas (helium). Regarding the metal particles used, they must be non-toxic, non-reactive, and have a diameter smaller than that of the target cell. Gold or tungsten are most commonly used. A great deal of information on the general use of gene guns and related systems is publicly available from manufacturers and suppliers.

[0089] In a particularly preferred embodiment of microprojectile bombardment, one or more compounds (ii.1), (ii.2) and (ii.3) are co-delivered with the genome engineering component (i) via a microcarrier comprising gold particles having a size in the range of 0.4-1.6 micrometers (μm), preferably in the range of 0.4-1.0 μm. In an exemplary method, 10-1000 μg of gold particles, preferably 50-300 μg, are used per bombardment.

[0090] Compound (ii) and genome engineering component (i) can be delivered into target cells using, for example, a Bio-Rad PDS-1000 / He particle gun or a handheld Helios gene gun system. When the PDS-1000 / He particle gun system is used, the bombardment rupture pressure is 450 psi to 2200 psi, preferably 450-1100 psi, while the rupture pressure of the Helios gene gun system is 100-600 psi. More than one chemical substance or construct can be co-delivered into cells with the genome engineering component at the same time.

[0091] Cultivation steps

[0092] In step b) of the method of the present invention, plant cells into which genome engineering components (i) and at least one compound (ii) have been co-introduced are cultured in the presence of at least one compound (ii) under conditions that allow genetic modification of the genome of the plant cells through the activity of the genome engineering components.

[0093] As used herein, "genetic modification of a genome" includes any type of manipulation such that endogenous nucleotides have been altered to include mutations such as deletions, insertions, transitions, transversions, or combinations thereof. For example, an endogenous coding region may be deleted. Such mutations may result in a polypeptide having an amino acid sequence that is different from the amino acid sequence encoded by the endogenous polynucleotide. Another example of a genetic modification is a change in a regulatory sequence such as a promoter to result in increased or decreased expression of an operably linked endogenous coding region.

[0094] "Suitable" conditions for genetic modification of the plant genome to occur, such as cleavage of polynucleotides, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions allow, enhance, promote and / or facilitate the event. Depending on the individual genome engineering components (i), these conditions may be different.

[0095] In the method of the present invention, preferably the plant cell is transiently transformed with the genome engineering component (i) and at least one compound (ii). As used herein, "transient transformation" refers to the transfer of foreign materials [i.e., nucleic acid fragments, proteins, ribonucleoproteins (RNPs), etc.] into host cells to cause gene expression and / or activity without integrating and stabilizing genetic foreign materials. Therefore, the genome engineering component (i) is transiently active and / or transiently present in the plant cell. The genome engineering component is not permanently incorporated into the cell genome but provides a temporary effect to cause the modification of the genome. For example, the transient activity and / or transient presence of the genome engineering component in the plant cell can lead to the introduction of one or more double-strand breaks in the genome of the plant cell, the introduction of one or more single-strand breaks in the genome of the plant cell, the introduction of one or more base editing events in the genome of the plant cell, or the introduction of one or more of the following in the genome of the plant cell: DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination.

[0096] Preferably, the introduction of the double strand break(s) or single strand break(s) is followed by non-homologous N-joining (NHIJ) and / or by homology-directed repair of the break(s) by homologous recombination mechanisms.

[0097] The resulting modification in the genome of the plant cell can be, for example, selected from the insertion of a transgene (preferably an expression cassette comprising a gene of interest), a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a change in DNA methylation, a change in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination, or a combination thereof. According to a particularly preferred aspect of the present invention, no exogenous genetic material associated with the applied gene editing mechanism / system is stably integrated into the genome of the plant cell.

[0098] A genetic modification may be a permanent and heritable change in the genome of a plant cell.

[0099] Optional pretreatment

[0100] According to a preferred aspect of the present invention, by culturing the plant material in vitro in a culture medium containing one or more compounds (ii), before the co-introduction step (a), the plant material may be pretreated with one or more chemical substances, such as one or more of compounds (ii.1), (ii.2) and (ii.3). Therefore, the method for genetic modification in plant cells may further comprise a step of pretreating the plant cells to be used in step (a), wherein the pretreatment comprises culturing the plant cells or the plant material comprising the same in a culture medium containing (ii.1) epigenetic regulatory chemicals or their active derivatives, in particular histone deacetylase inhibitors (HDACi) or DNA methyltransferase inhibitors, (ii.2) plant hormones or their active derivatives, (ii.3) proteins that cause improved plant regeneration from callus or embryonic tissue, or any combination thereof.

[0101] After the pretreatment step, the treated plant cells are extracted from the culture medium containing at least one of compounds (ii.1), (ii.2) and (ii.3) and used for the co-introduction step (a).

[0102] As an example, for the histone deacetylase inhibitor TSA, the duration of HDACi pretreatment is 10 minutes to 2 days, preferably 2.0 hours to 24 hours. The TSA concentration used for pretreatment is 1.0nM to 1000nM, preferably 10nM to 100nM. Thereafter, the treated plant material is transferred to a medium without HDACi and immediately used for TSA co-introduction (extended TSA pretreatment may cause non-selective enhancement of cell regeneration, which may increase the difficulty of recovering bombarded and modified cells).

[0103] Similar pretreatment conditions can be applied to all types of compounds (ii.1), (ii.2) and (ii.3). Plant tissue culture and genome engineering can be performed using currently available methods. Transient transformation and transgenic expression can be monitored using a red fluorescence reporter gene tdTomato (which encodes an abnormally bright red fluorescent protein with an excitation maximum of 554nm and an emission maximum of 581nm) or a green fluorescence reporter gene mNeonGreen (which encodes the brightest monomeric green or yellow fluorescent protein with a maximum excitation wavelength of 506nm and a maximum emission wavelength of 517nm). Genome editing efficiency can be analyzed, for example, by next generation sequencing (NGS).

[0104] particle

[0105] In the context of the present invention, microparticles coated with components (i) and (ii) have been found to be particularly suitable for co-introduction of components (i) and (ii) into plant cells. Thus, according to another embodiment, the present invention provides microparticles coated with at least

[0106] (i) Genome engineering components and

[0107] (ii) a second compound comprising

[0108] (ii.1) epigenetic regulatory chemicals, such as protein deacetylase inhibitors or their active derivatives, in particular histone deacetylase inhibitors (HDACi), and / or

[0109] (ii.2) plant hormones or their active derivatives, and / or

[0110] (ii.3) A protein which leads to improved plant regeneration from somatic cells, callus cells or embryonic cells, or an expression cassette comprising a nucleic acid encoding the protein.

