METHODS FOR INDUCING CALL FORMATION FROM AT LEAST ONE PLANT CELL OF Beta spp., REGENERATE INDIRECTLY A PLANT, TRANSFORM A PLANT CELL, PRODUCE A TRANSGENIC PLANT, MODIFY THE GENOME OF SAID PLANT CELL OF Beta spp. AS WELL AS TO PRODUCE CALMS WITH INCREASED CAPACITY OF BOOTH REGENERATION, USES OF A HISTONE DEACETYLASE INHIBITOR (HDACi), AS WELL AS FROM A PLANT, PLANT PART, PLANT CELL OR SEED OF A PLANT OBTAINED OR OBTAINABLE BY SUCH METHODS

Description

1 / 24 Methods for inducing callus formation from at least one plant cell of Beta spp., indirectly regenerating a plant, transforming a plant cell, producing a TRANSGENIC PLANT, MODIFYING THE GENOME OF SAID PLANT CELL OF Beta spp. AS WELL AS TO PRODUCE CALLUSES WITH INCREASED SHOOT REGENERATION CAPACITY, USING A HISTONE DEACETYLASE INHIBITOR (HDACi), AS WELL AS A PLANT, PLANT PART, PLANT CELL OR SEED OF A PLANT OBTAINED OR OBTAINABLE BY SAID METHODS Separated from BR112020012062-6, filed on December 21, 2018.

[0001] The present invention relates to the field of plant breeding and, in particular, to plant regeneration from cells and other tissues. More particularly, the invention provides methods and means for improving callus formation and plant regeneration from callus tissue using a histone deacetylase inhibitor.

[0002] Plant regeneration involves the in vitro culture of cells, tissues, and organs under defined physical and chemical conditions. Regeneration is known to occur in plants. In plants, differentiated cells are capable of regenerating into a wide variety of tissues under appropriate culture conditions. Regeneration can involve direct or indirect organogenesis. In direct regeneration, in vitro organs are induced directly from explant tissues; in indirect regeneration, a de novo organ is typically formed from an intermediate tissue, the callus. Plant calluses are undifferentiated structures that can give rise to new tissues. Leaves, shoots, roots, and plant embryos can be extracted from a growing callus by treating it with different proportions of hormones.

[0003] In general, three phases can be recognized during plant regeneration. First, somatic cells of explant tissues can respond to hormonal signals to acquire characteristics similar to meristematic cells, a process known as dedifferentiation. Second, callus cells with organogenic competence will be reprogrammed and determined for the formation of specific organs under the influence of hormonal balance. The third phase of regeneration, morphogenesis, is independent of exogenously supplied hormones. Thus, exogenous hormonal treatment is the critical factor that triggers the first Petition 870260084047, dated 08 / 19 / 2026, page 6 / 47 2 / 24 developmental events in in vitro regeneration.

[0004] However, obtaining dedifferentiated cells (calluses) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant to dedifferentiation and plant regeneration. These difficulties have been the main obstacles to obtaining transgenic sugar beets, for example, through an Agrobacterium-mediated transformation process. For decades, breeders and researchers have been working on developing more efficient protocols for the transformation and regeneration of plants recalcitrant to callus formation. Typically, these plants show genotypic variations, causing drastic differences in callus and shoot formation rates between different lines (Ivic-Haymes & Smigocki (2005), Identification of highly regenerative plants within sugar beet (Beta vulgaris L.).) breeding lines for molecular breeding, In Vitro Cellular and Developmental Biology-Plant, 41(4), 483-488; Mishutkina & Gaponenko (2006), Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants and plant genotype on shoot regeneration frequency, Russian Journal of Genetics, 42(2), 150-157; Tomita et al. (2013), Evaluation of the potential for somatic embryogenesis in sugar beet (Beta vulgaris L.) breeding lines and improvement of regeneration efficiency, Plant Biotechnology, 30(5), 479-487), A regeneração frequente para determinados genótipos não é possível em geral. Kishchenko et al. 2005 (Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin, Cell biology international, 29(1), 15-19) e Kagami et al. 2015, Sugar beet (Beta vulgaris L.(Agrobacterium Protocols, Volume 1, 335-347) describe well-known protocols for beet transformation; however, these protocols show strong genotype dependence.

[0005] In the context of inducing haploid embryogenesis in order to produce double haploid plants from microspores, it has been found that adding HDACi (histone deacetylase inhibitors), such as trichostatin A (TSA), to the culture medium greatly increases the proportion of cells derived from male gametophytes of various plant species that undergo embryogenic growth (document WO 2015 / 044199 A1). However, the use of TSA in callus formation and regeneration has been quite concerning. In Furuta et al. (2011), the chromatin remodeling factor CKH2 / PKL negatively regulates cytokine responses in Petition 870260084047, dated 08 / 19 / 2026, p. 7 / 47 3 / 24 Arabidopsis calli, Plant and Cell Physiology, 52(4), 618-628), characterization of the cytokinin 2 (ckh2) hypersensitive mutant in Arabidopsis showed that histone deacetylation is closely related to cytokine-induced callus growth. TSA application has been used as a partial substitute for cytokinins in promoting callus growth from hypocotyl explants. Cytokinin (kinetin) and TSA did not induce callus growth, either individually or in combination. Recently, Lee et al. (2016), Histone deacetylation-mediated cell dedifferentiation in Arabidopsis, Journal of Plant Physiology, 191, 95-100) described that histone deacetylation is necessary for callus formation from leaf explants in Arabidopsis. However, treatment with TSA led to the formation of defective calluses.In support of this, a subset of HDAC genes was upregulated in calluses, and some HDAC mutants showed reduced callus formation capacity.

[0006] Summarizing these findings, it appears that, in Arabidopsis, TSA has an opposite effect on callus induction from leaves and, in combination with cytokinin, TSA does not induce calluses in hypocotyl explants.

[0007] Surprisingly, the inventors discovered that histone deacetylase inhibitors (HDACi), such as TSA, have a positive effect on callus initiation in plants of the species Beta vulgaris, such as sugar beet. This effect of TSA and other HDACi has not been proven for indirect regeneration protocols or to overcome recalcitrance, for example, in beet genotypes before. It is important to note that neither TSA nor any other HDACi has been used in any crop transformation protocol aimed at improving efficiency.

