Method of transformation of a plant cell

Description

1 / 59 METHOD OF TRANSFORMING A PLANT CELL CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 63 / 493,523, filed March 31, 2023, which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The contents of the electronic sequence listing (207422000940seqlist.xml; Size: 15,225 bytes; and Creation date: March 26, 2024) are incorporated into this document by reference in their entirety. TECHNICAL FIELD

[003] The present invention relates generally to plant biotechnology. Aspects of the present invention relate to methods for transforming plants. More specifically, a method for transforming sugarcane (Saccharum spp.) is described. The present invention also relates to genetically modified sugarcane plants produced using these methods. BACKGROUND OF THE INVENTION

[004] There is a growing need to obtain new plant varieties to address major agricultural problems, such as pest control, disease susceptibility, resistance to adverse weather conditions, increased productivity with fewer natural resources, among others. In recent decades, these problems have been the focus of conventional genetic improvement programs, which seek to obtain plants with the desired traits to deal with these problems.

[005] Although such conventional breeding methods are important for continually supplying the market with new plant varieties, there is now a need to combine them with techniques of Petition 870250085581, dated 09 / 22 / 2025, page 9 / 89 2 / 59 Plant biotechnology (genetic engineering) to meet the needs of the modern market, since some of the desired traits are not found in the genetic background of the varieties being crossed.

[006] Due to the commercial success of incorporating desirable agronomic traits through genetic engineering (i.e., genetic transformation or gene editing) in various plant species in recent decades (soybean, corn, canola, sugar beet, and cotton, for example), the sugarcane industry has also gained interest in applying such techniques. Sugarcane (Saccharum spp.) is a grass belonging to the Poaceae botanical family, originating from Southeast Asia, the greater central region of New Guinea, and Indonesia (Daniels & Roach, 1987, Sugarcane Improvement Through Breeding, pp. 7-84). It is one of the most important cultivated plant species in tropical and subtropical regions, with an area exceeding 23 million hectares distributed across 121 countries (FAO Statistical Yearbook 2012, pp. 233).In addition to its culinary importance (especially sugar production), sugarcane provides a source of biofuel in the form of ethanol, which has a global market of approximately 50 billion dollars.

[007] The economic and social importance of sugarcane is highlighted, with significant research efforts aimed at defining better agricultural practices for cultivation and improving the quality of cultivated varieties. However, unlike other crops such as corn and soybeans, the introduction of transgenes (e.g., genetic transformation) and gene editing cannot be done only once in the donor germplasm and then backcrossed in the elite germplasm. Instead, each cell of the elite sugarcane germplasm must invariably be transformed or modified for the development of new commercial cultivars. Thus, Petition 870250085581, dated 09 / 22 / 2025, page 10 / 89 3 / 59 For the dissemination of the same trait in more than one germplasm, enabling productivity gains for different varieties cultivated in different geographic regions, it is necessary to carry out a new genetic transformation / modification process.

[008] Plant genetic engineering involves the transfer of nucleotide or polypeptide sequences of interest into plant cells so that agronomically superior offspring are produced by maintaining and stably expressing the sequences responsible for the desired trait and / or by stably maintaining a desired phenotype (hereditary traits), either by introducing heterologous sequences and / or modifying endogenous sequences in the plant genome, or by modifying the expression pattern of genes and / or cellular function of interest without altering the original DNA. In this sense, one option is the use of in vitro culture techniques.

[009] One of the in vitro culture techniques is somatic embryogenesis, which consists of producing embryos from a single cell or a small group of cells that, through in vitro culture, will give rise to somatic embryos and subsequently to a plant, without the fusion of gametes (Jimenez. 2001. Regulation of In Vitro Somatic Embryogenesis with Emphasis on the Role of Endogenous Hormones. Revista Brasileira de Fisiologia Vegetal, v. 13, pages 196-223).

[0010] Several types of explants have been used in the embryonic process of sugarcane. According to Lakshmanan et al. (2006. Developmental and Hormonal Regulation of Direct Shoot Organogenesis and Somatic Embryogenesis in Sugarcane (Saccharum spp. Interspecific Hybrids) Leaf Culture. Plant Cell Reports, v.25, pages 1007-1015), almost all plant tissues give rise to embryogenic calluses, but the youngest leaves and developing inflorescences are very prolific and constitute the preferred target tissues for the rapid production of embryogenic calluses. Petition 870250085581, dated 09 / 22 / 2025, page 11 / 89 4 / 59

[0011] Somatic embryogenesis is initiated by the addition of growth regulators to the culture medium, and among these, auxins stand out as the most widely used class of growth regulators in the embryonic process (Cooke et al. 1993. The Role of Auxin in Plant Embryogenesis. The Plant Cell, v. 5, pages 1494-1495, 1993). 2,4-D (2,4-dichlorophenoxyacetic acid) is the most widely used growth regulator in the process of inducing somatic embryogenesis in sugarcane.

[0012] The conversion of somatic embryos into plants is the final stage of the somatic embryogenesis process. Regeneration generally occurs in a medium devoid of growth regulators and in the presence of light (Genetic Transformation of the Euploid Saccharum officinarum Via Direct and Indirect Embryogenesis. Sugar Tech, v. 12, pages 21-25; Basnayake et al. 2011; Embryogenic Callus Proliferation and Regeneration Conditions for Genetic Transformation of Diverse Sugarcane Cultivars. Plant Cell Reports, v. 30, pages 439-448), however, this process can be enhanced with the use of different regulators (Ali et al. 2008. An Efficient Protocol for Large Scale Production of Sugarcane Through Micropropagation. Pakistan Journal of Botany, v.40, pages 139-149; Nieves et al. 2008. Effect of Exogenous Arginine on Sugarcane (Saccharum sp.) Somatic Embryogenesis, Free Polyamines and the Contents of the Soluble Proteins and Proline. Plant Cells, Tissue and Organ Culture, vol.95, pages 313-320; Wamaitha et al. 2010. Thidiazuron-Induced Rapid Shoot Regeneration Via Embryo-Like Structure Formation from Shoot Tip-Derived Callus Culture of Sugarcane. Plant Biotechnology, v. 27, pages 365-368). However, such improvements are limited to a few varieties, and few laboratories have been able to reproduce these pioneering works in sugarcane tissue culture techniques.

[0013] In recent decades, several scientific studies have been Petition 870250085581, dated 09 / 22 / 2025, page 12 / 89 5 / 59 studies were conducted to develop efficient methods for genetic engineering sugarcane. Different transformation techniques using electroporation, polyethylene glycol (PEG) treatment, microprojectile bombardment, and Agrobacterium tumefaciens were used to introduce transgenes or modified genes into sugarcane cells and calluses. However, improvements in the management and control of in vitro culture conditions are still needed, considering the optimal age, type, and stage of the embryonic culture to ensure an efficient genetic engineering protocol for this plant species.

[0014] Furthermore, some important sugarcane varieties exhibit recalcitrance to tissue culture and / or genetic manipulation, difficulties in tissue culture propagation, low rates of embryogenic callus induction and regeneration, and the inability to use the zygotic embryo as a target tissue in genetic transformation [(Anderson & Birch, 2012; Basnayake, Moyle, & Birch, 2011; Molinari et al., 2007)], which makes the development of a portfolio of genetically modified or edited varieties for this species even more challenging when compared to other cereal crops.

[0015] Another problem is that known genetic manipulation techniques, applicable to other plants, do not work for sugarcane. Although several genetic engineering approaches have been evaluated for this species, there are still no standard protocols that ensure the production of sugarcane plants modified through genetic engineering (Smith et al., 1992; Rathius & Birch, 1992; Chen et al., 1987; Arencibia, 1998; Manickavasagam et al., 2004; Elliott et al., 1998). This may be related to the complexity of the polyploid and aneuploid genome of modern sugarcane varieties, coupled with their relatively restricted genetic base (Souza et al., 2011; D'Hont & Glaszmann, 2005, Basel, v. 109, n° 1-3, pages 27-33; Petition 870250085581, dated 09 / 22 / 2025, page 13 / 89 6 / 59 Cheavegatti-Gianotto et al., 2011) and recalcitrance to current tissue culture and genetic modification processes.

[0016] Therefore, there is a clear need to develop methods and approaches for cellular genetic manipulation targeted at sugarcane to meet the needs of a high-throughput commercial biotechnology pipeline for this species.

[0017] In this sense, the present invention describes a new method of plant transformation and regeneration with a combination of steps that enable the efficient production of new varieties through genetic engineering. SUMMARY OF THE INVENTION

[0018] To meet such needs, the present invention provides a method for the efficient transformation of a monocotyledonous plant. More particularly, the method of the present invention is useful in agriculture for the transformation and regeneration of a monocotyledonous plant, such as sugarcane. The present invention also relates to genetically modified sugarcane plants produced using these methods and compositions. The methods of the present invention provide robust and genotype-independent transformation protocols.

[0019] One aspect of the present invention includes methods for transforming a plant cell or plant tissue to include a trait of interest, said method including: (a) cultivating a plant cell or plant tissue in vitro; (b) introducing a sequence of interest into the cell or tissue of step (a), thereby producing a transformed tissue or cell; (c) cultivating the cell or tissue of step (b) in a culture medium for at least 15 to 60 days at a temperature of 20 °C to 35 °C; (d) performing heat shock on the cell or tissue of step (c) for at least 1 to 3 days at a temperature of 30 °C to 45 °C; and (e) regenerating the cell or tissue of (d), wherein the regenerated cell Petition 870250085581, dated 09 / 22 / 2025, page 14 / 89 7 / 59 includes the trait of interest. In some embodiments of this aspect, in step (c), the cell or tissue is maintained in the culture medium without manipulation or subculture. Some embodiments of this aspect also include repeating steps (c) and (d) a second time to add a second selection step.In some embodiments of this aspect, which may be combined with any of the preceding embodiments, step (b) further includes at least one of the following additional steps: (i) preparing Agrobacterium strains that include the sequence of interest; (ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension from (i); (iii) co-culturing the plant cell or tissue in a co-culture medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or (iv) culturing the transformed plant cell and tissue in a resting medium that includes an agent (e.g., antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark. Some embodiments of this aspect also include step (f) which allows for the elongation of the plantlets regenerated from step (d).Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include screening cells or tissue between steps (b) and (c), screening seedlings after step (e), or screening plants after step (f) to identify the sequence introduced into the cells or tissues or the trait of interest. In some embodiments of this aspect, said sequence of interest includes at least one expression cassette containing a nucleic acid that confers resistance to a selection agent, and said selection agent is used to select the genetically altered plant cells and tissue at step (c) and / or after step (c). Some embodiments of this aspect further include selecting the genetically altered cells or tissues at step (c), selecting the genetically altered cells or tissues. Petition 870250085581, dated 09 / 22 / 2025, page 15 / 89 8 / 59 between steps (c) and (d) or select genetically altered seedlings after step (e), optionally using selectable markers. In some embodiments of this aspect, step (b) is performed by Agrobacterium transformation, microprojectile bombardment, nanoparticle administration, viral administration, or a combination thereof. In some embodiments of this aspect, the polynucleotide includes a recombinase sequence under the control of an inducible promoter and at least one polynucleotide sequence of interest, wherein both sequences are flanked by recombination sites. In some embodiments of this aspect, the inducible promoter is selected from the group consisting of a stress-inducible promoter and a chemically inducible promoter.Some embodiments of this aspect further include excision of polynucleotide sequences flanked by recombination sites by inducing site-specific recombinase expression under culture conditions in steps (c) and (d). Some embodiments of this aspect further include culturing the cells after step (c) in a culture medium that includes abscisic acid (ABA). In some embodiments of this aspect, ABA is present at a concentration of 20 μM to 150 μM, preferably at a concentration of 50 μM to 100 μM. In some embodiments of this aspect, the culture medium also includes polyethylene glycol (PEG) in a range of 20 μM to 100 μM.In some embodiments of this aspect, the sequence of interest is selected from the group consisting of CRISPR machinery genes, selectable markers, herbicide genes, silencer genes, inactivated nuclease genes, transcription factor genes, growth or development genes, morphogens, reporter genes, insecticide genes, DNA templates for homologous recombination, suppressor genes, agronomic trait genes, and a combination thereof. In some embodiments of this aspect, step (c) is performed in 20. Petition 870250085581, dated 09 / 22 / 2025, page 16 / 89 9 / 59 to 45 days, more preferably in 21 to 42 days, particularly in 30 days. In some embodiments of this aspect, step (c) is carried out at 25 °C to 30 °C, more preferably 25 °C to 29 °C, particularly at 27 °C. In some embodiments of this aspect, step (d) is carried out at 35 °C to 40 °C, more preferably 35 °C to 37 °C, particularly at 35 °C. In some embodiments of this aspect, step (d) is carried out in 2 to 3 days, more preferably in 3 days. In some embodiments of this aspect, the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disc, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts and any cells or tissues receptive to the introduction and absorption of a sequence.In some embodiments of this aspect, the plant cell or plant tissue of step (a) is a callus. In some embodiments of this aspect, the transformation efficiency is increased by at least 5% compared with a conventional cell transformation method. In some embodiments of this aspect, the transformation efficiency is increased by at least 10% to 30% compared with a conventional cell transformation method. In some embodiments of this aspect, the plant cell or plant tissue is derived from a sugarcane plant, seedling, plant part, or plant tissue.