[0111] The microparticles are composed of non-toxic, non-reactive materials. Preferably, the microparticles include metals such as gold or tungsten. The size of the microparticles may be in the range of 0.4-1.6 micrometers (μm), preferably in the range of 0.4-1.0μ.

[0112] The coating with components (i) and (ii) may comprise one or more coating layers. For example, the microparticle may comprise a first coating layer comprising the genome engineered component (i) and a second coating layer comprising compounds (ii.1), (ii.2) and / or (ii.3). Alternatively, the microparticle may comprise a coating layer comprising the genome engineered component (i) and at least one of compounds (ii.1), (ii.2) and (ii.3).

[0113] In addition, the present invention provides a kit for genetically modifying a plant genome by microprojectile bombardment, comprising

[0114] (I) one or more microparticles, and

[0115] (II) means for coating the microparticles with at least a genome engineering component and (1) an epigenetic regulatory chemical (e.g. a DNA methyltransferase inhibitor or a protein deacetylase inhibitor, in particular a histone deacetylase inhibitor (HDACi)) or an active derivative thereof, and / or (2) a plant hormone or an active derivative thereof, and / or (3) a protein that causes improved plant regeneration from callus or embryonic tissue or an expression cassette comprising a nucleic acid encoding the protein.

[0116] Another aspect of the invention is the use of microparticles as described above for biolistic transformation of plant cells.

[0117] The subject of the present invention is also a plant cell obtained or obtainable by the above method.Therefore, one embodiment of the present invention is a plant cell obtained or obtainable through genetic modification by the above method for genetic modification in plant cells.Compared with the original plant cell, the genetic modification in these plant cells can for example include the insertion of a transgenic (preferably an expression cassette comprising a gene of interest), the replacement of at least one nucleotide, the disappearance of at least one nucleotide, the insertion of at least one nucleotide, the change of DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination, or a combination thereof.Preferably, the plant cell through genetic modification does not include any exogenous genetic material that is stably integrated into the plant cell genome.

[0118] The genetically modified plant cell may be part of a whole plant or a part thereof. Therefore, the present invention also relates to a plant or plant part comprising the genetically modified plant cell described above.

[0119] According to another aspect of the present invention, the genetically modified plant cells can be regenerated into complete (fertile) plants. Therefore, in a preferred aspect of the present invention, the genetic modification of the plant cells is followed by a step of regenerating the plant. Therefore, the present invention provides a method for producing a genetically modified plant, comprising the following steps:

[0120] a) genetically modifying a plant cell according to the above method for genetic modification in a plant cell, and

[0121] b) regenerating a plant from the modified plant cell of step a),

[0122] Preferably, the plants produced do not comprise any genome engineering components, epigenetic regulatory chemicals or active derivatives thereof (particularly DNA methyltransferase inhibitors or histone deacetylase inhibitors (HDACi)), plant hormones or active derivatives thereof, or proteins that cause improved plant regeneration from callus or embryonic tissue, or expression cassettes comprising nucleic acids encoding said proteins, co-introduced in step a).

[0123] As used herein, "regeneration" refers to the process by which single or multiple cells proliferate and develop into tissues, organs, and ultimately entire plants.

[0124] Step b) of regenerating the plant may for example comprise culturing the genetically modified plant cells from step a) on a regeneration medium.

[0125] Regeneration techniques rely on the treatment of certain plant hormones in tissue culture growth media, and occasionally on biocide and / or herbicide markers that can be introduced. Regeneration can be obtained from plant somatic cells, callus cells or embryonic cells and protoplasts derived from different explants (e.g., callus, immature or mature embryos, leaves, buds, roots, flowers, microspores, embryonic tissues, meristems, organs or any part thereof). Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38: 467-486. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. To obtain a complete plant from the transformed or gene-edited cells, the cells are grown in a range of media containing nutrients and hormones under controlled environmental conditions, a process known as tissue culture. Once a complete plant is generated and produces seeds, the progeny are evaluated.

[0126] The present invention also provides a genetically modified plant obtained or obtainable by the above-mentioned method for producing a genetically modified plant or a progeny plant thereof.

[0127] Another subject of the present invention is a plant cell or seed derived from a genetically modified plant as described above. Such a plant cell or seed does not contain any genome engineering components, epigenetic regulatory chemicals or their active derivatives (in particular histone deacetylase inhibitors (HDACi)), plant hormones or their active derivatives, and proteins that cause improved plant regeneration from callus or embryonic tissue or expression cassettes containing nucleic acids encoding said proteins

[0128] Another subject of the present invention is the plant, plant cell or seed derived from the above-mentioned genetically modified cell without the need for selection based on marker genes. As used herein, "selection based on marker genes" refers to selecting from wild-type cells by using integrated selection markers (genes), such as antibiotic resistance genes (such as kanamycin resistance genes, hygromycin resistance genes) or herbicide resistance genes (such as phosphinothricin resistance genes, glyphosate resistance genes), identifying and / or purifying modified cells, particularly any process of cells transformed, gene-edited or base-edited. Without such selection, such plants, plant cells or seeds may not have any integrated genome engineering components, and thus may result in genetically modified plants without transgenics or only the modified plants of the transgenics of interest being integrated.

[0129] Another aspect of the present invention is the use of epigenetic regulating chemicals, such as protein deacetylase inhibitors or active derivatives thereof, in particular histone deacetylase inhibitors (HDACi), and / or plant hormones or active derivatives thereof, and / or proteins that cause improved plant regeneration from somatic cells, callus cells or embryonic cells, or expression cassettes comprising nucleic acids encoding said proteins, for increasing the efficiency of genetic modification in plant cells, preferably in the methods described herein above.