[0008] Thus, a first aspect of the present invention is the use of an HDACi in a method for inducing callus formation or producing calluses with an enhanced shoot regeneration capacity from plant cells, in particular from somatic or embryonic plant cells and, preferably, from an explant or an isolated part of a plant. The embryonic plant cells are preferably non-haploid cells. Greater shoot regeneration capacity is evaluated compared with the same method for callus production and the same genotype, but without the use of HDACi.

[0009] The present invention provides a method for inducing callus formation. Petition 870260084047, dated 08 / 19 / 2026, page 8 / 47 4 / 24 or produce calluses with an enhanced shoot regeneration capacity from at least one plant cell comprising the step of cultivating at least one plant cell in the presence of an HDACi. In principle, it is sufficient to use only one plant cell to perform the method according to the present invention. Thus, if the plural plant cells is used hereafter, the text should not be understood to mean that a minimum number of plant cells would be required.

[0010] Plant cells suitable for use in the method of the present invention include embryonic plant cells and somatic plant cells. The manner in which these plant cells are supplied is not important to the method according to the present invention. For example, embryonic or somatic plant cells can be supplied from an isolated explant of a plant. Which part of a plant is eligible for obtaining an explant depends on the particular plant species. In general, suitable plant cells can be obtained for instances of hypocotyl, petiole, bud and axial meristems, leaf blade, flower, parenchyma or parenchymatous cells, intermediate node, seeds, embryos, and roots of a plant.

[0011] In terms of the invention, histone deacetylase inhibitor or HDACi refers to any chemical compound that inhibits histone deacetylase. It should be understood that HDACi can be a single compound or a combination of several compounds. A preferred class of compounds suitable for conferring the desired histone deacetylase inhibitory activity are hydroxamic acids and hydroxamates, such as trichostatin A (TSA), vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589). According to the invention, it is preferred to use TSA as a histone deacetylase inhibitor. Other examples of HDACs for use according to the invention include cyclic tetrapeptides (such as trapoxin B) and depsipeptides, benzamides such as entinostat (MS-275), CI994 and mocetinostat (MGCD0103), electrophilic ketones and aliphatic acid compounds such as phenylbutyrate and valproic acid.

[0012] Cultivating plant cells can involve growing plant cells in a medium containing HDACi. Alternatively or additionally, HDACi can be introduced into plant cells, for example, via bombardment, Petition 870260084047, dated 08 / 19 / 2026, page 9 / 47 5 / 24 electroporation or microinjection or any other method known to those skilled in the art. According to the invention, it is preferable to cultivate the plant cells in a medium containing HDACi. The cultivation step can be carried out using any callus-inducing medium (CIM) well known in the art. In principle, various types of basal salt mixtures can be used for cell culture, but more preferably, the medium comprises modified Murashige and Skoog medium, White medium or medium for woody plants.

[0013] According to a preferred aspect of the invention, the CIM is supplemented with HDACi. The concentration of HDACi in the medium can vary from about 0.01 pM to about 5.0 pM. It has been found that different plants tolerate HDACi differently. At least in some plants, HDACi concentrations, particularly TSA, above 5.0 pM can be cytotoxic. In order to achieve the desired increase in callus formation, the concentration of HDACi in the medium is preferably in the range of 0.01 pM to 1.0 pM.

[0014] In addition to HDACi, one or more additional additives may be used in the culture medium. For example, the culture medium may be supplemented with plant growth regulators, such as auxins, cytokinins, and gibberellins, to initiate callus formation. Vitamins may be supplied to improve growth, such as Gamborg vitamin B5. Enrichment with nitrogen, phosphorus, and potassium has also proven useful.

[0015] Surprisingly, the method of the invention has been found to be suitable for inducing callus formation or producing calluses with an increased capacity for shoot regeneration, even in recalcitrant plant species or plant genotypes. Thus, using the method of the invention, it is possible to improve indirect regeneration in recalcitrant plant species or plant genotypes.

[0016] In a preferred embodiment of the present invention, the induction of callus formation can be followed by shoot regeneration from the callus tissue. Since the use of HDACi, in particular TSA, according to the present invention promotes callus formation, the result is an improved method for regenerating plants. The inventors found that more callus was frequently formed with the use of HDACi, in particular TSA; however, even if more or less callus tissue was not formed, the quality of the callus was clearly improved, i.e., the callus formed shows an enhanced shoot regeneration capacity. Petition 870260084047, dated 08 / 19 / 2026, page 10 / 47 6 / 24 Thus, the present invention provides a method for regenerating buds from callus tissue comprising the step of: (a) induce callus formation from at least one plant cell, as described above, and (b) cultivate the callus tissue obtained in step (a) under conditions that promote shoot growth from the callus tissue.

[0017] Suitable growing conditions are well known to those skilled in the art. Depending on the plant in question, these conditions may vary.

[0018] According to another aspect of the invention, the beneficial effect of HDACi on callus formation can be exploited in plant cell transformation methods, as well as in methods where the genome of a plant cell is modified. It has been found that, in recalcitrant plant species or plant genotypes, the efficiency of transformation can be improved using HDACi. Thus, the invention also relates to the use of an HDACi in a plant cell transformation method and to the use of an HDACi in a plant cell genome modification method.

[0019] Consequently, the invention provides a method for transforming a plant cell comprising the following steps: (a) induce callus formation from at least one plant cell, as described above, and (b) introduce into a plant cell to be used in step (a) and / or into a callus cell obtained in step (a) at least one nucleotide sequence of interest.

[0020] Step (a) of callus formation induction is performed using the method described above. Preferably, callus formation is induced in the presence of a TSA which can be added to the medium or directly introduced into the plant cells.

[0021] In step (b), a cell is transformed by introducing a nucleic acid molecule into the cell in a stable form to cause stable or transient expression of the nucleic acid sequence. Transformation of monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by those skilled in the art. The choice of method varies according to the type of plant to be transformed; those Petition 870260084047, dated 08 / 19 / 2026, page 11 / 47 7 / 24 skilled in the art will recognize the suitability of particular methods for certain types of plants. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG)-mediated transformation; transformation using viruses; plant cell microinjection; plant cell microprojectile bombardment; vacuum infiltration; and Agrobacterium-mediated transformation.