[0020] Some aspects of the present invention relate to a plant, part of a plant, seed or parent plant that includes a sequence or trace introduced by means of the method according to any of the previous embodiments.

[0021] A further aspect of the present invention relates to methods for increasing the regeneration rate of plant cells or plant tissues, which includes: (a) cultivating a plant cell or plant tissue Petition 870250085581, dated 09 / 22 / 2025, p. 17 / 89 10 / 59 in vitro; (b) cultivate the cell or tissue from step (a) in a culture medium for at least 15 to 60 days at a temperature of 20 °C to 35 °C; (c) perform heat shock treatment on the cell or tissue from step (b) for at least 1 to 3 days at a temperature of 30 °C to 45 °C in a culture medium; and (d) regenerate the cell or tissue from step (c). In some embodiments of this aspect, the regeneration efficiency is increased by at least 5% compared with a conventional method of cell and tissue regeneration. In some embodiments of this aspect, the regeneration efficiency is increased by at least 50%–100% compared with a conventional method of cell and tissue regeneration. In some embodiments of this aspect, in step (b), the cell or tissue is maintained in the culture medium without manipulation or subculture. Some variations of this aspect also include repeating steps (b) and (c) a second time to add a second selection step.In some embodiments of this aspect, step (b) further includes at least one of the following additional steps: (i) preparing Agrobacterium strains that include the sequence of interest; (ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension from (i); (iii) co-culturing the plant cell or tissue in a co-culture medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or (iv) culturing the transformed plant cell and tissue in a resting medium that includes an agent (e.g., antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark. Some embodiments of this aspect also include step (e) which allows for the elongation of the regenerated plantlets from step (d). Some embodiments of this aspect further include culturing the cells after step (b) in a culture medium that includes abscisic acid (ABA).In some embodiments of this aspect, abscisic acid (ABA) is present at a concentration of 20 μM to 150 μM, preferably at a concentration. Petition 870250085581, dated 09 / 22 / 2025, p. 18 / 89 11 / 59 from 50 μM to 100 μM. In some embodiments of this aspect, the composition also includes polyethylene glycol (PEG) in a range of 20 μM to 100 μM. In some embodiments of this aspect, step (b) is carried out for 20 to 45 days, more preferably for 21 to 42 days, particularly for 30 days. In some embodiments of this aspect, step (b) is carried out at 25 °C to 30 °C, more preferably for 25 °C to 29 °C, particularly for 27 °C. In some embodiments of this aspect, step (c) is carried out at 35 °C to 40 °C, more preferably for 35 °C to 37 °C, particularly for 35 °C. In some embodiments of this aspect, step (c) is carried out for 2 to 3 days, more preferably for 3 days.In some embodiments of this aspect, the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disc, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts and any cells or tissues receptive to the introduction and absorption of a sequence. In some embodiments of this aspect, the plant cell or plant tissue of step (a) is a callus. In some embodiments of this aspect, the plant cell or plant tissue is derived from a sugarcane plant, seedling, plant part or plant tissue.

[0022] Some aspects of the present invention relate to a plant, part of a plant, seed or parent plant that includes a sequence or trace introduced by means of the method according to any of the previous embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows expression cassettes for Agrobacterium transformation. The expression cassette sequences are provided in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Petition 870250085581, dated 09 / 22 / 2025, p. 19 / 89 12 / 59

[0024] Figures 2A-2B show representative images of the transformation protocol steps of the present invention. Figure 2A shows the plant tissue in the regeneration step of the transformation protocol of the present invention. Figure 2B shows the seedlings in the elongation step of the transformation protocol of the present invention. The plant tissue and seedlings shown in Figures 2A-2B are from a variety of sugarcane. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following description presents illustrative methods, parameters, and the like. It will be recognized, however, that such description is not intended to limit the scope of the present invention, but rather to describe illustrative embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that understood by those skilled in the art to which the invention relates. Unless otherwise indicated, all numbers expressing quantities, percentages, and proportions, and other numerical values ​​used in the descriptive report and claims, should be understood as modified, in all cases, by the term approximately. Therefore, unless otherwise indicated, the numerical parameters presented in the descriptive report and claims are approximations that may vary depending on the properties to be obtained.

[0026] The present invention provides a method for efficient plant transformation. More particularly, the method of the present invention is useful in agriculture for the transformation and regeneration of a monocotyledonous plant, such as sugarcane.

[0027] The term transformation refers to the transfer of nucleotide or polypeptide sequences of interest into plant cells, either transiently or stably, to introduce heterologous sequences and / or modify endogenous sequences in the plant genome or Petition 870250085581, dated 09 / 22 / 2025, p. 20 / 89 13 / 59 to modify the expression pattern of genes and / or cellular function of interest without altering the original DNA. The term transformation includes, among others, nanotube transformation, grafting, silica fiber vortexing, microparticle / nanoparticle bombardment, Agrobacterium-mediated transformation, microinjection, polyethylene glycol (PEG) procedures, liposome-mediated DNA uptake, electroporation, nanoparticle administration, among others.

[0028] By means of the present invention, the term genetically modified plants includes plants with stable expression of heterologous or modified sequences responsible for a desired trait or stable expression of a desired phenotype, either by introducing heterologous sequences and / or modifying endogenous sequences in the plant genome, or by modifying the gene expression pattern and / or cellular function without alterations to the original DNA.

[0029] The present invention relates to methods for producing such genetically modified plants, preferably sugarcane plants. In one embodiment, the present invention provides a genetically modified sugarcane plant that includes one or more transgenes (i.e., one or more heterologous genes) or one or more edited gene sequences in the sugarcane genome (i.e., one or more edited endogenous genes). In some embodiments, these methods use morphogens to further increase the efficiency of the transformation.

[0030] The terms polypeptide, peptide, and protein are used interchangeably in this document to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding natural amino acid, as well as to natural amino acid polymers.

[0031] The terms polynucleotide, nucleotide, nucleic acid Petition 870250085581, dated 09 / 22 / 2025, page 21 / 89 14 / 59 and gene are used interchangeably in this document to refer to a polymer of nucleotide residues (DNA or RNA). The terms apply to nucleotide polymers in which one or more nucleotide residues are an artificial chemical analogue of a corresponding natural nucleotide, as well as to natural nucleotide polymers.

[0032] One embodiment of the present invention provides a method of transforming a plant cell or plant tissue to comprise a trait of interest, said method comprising: a. to cultivate a plant cell or plant tissue in vitro; b. introduce a sequence of interest into the cell or tissue from step (a), thereby producing a transformed tissue or cell; c. Cultivate the cell or tissue from step (b) in a culture medium for at least 15 to 60 days at 20 to 35 °C; d. perform heat shock treatment on the cell or tissue from step (c) for at least 1 to 3 days at 30 to 45 °C; e. regenerate the cell or tissue of (d) that comprises the trait of interest.

[0033] The cells or tissues to be transformed may be leaf disc cells, callus cells, protoplast cells, or any cells or tissues receptive to the introduction and absorption of a sequence (DNA, RNA, or protein).

[0034] Any methodology known in the art for introducing a sequence of interest into a plant cell or tissue can be used in the practice of the inventions described herein. Any methodology known in the art for eliminating, inserting, or otherwise modifying cellular DNA (e.g., genomic DNA and organelle DNA) or the expression pattern of a DNA and / or a cellular function can be used in the practice of the inventions described herein. Petition 870250085581, dated 09 / 22 / 2025, page 22 / 89 15 / 59 The term introduction, in the context of the insertion of a nucleotide or polypeptide into a cell, means transfection, transformation or transduction and includes reference to the incorporation of a polynucleotide / polypeptide into a cell. Introduction includes reference to the stable or transient transfer of a nucleotide or protein sequence into a plant cell or tissue, as well as transfer or incorporation through crossing over. Therefore, introduced includes incorporation into the cell genome (e.g., chromosome DNA, plasmid, plastid or mitochondrial DNA), conversion into an autonomous replicon or transient expression (e.g., transfected mRNA). General molecular techniques used in the invention are provided, for example, by Sambrook et al. (eds.). 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.For example, an unarmed Ti plasmid, containing a genetic construct for deletion or insertion of a target gene in Agrobacterium tumefaciens, can be used to transform a plant cell, and subsequently, a transformed plant can be regenerated from the transformed plant cell using the present invention in combination with the procedures described in the art. The Ti plasmid vectors each contain the gene between the edge sequences, or at least located to the left of the right edge sequence, of the Ti plasmid T-DNA. Other types of vectors can also be used to transform the plant cell.

[0035] Recombinant DNA / RNA technology has enabled the isolation of genes and their stable insertion into the host genome or transient insertion and expression in the host cell. This technique can be defined as the controlled introduction of nucleic acids into the genome of a recipient, excluding introduction through fertilization. It is a controlled process in which a defined fragment of Petition 870250085581, dated 09 / 22 / 2025, page 23 / 89 16 / 59 DNA / RNA is introduced into the host (or recipient) and can be integrated into it. The stable or transient insertion of these molecules into the host genome gives rise to an individual that has a genome that is identical or substantially identical to that of the recipient (host) of the recombinant molecule, but with a new and particular trait. Substantially identical means a genome that has more than 80%, preferably 85%, 90%, 95%, 98%, 99% or 100% identity with respect to the recipient.

[0036] There are several techniques for genetic transformation of plants, grouped into two main categories: indirect and direct gene transfer. Indirect transfer occurs when an exogenous nucleic acid is inserted into the plant cell by the action of a biological vector, while direct transfer is based on physicochemical processes. Different tissues and / or cells can be used according to the genetic transformation technique and the species or genotypes to be transformed.In general, these tissues or cells include, without limitation, embryogenic calluses, calli, protoplasts, embryos, somatic embryos, meristematic tissues, and any other plant part, tissue, or cell with regenerative capacity.

[0037] Indirect transformation is based, for example, on the system mediated by bacteria of the genus Agrobacterium and has been the most widely used method for obtaining genetically modified plants. The advantages of this method include the ability to transfer relatively long DNA segments without rearrangement, maintaining integration with a low copy number of transgenes, thus ensuring greater genotypic stability for the generated events. Several species and strains of Agrobacterium, plasmids, and protocols have been developed and adapted for the genetic transformation of various plant species. The advantages of these methods include higher probabilities of single-copy events, stable integration, and genetic inheritance of the introduced genetic traits, as well as... Petition 870250085581, dated 09 / 22 / 2025, page 24 / 89 17 / 59 as consistent gene expression across generations and lower gene silencing rates. A variety of Agrobacterium species are known in the art and can be used in the methods of the invention. See, for example, Hooykaas. 1989. Plant Mol. Biol. 13: 327; Smith et al. 1995. Crop Science 35: 301; Chilton. 1993. Proc. Natl. Acad. Sci. USA 90: 3119; Mollony et al. 1993. Monograph Theoretical Appl Genet NY, Springer Verlag 19: 148, Ishida et al. 1996. Nature Biotechnol. 14: 745; Komari, et al. 1996. The Plant Journal 10: 165. In a preferred embodiment of the present invention, examples of Agrobacterium strains include, but are not limited to, LBA4404, EHA101, EHA105, AGL1, C58C1, GV3101, GV2260 and others.