[0130] Unless otherwise indicated in the examples, all recombinant DNA techniques were performed according to standard protocols as described in Sambrook et al. (1989), (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY; and Ausubel et al. (1994), Current Protocols in Molecular Biology, Volumes 1 and 2 of Current Protocols, USA. Standard materials and methods for plant molecular studies are described in Plant Molecular Biology Labfax (1993) by RDD Ray, jointly published by BIOS Scientific Publications (UK) and Blackwell Scientific Publications, UK. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax, Second Edition, Volumes I and II of Academic Press (UK). Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al., (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0131] All patents, patent applications, and publications or public disclosures (including publications on the internet) cited or referenced herein are incorporated by reference in their entirety.

[0132] The present invention is further illustrated by the following figures and examples. However, it should be understood that the present invention is not limited to these examples.

[0133] Attached photos

[0134] Figure 1 : Map of the pLH-Pat5077399-70Subi-tDt construct. tDT defines the tdTomato gene.

[0135] Figure 2 : 15 ng TSA was co-delivered with the construct pLH-Pat5077399-70Subi-tDt ( Figure 1 ).

[0136] A: Red fluorescent images show tdTomato expressing cells (white spots) in maize Hi II immature embryos 16 hours after bombardment. The top image was taken from a control bombardment without TSA (No TSA), while the bottom image was taken from a co-bombardment with 15 ng of TSA.

[0137] B: Average number of red fluorescent cells per embryo 16 hours post-bombardment without TSA (No TSA) or with 15 ng of TSA (15 ng TSA). Error bars = standard deviation.

[0138] FIG3 : Co-delivery of different amounts of TSA (no TSA, 15 ng, 30 ng and 45 ng) with the construct pLH-Pat5077399-70Subi-tDt ( Figure 1 ).

[0139] A: Average number of fluorescent cells per field in maize Hi II immature embryos 16 hours after bombardment with different amounts of TSA.

[0140] B: Percent increase in the mean number of fluorescent cells when co-bombarded with different amounts of TSA. Error bars = standard deviation.

[0141] Figure 4 : Map of pGEP359 construct. tDT defines the tdTomato gene. ZmLpCpf1 defines the maize codon-optimized CDS of the Lachnospiraceae bacterium CRISPR / Cpf1 (LbCpf1) gene.

[0142] Figure 5 : 15 ng TSA was co-delivered with the construct pGEP359 ( Figure 4 ).

[0143] A: 16 hours after bombardment, red fluorescent images show cells expressing tdTomato in maize Hi II type II callus. The left image was taken from a control bombardment without TSA (No TSA), while the right image was taken from a co-bombardment with 15 ng TSA.

[0144] B: Mean number of red fluorescent cells per field 16 hours post-bombardment without (no TSA) or with 15 ng of TSA. Error bars = standard deviation.

[0145] Figure 6 : 15 ng TSA was co-delivered with the construct pLH-Pat5077399-70Subi-tDt ( Figure 1 ).

[0146] A: Red fluorescent images show cells expressing tdTomato (white spots) in friable callus of sugar beet 24 hours after bombardment. The top image was taken from a control bombardment without TSA (No TSA), while the bottom image shows a co-bombardment with 15 ng TSA.

[0147] B: Mean number of fluorescent cells per field 24 hours after bombardment without (-TSA) or with 15 ng TSA (+TSA). Error bars = standard deviation.

[0148] Figure 7 : Map of pGEP284 construct. tDT defines the tdTomato gene. TaCRISPR defines the wheat codon-optimized CDS of the CRISPR nuclease. sgGEP14 targets the guide RNA to the first exon of the maize glossy 2 gene.

[0149] Figure 8 : Different amounts of TSA (no TSA, 15ng, 30ng, and 45ng) were co-delivered with the gene editing construct pGEP284 ( Figure 7 ). A: Site-specific InDel (insertion and deletion) rates in Hi II embryos 2 days after co-bombardment. B: Percent change in InDel rate when different amounts of TSA (no TSA, 15ng, 30ng and 45ng, from left to right) in maize Hi II embryos were co-bombarded with the genome editing construct pGEP284.

[0150] Fig. 9: Map of pGEP353 constructs. crGEP46 defines crRNA46, which targets the maize glycerate kinase gene (GLYK).

[0151] Fig.10 : Gene editing construct pGEP359 (ZmLbCpf1, Figure 4 ) and pGEP353 (crRNA46, Fig. 9 ) were co-transferred into maize Hi II callus with 15 ng TSA (right, 15 ng TSA) or without TSA (left, no TSA).

[0152] Fig.11 : pGEP362 construct map. mNeonGreen defines the mNeonGreen gene, which encodes the brightest monomeric green or yellow fluorescent protein with an excitation maximum of 506 nm and an emission maximum of 517 nm. ZmLpCpf1 defines the maize codon-optimized CDS of the Lachnospiraceae bacterial CRISPR / Cpf1 (LbCpf1) gene.

[0153] Fig.12 : 250 ng of 2,4-D was mixed with the construct pGEP362 ( Fig.11 ) were co-delivered into maize HiII immature embryos.

[0154] A: Green fluorescent images show cells expressing the mNeonGreen reporter gene in maize Hi II immature embryos 16 hours after bombardment. The top image was taken from a control bombardment without 2,4-D (No 2,4-D), while the bottom image shows a co-bombardment with 250 ng 2,4-D.

[0155] B: Average number of green fluorescent cells per embryo 16 hours after bombardment. Error bars = standard deviation.

[0156] Fig.13 : Different amounts of 2,4-D (0 ng, 125 ng, 250 ng and 500 ng) were co-delivered with the construct pGEP362 ( Fig.11 ).

[0157] A: Green fluorescence images show cells expressing the mNeonGreen reporter gene in maize Hi II type II callus cells 16 hours after co-bombardment with different amounts of 2,4-D (0 ng, 125 ng, 250 ng, and 500 ng).

[0158] B: Average number of green fluorescent cells per field 16 hours after bombardment with different amounts of 2,4-D (0 ng, 125 ng, 250 ng and 500 ng). Error bars = standard deviation.

[0159] Fig.14 : 2,4-D was combined with the construct pGEP359 ( Figure 4 ) were co-delivered into leaves of corn plants (top: without 2,4-D, bottom: with 250 ng of 2,4-D) (exemplary tdT expression is indicated by arrows).

[0160] Fig.15 : 250 ng of 6-BA or zeatin were combined with the construct pGEP359 ( Figure 4 ) were co-delivered into maize Hi II type II callus tissue.