[0022] According to one embodiment of the present invention, at least one nucleotide sequence of interest is introduced into the plant cell to be used in step (a) to induce callus formation. It should be understood that, in this case, step (b) is performed before step (a). According to another embodiment of the invention, at least one nucleotide sequence of interest is introduced into a callus cell obtained in step (a).It should be understood that, in this case, step (b) is performed after step (a). According to another embodiment of the invention, at least one nucleotide sequence of interest is introduced into a plant cell during callus induction / formation, i.e., steps (a) and (b) are performed in parallel or simultaneously. Furthermore, it is possible to introduce nucleotide sequences of interest both into the cell to be used for callus formation and into the callus cell resulting from step (a). According to this embodiment, the method includes the following steps:. (i) introduce at least one nucleotide sequence of interest into a plant cell, (ii) induce callus formation from the cell obtained in step (i), and (iii) introduce at least one nucleotide sequence of interest into a callus cell obtained in step (ii).

[0023] The step of introducing at least one nucleotide sequence of interest can be performed using any suitable method commonly known in the art. Several methods are available for transferring nucleic acids of interest into plant cells. An exemplary vector-mediated method is Agrobacterium-mediated transformation as described, for example, by Lindsay & Gallois, 1990, Journal of Experimental Botany, and Kischenko et al., 2005, Cell Biology International for Sugar Beet, by Ishida et al., 2007, (Agrobacterium-mediated transformation of maize, Nature Protocols, 2 (7), 1614-1621) for maize or by in Petition 870260084047, dated 08 / 19 / 2026, page 12 / 47 8 / 24 press PureWheat Technology from Japan Tobacco for wheat. Other suitable techniques include particle bombardment, vacuum infiltration, floral immersion, and electroporation.

[0024] The nucleotide sequence of interest according to the invention may be a DNA or RNA sequence, for example, mRNA, siRNA, miRNA, etc. More particularly, the nucleotide sequence of interest encodes at least one phenotypic characteristic.Preferably, the phenotypic characteristic conferred by DNA or RNA can be selected from the group consisting of resistance / tolerance to biotic stress, including resistance / tolerance to pathogens, where the pathogen can be a viral, bacterial, fungal or animal pathogen, resistance / tolerance to abiotic stress, including resistance / tolerance to cold, resistance / tolerance to drought stress, osmotic resistance / tolerance, resistance / tolerance to heat stress, resistance / tolerance to cold or frost stress, resistance / tolerance to oxidative stress, resistance / tolerance to heavy metal stress, salt stress or resistance / tolerance to water extraction, resistance / tolerance to lodging, resistance / tolerance to damping-off or resistance / tolerance to one or more herbicides, such as glyphosate, glufosinate, 2,4-D, Dicamba, ALS inhibitors, etc.At least one phenotypic trait of interest may also be selected from the group consisting of the modification of another agronomic trait of interest, including increased yield, modification of flowering time, modification of seed color, modification of endosperm composition, modification of nutritional content, or metabolic engineering of a pathway of interest.

[0025] A nucleic acid (molecule) or nucleotide (sequence) or polynucleotide, as used herein, refers to DNA and RNA. DNA also includes cDNA and genomic DNA. A nucleic acid molecule can be single-stranded or double-stranded and can be synthesized chemically or produced through biological expression in vitro or even in vivo.

[0026] It will be evident that whenever the nucleotide sequences of RNA molecules are defined by reference to the nucleotide sequence of the corresponding DNA molecules, thymine (T) in the nucleotide sequence must be replaced by uracil (U). If reference is made to RNA or DNA molecules, this will be clear from the context of the request. Petition 870260084047, dated 08 / 19 / 2026, p. 13 / 47 9 / 24

[0027] In addition, the invention also provides a method for modifying the genome of a plant cell comprising the following steps: (a) induce callus formation from at least one plant cell, as described above, and (b) modify the genome of a plant cell to be used in step (a) and / or of a callus tissue cell obtained in step (a) by introducing into said cell a location-specific effector enzyme that preferably recognizes a predetermined location in the genome of said cell and, optionally, a repair nucleic acid molecule,

[0028] wherein the modification of said genome is selected from: i. a substitution of at least one nucleotide; ii. the elimination of at least one nucleotide; iii. an insertion of at least one nucleotide; or iv. any combination of i-iii.

[0029] Step (a) of callus formation induction is performed using the method described above. Preferably, callus formation is induced in the presence of TSA as HDACi, which can be added to the medium or introduced directly into the plant cells.

[0030] In step (b), modification of the cell genome is achieved by means of a double-stranded DNA (DSB) break-inducing enzyme or a single-stranded DNA (SSB) break-inducing enzyme (nickase), which preferably recognizes a predetermined location in the genome of said cell.

[0031] The genome modification step can be performed before and / or after callus formation induction. Thus, according to a first aspect of the invention, the genome of a plant cell is modified as described in step (b) and the resulting modified plant cell is then used in a subsequent step (a) to induce callus formation. According to another aspect of the invention, the callus formation induction step (a) is performed first and subsequently at least one cell of the resulting callus tissue is modified in step (b) by means of a specific effector enzyme for localization. According to another embodiment of the invention, the genome of a plant cell is modified as described in step (b) during callus induction / formation, i.e., steps (a) and (b) are Petition 870260084047, dated 08 / 19 / 2026, page 14 / 47 10 / 24 performed in parallel or simultaneously. Furthermore, it is possible to modify the genome of the plant cell to be used in the callus formation step and of a callus tissue cell resulting from the callus formation induction step. According to this aspect of the invention, the method includes the steps of: (i) modify the genome of a plant cell, (ii) induce callus formation in the cell resulting from step (i), and (iii) modify the genome of a cell from the callus tissue obtained in step (ii).

[0032] Examples of specific effector enzymes are, in particular, enzymes such as nucleases, nickases, recombinases, transposases, base editors or molecular complexes, including these tools. These effectors have the ability to introduce a double-strand cleavage (double-strand DNA break-inducing enzyme (DSBI)) or a single-strand cleavage (single-strand DNA break-inducing enzyme (SSBI)) at a target genomic location or have the ability to introduce a targeted modification, including a point mutation, an insertion or a deletion at an allo genomic location of interest. A location-specific effector enzyme may act alone or in combination with other molecules as part of a molecular complex.The location-specific effector enzyme may be present as a fusion molecule or as individual molecules associated or associated through at least one covalent or non-covalent interaction, such that the components of the location-specific effector complex are maintained in close physical proximity. The complex may include a repair template to perform a targeted sequence conversion or substitution at the target location. A repair template (RT) represents a single- or double-stranded nucleic acid sequence that may be provided during any genome editing, causing a double- or single-stranded DNA break to aid targeted repair of said DNA break by providing an RT as a known sequence template that aids homology-targeted repair.