[0038] Agrobacterium tumefaciens and A. rhizogenes are bacteria Gram-negative, phytopathogenic soil bacteria belonging to the Rhizobiaceae family cause diseases in dicotyledons, known as crown gall and hairy root gall, respectively. In this plant-pathogen interaction, there is a natural gene transfer process between Agrobacterium and the plant cell, in which fragments of bacterial DNA are transferred to the plant cell (T-DNA), integrating into the nuclear genome. In its natural form, the bacterium transfers T-DNA (transferred DNA), which is part of the bacterial plasmid called Ti (tumor inducer) and integrates into the genome of infected plant cells. The T-DNA fragment that is transferred to the plant cell is composed of genes involved in the constitutive biosynthesis of phytohormones (auxins and cytokinins), which alter the normal developmental program of the infected tissue and cause tumor formation.Furthermore, it also contains oncogenes for the synthesis of sugars and amino acids called opines, which serve as carbon and nitrogen sources for bacteria (Oger et al. 1997). Repeated 25 base pair (bp) ends on the right and left borders delimit the T-DNA and are essential for its transfer. Petition 870250085581, dated 09 / 22 / 2025, page 25 / 89 18 / 59 Phenolic compounds released by damaged plant tissues activate specific regions (vir regions), initiating the process of transferring T-DNA into the plant cell. Agrobacterium also has chromosomal genes (chv) that promote binding between bacterial and host cells, allowing the formation of the pore passage for the T-DNA-containing complex (Sheng & Citovsky, 1996).

[0039] Since the segment to be transferred is defined by its edges, any sequence flanked by them can be transferred to a plant via Agrobacterium, enabling the manipulation of these sequences for the transfer of coding sequences of interest. The substitution or elimination of coding regions of wild-type T-DNA (oncogenes) allows the generation of non-oncogenic (disarmed) Agrobacterium strains, which can carry the sequences of interest. The modified T-DNA is able to transfer the sequences of interest to plants, since the virulence genes (vir region) remain intact.

[0040] Furthermore, the Agrobacterium indirect transformation system allows the transfer of artificial plasmid constructs into plants, provided that the constructs contain such TDNA borders, which allows the flexibility to use molecular tools and materials developed for other bacterial strains. These artificial plasmid constructs have promoters of different origins such as, for example, plant promoters, viral promoters, bacterial and / or chimeric promoters, as well as genes that confer antibiotic resistance, herbicide resistance or tolerance, or enzymatic activity (phosphomannose isomerase (PMI) / mannose (Man)), so that these markers can be used for the selection of transformed cells or plants. These constructs may also contain auxiliary genes that interfere with relevant morphogenesis signaling pathways, increasing the efficiency of the genetic transformation process. Petition 870250085581, dated 09 / 22 / 2025, p. 26 / 89 19 / 59 and the regeneration of plant tissues.

[0041] In one aspect of the present invention, foreign or exogenous nucleic acids to be introduced into the plant are cloned into a binary plasmid between the consensus sequences of the left and right edges (T-DNA). The modified T-DNA, comprising foreign DNA (the nucleotide sequence to be transferred), is constructed into a plasmid that is replicated in E. coli cells. The binary plasmid is then extracted, purified, and transferred into an Agrobacterium cell, which is subsequently used to infect plant tissue or cells. The vector T-DNA region, comprising the exogenous DNA, is inserted into the plant genome. The marker gene expression cassette and the trait gene expression cassette may be present in the same T-DNA region, in different T-DNA regions in the same plasmid, or in different T-DNA regions in different plasmids. In one embodiment of the present invention, the cassettes are present in the same region as the T-DNA.Those versed in the technique are familiar with the indirect transformation methods using Agrobacterium.

[0042] In one embodiment, the method of transforming a plant cell or plant tissue of the present invention comprises introducing a sequence into a plant cell or tissue mediated by an Agrobacterium strain (step b). A plant cell or plant tissue is placed in contact with an Agrobacterium strain. This is the inoculation phase and can last from at least about one minute to about 12 hours, more preferably from about 5 minutes to about 2.5 hours, even more preferably from about 25 minutes to about 40 minutes at room temperature and with or without agitation. During or after inoculation, it is possible to apply some treatments to aid infection, such as vacuum infiltration and sonication of the Agrobacterium solution. For example, in vacuum infiltration, Petition 870250085581, dated 09 / 22 / 2025, p. 27 / 8920 / 59 The plant tissue or cell in contact with the bacterial suspension is subjected to vacuum pressure, preferably from -300 mmHg to 1000 mmHg, more preferably from 400 mmHg to 800 mmHg, even more preferably from -500 mmHg to -700 mmHg, generally for a period of 1 to 10 minutes, more preferably from 1 to 7 minutes, even more preferably from 1 to 5 minutes. In another non-limiting example, vacuum infiltration occurs at a vacuum pressure of -700 mmHg for 5 minutes. Also in this inoculation phase, to improve the efficiency of the transformation, it is possible to incorporate additives such as acetosyringone and surfactants into the Agrobacterium suspension.

[0043] Optionally, in some embodiments, the plant cell or tissue to be infected in step (b), before inoculation with Agrobacterium, may be subjected to a heat shock pretreatment, in which said tissue or cell is placed in a liquid plant culture medium, such as Murashige and Skook, Gamborg's, Chu (Ne), Schenk and Hildebrand and others known to those skilled in the art, preheated to the temperature at which the heat shock pretreatment will be conducted. The plant tissue or cell is then incubated in an incubator or water bath at a temperature above that at which inoculation will occur (e.g., room temperature).Thus, for example, the temperature of the thermal shock pretreatment may occur at a temperature of about 30 °C to about 55 °C, preferably from about 35 °C to about 50 °C, even more preferably from about 40 °C to 45 °C, during a period of about 1 minute to about 60 minutes, about 1 minute to about 50 minutes, about 1 minute to about 40 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes. In another example, not limited... Petition 870250085581, dated 09 / 22 / 2025, p. 28 / 89 21 / 59 tativo, the thermal shock treatment comprises placing and maintaining the plant tissue or cell in a pre-heated liquid plant culture medium at a temperature of about 45 °C for about 5 minutes.

[0044] After this period of time, the liquid culture medium is discarded and replaced with the Agrobacterium suspension prepared as described below. The useful concentration of Agrobacterium in the methods of the invention may vary depending on the Agrobacterium strain used, the tissue or cell to be transformed, the genotype to be transformed, among other factors. Although the concentration of Agrobacterium may vary, generally the OD600 used ranges from about 0.001 to about 5, more preferably from about 0.05 to about 2 and, even more preferably, from about 0.1 to about 1.0.

[0045] In step b), the period between the moment immediately after inoculation (contact of Agrobacterium with plant tissue) and the moment when the bacterium is removed or inactivated after inoculation, the plant tissue or infected tissue is incubated on a support to allow the transfer of Agrobacterium T-DNA to the plant cells (co-culture phase). In one embodiment, co-culture of plant tissue with Agrobacterium occurs on a culture medium, filter paper, or any other appropriate support.

[0046] The inoculated tissue can be co-cultured for about 1 to 30 days, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5 days. During the co-culture stage, the temperature can be any temperature suitable for the target plant known in the art. Illustratively, for sugarcane, the temperature can range from about 15 °C to about 30 °C, from about 16 °C to about 29 °C, from about 20 °C to about 25 °C, from about 21 °C to about 24 °C, or from about 22 °C to about 23 °C. In some embodiments, the co-culture stage occurs in the absence of light. Petition 870250085581, dated 09 / 22 / 2025, p. 29 / 89 22 / 59

[0047] For the purposes of the present invention, culture medium refers to any medium used in the art to support the viability and growth of a plant cell or tissue or the growth of a whole plant, such as Murashige and Skook, Gamborg's, Chu (Ne), Schenk and Hildebrand, and others known to those skilled in the art.Such media commonly include defined, but not limited to, the following components: macronutrients, which provide nutritional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, and iron; micronutrients, such as boron, molybdenum, manganese, cobalt, chlorine, iodine, and zinc; carbohydrates, such as maltose, sorbitol, and saccharide; phytohormones; vitamins; selection agents, such as antibiotics or herbicides, to select transformed cells or tissues; phenolic compounds (preferably those found in plant lesion exudates, such as acetosyringone, sinapinic acid, syringic acid, ferulic acid, catechol, gallic acid, among others); antioxidants (e.g., dithiothreitol); and gelling agents. It may also include undefined complex components, such as casein hydrolysate, coconut water, yeast extract, and activated charcoal.

[0048] In one aspect, the culture media for each step of the transformation method of the present invention have particular characteristics and can be any plant tissue culture media known in the art. Preferably, the culture media of the present invention are semi-solid and comprise a gelling agent. Gelling agent means any substance that increases the viscosity of a solution without substantially altering its properties, and includes those gelling agents commonly used in plant tissue culture, such as agar, Agargel™, Phytablend™, Agargellan™, carrageenan and gellan gum (Gelzan™, Gelrite™, Phytagel™).

[0049] Alternatively, direct nucleic acid / protein transfer can be used to directly introduce a molecule into Petition 870250085581, dated 09 / 22 / 2025, page 30 / 89 23 / 59 a plant cell. One method of direct nucleic acid transfer is to bombard plant cells with a vector comprising DNA for insertion using a particle gun (particle-mediated biolistic transformation). Other methods for plant cell transformation include protoplast transformation (optionally in the presence of polyethylene glycols (PEGs)); ultrasound treatment of plant tissues, cells, or protoplasts in a medium comprising the polynucleotide / polypeptide or vector; microinjection of the polynucleotide / polypeptide or vector into plant material; microinjection, vacuum infiltration, sonication, use of silicon carbide, chemical transformation with PEG, electroporation of plant cells, and the like. Disadvantages of direct transformation include challenges related to plant tissue regeneration and low transgene expression.

[0050] Furthermore, plant transformation can be carried out through direct insertion into the site via homologous recombination mediated by nucleases (genome editing). In recent years, genome editing technology based on the use of engineered or chimeric nucleases has allowed the generation of genetically modified organisms in a more precise and specific way. The introduction of exogenous or foreign genes occurs through homologous recombination by introducing a homologous recombination (HR) template that has exogenous DNA linked to a DNA fragment homologous to the genome of the recipient organism. Available tools include the chimeric CRISPR (clustered regularly interspaced short palindromic repeats) - Cas enzyme system, zinc finger nucleases (ZFNs), and TAL effector nucleases (TALENs).CRISPR-Cas systems are enzymatic systems that include two main components: an endonuclease (Cas) and a guide RNA (single guide RNA - sgRNA; a guide to the specific cleavage site of the Cas endonuclease). The guide RNA may also include two components: an RNA. Petition 870250085581, dated 09 / 22 / 2025, p. 31 / 89 24 / 59 CRISPR (crRNA) - a 17-20 mer sequence complementary to specific genomic DNA sequences and, optionally, a tracr RNA. The specific cleavage performed by the endonuclease and guided by sgRNA is homologous recombination repair, specifically inserting exogenous DNA flanked by homologous sequences into the cleavage site. The introduction of this enzymatic system into the cell can occur through various methods, including the use of plasmids, through direct or indirect transformation, or using supports such as proteins and other chemical agents. The expression of the system components can occur transiently or stably using the cellular machinery of the recipient organism or used exogenously, in vitro, distributing to the target cell or tissue all the components ready for use (endonucleases + sgRNA, transcripts in vitro and combined before cellular distribution).The description presented in this document is not exhaustive and should not limit the use of the different variations, systems, and methods of genome editing within the scope of the present invention known in the state of the art, and even those yet to be discovered.