[0161] A: Red fluorescent images from left to right show cells expressing the tdTomato reporter gene in maize Hi II type II callus cells 16 hours after bombardment without hormones (No Hormone), with 250 ng 6-BA, or with 250 ng zeatin.

[0162] B: Average number of red fluorescent cells per field 16 hours after bombardment. Error bars = standard deviation.

[0163] Fig.16 : Map of the pABM-BdEF1_ZmPLT5 construct. The maize PLT5 gene (ZmPLT5) is driven by the strong constitutive EF1 promoter (BDEF1) from Brachypodium.

[0164] Fig.17 : Map of the pABM-BdEF1_ZmPLT7 construct. The maize PLT7 gene (ZmPLT7) is driven by the strong constitutive EF1 promoter (BDEF1) from Brachypodium.

[0165] Fig.18 : Map of the pABM-BdEF1_TaRKD construct. The wheat RKD gene (TaRKD) is driven by the strong constitutive EF1 promoter (BDEF1) from Brachypodium.

[0166] Fig.19 100 ng of the booster gene construct was mixed with the construct pGEP359 ( Figure 4 ) were co-delivered into maize Hi II immature embryos.

[0167] A: Red fluorescent images show cells expressing the tdTomato reporter gene in maize Hi II immature embryos 16 hours after bombardment. Images from left to right are from control bombardments without boost (tDT only) or with ZmPLT5 ( Fig.16 )(tDT+ZmPLT5) or wheat RKD (TaRKD, Fig.18 )(tDT+TaRKD) was photographed during the bombardment of the booster construct.

[0168] B: Average number of red fluorescent cells per embryo 16 hours after bombardment. Error bars = standard deviation.

[0169] Fig. 20 : 12 days after co-bombardment with ZmPLT5 or ZmPLT7 gene constructs, tdTomato fluorescent embryogenic callus was observed. The red fluorescent images shown in the figure show the tdTomato reporter gene expressed in callus cells of embryogenesis induced from immature embryos 12 days after bombardment. Images from left to right show embryos bombarded with only the tDTomato reporter gene (tDT only) or with 100ng of boosted ZmPLT5 (tDT+ZmPLT5) or ZmPLT7 gene constructs (tDT+ZmPLT7).

[0170] Fig.21 : Callus induction in A188 immature embryos 17 days after co-bombardment of tdTomato with the wheat RKD booster construct.

[0171] A: Bright field images showing callus induction from immature embryos bombarded with the tDTomato reporter construct alone.

[0172] B: Bright field image showing callus induction from immature embryos co-bombarded with tDTomato reporter and wheat RKD constructs. Example

[0173] Example 1: Co-delivery of trichostatin A (TSA) with a construct containing the tdTomato reporter gene (ie, pLH-Pat5077399-70Subi-tDt) by microprojectile bombardment in maize immature embryos without TSA pretreatment improves transient transformation efficiency.

[0174] Procedure: Preparation of immature embryos of maize for bombardment: 8-10 days after pollination, harvest maize ears (i.e. A188 or Hi II) with immature embryos of 0.8 to 1.8 mm in size. The ears are sterilized with 70% ethanol for 10-15 minutes. After a short air-drying in a laminar hood, the upper ~1 / 3 of the corn kernels are removed from the ears with a shark scalpel, and the immature embryos are carefully removed from the corn kernels with a spatula. The freshly separated embryos are placed on the bombardment target area in the infiltration culture plate (see below) with the embryo cotyledon facing upward. Before bombardment, the plate is wrapped with parafilm and incubated in the dark at 25°C for 4-20 hours.

[0175] For the use of 100 μg gold particles (approximately 4.0-5.0 x 10 7 The amount of TSA for bombardment (0.6 micron gold particles) is in the range of 0.01 ng to 500 ng, preferably in the range of 0.1 to 50 ng. Co-coat plasmid DNA and TSA onto gold particles for bombardment: For 10 shots, pipette 1 mg of 0.6 micron (μm) gold particles (100 μg gold particles per shot) in a total volume of 100 microliters (μl) of 50% (v / v) glycerol into a clear low retention microcentrifuge tube. Sonicate for 15 seconds to suspend the gold particles. Under low speed vortexing, add the following in order to each 100 μl of gold particles:

[0176] - Up to 10 μl of DNA (1.0 μg total DNA, 100 ng per shot)

[0177] -100 μl 2.5M CaCl2 (pre-chilled on ice)

[0178] - 40 μl of 0.1 M cold spermidine

[0179] Cover the tube and vortex the centrifuge tube at 0-10°C for 2-30 minutes, and spin down the DNA-coated gold particles. After washing twice with 500 μl of 100% ethanol, resuspend the pellet in 120 μl of 100% ethanol. Finally, carefully add an appropriate amount of TSA (for the bombardment of 100 μg of 0.6 μm gold particles, the amount of TSA is 0.01 to 500 ng, preferably 0.1-50 ng; TSA is dissolved in DMSO) to the resuspended gold particles. While vortexing at low speed, add 10 μl of plasmid DNA (pLH-Pat5077399-70Subi-tDt construct; Figure 1 ) and TSA-coated gold particles were evenly pipetted from the tube to the center of the macrocarrier using a wide-mouth 20 μl pipette tip. Since the particles tend to form clumps at this time, the gold particles were placed on the macrocarrier as quickly as possible. Air dry.

[0180] Bombardment was performed using a Bio-Rad PDS-1000 / He particle gun. Bombardment conditions were: 27-28 mm / Hg vacuum, 450 or 650 psi rupture disc, 6 mm gap distance, and the sample platform was at a second position 60 mm away from the chamber bottom. After bombardment, embryos were retained on the osmotic medium for another 16 hours, and then moved to the II type callus induction medium flat plate (see below). After bombardment, 16-48 hours, transient conversion was checked for tdTomato gene expression using a fluorescent microscope at an excitation maximum of 554 nm and an emission maximum of 581 nm.

[0181] Type II callus induction medium: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 2.9 g / L L-proline, 20 g / L sucrose, 5 g / L glucose, 5 mg / L AgNO3, 8 g / L Bacto-agar, pH 5.8.

[0182] Osmotic medium: N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 0.7 g / L L-proline, 0.2 M mannitol (36.4 g / L), 0.2 M sorbitol (36.4 g / L), 20 g / L sucrose, 15 g / L bacterial agar, pH 5.8.