[0033] As used herein, a double-strand DNA break-inducing enzyme or DSBI enzyme is an enzyme capable of inducing a break in double-stranded DNA at a specific nucleotide sequence, called a recognition site. The double-strand DNA break-inducing enzyme (DSB) can, for example, be selected from the group consisting of meganuclease, effector nuclease Petition 870260084047, dated 08 / 19 / 2026, p. 15 / 47 11 / 24 TAL, zinc finger nuclease, CRISPR systems, such as CRISPR / Cas9, CRISPR / Cpfl, CRISPR / Csm1, CRISPR / MAD7, CRISPR / CasX, or CRISPR / CasY. Rare cleavage endonucleases are DSBI enzymes that have a recognition site, preferably from about 14 to 70 consecutive nucleotides, and therefore have a very low cleavage frequency, even in larger genomes, such as most plant genomes. Homing endonucleases, also called meganucleases, constitute a family of such rare cleavage endonucleases. They can be encoded by introns, independent genes, or intervening sequences, and exhibit impressive structural and functional properties that differentiate them from more classical restriction enzymes, usually from bacterial type II restriction modification systems.Their recognition sites have an overall asymmetry that contrasts with the dyad symmetry characteristic of most restriction enzyme recognition sites. Several intron- or whole-encoded homing endonucleases have been shown to promote the lodging of their respective genetic elements at allelic sites without introns or intervening sequences. By making a specific double-strand break for localization at alleles without introns or intervening sequences, these nucleases create recombinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or intervening sequence at the DNA level. A list of other rare cleavage meganucleases and their respective recognition sites is provided in Table I of WO 03 / 004659 (pages 17 to 20) (incorporated herein by reference).

[0034] In addition, methods are available for designing customized rare cleavage endonucleases that recognize virtually any target nucleotide sequence of choice. Briefly, chimeric restriction enzymes can be prepared using hybrids between a zinc finger domain designed to recognize a specific nucleotide sequence and the non-specific DNA cleavage domain of a natural restriction enzyme, such as Fokl. Such methods have been described, for example, in documents WO 03 / 080809, WO 94 / 18313 or WO 95 / 09233 and in Isalan et al., 2001, Nature Biotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA 94, 5525-5530). Petition 870260084047, dated 08 / 19 / 2026, p. 16 / 47 12 / 24

[0035] Another example of customized endonucleases includes the so-called TALE nucleases (TALENs), which are based on transcription activator-like effectors (TALEs) of the bacterial genus Xanthomonas fused to the catalytic domain of a nuclease (e.g., FokI or a variant thereof). The DNA binding specificity of these TALEs is defined by repeat-variable dires (RVDs) of 34 / 35 amino acid repeat units arranged in tandem, such that an RVD specifically recognizes a nucleotide in the target DNA. Repeat units can be assembled to recognize virtually any target sequence and fused to a catalytic domain of a nuclease to create sequence-specific endonucleases (see, for example, Boch et al., 2009, Science 326: pages 1509-1512; Moscow and Bogdanove, 2009, Science 326: p. 1501; and WO papers). 2010 / 079430, WO 2011 / 072246, WO 2011 / 154393, WO 2011 / 146121, WO 2012 / 001527, WO 2012 / 093833, WO 2012 / 104729, WO 2012 / 138927, WO 2012 / 138939). Document WO2012 / 138927 further describes TALENs and monomeric (compact) TALENs with multiple catalytic domains and combinations thereof.

[0036] Recently, a new type of customizable endonuclease system has been described; the so-called CRISPR / Cas system. A CRISPR system, in its natural environment, describes a molecular complex comprising at least one small, single non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease, such as a Cpf1 nuclease (Zetsche et al., Cpf1 Is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, p. 113, October 2015), which can produce a specific double-strand break in DNA. Currently, CRISPR systems are classified into 2 classes, which comprise five types of CRISPR systems, for example, the type II system, which uses Cas9 as an effector, and the type V system which uses Cpf1 as an effector molecule (Makarova et al., Nature Rev. Microbiol., 2015).In artificial CRISPR systems, a synthetic non-coding RNA and a CRISPR nuclease, and / or optionally a CRISPR nuclease modified to act as a nickase or without any nuclease function, can be used in combination with at least one synthetic or artificial guide RNA or gRNA that combines the function of a crRNA and / or a tracrRNA (Makarova et al.,). Petition 870260084047, dated 08 / 19 / 2026, p. 17 / 47 13 / 24 (2015, above). The CRISPR / Cas-mediated immune response in natural systems requires CRISPR-RNA (crRNA), where the maturation of this guide RNA, which controls the specific activation of the CRISPR nuclease, varies significantly among the various CRISPR systems that have been characterized to date. First, the invading DNA, also known as a spacer, is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. Type II CRISPR systems, for example, may encode a Cas9 nuclease as an essential enzyme for the interference step, a system that contains a crRNA and also a transactivating RNA (tracrRNA) as a guide motif. These hybridize and form double-stranded RNA (ds) regions that are recognized by RNase III and can be cleaved to form mature crRNAs. These, in turn, associate with the Cas molecule in order to direct the nuclease specifically to the target nucleic acid region.Recombinant gRNA molecules can comprise both the DNA recognition variable region and the Cas interaction region, and therefore can be specifically designed independently of the specific target nucleic acid and the desired Cas nuclease. As an additional safety mechanism, PAMs (proto-adjacent spacer motifs) must be present in the target nucleic acid region; these are DNA sequences that directly track the DNA recognized by the Cas9 / RNA complex. The PAM sequence for Streptococcus pyogenes Cas9 has been described as NGG or NAG (IUPAC standard nucleotide code) (Jinek et al., Science 2012, supra) for a Streptococcus pyogenes-derived Cas9. The PAM sequence for Staphylococcus aureus Cas9 is NNGRRT or NNGRR(N). Other variant CRISPR / Cas9 systems are known. Thus, a Neisseria meningitidis Cas9 cleaves at the PAM sequence NNNNGATT. A Cas9 strain of Streptococcus thermophilus cleaves in the PAM NNAGAAW sequence.Recently, another PAM motif, NNNNRYAC, was described for a Campylobacter CRISPR system (document WO 2016 / 021973 A1). For Cpf1 nucleases, it has been described that the Cpf1 crRNA complex, without a tracrRNA, efficiently recognizes and cleaves target DNA preceded by a short T-rich PAM, in contrast to the G-rich PAMs generally recognized by Cas9 systems (Zetsche et al. supra). Furthermore, using modified CRISPR polypeptides, single-strand breaks can be achieved. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific double-strand DNA breaks via double penetration. Petition 870260084047, dated 08 / 19 / 2026, page 18 / 47 14 / 24 DNA. Furthermore, by using two gRNAs, the specificity of DNA binding and therefore DNA cleavage can be optimized. However, other CRISPR effectors, such as CasX and CasY originally described for bacteria, are available and represent other effectors that can be used for genomic engineering purposes (Burstein et al., New CRISPR-Cas Systems from Uncultivated Microbes, Nature, 2017, 542, 237-241).