[0051] In another embodiment, plant transformation also involves the administration of genome editing reagents for modification of endogenous genes (knock-out, correction, overexpression, etc.) through base editing or template editing (HR or prime editing). According to this embodiment, the editing reagents are administered in step (b) via a plasmid containing a nuclease gene (e.g., Cas9 or Cpf1) and its crRNA. In another embodiment, genome editing reagents are administered using a ribonucleoprotein (RNP) complex. For homologous recombination (HR)-mediated editing, a homologous template in the form of a plasmid is administered, in addition to Cas and crRNA. In another embodiment Petition 870250085581, dated 09 / 22 / 2025, page 32 / 89 25 / 59 In this case, a homologous template in dsDNA or ssDNA format is administered, in addition to Cas and crRNA. The HR template can be administered on the same plasmid or on a separate plasmid from that of the genome editing reagent. The genome editing reagents on the plasmid(s) can be administered via Agrobacterium transformation or particle bombardment. When RNP is used and / or when an HR template is used on a separate plasmid, particle bombardment can be used for delivery. A combination of plasmid and / or RNP delivery methods is also provided, combining sequentially or simultaneously different plant cell or tissue transformation methods.

[0052] Some aspects of the invention relate to editing or modifying the plant genome. Suitable plant material for genome editing includes cells (e.g., in cell cultures) or tissues (e.g., in plants). Genome editing targets include genes, introns, non-coding sequences (e.g., miRNAs), and regulatory elements (e.g., promoters). Several types of genome editing can be used, including knock-out editing, knock-in editing, homologous recombination, site-directed integration, base editing, or primer editing. Similarly, various genome editing components can be used. In a preferred embodiment, the genome engineering component includes a CRISPR system, preferably a CRISPR / Cas9 or CRISPR / Cpf1 system, and a targeting sequence. The genome engineering components can be delivered in various formats, including via plasmids or using a ribonucleoprotein (RNP) complex.

[0053] It is evident that plant transformation may involve the construction of an expression cassette or an expression vector that will act on a specific cell. Said cassette or expression vector may comprise a nucleotide sequence that includes a Petition 870250085581, dated 09 / 22 / 2025, page 33 / 89 26 / 59 gene under the control of, or operatively linked to, a regulatory element (e.g., a promoter). The expression cassette or vector may contain one or more genes, such as combinations of operatively linked genes and regulatory elements. The vector may be a plasmid and may be used alone or in combination with other plasmids to provide transformed cells using transformation methods, as provided in the present invention, to incorporate the genetic sequences of interest or traits into the plant cell or tissue.

[0054] Genetic elements introduced, either in an expression vector or in an expression cassette, that result in the expression of an introduced gene typically comprise, in the 5'-3' direction of transcription: a transcription and translation start region, a DNA sequence of interest, and a functional transcription and translation termination region in plants. The transcription start region, the promoter, may be native, homologous, foreign, or heterologous to the host. Such a region generally comprises a plant-expressible promoter. A plant-expressible promoter, as used herein, refers to a promoter that ensures the expression of the genetic alteration(s) of the invention in a plant cell. Promoters suitable for plant expression may be isolated from plants or other organisms.Several promoters have been isolated or developed, including constitutive promoters, inducible promoters, and promoters that respond to abiotic stresses specific to tissues, specific to cells, among others. Many of these promoters have intronic sequences described as relevant for proper gene expression. In a preferred aspect of the invention, the promoters are constitutive promoters and can be selected from the non-limiting group consisting of CaMV. Petition 870250085581, dated 09 / 22 / 2025, page 34 / 89 27 / 59 35S, CoYMV (Commelina Yellow Mosaic Virus), FMV 35S, ubiquitin (Ubi), rice actin promoter (Act-1), Act-2, nopalin synthase (NOS) promoter, octopine synthase (OCS) promoter, maize alcohol dehydrogenase promoter (Adh-1), PvUbi1, among others. In one embodiment of the invention, the promoter is the Brachypodium distachyon ubiquitin gene promoter (BdUbi10). In another embodiment of the invention, the promoter is the Zea mays ubiquitin gene promoter (ZmUbi1). Examples of promoters that direct constitutive expression in plants are known in the art and include: the strong constitutive 35S promoters (the 35S promoters) of cauliflower mosaic virus (CaMV), for example, from isolates CM 1841 (Gardner et al., Nucleic Acids Res., (1981) 9, 2871-2887), CabbB S (Franck et al., Cell (1980) 21, 285-294) and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters of the ubiquitin family (for example, the maize ubiquitin promoter (Christensen et al.), Plant Mol Biol, (1992) 18, 675689), the gos2 promoter (from Pater et al., The Plant J (1992) 2, 834-844), the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581-588), actin promoters, such as the promoter described by An et al. (The Plant J, (1996) 10, 107), the rice actin promoter described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); promoters of cassava vein mosaic virus (document WO 97 / 48819, Verdaguer et al. (Plant Mol Biol, (1998) 37, 1055-1067), the pPLEX series of promoters of Subterranean Clover Stunt virus (document WO 96 / 06932, particularly the S4 or S7 promoter), an alcohol dehydrogenase promoter, for example, pAdhlS (GenBank accession numbers X04049, X00581) and the TRT promoter and the TR2' promoter (the TRT promoter and the TR2' promoter, respectively) that control the expression of the G and 2' genes, respectively, of T-DNA (Velten et al., EMBO J, (1984) 3, 2723 2730).Alternatively, a promoter capable of expression in plants can be a tissue-specific promoter, that is, a... Petition 870250085581, dated 09 / 22 / 2025, page 35 / 89 28 / 59 promoters that direct a higher level of expression in certain plant cells or tissues. These plant promoters can be combined with enhancer elements, can be combined with minimal promoter elements, or can include repeat elements to ensure the desired expression profile.

[0055] In some embodiments, genetic elements can be used to increase expression in plant cells. For example, an intron at the 5' or 3' end of an introduced gene, or in the coding sequence of the introduced gene, for example, the hsp70 intron, can be used. Other genetic elements may include, but are not limited to, enhancer promoter elements, duplicated or triplicated promoter regions, 5' leader sequences different from another transgene or different from an endogenous (host plant) leader sequence, 3' trailer sequences different from another transgene used in the same plant or different from an endogenous (host plant) trailer sequence. Additional elements incorporated into the expression cassette for the purpose of increasing gene expression levels, for example, transcriptional or translational enhancers, such as the CaMV 35S, FMV 35S, Nos, supP enhancers, among others.

[0056] Terminator sequences are also considered in the expression cassette. Examples of suitable and functional plant polyadenylation signals include those from the Agrobacterium tumefaciens nopalin synthase gene (nos), the pea proteinase inhibitor II gene rbcS (small subunit of ribulose-1,5-bisphosphate carboxylase), tobacco Lhcb1 (tobacco chlorophyll a / b binding proteins), heat shock protein (Hsp), CaMV 35S, octopine synthases, and alpha-tubulin genes, among others.

[0057] An introduced gene of the present invention can be inserted into the host cell DNA such that the inserted gene portion is upstream (i.e., 5') of transcriptional regulatory signals. Petition 870250085581, dated 09 / 22 / 2025, page 36 / 89 29 / 59 suitable at the 3' end (e.g., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the gene into the plant cell genome. Preferred signals for polyadenylation and transcript formation include those from the nopaline synthase gene (Depicker et al., J. Molec Appl Gen, (1982) 1, 561-573), the octopine synthase gene (Gielen et al., EMBO J, (1984) 3: 835-845), the SCSV or malic enzyme terminators (Schunmann et al., Plant Funct Biol, (2003) 30: 453-460), and the T DNA 7 gene (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981-6998), which act as untranslated 3' DNA sequences in transformed plant cells. In some embodiments, one or more of the introduced genes are stably integrated into the nuclear genome.Stable integration occurs when the nucleic acid sequence remains integrated into the nuclear genome and continues to be expressed (e.g., when a detectable mRNA or protein transcript is produced) across subsequent plant generations. Stable integration and / or nuclear genome editing can be performed using any method known in the art (e.g., microparticle bombardment, Agrobacterium-mediated transformation, CRISPR / Cas9, protoplast electroporation, microinjection, etc.).

[0058] Under certain circumstances, it may be desirable to use an inducible promoter. An inducible promoter is responsible for gene expression in response to a specific signal, such as physical stimulus (e.g., heat shock genes), light (e.g., ribulose-bis-phosphate carboxylase 1.5), hormones (e.g., glucocorticoid), antibiotics (e.g., tetracycline), metabolites, and stress (e.g., drought). Other functional elements of transcription and translation in plants may be used, such as, for example, untranslated 5' leader sequences, 3' transcription termination sequences, and polyadenylate addition signal sequences. Petition 870250085581, dated 09 / 22 / 2025, page 37 / 89 30 / 59

[0059] The term recombinant or modified nucleic acids refers to polynucleotides that are produced by combining two separate sequence segments through the artificial manipulation of isolated polynucleotide segments using genetic engineering techniques or chemical synthesis. In this way, it is possible to join polynucleotide segments with desired functions to generate a desired combination of functions.

[0060] As used in this document, the terms overexpression and upregulation refer to increased expression (e.g., of an mRNA, polypeptides, etc.) relative to expression in a wild-type organism (e.g., plant) as a result of genetic modification. In some embodiments, the increase in expression is a slight increase of about 10% more than wild-type expression. In some embodiments, the increase in expression is an increase of 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) relative to wild-type expression. In some embodiments, an endogenous gene is overexpressed. In some embodiments, an exogenous or heterologous gene is overexpressed by virtue of being expressed.Overexpression of a gene in plants can be achieved through any method known in the art, including, but not limited to, the use of constitutive promoters, inducible promoters, high-expression promoters (e.g., PsaD promoter), enhancers, transcription and / or translation regulatory sequences, codon optimization, modified transcription factors, and / or mutant or modified genes that control the expression of the gene to be overexpressed.

[0061] When a recombinant nucleic acid is intended for the expression, cloning, or replication of a specific sequence, the DNA constructs prepared for introduction into a host cell will typically comprise a replication system (e.g., Petition 870250085581, dated 09 / 22 / 2025, page 38 / 89 31 / 59 vector) recognized by the host that includes the intended DNA fragment encoding a desired polypeptide, and may also include transcription and translation initiation regulatory sequences operatively linked to the segment encoding the polypeptide. In addition, such constructs may include cell localization signals (e.g., plasma membrane localization signals). In preferred embodiments, such DNA constructs are introduced into the genomic DNA, chloroplast DNA, or mitochondrial DNA of a host cell.

[0062] In some embodiments, a non-integrated expression system can be used to induce the expression of one or more introduced genes. Expression systems (expression vectors) may include, for example, an origin of replication or autonomous replication sequence (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included, when appropriate, from secreted polypeptides of the same or related species, which allow the protein to cross and / or lodge in cell membranes, cell walls, or be secreted by the cell.A non-integrated expression system allows transient expression, for example, of morphogens, so that heterologous sequences are expressed only during a limited period of time. In some embodiments of the present invention, morphogens are transiently expressed at one or more stages of the transformation process, and then the plant produced using the transformation process does not include the morphogen.