[0183] exist Figure 2 In the experiment, 15 ng of TSA was co-delivered with the construct pLH-Pat5077399-70Subi-tDt ( Figure 1 ) to improve transient DNA transformation in maize Hi II immature embryos. Figure 2 In A, red fluorescent images show cells expressing tdTomato in maize Hi II immature embryos 16 hours after bombardment with 15 ng TSA, compared to controls bombarded without TSA. By co-delivering 15 ng TSA, the average number of red fluorescent cells (i.e., positive transiently transformed cells) per embryo 16 hours after bombardment was increased by 98.2% ( Figure 2 B).

[0184] This co-delivery experiment has been repeated using different amounts of TSA - no TSA, 15 ng TSA, 30 ng TSA and 45 ng TSA (Figure 3). The presence of TSA always improves transient transformation in maize Hi II immature embryos. The average number of fluorescent cells (i.e., positive transiently transformed cells) per field of view in maize Hi II immature embryos at 16 hours shows an optimum around 30 ng of TSA ( Figure 3AHowever, even lower and higher concentrations resulted in a significant increase in transiently transformed cells ( Figure 3B ).

[0185] Example 2: In type II maize callus without TSA pretreatment, TSA and tdTomato reporter construct pGEP359 ( Figure 4 ) co-delivery promotes transformation efficiency

[0186] Type II callus induction and selection: As described in Example 1, Hi II immature embryos of 0.8-1.8 mm in size were isolated and immediately placed on Type II callus induction medium (see below) with the embryo cotyledon side facing upwards at a density of 10-15 embryos per plate (100 mm in diameter). The plates were wrapped with parafilm and embryos were cultured in the plates at 27°C in the dark until Type II callus appeared (~2 weeks). Loose Type II callus was selected under a stereoscope and moved to Type II callus selection medium (see below). This process was repeated 2-3 times and embryos were discarded 4 weeks after induction. Based on looseness (highly loose type), morphology (no embryo-like structure), color (fresh, white, translucent), the pre-embryonic stage of Type II callus was carefully selected under a stereoscope. Type II callus was selected and subcultured in callus selection medium every 1-2 weeks (see below) until the callus line was stable (about 3-5 rounds of selection). Stable Type II callus lines were cultured every 1-2 weeks on Type II callus subculture medium (see below).

[0187] Preparation of Type II callus for bombardment: Highly loose Type II callus was selected at the pre-embryonic stage and transferred to the bombardment target area (see Example 1) in an osmotic medium plate (single layer, no overlap). Before bombardment, the plate was wrapped with parafilm and incubated at 25° C. in the dark for 4-20 hours (preferably 4 hours).

[0188] The same procedures as described in Example 1 were used for microprojectile bombardment and post-bombardment treatment.

[0189] Type II callus induction medium: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 2.9 g / L L-proline, 20 g / L sucrose, 5 g / L glucose, 5 mg / L AgNO3, 8 g / L bacterial agar, pH 5.8

[0190] Type II callus selection medium: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 2.9 g / L L-proline, 20 g / L sucrose, 2 mg / L AgNO3, 8 g / L bacterial agar, pH 5.8

[0191] Type II callus subculture medium: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 0.7 g / L L-proline, 20 g / L sucrose, 8 g / L bacterial agar, pH 5.8

[0192] exist Figure 5 In , co-delivery of 15 ng TSA with construct pGEP359 by microprojectile bombardment of 100 μg gold particles (0.6 μm size) enhanced transient transformation in maize Hi II type II callus. Figure 5 In A, the red fluorescent image shows cells expressing tdTomato in maize Hi II II callus 16 hours after bombardment with 15 ng TSA compared to a control bombardment without TSA. The average number of fluorescent cells (i.e., positive transiently transformed cells) per field in maize Hi II II callus 16 hours after bombardment was increased by 43.3% by co-delivery of 15 ng TSA ( Figure 5 B).

[0193] Example 3: Co-delivery of trichostatin A (TSA) and construct pLH-Pat5077399-70Subi-tDt by microprojectile bombardment improves transient transformation of sugar beet loose callus.

[0194] Beet callus induction: Young leaves from beet sprouts cultured in vitro in sprout medium (see below) were cut into small pieces (square, size 3-5 mm) in a laminar flow hood and placed on callus induction medium (see below) at a density of 10-15 pieces per plate (100 mm diameter), adaxial side facing up. The plates were wrapped with parafilm and the leaf segments were cultured on the plates in the dark at 23°C for 6-8 weeks until callus appeared.

[0195] Preparation of sugar beet callus for bombardment: Fresh loose callus were harvested under a stereoscope and transferred to the bombardment target area (see below) in sugar beet osmotic medium (single layer, no overlap). Prior to bombardment, plates were wrapped with parafilm and incubated at 25°C in the dark for 4-20 hours.

[0196] Microprojectile bombardment and post-bombardment treatment were performed using the same procedures described in Example 1, except that the amount of gold particles used for bombardment was 300 μg.

[0197] Beet sprout culture medium: MS, 0.25 mg / L BAP, 30 g / L sucrose, 8 g / L plant agar, pH 6.0

[0198] Beet callus induction medium: MS, 2.0 mg / L BAP, 15 g / L sucrose, 8 g / L plant agar, pH 6.0

[0199] Beet callus infiltration medium: MS, 2.0 mg / L BAP, 15 g / L sucrose, 0.2 M mannitol (36.4 g / L), 0.2 M sorbitol (36.4 g / L), 8 g / L plant agar, pH 6.0

[0200] exist Figure 6 In the experiment, 15 ng of TSA was co-delivered with the construct pLH-Pat5077399-70Subi-tDt ( Figure 1 ) improved transient transformation in loose callus of sugar beet. Figure 6 In A, the red fluorescent image shows cells expressing tdTomato in loose beet callus 24 hours after bombardment with 15 ng TSA compared to control bombardment without TSA. The average number of fluorescent cells (i.e., positive transiently transformed cells) per field 24 hours after bombardment was increased by 193.7% ( Figure 6 B).

[0201] Example 4: Co-delivery of trichostatin A (TSA) with gene editing constructs improves genome editing efficiency in maize immature embryos.

[0202] The same procedures as described in Example 1 were used for embryo isolation, microprojectile bombardment, and post-bombardment treatment.