[0037] Furthermore, modified Cas or Cpf1 variants or any other modified CRISPR effector variants, for example, Cas9 variants, can be used according to the methods of the present invention as part of a base-editing complex, for example, BE3, VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, SaKKH-BE3 (see Kim et al., Nat. Biotech., 2017, doi: 10.1038 / nbt.3803). Therefore, according to the present invention, artificially modified CRISPR nucleases are provided that may not actually be nucleases in the sense of double-strand cleavage enzymes, but are nickases or dead variants of nuclease, which still possess inherent DNA recognition and therefore binding capability.

[0038] A base editor, as used herein, refers to a protein or a fragment thereof that has the same catalytic activity as the protein from which it is derived; the protein or fragment thereof, individually or when provided as a molecular complex, referred to as a base-editing complex herein, has the ability to mediate a targeted base modification, i.e., the conversion of a base of interest, resulting in a point mutation of interest. Preferably, the at least one base editor, in the context of the present invention, is temporarily or permanently linked to at least one specific effector for localization or, optionally, to a component of at least one specific effector complex for localization. The linkage may be covalent and / or non-covalent.

[0039] The cleavage site of a DSBI enzyme or an SSBI enzyme refers to the exact location on the DNA where double-strand DNA breakage is induced. The cleavage site may or may not be encompassed (overlapping) the recognition site of the DSBI or SSBI enzyme, and therefore the cleavage site of a DSBI or SSBI enzyme is said to be located at or near the recognition site. The recognition site of a DSBI or SSBI enzyme, also called the binding site, is the nucleotide sequence that is (specifically) recognized by Petition 870260084047, dated 08 / 19 / 2026, p. 19 / 47 15 / 24 DSBI or SSBI enzyme and determines its binding specificity. For example, a TALEN or ZNF monomer has a recognition site that is determined by its RVD or ZF repeats, respectively, while the cleavage site is determined by the nuclease domain (e.g., FokI) and is usually located outside the recognition site. In the case of dimeric TALENs or ZNFs, the cleavage site is located between the two recognition / binding sites of the respective monomers, that intervening DNA region where cleavage occurs being called the spacer region.

[0040] Those skilled in the art will be able to select a DSBI or SSBI enzyme that recognizes a particular recognition site and induce a DSB or SSB at a cleavage site in or near the pre-selected location or design a DSBI or SSBI enzyme. Alternatively, a DSBI or SSBI enzyme recognition site can be introduced into the target genome using any conventional transformation method or by crossing with an organism with a DSBI or SSBI enzyme recognition site in its genome, and any desired DNA can then be introduced into or near the cleavage site of this DSBI or SSBI enzyme.

[0041] In a particularly preferred aspect of this embodiment, a repair nucleic acid molecule is additionally introduced into the plant cell.

[0042] As used herein, a repair nucleic acid molecule is a single- or double-stranded DNA molecule or RNA molecule that is used as a template for genomic DNA modification at the pre-selected location near or at the cleavage site. As used herein, use as a template for genomic DNA modification means that the repair nucleic acid molecule is copied or integrated at the pre-selected location by homologous recombination between the flanking region(s) and the corresponding homology region(s) in the target genome flanking the pre-selected location, optionally in combination with non-homologous end-joining (NHEJ) at one of the two ends of the repair nucleic acid molecule (e.g., in the case of there being only one flanking region).Integration through homologous recombination will allow for the precise joining of the repair nucleic acid molecule to the target genome down to the nucleotide level, while... Petition 870260084047, dated 08 / 19 / 2026, page 20 / 47 16 / 24 that NHEJ can result in small insertions / deletions at the junction between the repair nucleic acid molecule and the genomic DNA.

[0043] As used herein, a genomic modification means that the genome has changed by at least one nucleotide. This can occur by the substitution of at least one nucleotide and / or the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, provided that it results in a total alteration of at least one nucleotide compared with the nucleotide sequence of the pre-selected target genomic location prior to the modification, thus allowing the identification of the modification, for example, by means of techniques such as sequencing or PCR analysis and so forth, of which those skilled in the art will be well aware.

[0044] As used herein, a pre-selected location or predefined location indicates a specific nucleotide sequence in the genome (e.g., the nuclear genome) at the site where one or more nucleotides are to be inserted, replaced, and / or deleted. This may, for example, be an endogenous locus or a specific nucleotide sequence or one linked to a previously introduced foreign DNA or transgene. The pre-selected location may be a specific nucleotide position (after) where one or more nucleotides are to be inserted. The pre-selected location may also comprise a sequence of one or more nucleotides that are to be exchanged (replaced) or deleted.

[0045] As used in the context of this application, the term approximately means + / - 10% of the quoted value, preferably + / - 5% of the quoted value. For example, approximately 100 nucleotides (nt) should be understood as a value between 90 and 110 nt, preferably between 95 and 105.