[0063] Selectable markers useful in the practice of the methodologies of the invention described herein may be markers. Petition 870250085581, dated 09 / 22 / 2025, page 39 / 89 32 / 59 Selectable positive markers. Typically, positive selection refers to the case where a genetically altered cell can survive in the presence of a toxic substance only if the recombinant polynucleotide of interest is present in the cell. Selectable negative markers and traceable markers are also well known in the art and are considered by the present invention. Those skilled in the art will recognize that any relevant markers available can be used in the practice of the inventions described herein.Thus, useful plant expression cassettes in the practice of the invention may include at least one genetic marker operatively linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be recovered by means of negative selection (i.e., inhibiting the growth of cells that do not contain the selective marker gene) or by means of positive selection (i.e., screening the product produced by the genetic marker). Many of the genetic marker genes suitable for plant transformation are known and include, for example, genes encoding enzymes that metabolically detoxify a selective chemical agent, which may be an antibiotic or a herbicide, or genes encoding an altered target that may be sensitive to the inhibitor. Some methods of positive selection are known in the art.The genetic selection marker can therefore allow the selection of transformed cells, while the growth of cells that do not contain the inserted DNA can be suppressed by the selection compound. The preference for a selection marker gene is at the technician's discretion; however, any of the following selection markers may be used, as well as any other gene not listed in this document. Examples of selection markers include, but are not limited to, resistance or tolerance to kanamycin (e.g., nptII). Petition 870250085581, dated 09 / 22 / 2025, page 40 / 89 33 / 59 hygromycin (HyG), bleomycin, G418, methotrexate, phosphinothricin (Bialaphos, Bar gene), imidazolinone, glyphosate (EPSPS), sulfonylureas and triazolopyrimidine herbicides such as chlorosulforon, bromoxynil and dalapon, lethal genes, PMI, ALS gene, GUS or fluorescent markers or reporter genes (e.g. GUS, GFP, CFP, YFP, RFP, dsRED, TdTomato, mNeonGreen, AmCyan, mCherry, Ruby, etc.).

[0064] Some embodiments of this aspect further include selection of genetically altered cells or tissues in step (c) or selection between steps (c) and (d) or selection of genetically altered plantlets after step (e), optionally using selectable markers. These selectable markers may be nptII or EPSPS. In some embodiments of this aspect, step (b) is performed by Agrobacterium transformation, microprojectile bombardment, nanoparticle administration, viral administration, or any other methods.

[0065] Optionally, in some embodiments, after the co-culture step (b-iii), the transformed cells may be subjected to a resting step. As used in this document, resting refers to a step in which plant cells, for example, embryogenic calluses, are incubated after the introduction of the sequence of interest by Agrobacterium-mediated infection. Resting allows preferential growth of a callus from transformed cells containing the sequence of interest and is generally performed in the absence of selective pressure. The transformed plant tissue is subjected to a resting medium that typically includes an agent (e.g., antibiotic) that inhibits Agrobacterium growth. Such agents are known in the art and include cefotaxime, thymetin, vancomycin, carbenicillin, and the like. The concentrations of said agent will vary according to the standard for each antibiotic.Those versed in the art will recognize the concentration of the Agrobacterium inhibitory agent. Petition 870250085581, dated 09 / 22 / 2025, p. 41 / 89 34 / 59 can be optimized for a specific transformation protocol without undue experimentation.

[0066] The resting period may vary from about 1 to about 20 days, preferably from about 1 to about 20 days, and even more preferably from about 5 to about 15 days. During the resting period, the temperature may be any temperature suitable for the target plant known in the art. Illustratively, for sugarcane, the temperature may vary from about 15 °C to about 30 °C, from about 16 °C to about 29 °C, from about 17 °C to about 28 °C, from about 21 °C to about 27 °C, or from about 26 °C to about 27 °C. In some embodiments, the resting period occurs in the absence of light.

[0067] When there is no resting stage, it is possible to carry out an extended co-culture stage before adding the selective agent to the transformed plant cells.

[0068] The method provided in this document further includes the selection of genetically altered plant cells or tissues comprising at least one copy of the genetic sequence of interest (step c) or the protein of interest. Selection, as used in this document, means the situation in which a selective agent is used for the transformants, wherein said selective agent will permit the preferential growth of genetically altered plant cells or tissues. Selection also means the step in the process in which the genetically altered cells or tissues are maintained under culture conditions ideal for the expression of the trait of interest, so that the trait can be used to select the genetically altered cells or tissues. As indicated above, any suitable selection marker, selection condition, or selection method may be used. In some embodiments, an agent is also added to inhibit the growth of Agrobacterium.The selection. Petition 870250085581, dated 09 / 22 / 2025, page 42 / 89 35 / 59 can occur under light or dark conditions, depending on the plant species to be transformed and, for example, the genotype. In the case of callus transformation, it is possible to keep individual calluses separate to ensure that only one plant is regenerated per callus and, therefore, all regenerated plants are derived from independent transformation events. In one embodiment, the selection step is step c) of the transformation method described in this document. In a preferred embodiment, the selection step occurs in the dark. The selection step is preferably carried out in a sealed culture plate or container for at least 15 to 60 days, between 20 °C and 35 °C. In a preferred embodiment, the selection step is carried out for at least 20 to 45 days, more preferably for at least 21 to 42 days, and even more preferably for 30 days. In one embodiment, the selection step is carried out between 25 and 30 °C.In a preferred embodiment, the selection step is carried out between 25 and 29 °C, particularly at 27 °C. In one embodiment, the selection culture medium is a semi-solid or solid medium. In another embodiment, the selection medium is liquid and the cells or tissues are grown on a solid support, the solid support being filter paper, paper, a polymeric / nylon membrane, a common Petri dish, among others. The liquid selection medium is added in sufficient quantity to form a thin film on the solid support or to moisten the filter paper or membrane, without covering the genetically modified plant cells or tissues. In some embodiments, the selection medium is a dehydration medium comprising an osmotic agent such as, for example, a high concentration of salts.

[0069] After the selection phase (step (c)), the selected genetically altered cells or tissues must be subjected to a heat shock treatment to ensure efficient regeneration and the efficiency of the transformation method. The combination of steps (c). Petition 870250085581, dated 09 / 22 / 2025, page 43 / 89 36 / 59 and (d) is crucial for the efficiency of the transformation method of the invention, promoting a high rate of regeneration of genetically altered cells or tissues. The gain is even more evident when observed for recalcitrant plant species or varieties.

[0070] By thermal shock, the present invention relates to the controlled temperature variation to which the plant cell or tissue is subjected after or shortly after the selection phase (step (c)). This controlled temperature variation can be obtained by any means and equipment, provided that the plant cell or tissue is subjected for 1 to 5 days at a temperature of 30 °C to 45 °C. Preferably, the plant cell or tissue is subjected for 1 to 3 days at a temperature of 30 °C to 45 °C. More preferably, the thermal shock (step (c)) is carried out at 35 °C to 40 °C, more preferably 35 to 37 °C, particularly at 35 °C. Additionally, the thermal shock is carried out for 2 to 3 days, more preferably for 3 days. More preferably, the plant cell or tissue is subjected for 3 days at 35 °C.Optionally, the plant cell or tissue may first be subjected to a cold shock by incubating the plant cell or tissue from step c) at a reduced temperature for less than 1 day to 3 days at a temperature of 1 to 10 °C. After the cold shock, the plant cell or tissue is subjected to the heat shock treatment described in step d) of the invention.

[0071] By transformation efficiency or transformation frequency, in the present invention, is meant a parameter that can be measured by the number of transformed and regenerated plant cells that are recovered under experimental conditions. For example, when calluses are used as starting material for transformation, the transformation frequency can be expressed as the number of positive events obtained per gram of callus subjected to transformation.

[0072] In some embodiments of this aspect, the transformation efficiency is increased by at least 5% compared to a Petition 870250085581, dated 09 / 22 / 2025, p. 44 / 89 37 / 59 sugarcane cell transformation method that does not use at least one morphogen nucleotide sequence. In some embodiments of this aspect, the transformation is increased by 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 30%. In some embodiments of this aspect, the transformation efficiency is increased by 50% to 100% compared with a sugarcane cell transformation method that does not use at least one morphogen nucleotide sequence.

[0073] In a specific embodiment, the present invention describes a novel transformation method in which a polynucleotide used in the transformation process is efficiently excised by a recombinase / extraction system activated by a thermal / stress stimulus, allowing the production of transformed events without unwanted integration of such polynucleotide sequences. The present invention involves methods for excising a polynucleotide of interest from plant cells or tissues by means of a thermal shock treatment, allowing excision without compromising regeneration rates.Thus, such a transformation method further comprises, in step (b), the introduction of at least one polynucleotide sequence of interest into the cell or tissue of step (a), wherein said polynucleotide comprises a recombinase sequence under the control of an inducible promoter and at least one other polynucleotide, wherein both sequences are flanked by recombination sites. In a preferred aspect, the inducible promoter is selected from the group consisting of a stress-inducible promoter and a chemically inducible promoter. Said method comprises the excision of the polynucleotide sequences flanked by recombination sites during the induction of site-specific recombinase expression by the culture conditions in steps (c) and (d). In a preferred embodiment, the thermally inducible promoter is P-rab17. Petition 870250085581, dated 09 / 22 / 2025, p. 45 / 89 38 / 59

[0074] In another embodiment of the invention, the recombinase gene present in the polynucleotide of interest can be any gene widely known to those skilled in the art, such as, but not limited to, LoxP / Cre, FLP / FRT, R-RS, Bxb11, among others. The preferred aspect of this embodiment consists of using the LoxP / Cre recombinase system. Any inducible promoter known to those skilled in the art can be used in this document, preferably those controlled by a thermal stimulus, such as, but not limited to, pHSP18.2, pHSP26, pHSP82, pHSP18, pRab17, among others. In a preferred embodiment of the invention, the inducible promoter is pRab17.

[0075] In addition, the invention includes the use of a composition comprising at least one additional compound that promotes the activation of the thermally inducible promoter and stimulates the regeneration of a plant cell or tissue. Non-limiting examples of such compounds are ABA and PEG. In a preferred embodiment of the invention, the culture medium composition in step (d) comprises abscisic acid (ABA) in concentrations ranging from 20 to 200 μM, particularly from 50 to 100 μM. When PEG is added, alone or in combination with ABA, its concentration may range from 20 to 150 μM.

[0076] In another embodiment, the first and second polynucleotides of interest may be operatively linked or be in the same expression cassette as a selectable gene or marker that indicates the presence / absence of such polynucleotides in the transformed plant or plant part. Furthermore, the selectable gene or marker may be in another expression cassette, provided that it is expressed only after the excision of the second polynucleotide of interest or with the maintenance of the second polynucleotide of interest, indicating whether the excision occurred or not.

[0077] The polynucleotide of interest is flanked by the sites of Petition 870250085581, dated 09 / 22 / 2025, page 46 / 89 39 / 59 recombinase, which may be genes from the CRISPR machinery, genes from the primary editing machinery, selectable markers, herbicide genes, silencing genes, genes for inactivated nucleases, transcription factors, growth or development genes, antibody resistance genes, morphogens, reporter genes, among others. In a preferred embodiment of the invention, the second polynucleotide of interest is a selectable marker, such as an antibody or herbicide resistance gene or a growth or development stimulating gene, such as a morphogen.

[0078] Although well known, the combination of a stress-inducible promoter with a recombinase / extraction system is not yet mastered for transforming / modifying any monocotyledon, especially for sugarcane varieties, since there is no data showing that it is possible to proceed with an efficient excision with a heat shock step without compromising the regeneration capacity of this species.

[0079] In one embodiment of the invention, after heat shock treatment (step (d)), the genetically altered plant cell or tissue is regenerated into a seedling by culturing the cells or tissue in a growth regulator-free culture medium in the presence of light; and (e) growing the genetically altered seedling into a genetically altered plant. When a callus culture is established in step (a) and subjected to the subsequent steps of the transformation methods of the present invention, the conditions applied in the selection step (c), in combination with the heat shock treatment in step (d), synchronize the development of the cells / tissues so that they respond to the regeneration culture medium and conditions, rapidly converting the callus culture into seedlings.

[0080] Some embodiments of this aspect further include screening the sugarcane cells between steps (c) and (d), screening Petition 870250085581, dated 09 / 22 / 2025, page 47 / 89 40 / 59 of the seedlings after step (e) or screening of the plants after step (f) to identify the modification introduced. Some embodiments of this aspect also include selection by any means, as described in this document. In some embodiments of this aspect, step (b) is carried out by means of transformation by Agrobacterium, microprojectile bombardment, nanoparticle administration, viral administration or a combination thereof.

[0081] As used in this document, the term regenerable plant or plant part or regenerated plant or plant part or any other term referring to the regeneration process and results refers to plant cells or tissues in which a genetic alteration, such as transformation, has been performed in relation to a trait of interest or is a plant or plant part, such as plant cells, that descends from a plant or plant part that has been genetically altered.