[0203] Two days after bombardment, embryos were harvested and used for genomic DNA isolation using the Plant DNA Isolation kit from Qiagen (Venlo, Netherlands). NGS (Next Generation Sequencing) was performed by the Miseq platform of Illumina Inc. (San Diego, California, USA). InDel (insertion and deletion) rates were analyzed by the tool of CRISPResso (http: / / crispresso.rocks / ).

[0204] exist Figure 8 In the figure, co-bombardment with TSA (from left to right, no TSA, 15 ng, 30 ng, and 45 ng, respectively) resulted in improved gene editing efficiency in Hi II embryos 2 days after bombardment. Figure 8 In A, the expression of different amounts of TSA and the gene editing construct pGEP284 ( Figure 7) Site-specific InDel rates in Hi II embryos 2 days after co-bombardment, where the site-specific InDel rate indicates gene editing efficiency. The presence of TSA always increases the frequency of gene editing events in maize Hi II immature embryos. The rate of InDel events (i.e., embryos positive for gene editing) shows an optimal value at around 30ng of TSA. However, even lower and higher concentrations result in significantly increased InDel rates compared to the absence of TSA. When different amounts of TSA were co-bombarded with the gene editing construct pGEP284 in maize Hi II embryos, the percentage change in the InDel rate is shown in Figure 2. Figure 8 As shown in B.

[0205] Example 5: Trichostatin A (TSA) and gene editing construct pGEP359 ( Figure 4 ) and pGEP353( Fig. 9 ) improves genome editing efficiency in maize Hi II type II callus

[0206] The same procedures as described in Example 2 were used for Type II callus culture and microprojectile bombardment and post-bombardment treatment.

[0207] 2-15 days after bombardment, embryos were harvested and used for genomic DNA isolation using the Plant DNA Isolation kit from Qiagen. NGS (Next Generation Sequencing) was performed by the Illumina Miseq platform. InDel (Insertion and Deletion) rates were analyzed by the tool of CRISPResso.

[0208] exist Fig.10 In the study, the gene editing construct pGEP359 (ZmLbCpf1, Figure 4 ) and pGEP353 (crRNA46, Fig. 9 ) with 15 ng TSA (on the right, 15 ng of TSA) or no TSA (on the left, no TSA) in maize Hi II callus shows a 6.75-fold or 575% increase in site-specific InDel (insertion and deletion) rate 13 days after co-bombardment.

[0209] Example 6: Auxin 2,4-D and mNeonGreen reporter construct pGEP362 ( Fig.11 ) improves its transient transformation efficiency in maize immature embryos

[0210] The same procedures as described in Example 1 were used for embryo isolation and microprojectile bombardment and post-bombardment treatment.

[0211] For the use of 100 μg gold particles (approximately 4.0 -5.0 x 107 The amount of 2,4-D bombarded with 0.6 μm gold particles (0.6 μm gold particles) is in the range of 1.0 ng to 1000 ng, preferably in the range of 10 ng to 500 ng. Co-coating of plasmid DNA and 2,4-D onto gold particles for bombardment is performed as described in Example 1. A 2,4-D stock solution (e.g., 1 mg / ml) is prepared in 100% DMSO.

[0212] exist Fig.12 In the experiment, 250 ng of 2,4-D was co-delivered with the construct pGEP362 ( Fig.11 ) improved transient DNA transformation in maize Hi II immature embryos. Fig.12 In A, green fluorescence images show cells expressing the mNeonGreen reporter gene in maize Hi II immature embryos 16 hours after bombardment. B: Average number of green fluorescent cells per field 16 hours after bombardment with 250 ng of 2,4-D compared to control bombardment without 2,4-D. Co-bombardment with 250 ng of 2,4-D resulted in a 187% increase in the average number of fluorescent cells per embryo ( Fig.12 B).

[0213] Example 7: Auxin 2,4-D and mNeonGreen reporter construct pGEP362 ( Fig.11 ) co-delivery improves its transient transformation efficiency in maize Hi II type II callus

[0214] The same procedures as described in Example 2 were used for Type II callus culture and microprojectile bombardment and post-bombardment treatment.

[0215] For the use of 100 μg gold particles (approximately 4.0 -5.0 x 10 7 The amount of 2,4-D bombarded with 0.6 μm gold particles (0.6 μm gold particles) is in the range of 1.0 ng to 1000 ng, preferably in the range of 10 ng to 500 ng. Plasmid DNA and 2,4-D are co-coated onto gold particles for bombardment as described in Example 6.

[0216] exist Fig.13 In the experiment, different amounts of 2,4-D (0 ng, 125 ng, 250 ng and 500 ng) were co-delivered with the construct pGEP362 ( Fig.11) improves transient transformation of maize Hi II II callus. Green fluorescence images show cells expressing the mNeonGreen reporter gene in maize Hi II II callus cells 16 hours after co-bombardment with different amounts of 2,4-D (from top left to bottom right: 2,4-D 0 ng, 125 ng, 250 ng, and 500 ng), showing that co-bombardment with 2,4-D significantly increased fluorescence ( Fig.13 A) In Fig.13 In B, the average number of green fluorescent cells per field of view is shown 16 hours after bombardment with different amounts of 2,4-D (0 ng, 125 ng, 250 ng and 500 ng). By adding 2,4-D, the average number of fluorescent cells has been increased by at least 34.8%.

[0217] Example 8: Auxin 2,4-D and tDTomato reporter construct pGEP359 ( Figure 4 ) improves its transient transformation efficiency in maize plant leaves

[0218] Corn plants have been grown in a greenhouse. Microprojectile bombardment was performed in stage V8 using a Bio-Rad PDS-1000 / He particle gun. Bombardment conditions were: 27-28 mm / Hg vacuum, 450 or 650 psi rupture disc, 6 mm gap distance. 20 hours after bombardment, transient conversion was checked for tdTomato gene expression using a fluorescence microscope at excitation maximum 554 nm and emission maximum 581 nm. Plasmid DNA and 2,4-D were co-coated onto gold particles for bombardment as described in Example 1. 2,4-D stock solution (e.g., 25 mg / ml in DMSO).

[0219] exist Fig.14 In this study, 2,4-D was co-delivered with the construct pGEP359 ( Figure 4 ) improved transient transformation in maize leaves.