[0046] As used herein, a flanking region is a region of the repair nucleic acid molecule that has a nucleotide sequence homologous to the nucleotide sequence of the flanking region of the DNA region (i.e., upstream or downstream) of the pre-selected location. It will be evident that the length and percentage of sequence identity of the flanking regions must be chosen so as to allow homologous recombination between said flanking regions and their corresponding DNA region upstream or downstream of the pre-selected location. The flanking region or regions of DNA Petition 870260084047, dated 08 / 19 / 2026, page 21 / 47 17 / 24 The pre-selected location that has homology with the flanking region or regions of DNA or regions of the repair nucleic acid molecule are also referred to as homology regions or regions in genomic DNA.

[0047] To have sufficient homology for recombination, the flanking DNA regions of the repair nucleic acid molecule can vary in length and must be at least about 10 nt, about 15 nt, or about 20 nt in length. However, the flanking region can be as long as possible (e.g., up to about 100-150 kb, such as complete bacterial artificial chromosomes (BACs). Preferably, the flanking region will have from 50 to 2000 nt, for example, about 100, 200, 500, 1000 or 1000 nt. Furthermore, the regions flanking the DNA of interest do not need to be identical to the homology regions (the DNA regions flanking the pre-selected location) and can have between about 80% and about 100% sequence identity, preferably about 95% to about 100% sequence identity with the DNA regions flanking the pre-selected location.The larger the flanking region, the less stringent the homology requirement becomes. Furthermore, to achieve the exchange of the target DNA sequence at the pre-selected location without altering the DNA sequence of adjacent DNA sequences, the flanking DNA sequences should preferably be identical to the upstream and downstream DNA regions flanking the pre-selected location.

[0048] As used herein, upstream indicates a location on a nucleic acid molecule that is closer to the 5' end of said nucleic acid molecule. Similarly, the term downstream refers to a location on a nucleic acid molecule that is closer to the 3' end of said nucleic acid molecule. For the avoidance of doubt, nucleic acid molecules and their sequences are typically represented in the 5' to 3' direction (from left to right).

[0049] To achieve sequence modification at the pre-selected location, the flanking regions must be chosen so that the 3' end of the upstream flanking region and / or the 5' end of the downstream flanking region align with the ends of the predefined location. As such, the 3' end of the upstream flanking region determines the end Petition 870260084047, dated 08 / 19 / 2026, page 22 / 47 18 / 24 5' from the predefined location, while the 5' end of the downstream flanking region determines the 3' end of the predefined region.

[0050] As used herein, the said pre-selected location that is located outside or distant from the cleavage (and / or recognition) site means that the location where the genomic modification is intended to be made (the pre-selected location) does not include the cleavage site and / or recognition site of the DSBI or SSBI enzyme, i.e., the pre-selected location does not overlap with the cleavage (and / or recognition) site. Outside / distant in this respect thus means upstream or downstream of the cleavage (and / or recognition) site.

[0051] The modified plant cell that has undergone transformation or gene editing according to the methods of the present invention and possibly has a modified genome can be regenerated into a whole (fertile) plant. Thus, in a preferred aspect of the invention, the transformation of a plant cell or the modification of a plant cell genome, respectively, is followed by a plant regeneration step.

[0052] Consequently, the present invention provides a method for producing a transgenic plant comprising the following steps: (a) transform a plant cell according to the method described above, and (b) regenerate a transgenic plant from the transgenic cell resulting from step (a) or from a transgenic cell derived from it.

[0053] The transgenic plants or transgenic cells of step (b) comprise at least one nucleotide sequence of interest introduced in step (a) as a transgene in a stable or transient manner.

[0054] In addition, the present invention also provides a method for producing a genetically modified plant comprising the following steps: (a) modify the genome of a plant cell according to the method described above, and (b) regenerate a plant from the cell resulting from step (a) or from a cell (comprising the genome modification generated in step (a)) derived therefrom.

[0055] Regeneration techniques depend on the manipulation of certain Petition 870260084047, dated 08 / 19 / 2026, page 23 / 47 19 / 24 phytohormones in a tissue culture growth medium, occasionally depending on a biocidal and / or herbicidal marker that can be introduced along with the desired nucleotide sequence(s) of interest. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pages 124-176, MacMilllan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pages 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant calluses, explants, protoplasts, immature or mature embryos, embryonic tissue, meristematic tissues, organs or parts thereof. Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38: 467486.To obtain whole plants from transgenic tissues, such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seeds, progeny evaluation begins.

[0056] The present invention is applicable to any plant species, whether monocotyledonous or dicotyledonous. Preferably, plants that can be subjected to the methods and uses of the present invention are plants that do not belong to the genus Arabidopsis or that are not plants of the species Arabidopsis thaliana. More preferably, the plants that can be subjected to the methods and uses of the present invention are selected from the group consisting of Hordeum vulgare, Hordeum bulbusum, Sorghum bicolor, Saccharum officinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica ni. Petition 870260084047, dated 19 / 08 / 2026, p. 24 / 47 20 / 24 gra, Eruca vesicaria subsp. 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, Helianthus tuberosus and / or Allium tuberosum. Particularly preferred are Beta vulgaris, Zea mays, Triticum aestivum, Hordeum vulgare, Secale cereale, Helianthus annuus, Solanum tuberosum, Sorghum bicolor, Brassica rapa, Brassica napus, Brassica juncacea, Brassica oleracea, Glycine max and / or Gossypium sp.

[0057] A plant of the species Beta vulgaris is, in particular, a plant of the subspecies Beta vulgaris subsp. maritima (Seemangold) or Beta vulgaris subsp. vulgar. This includes, for example, Beta vulgaris subsp. vulgaris var. altissima (sugar beet in the strictest sense), Beta vulgaris ssp. vulgaris var. vulgar (Mangold), Beta vulgaris ssp. vulgaris var. condditiva (beetroot), Beta vulgaris ssp. vulgaris var. crassa / alba (pound beet).

[0058] The object of the present invention is also plants that are obtained or obtainable by means of the methods described above, or parts or seeds of plants. Consequently, one embodiment of the invention is a transgenic plant obtained or obtainable by means of the above method of transforming a plant cell and regenerating a plant from said cell, as well as progeny, seeds or parts thereof, wherein the progeny, seed or part thereof comprises at least one nucleotide sequence of interest as a transgene, in a stable or transient manner. Another embodiment of the invention is a genetically modified plant obtained or obtainable by means of the above method for modifying the genome of a plant cell and regenerating a plant from said cell, as well as progeny, seeds or parts thereof, wherein the progeny, seed or part thereof comprises the genomic modification introduced by means of the method of the invention.