[0082] The screening and molecular analysis of genetically modified plants, plant cells, or tissues of the present invention can be performed during the selection step (steps ced) or later in the regeneration phase (step (e)) using nucleic acid hybridization techniques. Hybridization procedures are useful for identifying polynucleotides, such as those modified using the techniques described herein, with sufficient homology to the regulatory sequences in question to be useful as taught herein. Specific hybridization techniques are not essential to the present invention. As hybridization techniques are improved, they can be easily applied by those skilled in the art. Hybridization probes can be labeled with any appropriate marker known to those skilled in the art.The hybridization and washing conditions, for example, temperature and salt concentration, can be altered to... Petition 870250085581, dated 09 / 22 / 2025, p. 48 / 89 41 / 59 change the severity of the detection threshold. See, for example, Sambrook et al. (1989) see below or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, for further guidance on hybridization conditions.

[0083] Similarly, screening can be performed using polypeptide-based techniques, including enzyme-linked immunosorbent assays (ELISAs), fluorescence detection (if a fluorescent marker has been used), or Western blots. Those skilled in the art will recognize that any available polypeptide-based techniques can be used in screening the inventions described herein.

[0084] Furthermore, screening and molecular analysis of genetically altered strains, as well as the creation of desired isolated nucleic acids, can be performed using the Polymerase Chain Reaction (PCR). PCR is a repetitive, enzymatic, and preparative synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those versed in this technique (see Mullis, U.S. Patents Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230: 1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize with opposite strands of the target sequence. The primers are oriented with their 3' ends pointing towards each other.Repeated cycles of thermal denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of template DNA produced in the previous cycle. This results in exponential accumulation. Petition 870250085581, dated 09 / 22 / 2025, page 49 / 89 42 / 59 of the specific target fragment, up to several million times in a few hours. Using a thermostable DNA polymerase, such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes that can be used are known to those skilled in the art.

[0085] In some modalities, screening can be done using PCR, ELISA, fluorescence detection, or other screening methods known in the art.

[0086] The nucleic acids and proteins of the present invention may also include homologs of the specifically described sequences. The homology (e.g., sequence identity) may be from 50% to 100%. In some cases, this homology is greater than 80%, greater than 85%, greater than 90%, or greater than 95%. The degree of homology or identity required for any intended use of the sequence(s) is readily identified by those skilled in the art. As used in the present document, the percentage of sequence identity of two nucleic acids is determined using an algorithm known in the field, such as that described by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as per Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs by Altschul et al. (1990) J. Mol. Biol. 215: 402-410.Nucleotide searches using BLAST are performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences that have the desired percentage of sequence identity. To obtain gap alignments for comparison purposes, Gapped BLAST is used, as described in Altschul et al. (1997) Nucl. Acids. Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used (NBLAST). Petition 870250085581, dated 09 / 22 / 2025, pp. 50 / 89 43 / 59 and XBLAST). See www.ncbi.nih.gov.

[0087] The preferred host cells or explants are plant cells or tissues. Plant cells can be derived from plants including maize (e.g., sweet corn, Zea mays), barley (e.g., Hordeum vulgare), millet (e.g., finger millet, foxtail millet, pearl millet, rice millet, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), oats (e.g., Avena sativa), rice (e.g., rice indica, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), rye (e.g., Secale cereale, Secale cereanum), sugarcane (e.g., Saccharum sp.), setaria (e.g., Setaria italica, Setaria viridis), Brachypodium sp., sorghum (e.g., Sorghum bicolor), truffle (e.g., Eragrostis tef), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A.Camus, Triticosecale neoblaringhemii A. Camus), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), foxtail grass (e.g., Panicum virgatum), Brassica sp., or tobacco (e.g., Nicotiana benthamiana, Nicotiana tabacum). Plant cells can also be derived from other monocotyledonous and dicotyledonous plant species. Preferably, the plant cells are from monocotyledonous species, especially sugarcane (e.g., Saccharum spp.). The methods of the present invention may be particularly suitable for the transformation of recalcitrant species.

[0088] The sugarcane plants of the present invention include species and hybrids of the genus Saccharum, for example, Saccharum officinarum, Saccharum sinense, Saccharum barberi, Saccharum robus Petition 870250085581, dated 22 / 09 / 2025, p. 51 / 89 44 / 59 tum, Saccharum spontaneum, Saccharum spp., Saccharum spp. hybrid, S. edule, S. aegyptiacum, S. esculentum, S. aenicol, S. arundinaceum, S. bengalense, S. biflorum, S.ciliare, S. cylindricum, S. Elephantinum, S. exaltatum, S. fallax, S. floridulum, S. giganteum, S. japonicum, S. koenigii, S. laguroides, S. munja, S. narenga, S. paniceum, S. pophyrocoma, S. purpuratum, S. ravennae, S. roseum, S. sanguineum, S. sara, S. chinense, S. tinctorium, S. versicolor, S. violaceum. Even more preferably, these are interspecific hybrids produced by crossing commercial species and varieties thereof. The methods of the present invention are genotype independent.

[0089] Plant cells and tissues can be derived from tissue types, including embryo, callus, leaf disc, shoots, axillary buds, section or fragment of plant parts, leaf blade, stem, shoot apex, leaf sheath, internodes, petioles, flower stalks, root, inflorescence, and other explants. Properly, an explant is a segment, slice, or section of tissue. Plant cells can be differentiated or undifferentiated (e.g., callus, undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf sheath, leaf cells, root cells, phloem cells, and pollen).Plant cells include, but are not limited to, seed cells, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic calluses, meristems, somatic meristems, organogenic calluses, protoplasts, leaf bases, mature plant leaves, leaf tips, immature inflorescences, cotyledons, immature cotyledons, embryonic axes, meristematic regions, callus tissue, leaf cells, stem cells, root cells, budding cells, gametophytes, sporophytes, pollen, and microspores. Plant cells also include various forms of cells in culture (e.g., isolated cells, protoplasts, embryos, and callus tissue). Petition 870250085581, dated 09 / 22 / 2025, page 52 / 89 45 / 59 that protoplasts or cells are produced from a part of the plant selected from the group of leaves, stems, anthers, pistils, roots, fruits, flowers, seeds, cotyledons, hypocotyls, embryos or meristematic cells. Genetically altered plant cells, in the present context, are those that have been modified to contain a protein molecule or nucleic acids containing one or more deleted or non-functional genes, normally present and functional in the host cell, or a different pattern of expression of a characteristic (such as an altered cellular function without modification of the genome, for example, epigenetic variations).Nucleic acid(s) and / or proteins may be introduced by any means known in the art that is appropriate for the specific cell type, including, without limitation, Agrobacterium transformation or bombardment, lipofection, electroporation, or any other methodology known to those skilled in the art.

[0090] More preferably, the plant cell or tissue of step (b) of the method of the present invention is a callus. More preferably, the embryogenic callus is of type II or III. Embryogenic calluses can be formed from any suitable plant tissue, preferably from a sugarcane plant. Sugarcane tissue culture is well known and follows a conventional model of callus production and plant regeneration, initially described by Ho & Vasil. 1983. Protoplasma, 118: 169-180; Brisibe et al. 1993. Plant Science, 89: 85-92, and subsequently by Falco et al. 1996. R. Bras. Fisiol. Veg., 8(2): 93-97. Ideally, immature tissue is used to initiate callus formation, such as the core of sugarcane or meristems.

[0091] Type I, II, and III calluses can be initiated from tissues including, but not limited to, immature embryos, axial meristems, axillary meristems, microspores, and others. These cells capable of proliferating as calluses are also target cells for transformation of Petition 870250085581, dated 09 / 22 / 2025, page 53 / 89 46 / 59 plant. Target cells can also be somatic cells, which are those cells that, during normal plant development, do not contribute to the plant's reproductive processes. Meristem cells (i.e., capable of continuous cell division and characterized by an undifferentiated cytological appearance, normally found at growth points such as root tips, axillary meristems, shoot apices, lateral buds, and others) can represent another type of target cell. By virtue of their undifferentiated state and capacity for differentiation and totipotency, a single transformed meristematic cell has the potential to regenerate an entire transformed plant.

[0092] Suitable cell cultures can be started from various types of explants. For example, for sugarcane varieties, explants can be obtained from suitable plant tissue, including the heartwood or core of sugarcane (a cluster of young, coiled leaves containing apical meristem), leaf blade, axillary buds, stem, shoot apex, leaf sheath, internodes, petioles, flower stalks, seeds, roots, or inflorescence. Appropriately, an explant is a segment, slice, or section of tissue. More preferably, the explant is a section of the apical portion of the heartwood of sugarcane from sugarcane seedlings. Explants can be obtained from plants grown in vitro, in greenhouses, or in the field.Preferably, the age of the plant is less than about 24 months, less than about 23 months, less than about 22 months, less than about 21 months, less than about 20 months, less than about 19 months, less than about 18 months, less than about 17 months, less than about 16 months, less than about 15 months, less than about 14 months, less than about 13 months, less than about 12 months, less than about 11 months, less than about 10 months, less than about 9 months, less than about 8 months, less than about 7 months, less than about 6 months, less than about 5 months, less than about 4 months, less than about. Petition 870250085581, dated 09 / 22 / 2025, p. 54 / 89 47 / 59 of 3 months, less than about 2 months, or less than about 1 month. Preferably, the age of the plant is about 24 to 12 months, more preferably 12 to 8 months, even more preferably 6 to 2 months. Said tissue culture is generally initiated from sterile pieces of a plant, as described above. Many traits of the explant are known to affect the efficiency of culture initiation; however, it is generally considered that young, faster-growing tissues or tissue in an early stage of development are more efficient. Explants grown in appropriate media may give rise to a disorganized mass of dividing cells (calluses) which, in culture, can be maintained more or less indefinitely, provided that periodic subcultures are carried out in a fresh culture medium.

[0093] As used in this document, plant refers to the whole plant, plant tissue, a part of the plant (such as an embryo), a plant cell or a group of plant cells. More preferably, plants are monocotyledons and, even more preferably, they are those used for food or energy generation, such as rice, maize, wheat, barley, millet, sorghum, rye, triticale, sugarcane and other species such as Erianthus, Miscanthus, Narenga, Sclerostachya and Brachypodium.All genera of the subfamilies Bambusoideae (e.g., the genus Bambusa), Andropogonoideae (e.g., the genera Saccharum, Sorghum, and Zea), Arundineae (e.g., the genus Phragmites), Oryzoideae (e.g., the genus Oryza), Panicoideae (e.g., the genera Panicum, Pennisetum, and Setaria), and Pooideae (Festuciadeae) (e.g., the genera Poa, Festuca, Lolium, Trisetum, Agrostis, Phleum, Dactylis, Alopecurus, Avena, Triticum, Secale, and Hordeum) are included. More specifically, a plant that can be transformed according to the present invention is sugarcane. Petition 870250085581, dated 09 / 22 / 2025, pp. 55 / 89 48 / 59

[0094] Through genetic modification, a plant, preferably a sugarcane plant or plant cell, can be modified to exhibit improved or superior agronomic traits compared to non-transformed plants of the same genotype. For example, transgenic plants can be modified to express genes that provide resistance to diseases and insects, herbicide tolerance, confer nutritional value, increase sucrose content, fiber content, influence plant growth, tolerance to abiotic stresses, increase biomass production, modify lignin content (composition / content), sterility, among others.

[0095] When appropriate, the sequence of interest to be transferred to a plant can be modified to optimize expression. For example, a sequence can be modified to improve expression in a monocotyledonous plant, most preferably sugarcane. Methods for synthetic optimization are available in the art, for example, in US documents 5,380,831; US ​​5,436,391 and Murray et al. 1989. Nucleic Acids Res. 17: 477-498. Preferred codons of the target plant can be determined from codons of higher frequency in the target plants of interest. Other modifications can be made to increase gene expression in the target plant including, for example, the elimination of spurious polyadenylation signals, exon-intron splicing signals, similar transposon repeats, among others. The GC content of the sequence can be adjusted to average levels for a given target plant, calculated with reference to known genes expressed in the target plant.Furthermore, the sequence can be modified to avoid hairpin-like structures in the mRNA.