[0220] Example 9: Cytokinins such as 6-BA or zeatin and tDTomato reporter construct pGEP359 ( Figure 4 ) co-delivery improves its transient transformation efficiency in maize Hi II type II callus

[0221] The same procedures as described in Example 2 were used for Type II callus culture and microprojectile bombardment and post-bombardment treatment.

[0222] For the use of 100 μg gold particles (approximately 4.0 -5.0 x 10 7The amount of 6-BA or zeatin bombarded with 0.6 μm gold particles (0.6 μm gold particles) is in the range of 1.0 ng to 1000 ng, preferably in the range of 10 ng to 1000 ng. Plasmid DNA and cytokinin are co-coated onto gold particles for bombardment as described in Example 6.

[0223] exist Fig.15 In maize Hi II type II callus, 250 ng of 6-BA or zeatin were combined with the construct pGEP359 ( Figure 4 ) co-delivery. 16 hours after bombardment ( Fig.15 A), Red fluorescence images showing cells expressing the tdTomato reporter gene in maize Hi II type II callus cells, from left to right: control bombardment without hormones (no hormone), bombardment with 250 ng of 6-BA, and bombardment with 250 ng of zeatin. Fig.15 In B, the average number of red fluorescent cells per field 16 hours after bombardment is shown. Co-bombardment with 250 ng 6-BA increased the average number of fluorescent cells by 35.8%, and co-bombardment with 250 ng zeatin increased the average number of fluorescent cells by 31.2%.

[0224] Example 10: Booster gene and tDTomato reporter construct by microprojectile bombardment ( Figure 4 ) improves its transient transformation efficiency in maize immature embryos

[0225] The same procedures as described in Example 1 were used for embryo isolation, microprojectile bombardment, and post-bombardment treatment.

[0226] Enhance gene and fluorescent reporter construct (tdTomato gene, Figure 4 ) were co-bombarded with 100 μg of gold particles (approximately 4.0 -5.0 x 10 7 The booster gene construct ( Fig.16 , Fig.17 , Fig.18 ) is in the range of 10.0 ng to 1000 ng, preferably in the range of 50 ng to 100 ng. Plasmid DNA is coated on gold particles for bombardment as described in Example 1.

[0227] The boosting effect was measured by its ability to increase the transient transformation frequency of a reporter gene 16-20 days after bombardment of maize Hi II immature embryos.

[0228] 100 ng of the booster gene construct was co-delivered with 100 ng of the tDTomato reporter construct ( Figure 4 ) improved transient transformation of the tDTomato gene in maize Hi II immature embryos.

[0229] exist Fig.19 In A, red fluorescent image shows cells expressing the tDTomato reporter gene in maize Hi II immature embryos 16 hours after bombardment. Fig.19 B: Average number of red fluorescent cells per embryo 16 hours after bombardment with booster constructs compared to control bombardments with reporter only (tDT only). Fig.16 Co-bombardment of )(tDT+ZmPLT5) resulted in a 102% increase in the average number of fluorescent cells per embryo, or 100 ng of wheat RKD (TaRKD) ( Fig.18 Co-bombardment of )(tDT+TaRKD) resulted in a 144% increase in the average number of fluorescent cells per embryo ( Fig.19 B).

[0230] Example 11: Transient overexpression of enhanced genes promotes transformation frequency (TF)

[0231] Embryo isolation, microprojectile co-bombardment, and post-bombardment treatments were performed using the same procedures as described in Example 10. The enhancement of transformation was measured by its ability to increase the transformation frequency of a reporter gene 12 days after bombardment of maize Hi II immature embryos (IE) without selection.

[0232] As shown in Table 1, co-bombardment of the tdTomato construct with ZmPLT5 resulted in a 42.9% increase in transformation frequency of the tdTomato gene (more than 16-fold increase compared to the control), while co-bombardment with ZmPLT7 resulted in a 53% increase in transformation frequency of the tdTomato gene 12 days after bombardment without selection (more than 16-fold increase compared to the control) ( Fig. 20 ).

[0233] Table 1: tDT transformation frequency (FT) 12 days after bombardment: FT is defined as the number of embryos with at least one embryogenic structure expressing tDT (number of tDT-positive IEs) out of 100 bombarded embryos.

[0234] Only tDT tDT+ZmPLT5 tDT+ZMPLT7 Number of tDT-positive IE / total IE 1 / 40 21 / 49 26 / 49 TTW 2.5％ 42.9％ 53.1％

[0235] Example 12: Transient overexpression of wheat RKD enhancing gene (SEQ ID NO: 6) promotes callus induction in A188 immature embryos

[0236] Embryo isolation, microprojectile co-bombardment, and post-bombardment treatment were performed using the same procedures as described in Example 10, and callus induction was performed as described in Example 2.

[0237] Transient overexpression of the wheat RKD gene resulted in a significant improvement in callus induction, with the induction rate increasing from 38% without TaRKD to 75% with 100 ng of TaRKD (almost doubling the callus induction rate) ( Fig.21 ).

Claims

1. A method for genetic modification in a sugar beet plant cell, the method comprising steps (a) and (b) (a) introducing the following (i) and (ii) together into the beet plant cell, (i) a genome engineering component, and (ii) a second compound comprising (ii.1) trichostatin, or (ii.2) a plant hormone selected from 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine or zeatin, or (ii.3) a protein which leads to improved regeneration of sugar beet plants or an expression cassette comprising a nucleic acid encoding said protein, (b) culturing the sugar beet plant cell under conditions that allow the genome of the sugar beet plant cell to be genetically modified by the activity of a genome engineering component in the presence of the second compound; And among them, The protein causing improved regeneration of sugar beet plants comprises an amino acid sequence selected from one of the following: a) a sequence as shown in any one of SEQ ID NOs: 1, 3, 5, 13, 15, 29 or 31, or b) an amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 14, 16, 30 or 32.

2. The method of claim 1, wherein the genome engineered component (i) and / or the second compound (ii) are transiently active and / or transiently present in the sugar beet plant cell.