[0059] Parts of a plant include plant organs such as leaves, calluses, stems, roots, vegetative buds, meristems, embryos, anthers, ovules or fruits, plant tissues such as callus tissue, storage tissue, meristematic tissue, embryogenic tissue, leaf tissue, bud tissue, root tissue, plant tumor tissue or reproductive tissue, including plant cells such as cells of Petition 870260084047, dated 08 / 19 / 2026, page 25 / 47 21 / 24 isolated plants with a cell wall or aggregates or protoplasts, for example, and may signify a fusion of several organs, for example, a flower or seed or part of an organ, for example, a cross-section of the stem.

[0060] Another objective of the present invention is a plant cell or seeds derived from the transgenic plant or genetically modified plant described above. A plant cell derived from the transgenic plant described above comprises at least one nucleotide sequence of interest as a transgene, whereas a plant cell derived from the genetically modified plant described above comprises the modification in its genome.

[0061] The invention will be further described with reference to the following figures and examples described herein. However, it should be understood that the invention is not limited to such examples.

[0062] Unless otherwise indicated in the Examples, all recombinant DNA techniques are performed according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by RDD Cray, published jointly by BIOS Scientific Publications Ltd. (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK).Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson et al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0063] All patents, patent applications, and public publications or descriptions (including Internet publications) mentioned or cited herein are incorporated by reference in full. Figures

[0064] Figure 1 shows the results of a qualitative analysis of the induction of Petition 870260084047, dated 08 / 19 / 2026, page 26 / 47 22 / 24 calluses in media supplemented with TSA at 0.5, 1.0, or 5.0 μM. Induction of a control in media without TSA is also shown. Ten explants per condition were randomly photographed.

[0065] Figure 2 shows the results of a qualitative analysis of callus induction in media supplemented with TSA at 0.01 or 0.1 μM. Control induction in medium without TSA is also shown. Ten explants per condition were randomly photographed.

[0066] Figure 3 shows bar diagrams that demonstrate callus induction and plant regeneration using different amounts of TSA. A: Frequency of callus induction from leaf explants incubated in medium supplemented with TSA at 0.5, 1.0 and 5.0 μM. B: quantity of calluses produced under each condition. The quantity was estimated based on the number of plates with harvested calluses obtained in each variant. C: shoot regeneration capacity based on the number of shoots developed per leaf explant used for each experimental condition.

[0067] Figure 4 shows bar diagrams that demonstrate callus induction and plant regeneration using different amounts of TSA. A: frequency of callus induction from leaf explants incubated in medium supplemented with TSA at 0.01 and 0.1 μM. B: quantity of calluses produced in each condition. The quantity was estimated based on the number of plates with harvested calluses obtained in each variant. C: shoot regeneration capacity based on the number of shoots developed per leaf explant used for each experimental condition.

[0068] Figure 5 is a diagram showing the quantification of leaf explants with friable callus development at 3 time points during callus induction. The medium was supplemented with different concentrations of TSA.

[0069] Figure 6. Shoot regeneration from callus induced in medium supplemented with TSA is enhanced in recalcitrant genotypes. A: Average frequency of callus induction of the control genotype (1) and two genotypes with medium (2) or high (3) levels of recalcitrance in shoot regeneration. Callus induction was performed in medium without TSA (white bar) or supplemented with TSA at 0.01 μM Petition 870260084047, dated 08 / 19 / 2026, page 27 / 47 23 / 24 (gray bar). B: frequency of shoot regeneration of calluses produced in both the control medium (white bar) and the medium supplemented with TSA at 0.01 μM (gray bar). Two experiments were performed with three replicates per genotype. Note that the very recalcitrant genotype 3 is able to regenerate shoots only when the calluses were produced in a medium containing TSA. Examples 1. Technical description of the sugar beet callus induction protocol

[0070] This method is based on the publication by Kischenko et al., 2005 Cell Biology International.

[0071] 1. Micropropagated shoots of the S706 genotype were used as raw material. The shoots were multiplied in MS salts supplemented with 30 g / l of sucrose and 0.25 mg / l of benzyladenine (BAP).

[0072] 2. To induce friable calluses, leaf explants were isolated from micropropagated shoots and incubated in a medium containing MS salts, including 15 g / l sucrose and 2 mg / l BAP as a control, and in the same medium supplemented with TSA at 0.01 μM (B1), TSA at 0.1 μM (B2), TSA at 0.5 μM (B3), TSA at 1.0 μM (B4) and TSA at 5.0 μM (B5), at 28 °C in the dark for 7 weeks.

[0073] 3. Callus development from leaf explants was monitored during incubation in callus induction medium at 4, 5, 6 and 7 weeks.

[0074] 4. Leaf explants that produce friable calluses were scored to calculate the frequency of callus induction (percentage of leaf explants that produced friable calluses).

[0075] An increased callus induction frequency was observed when TSA is supplemented to the callus induction medium in a concentration range from 0.01 μM to 1.0 μM (Figure 1, Figure 2, Figure 3A and Figure 4A). The effect depends on the TSA concentration, as higher TSA concentrations (e.g., 5.0 μM) appear to be cytotoxic. Furthermore, TSA increases the amount of callus per leaf explant (Figures 3B and 4B). 2. Technical description of the shoot regeneration protocol

[0076] 1. Friable calluses from step 4 were harvested in a medium containing MS salts, g / l of sucrose, 1 mg / l of GA3 and 1 mg / l of TDZ and transferred to separate dishes.

[0077] 2. The dishes were incubated under light (16 h) at 24 °C for 10 days. Petition 870260084047, dated 08 / 19 / 2026, page 28 / 47 24 / 24

[0078] 3. Developing shoots were counted under a stereomicroscope to estimate regeneration capacity (number of shoots per initial leaf explant). 3. Results

[0079] An increased number of regenerated shoots per explant was observed (Figures 3C and 4C). Furthermore, TSA accelerates callus formation and therefore reduces the time to produce transgenic events (Figure 5). After 28 days, a large number of leaf explants with developing calluses were observed. Without the application of TSA, this number was not reached, even after 49 days. In addition, initial tests showed that by adding TSA, genotype-dependent recalcitrance to callus formation could be reduced.