[0096] In some embodiments of this aspect, step (b) is achieved by means of transformation by Agrobacterium, microprojectile bombardment, nanoparticle administration, viral administration Petition 870250085581, dated 09 / 22 / 2025, pp. 56 / 89 49 / 59 or any other transformation method, as described in this document. In some embodiments of this aspect, at least one introduced nucleotide sequence encodes a nucleotide sequence or protein capable of exhibiting a trait selected from the group consisting of expressing a fluorescent protein (e.g., GFP, CFP, dsRED, etc.), herbicide resistance or tolerance (e.g., CP4-EPSPS, BAR, ALS, etc.), an agronomic trait and a disease / pest resistance or tolerance protein (e.g., BT, Cry, VIP, etc.) or a morphogen. In some embodiments, the agronomic trait includes a biomass trait, a sucrose trait, a flowering trait, and / or an aluminum tolerance trait.

[0097] Morphogenes are genes that have been shown to functionally enhance embryogenesis and somatic transformation. Any combination of morphogens can be used in the methods of the present invention. Thus, in some embodiments of this aspect, at least one morphogen is introduced in step (b). In some embodiments of this aspect, a nucleotide sequence, other than a morphogen, is introduced in step (b). In some embodiments of this aspect, at least one nucleotide sequence is introduced at the same time as the morphogen. In some embodiments of this aspect, at least one morphogen sequence is introduced before at least one nucleotide sequence. In some embodiments of this aspect, at least one morphogen sequence is introduced after at least one nucleotide sequence. In some embodiments of this aspect, the morphogen and the nucleotide sequence are introduced by separate vectors.In one aspect, the vector includes a promoter operatively linked to at least one nucleotide sequence. In some embodiments of this aspect, the promoters are selected from the group of a constitutive promoter, an inducible promoter, or a tissue- or cell-type-specific promoter. Petition 870250085581, dated 09 / 22 / 2025, pp. 57 / 89 50 / 59 In some embodiments of this approach, the morphogene and the nucleotide sequence are introduced by the same vectors into different expression cassettes.

[0098] In some embodiments of this aspect, the introduction of at least one morphogen sequence is transient. In some embodiments of this aspect, the genetically modified plant of step (d) does not include at least one morphogen sequence. In a specific embodiment, the morphogen sequence is excised by a recombinase / extraction system activated by a thermal stimulus, according to the method described in the present invention, allowing the production of transformed events without the integration of such polynucleotide sequences.

[0099] After the selection period, the plant tissue that continued to grow in the presence of the selection agent and which was therefore genetically modified can be manipulated and regenerated by placing it in culture media and under suitable growth conditions. The transgenic plants thus obtained can be tested for the presence of the DNA of interest. The term regenerate, for the purposes of the present invention, refers to the formation of a seedling or plant, including an aerial part and roots. The regeneration of various species is well known in the art. Regenerated plants can be planted in a suitable substrate such as, for example, soil. As used herein, genetically modified or transgenic or stably transformed means a plant cell, plant part, plant tissue or plant comprising a DNA sequence of interest that is introduced into its genome by means of transformation.

[00100] In some respects, the transformation method of the present invention further comprises step (f) for elongating the regenerated plantlets from step (d). The elongation step comprises cultivating the plantlets from step (e) in a culture medium and maintaining them. Petition 870250085581, dated 09 / 22 / 2025, pp. 58 / 89 51 / 59 las for 14 to 21 days under light at 27 °C ± 2 °C. Optionally, this culture cycle can be repeated for another 14 to 21 days under light at 27 °C ± 2 °C.

[00101] After the elongation stage, the plants were subjected to molecular and morphological analyses to confirm the incorporation of the trait of interest.

[00102] In some aspects, the present invention relates to a seed, plant part or plant tissue genetically modified according to any of the embodiments above. In some embodiments, the plant part is selected from the leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo or meristematic cell group. Plant parts include differentiated and undifferentiated tissues including, among others, roots, stems, shoots, leaves, pollen and seeds.

[00103] In some respects, the present invention relates to a pollen grain or an ovule of the sugarcane plant genetically modified according to any of the embodiments above.

[00104] In some respects, the present invention relates to a protoplast of the sugarcane plant genetically modified according to any of the embodiments above.

[00105] In some respects, the present invention relates to a tissue culture produced from protoplasts or cells of a genetically modified plant in accordance with any of the above embodiments.

[00106] In one aspect of the invention, a method is provided for increasing the regeneration of plant cells or plant tissues comprising: a) to cultivate a plant cell or plant tissue in vitro; b) cultivate the cell or tissue from step (a) in a culture medium for at least 15 to 60 days at 20 to 35 °C; Petition 870250085581, dated 09 / 22 / 2025, pp. 59 / 89 52 / 59 c) perform heat shock treatment on the cell or tissue from step (b) for at least 1-3 days at 30 to 45 °C in a culture medium; d) regenerate the cell or tissue from step (c).

[00107] The method described in this document increases regeneration efficiency by at least 5% compared to a conventional method of cell and tissue regeneration. Preferably, said method increases regeneration efficiency by at least 50% to 100% compared to a conventional method of cell and tissue regeneration.

[00108] Having described the present invention in a general way, it will be better understood by reference to certain specific examples, which are included herein to better illustrate the invention and are not intended to limit the scope of the invention as defined by the claims. EXAMPLES

[00109] The following description presents exemplary methods, parameters and the like. It will be recognized, however, that such description is not intended to limit the scope of the present invention, but rather to describe exemplary embodiments.

[00110] The combination of the selection step and heat shock treatment, as described in the present invention, significantly increases the regeneration rate of genetically altered cells or tissues, improving the efficiency of the transformation method. To demonstrate the present invention, a recalcitrant sugarcane variety was subjected to genetic transformation by Agrobacterium using a conventional protocol (Control) and the Method of the Invention.

[00111] Three experiments were conducted with the recalcitrant sugarcane variety using a 3-day heat shock at 35 °C (step d) after a 21-day selection phase without culture medium change or cell manipulation at 27 °C. Petition 870250085581, dated 09 / 22 / 2025, pp. 60 / 89 53 / 59

[00112] The plasmid used in these examples contains a herbicide tolerance gene (expression cassette 3, SEQ ID NO: 3) and an insecticide gene (expression cassette 4, SEQ ID NO: 4) as traits to be incorporated into the plant cell and a recombinase gene (moCRE, expression cassette 2, SEQ ID NO: 2) in combination with a selection marker gene (nptII, cassette 1, SEQ ID NO: 1) both flanked by recombination sites (LoxP) and planned to be excised during steps (c) and (d) of the transformation method. 1. Expression Cassette 1: pBdUbi10::LoxP::nptII::T-35S 2. Expression Cassette 2: P-rab17::moCRE::T-NOS::LoxP 3. Expression cassette 3: CTP2::Herbicide gene::T35S 4. Expression cassette 4: P-2X35S::L-CAB::I- OsAct1::gene insecida::T-35S.

[00113] Recombination site-flanked cassettes (LoxP) were included to exemplify the possibility of using stress / thermal promoter-inducible (such as rab-17)-controlled recombinase excision systems efficiently, even for a recalcitrant strain, without affecting the viability of the transformed cells and increasing the regeneration rate. Example 1: Plant Material: Explant for Transformation (Step a)

[00114] Tissue culture is commonly used for plant transformation, generating potentially transformable cells. Maintaining tissue cultures requires the use of culture media (a mixture of nutrients and plant growth regulators for in vitro cell growth and maintenance) and controlled environmental conditions. The tissue explant chosen to exemplify the present invention is the embryogenic callus of sugarcane.

[00115] To obtain embryogenic calli, young, rolled-up (heart) sugarcane leaves, grown in the field or in a greenhouse for 3 to 12 months, were collected for isolation of Petition 870250085581, dated 09 / 22 / 2025, pp. 61 / 89 54 / 59 initial explants.

[00116] After surface disinfection, cross-sections approximately 0.05–5 mm thick were cut from the region above the meristem under aseptic conditions. The sections were placed on the surface of SCIM culture medium (Table 1). The cultures were kept in the dark at a temperature of 26 °C ± 2 °C and subcultured every 15 days for three to five cycles of 7 to 28 days. Example 2: Preparation of Agrobacterium and Callus Infection (Steps b-i and b-ii)

[00117] The Agrobacterium culture, comprising the EHA105 strain (Hood et al. 1993. New Agrobacterium Helper Plasmids for Gene Transfer to Plants. Transgenic Research, v. 2, pp. 208-218) comprising the plasmid with the expression cassettes as described below (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; Figure 1), was initiated from a glycerol stock maintained at -80 °C in solid LB medium plus appropriate antibiotics. This culture was kept in the dark at 28 °C for two to three days. The Agrobacterium suspension for infecting plant material was prepared by resuspending the culture in a ½ MS liquid medium plus acetosyringone at 200 μM, adjusting to a final OD600 of 0.1-1.0.

[00118] The calluses were transferred directly to the Agrobacterium suspension, where they remained for 30 minutes in the dark, under constant agitation at 50 rpm.

[00119] After this period, the calluses were separated from the Agrobacterium and the excess suspension was removed by drying on filter paper sheets.

[00120] Alternatively, prior to infection, calluses can be treated in ½ MS liquid medium at approximately 45°C for about 5 minutes. Petition 870250085581, dated 09 / 22 / 2025, pp. 62 / 89 55 / 59

[00121] Another optional treatment is to subject the infected plant material to a vacuum pressure of approximately -700 mmHg for about 5 minutes. Example 3: Co-culture and Callus Rest (Steps b-iii and b-iv)

[00122] This step was performed in liquid or solid SCIM culture medium (Table 1) with 7; 14; 21; 28; 35; 42 or 49 g / L of Agargel™, weighing between 0.5 and 10 g of callus per plate (100 x 20 mm). Co-culture was carried out for a period of 1 to 5 days at a temperature of 22 °C and in the dark.

[00123] After co-culture, the calluses were transferred to DT resting medium (Table 1) supplemented with the bacteriostatic agent Timentim® at a concentration of 200 mg / L to control the unwanted growth of Agrobacterium. The resting period was 5 to 14 days at 26 °C in the dark. Example 4: Selection of Genetically Altered Cells or Tissues (Step c) and Heat Shock Treatment (Step d)

[00124] Calluses were transferred to SGT selection medium (Table 1), supplemented with 200 mg / L of Timentim® + 50 mg / L of the geneticin selection agent. Calluses remained in this condition for 21 days at 26 °C ± 2 °C in the dark (Selection 1, optional step) and then were transferred to a new SGT selection medium and remained for another 21 days at 26 °C ± 2 °C in the dark, without any further culture replacement or cell manipulation.

[00125] After the Selection stage, the culture plates subjected to the method of the present invention were incubated in a Biodegradable Oxygen (BOD) chamber (Thermolab Scientific Equipments) at approximately 35 °C ± 2 °C for 3 days (heat shock treatment - stage d). The heat shock can be performed with the structures still in selection medium or after changing to Regene medium. Petition 870250085581, dated 09 / 22 / 2025, pp. 63 / 89 56 / 59 feed (Table 1). The plates were sealed with micropore tape or plastic film according to the material profile.