3. The method of claim 1, wherein the genome engineering component comprises a. a double-strand DNA break inducing enzyme or a nucleic acid encoding the same, and optionally comprising a repair nucleic acid molecule, or b. a single-stranded DNA or RNA break-inducing enzyme or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, or c. a base editor enzyme, optionally fused to a disarmed double-stranded DNA break-inducing enzyme or a single-stranded DNA or RNA break-inducing enzyme, or d. An enzyme that affects DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination, optionally fused to a disarmed double-strand DNA break-inducing enzyme or a single-strand DNA or RNA break-inducing enzyme.

4. The method of claim 3, wherein the double-strand DNA break-inducing enzyme recognizes a predetermined site in the genome of the cell.

5. The method of claim 3, wherein the single-stranded DNA or RNA break-inducing enzyme recognizes a predetermined site in the genome of the cell.

6. The method of claim 3, wherein in c, the disarmed double-stranded DNA break-inducing enzyme or single-stranded DNA or RNA break-inducing enzyme recognizes a predetermined site in the genome of the cell.

7. The method of claim 3, wherein in d, the disarmed double-stranded DNA break-inducing enzyme or single-stranded DNA or RNA break-inducing enzyme recognizes a predetermined site in the genome of the cell.

8. The method of claim 1, wherein the genome engineering component comprises a double-stranded DNA break-inducing enzyme or a single-stranded DNA or RNA break-inducing enzyme selected from the group consisting of CRISPR / Cas endonucleases, zinc finger nucleases, homing endonucleases, meganucleases, and TAL effector nucleases.

9. The method of claim 8, wherein the CRISPR / Cas endonuclease is a CRISPR / Cpf1 endonuclease.

10. The method of claim 2, wherein the transient activity of the genome engineering component in step (b) comprises inducing one or more double-strand breaks in the genome of the beet plant cell, inducing one or more single-strand breaks in the genome of the beet plant cell, inducing one or more base editing events in the genome of the beet plant cell, or inducing one or more DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination in the genome of the beet plant cell.

11. The method of claim 10, wherein the induction of one or more double-strand breaks or one or more single-strand breaks is followed by non-homologous end joining and / or homology-directed repair of the breaks by homologous recombination mechanisms.

12. The method of claim 1, wherein in step (b) the modification of the genome is selected from a. replace at least one nucleotide; b. at least one nucleotide is missing; c. insert at least one nucleotide; d. Changes in DNA methylation, e. changes in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination, or Any combination of fa-e.

13. The method of claim 1, wherein the beet plant cell in step (a) is a cell derived from callus, immature or mature embryo, leaf, shoot, root, flower, microspore, embryonic tissue or meristem.

14. The method described in claim 1 further comprises the step of pretreating the beet plant cells to be used in step (a), wherein the pretreatment comprises culturing the beet plant cells or the beet plant material comprising the beet plant cells in a culture medium containing the trichostatin, the phytohormone, and the protein that causes improved regeneration of the beet plant.

15. The method of claim 1, wherein the co-introduction is performed by particle bombardment, microinjection, Agrobacterium-mediated transformation, electroporation, physical delivery via nanoparticles or cell penetrating peptides, or vacuum infiltration.

16. The method of claim 1, wherein the co-introduction is performed by agroinfiltration or based on physical delivery.

17. The method of claim 16, wherein the physical delivery is particle bombardment, microinjection, or electroporation.

18. The method of claim 15, wherein the co-introduction is by particle bombardment.

19. The method of claim 1, wherein the method comprises a further step (c) of obtaining and / or selecting genetically modified sugar beet plant cells or sugar beet plants or parts thereof, said sugar beet plants or parts thereof comprising genetically modified sugar beet plant cells or derived from genetically modified sugar beet plant cells and comprising a genetic modification of the genome in at least one cell.

20. The method of claim 19, wherein the selection is performed by means of detecting a genetic modification.

21. The method of claim 20, wherein the selection is performed by a PCR-based method, by a phenotypic characteristic, or by a morphological characteristic.

22. The method of claim 21, wherein the phenotypic characteristic is herbicide resistance or a color marker.

23. The method of claim 21, wherein the phenotypic characteristic is a fluorescent marker.

24. The method of claim 23, wherein the fluorescent marker is tdTomato or mNeonGreen.

25. A microparticle for genetically modifying the genome of a sugar beet plant by microprojectile bombardment, the microparticle being coated with at least (i) a genome engineering component, and (ii) a second compound comprising (ii.1) trichostatin, or (ii.2) a plant hormone selected from 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine or zeatin, or (ii.3) a protein which leads to improved regeneration of sugar beet plants or an expression cassette comprising a nucleic acid encoding said protein, The protein causing improved regeneration of sugar beet plants comprises an amino acid sequence selected from one of the following: a) a sequence as shown in any one of SEQ ID NOs: 1, 3, 5, 13, 15, 29 or 31, or b) an amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 14, 16, 30 or 32.

26. A kit for genetically modifying the genome of a sugar beet plant by microprojectile bombardment, comprising (I) one or more microparticles having a genome engineered component and a second compound comprising (1) Trichostatin, or (2) a plant hormone selected from 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, and zeatin, or (3) a protein which leads to improved regeneration of sugar beet plants or an expression cassette comprising a nucleic acid encoding said protein, The protein causing improved regeneration of sugar beet plants comprises an amino acid sequence selected from one of the following: a) a sequence as shown in any one of SEQ ID NOs: 1, 3, 5, 13, 15, 29 or 31, or b) an amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 14, 16, 30 or 32; and (II) means for coating the microparticles.

27. A method for producing a genetically modified sugar beet plant comprising the following steps: 1). Genetically modifying a sugar beet plant cell according to any one of claims 1 to 24, and 2). Regenerating a sugar beet plant from the modified sugar beet plant cell of step 1).

28. The method of claim 27, wherein the produced sugar beet plant does not contain any of the genome engineering components and the second compound co-introduced in step (a).

29. Use of trichostatin, or a plant hormone, or a protein causing improved regeneration of sugar beet plants, or an expression cassette comprising a nucleic acid encoding said protein, in a method according to any one of claims 1 to 24, 27 or 28, wherein the plant hormone is selected from 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, or zeatin, The protein causing improved regeneration of sugar beet plants comprises an amino acid sequence selected from one of the following: a) a sequence as shown in any one of SEQ ID NOs: 1, 3, 5, 13, 15, 29 or 31, or b) an amino acid sequence encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 14, 16, 30 or 32.

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