[0080] Further experiments show that shoot regeneration induced in medium (CIM) supplemented with TSA is enhanced in recalcitrant Beta vulgaris genotypes. Genotypes 1 and 2 represent recalcitrant Beta vulgaris genotypes from which only a small number of plants can be regenerated from callus tissue using standard protocols. Genotype 3 is absolutely recalcitrant, whose regeneration through known protocols is not possible. Callus induction was performed in medium without TSA (white bar) or supplemented with TSA at 0.01 μM (gray bar) (Figure 6A). Frequency of shoot regeneration from callus produced in the control medium (white bar) or in the medium supplemented with TSA at 0.01 μM (gray bar) (Figure 6B). Two experiments were performed with three replicates per genotype.In genotype 1, the addition of TSA resulted in increased callus formation and an improved capacity for shoot regeneration from this callus: the average frequency of callus induction increased from 66.3% to 82%, and the average number of shoots per explant increased from 4.7 to 7.7. For genotype 2, no significant increase in the frequency of callus induction was observed; however, the callus produced was obviously of better quality, so the shoot regeneration capacity was clearly improved: the average number of shoots per explant increased from 2.4 to 4.6. For genotype 3, with a high level of recalcitrance, the frequency of callus induction was very low both with and without TSA, perhaps slightly higher with TSA. However, shoot regeneration from the produced calluses was only possible if the callus was induced in the presence of TSA. Petition 870260084047, dated 08 / 19 / 2026, p. 29 / 47

Claims

1 / 3 CLAIMS 1. A method for inducing callus formation from at least one plant cell of Beta spp., characterized in that it comprises the step of cultivating at least one plant cell in callus induction medium in the presence of a histone deacetylase inhibitor (HDACi); wherein the HDACi concentration is from 0.01 to 1.0 μM; and wherein the at least one plant cell of Beta spp. is of a species other than Beta vulgaris.

2. Method according to claim 1, characterized in that at least one plant cell is a somatic or embryonic cell and, preferably, an explant or a part thereof isolated from a plant.

3. Method according to claim 1 or 2, characterized in that HDACi is trichostatin A (TSA).

4. Method, according to any one of claims 1 to 3, characterized in that the step of cultivating at least one cell comprises: (i) growing at least one cell in a medium comprising HDACi; and / or (ii) introducing HDACi into at least one cell, for example, via bombardment, electroporation or microinjection.

5. Method for indirectly regenerating a Beta spp. plant, characterized in that it comprises the following steps: (a) inducing callus formation from at least one cell of a Beta spp. plant by means of the method as defined in any one of claims 1 to 4; and (b) cultivating the callus tissue obtained in Step (a) under conditions that promote shoot growth from the callus tissue.

6. Method for transforming a Beta spp. plant cell, characterized in that it comprises the following steps: (a) inducing callus formation from at least one Beta spp. plant cell by means of the method as defined in any one of claims 1 to 4; and (b) introducing into a plant cell to be used in Step (a), and / or into a callus cell obtained in Step (a), at least one nucleotide sequence of interest.

7. Method for producing a transgenic Beta spp. plant, characterized in that it comprises the following steps: Petition 870260084047, dated 19 / 08 / 2026, page 30 / 47 2 / 3 (a) transforming a Beta spp. plant cell by means of the method as defined in claim 6; and (b) regenerating a transgenic plant from the transgenic cell resulting from Step (a) or from a transgenic cell derived therefrom.

8. Method for modifying the genome of a Beta spp. plant cell, characterized in that it comprises the following steps: (a) inducing callus formation from at least one cell of a Beta spp. plant by means of the method as defined in any one of claims 1 to 4; and (b) modifying the genome of a plant cell to be used in Step (a) and / or a callus tissue cell obtained in Step (a) by introducing into said cell a double-stranded DNA (DSB) break-inducing enzyme that preferentially recognizes a predetermined site in the genome of said cell and, optionally, a repair nucleic acid molecule, wherein the modification of said genome is selected from: (i) a substitution of at least one nucleotide; (ii) a deletion of at least one nucleotide; (iii) an insertion of at least one nucleotide; or (iv) any combination of (i) - (iii).

9. Method for producing a genetically modified Beta spp. plant, characterized in that it comprises the following steps: (a) modifying the genome of a cell of a Beta spp. plant by means of the method as defined in claim 8; and (b) regenerating a plant from the cell resulting from Step (a) or from a cell derived therefrom.

10. Use of HDACi, characterized by the fact that it is a method for inducing callus formation from at least one cell of a Beta spp. plant, wherein the HDACi concentration is from 0.01 to 1.0 μM, and wherein the Beta spp. plant is of a species other than Beta vulgaris.

11. Use of HDACi, according to claim 10, characterized in that at least one cell is from an explant isolated from a Beta spp. plant.

12. Use of HDACi, characterized by the fact that it is in a method for indirect regeneration of a Beta spp. plant, in a method of transforming a cell of a Beta spp. plant or in a method of modifying the genome of a cell of a Beta spp. plant, wherein the concentration of HDACi is from 0.01 to 1.0 μM, and wherein the Beta spp. plant is of a species other than Beta vulgaris.

13. Method for producing calluses with increased shoot regeneration capacity from at least one plant cell, characterized in that it comprises the step of cultivating at least one plant cell in the presence of a histone deacetylase inhibitor (HDACi); wherein the step of cultivating at least one cell comprises: (i) introducing HDACi into at least one cell, (ii) inducing callus formation from at least one cell of (i) using callus-inducing medium, and (iii) obtaining callus with enhanced shoot regeneration capacity; wherein the enhanced shoot regeneration capacity is evaluated in comparison with the same method for callus production and the same genotype, but without the use of HDACi, wherein the HDACi concentration is from 0.01 to 1.0 μM.

14. Use of a plant, part of a plant, plant cell or seed of a plant obtained or obtainable by the method as defined in claim 7 or 9, or of a progeny plant thereof, characterized in that said use is for crossing with a second plant, regeneration of a plant, planting or cultivation of a field of plants, or production of plant products. Petition 870260084047, dated 19 / 08 / 2026, pp. 32 / 47

Images