[00126] In the control treatment, after the selection stage, the calluses were transferred to the RG1 regeneration medium. Table 1: Culture media recipes Reagents SCIM Induction CC Coculture DT Rest SGT Selection RG1 RG2 (optional) AGT Elongation Sais MS (g / L) 4.3 4.3 4.3 4.3 4.3 4.3 4.3 Vitamins 1000x (mL / L) 25 25 25 25 25 25 25 2.4D (mg / L) 3 3 3 3 - - - BAP (mg / L) - - - - 1 0.1 - Casein (g / L) 0.5 - 0.5 0.5 - - - Agar (g / L) 7 7 - 7 7 7 7 Timent (mg / L) - - 200 200 200 200 200 Geneticin (mg / L) - - - 50 30 30 30 Acetossyringone (μΜ) - 200 - - - - - PH 5.7 5.4 5.7 5.7 5.7 5.7 5.7 Example 5: Regeneration of Genetically Modified Plants (Step e)

[00127] After the selection and / or heat shock treatment step, the calluses were transferred to RG1 regeneration medium, supplemented with 200 mg / L of Timentim® + 30 mg / L of geneticin, and cultivated under a 15-hour photoperiod at 4,000 lux at 27 °C ± 2 °C for 14 to 21 days. After 14 to 21 days, the calluses that showed plantlet formation (Figures 2A-2B) were transferred to RG2 medium, supplemented with 200 mg / L of Timentim® + 30 mg / L of geneticin and maintained for another 14 to 21 days under a 15-hour photoperiod at 4,000 lux at 27 °C ± 2 °C.

[00128] It was observed that the application of said thermal shock in combination with the conditions applied in the selection step (c) is capable of increasing the cell or tissue regeneration rate by 4.6X Petition 870250085581, dated 09 / 22 / 2025, pp. 64 / 89 57 / 59 genetically modified plant (Table 2). Table 2. Regeneration rate Treatments Control Treatment Selection Step (c) Method of the Invention Selection Step (c) + Thermal Shock (Step d) Regeneration Rate (%) 10.40% 48%

[00129] This indicates that the combination of a selection step as defined by the present invention, followed by a heat shock treatment, not only did not affect the viability of the transformed sugarcane cells, but is also necessary to achieve an increase in the regeneration rate.

[00130] When the plants reached an average height of five centimeters, they were transferred to the stretching medium. Example 6: Stretching Stage (Stage f)

[00131] When the plants reached an average height of five centimeters, they were transferred to the elongation medium and maintained for 14 to 21 days under light at 27 °C ± 2 °C (Elongation 1). Then, the plants were subcultured in the same culture medium and maintained for another 14 to 21 days under light at 27 °C ± 2 °C (Elongation 2, optional). After the elongation step, the plants were subjected to molecular and morphological analyses to confirm the incorporation of the trait of interest. Example 7: ABA-Mediated Thermal Excision using the Cre-LOX Recombinase System

[00132] Part of the calluses subjected to the Thermal Shock step (Example 4) were transferred to a regeneration medium containing ABA (RG1 plus ABA) with concentrations in the approximate range of 25 to 100 μM and maintained for a period of 24 to 72 hours in a dark chamber at 27 ± 2 °C. Subsequently, the calluses were transferred to a new regeneration medium without ABA and in the presence of light and subjected to the conditions described in the regeneration step (Example 5). Petition 870250085581, dated 09 / 22 / 2025, pp. 65 / 89 58 / 59

[00133] Three analyses were performed based on this methodology, with three repetitions of 40 callus groups in each treatment / replication, totaling 120 groups per treatment. The excision treatments were: • Treatment 1: 3 days of thermal shock treatment in the BOD; • Treatment 2: Treatment 1 plus ABA at 50 μM for 24 hours; and • Treatment 3: Treatment 1 plus ABA at 100 μM for 24 hours.

[00134] After the regeneration stage, a total of 1,163 plants were analyzed. It was found that the application of ABA in the excision phase for 24 hours was effective in increasing the percentage of excised plants by 15.6% and 17.5% (ABA at 50 and 100 μM, respectively) compared to the method without ABA. Table 3: Percentage of plant excision under different excision treatments Treatment 1 Treatment 2 ABA at 50 μM Treatment 3 ABA at 100 μM Excised plants 31.7% 47.3% 49.2% Example 8: ABA / PEG-mediated excision by the Cre-Lox recombinant system.

[00135] To verify the effect of 50 μM ABA plus 50 μM PEG for 3 days on excision rates, two replicates were performed with 20 callus groups each. The excision treatments were: • Treatment 1: 3 days of thermal shock treatment in the BOD; • Treatment 2: Treatment 1 plus 50 μM ABA + 50 g / L PEG for 72 hours Petition 870250085581, dated 09 / 22 / 2025, pp. 66 / 89 59 / 59

[00136] The results showed that the addition of PEG to ABA treatment is also effective for moCre recombinase-mediated excision. The same increase in excision rate observed with ABA treatment alone was seen, compared to Treatment 1 (15.6%).

[00137] Although the prior invention has been described in some detail by way of examples for the sake of clarity, it will be obvious that certain alterations and modifications may be made within the scope of the appended claims. Petition 870250085581, dated 09 / 22 / 2025, pp. 67 / 89

Claims

1 / 7 CLAIMS 1. A method for transforming a plant cell or plant tissue to comprise a trait of interest, characterized in that it comprises: a) cultivating a plant cell or plant tissue in vitro; b) introducing a sequence of interest into the cell or tissue of step (a), thereby producing a transformed tissue or cell; c) cultivating the cell or tissue of step (b) in culture medium for at least 15 to 60 days at 20 °C to 35 °C; d) performing heat shock on the cell or tissue of step (c) for at least 1-3 days at 30 °C to 45 °C; ee) regenerating the cell or tissue of (d), wherein the regenerated cell comprises the trait of interest.

2. Method according to claim 1, characterized in that, in step (c), the cell or tissue is maintained in the culture medium without manipulation or subculture and / or that it further comprises repeating steps (c) and (d) a second time to add a second selection step.

3. Method according to any one of claims 1 or 2, characterized in that step (b) further comprises at least one of the following additional steps: i) preparing Agrobacterium strains comprising the sequence of interest; ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension from (i); iii) co-culturing the plant cell or tissue in a co-culture medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or Petition 870250085581, dated 22 / 09 / 2025, page 68 / 89 2 / 7 iv) culturing the transformed plant cell and tissue in a resting medium containing an agent (e.g., antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark.

4. Method according to claim 1, characterized in that it further comprises step (f) allowing the elongation of the regenerated seedlings from step (d).

5. Method according to any one of claims 1 to 4, characterized in that it further comprises: (i) screening cells or tissue between steps (b) and (c), screening seedlings after step (e) or screening plants after step (f) to identify the sequence introduced into the cells or tissues or the trait of interest; and / or (ii) selecting the genetically altered cells or tissues in step (c), selecting the genetically altered cells or tissues between steps (c) and (d) or selecting the genetically altered seedlings after step (e), optionally using selectable markers.

6. Method according to claim 1, characterized in that the sequence of interest comprises at least one expression cassette comprising a nucleic acid that confers resistance to a selection agent and wherein the selection agent is used to select the genetically altered plant cells and tissues in step (c) and / or after step (c).

7. Method according to claim 1, characterized in that step (b) is achieved by means of Agrobacterium transformation, microprojectile bombardment, nanoparticle administration, viral administration or a combination thereof.

8. Method according to claim 1, characterized in that the polynucleotide comprises a recombinase sequence under the control of an inducible promoter and at least one polynucleotide sequence of interest, wherein both sequences are flanked by recombination sites, optionally wherein the inducible promoter is selected from the group consisting of a stress-inducible promoter and a chemical-inducible promoter.

9. Method according to claim 8, characterized in that it further comprises excising the polynucleotide sequences flanked by the recombination sites by inducing site-specific recombinase expression under culture conditions in steps (c) and (d); and / or that it further comprises culturing the cells after step (c) in a culture medium comprising abscisic acid (ABA); optionally wherein the ABA is present at a concentration of 20 μM to 150 μM, preferably at a concentration of 50 μM to 100 pM; optionally wherein the culture medium further comprises polyethylene glycol (PEG) in a range of 20 μM to 100 μM.

10. A method according to claim 1, characterized in that the sequence of interest is selected from the group consisting of CRISPR machinery genes, selectable markers, herbicide genes, silencer genes, inactivated nuclease genes, transcription factor genes, growth or development genes, morphogens, reporter genes, insecticide genes, DNA templates for homologous recombination, suppressor genes, agronomic trait genes, and a combination thereof.

11. Method according to claim 1, characterized in that: (i) step (c) is carried out in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days; (ii) step (c) is carried out at 25 °C to 30 °C, more preferably at 25 °C to 29 °C, particularly at 27 °C; Petition 870250085581, dated 22 / 09 / 2025, p. 70 / 89 4 / 7 (iii) step (d) is carried out at 35 °C to 40 °C, more preferably at 35 °C to 37 °C, particularly at 35 °C; and / or (iv) where step (d) is carried out in 2 to 3 days, more preferably 3 days.

12. Method according to claim 1, characterized in that the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disc, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts and any cells or tissues receptive to the introduction and absorption of a sequence.

13. Method according to claim 1, characterized in that the transformation efficiency is increased by at least 5% compared with a conventional cell transformation method or in that the transformation efficiency is increased by at least 10% - 30% compared with a conventional cell transformation method.

14. Method according to claim 1, characterized in that the plant cell or plant tissue is derived from a sugarcane plant, seedling, plant part or plant tissue.

15. Plant, plant part, seed or parent plant characterized in that they comprise a sequence or trait introduced by means of the method as defined in any one of claims 1 to 14.

16. Method for increasing the regeneration rate of plant cells or plant tissues characterized in that it comprises: a) cultivating a plant cell or plant tissue in vitro; Petition 870250085581, dated 22 / 09 / 2025, page 71 / 89 5 / 7 b) cultivating the cell or tissue from step (a) in a culture medium for at least 15 to 60 days at 20 °C to 35 °C; c) performing heat shock treatment on the cell or tissue from step (b) for at least 1-3 days at 30 °C to 45 °C in a culture medium; d) regenerating the cell or tissue from step (c).

17. Method, according to claim 16, characterized in that the regeneration efficiency is increased by at least 5% compared with a conventional method of cell and tissue regeneration or in that the regeneration efficiency is increased by at least 50% - 100% compared with a conventional method of cell and tissue regeneration.

18. Method according to any one of claims 16 or 17, characterized in that in step (b) the cell or tissue is maintained in the culture medium without manipulation or subculture and / or that it further comprises repeating steps (b) and (c) a second time to add a second selection step.

19. Method according to any one of claims 16 to 18, characterized in that step (b) further comprises at least one of the following additional steps: i) preparing Agrobacterium strains comprising the sequence of interest; ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension from (i); iii) co-culturing the plant cell or tissue in a co-culture medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or iv) culturing the transformed plant cell and tissue in a resting medium containing an agent (e.g., antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark. Petition 870250085581, dated 22 / 09 / 2025, pp. 72 / 89 6 / 7 20. Method according to claim 16, characterized in that it further comprises step (e) allowing the elongation of the regenerated seedlings from step (d).

21. Method according to claim 16, characterized in that it further comprises cultivating the cells after step (b) in a culture medium comprising abscisic acid (ABA); optionally wherein the abscisic acid (ABA) is present in a concentration of 20 μM to 150 μM, preferably in a concentration of 50 pM to 100 μM; optionally wherein the composition further comprises polyethylene glycol (PEG) in a range of 20 μM to 100 μM.

22. Method according to claim 16, characterized in that: (i) step (b) is carried out in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days; (ii) step (b) is carried out at 25 °C to 30 °C, more preferably at 25 °C to 29 °C, particularly at 27 °C; (iii) step (c) is carried out at 35 °C to 40 °C, more preferably at 35 °C to 37 °C, particularly at 35 °C; and / or (iv) step (c) is carried out in 2 to 3 days, more preferably 3 days.

23. Method according to claim 16, characterized in that the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disc, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts and any cells or tissues receptive to the introduction and absorption of a sequence. Petition 870250085581, dated 22 / 09 / 2025, p. 73 / 89 7 / 7 24. Method according to claim 16, characterized in that the plant cell or plant tissue is derived from a sugarcane plant, seedling, plant part or plant tissue.

25. Plant, part of plant, seed or parent plant characterized in that they comprise a sequence or trace introduced by means of the method as defined in any of claims 16 to 24. Petition 870250085581, dated 22 / 09 / 2025, pp. 74 / 89

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