Biocomplexes for genetic modification in plants

Description

1 / 50 BIOCOMPLEXES FOR IN PLANT GENETIC MODIFICATION Field of invention [1] The present invention relates to in planta transformation methods in which exogenous DNA is administered to a floral structure in order to produce seeds exhibiting transient expression or stable integration of the exogenous DNA into the genome of the seeds. Methods for producing a genetically modified (GM) plant based on the in planta transformation methods according to the present invention are also provided. In addition, a GM plant produced according to the transformation and / or production methods according to the present invention or descended from it is provided. Fundamentals of the invention [2] In recent decades, many techniques have been developed for the genetic transformation of plants. All of these methods have the ultimate goal of obtaining a transgenic plant containing, in all or part of its cells, a foreign nucleic acid comprising a gene or a characteristic of interest, in the case of a gene, a transgene, but with the increase in new genome engineering techniques, for example, CRISPR / Cas technology (see, for example, US 8,697,359 B1, EP 2,800,811 A1 or WO 2013 / 142578 A1), the insertion of ribonucleic acids or sequences that encode them and amino acid sequences is also becoming more and more relevant.In the case of nucleic acid molecules encoding a transgene and, optionally, additional elements, stable integration into a plant genome, particularly the nuclear genome, but also into the genome of, for example, plant plastids, is one of the main objectives of the previous transformation process to generate a stable knockin'1, but also a stable heritable knockout of a gene or region of interest. For other applications, for example, when designing experiments using genome editing tools such as CRISPR / Cas or CRISPR / Cpf1, the transient expression of the construct of interest to be transformed or transfected may be of interest to temporarily influence the genetic material of a plant, i.e., the genome, or the transcriptome, i.e., all the RNA material, in a controlled manner. [3] The CRISPR system means that a suitable individual small non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease such Petition 870250016683, dated 28 / 02 / 2025, p. 10 / 73 2 / 50 A Cpf1 nuclease (Zetsche et al., Cpf1 is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, pp. 1-13, October 2015) can produce a specific double-strand DNA break in a natural environment. In artificial CRISPR systems, a modified CRISPR nuclease, modified to act as a nickase endonuclease or lacking any nuclease function, can be used in combination with at least one artificial guide RNA or gRNA combining the function of a crRNA and / or a tracrRNA (Makarova et al., Nature Rev. Microbiol. 13, “An updated evolutionary classification of CRISPR-Cas systems”, 722736 (2015)). The CRISPR / Cas-mediated immune response in natural systems requires CRISPR-RNA (crRNA), where the maturation of this guide RNA, which controls the specific activation of CRISPR nuclease, varies significantly among the various CRISPR systems that have been characterized in this way.First, the invading DNA, also known as a spacer, is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. Type II CRISPR systems code for a Cas9 nuclease as the key enzyme for the interference step, which system contains both a crRNA and a trans-activating RNA (tracrRNA) as the guide motif. They hybridize and form double-stranded RNA (ds) regions that are recognized by RNase III and can be cleaved to form mature crRNAs. These, in turn, associate with the Cas molecule to direct the nuclease specifically to the target nucleic acid region. Recombinant gRNA molecules can comprise both the variable DNA recognition region and the Cas interaction region and, therefore, can be specifically designed regardless of the specific target nucleic acid and the desired Cas nuclease.As an additional safety mechanism, PAMs (protospacer adjacent motifs) must be present in the target nucleic acid region; these are DNA sequences that result directly from the DNA recognized by the Cas9 / RNA complex. The PAM sequence for Cas9 of Streptococcus pyogenes has been described as NGG or NAG (standard IUPAC nucleotide code) (Jinek et al., A programmable double RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 2012 337: 816-821). The PAM sequence for Cas9 of Staphylococcus aureus is NNGRRT or NNGRR(N). Other variable CRISPR / Cas9 systems are known. Thus, a Neisseria meningitidis Cas9 cleaves at the PAM sequence NNNNGATT. A Streptococcus thermophilus Cas9 cleaves at the PAM sequence NNAGAAW. Recently, another PAM motif, NNNNRYAC, was described for a Campylobacter CRISPR system (WO 2016 / 021973 A1). For Cpf1 nucleases, the Cpf1-crRNA complex has been shown to efficiently cleaves the... Petition 870250016683, dated 28 / 02 / 2025, page 11 / 73 3 / 50 Target DNA is preceded by a short T-rich PAM, in contrast to the G-rich PAMs commonly recognized by Cas9 systems (Zetsche et al., Cpf1 is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, pp. 113, October 2015). [4] Furthermore, using modified Cas polypeptides, specific single-strand breaks can be achieved. The combined use of Cas nickase endonucleases with various recombinant gRNAs can also induce highly specific double-strand DNA breaks via double DNA cleavage. Using two gRNAs, moreover, the specificity of DNA binding and therefore DNA cleavage can be optimized. [5] Currently, there are a variety of plant transformation methods to introduce genetic material in the form of a genetic construct into a plant cell of interest. A preferred technique is transformation with Agrobacterium spp., which has been used for decades for a variety of different plant materials. Agrobacterium transformation, however, is much better established for dicotyledonous plants than for monocotyledonous plants, the latter comprising economically important plants, for example, in the Poaceae / Graminaceae family, including inter alia plants such as maize, wheat, barley, rye, sorghum or sugarcane.Over the past few years, some progress has been made in other methods of plant transformation by choosing direct delivery techniques for the introduction of genetic material into a plant cell, for example, through the choice of direct delivery techniques ranging from the treatment of protoplasts with polyethylene glycol (PEG) (Potrykus et al, Mol. Gen. Genet. 199, “Direct gene transfer to cells of a graminaceous monocot” 183-188, (1985)), procedures such as electroporation (D'Halluin et al, Plant Cell 4, “Transgenic maize plants by tissue electroporation”, 12:1495-1505 (1992)), microinjection (Neuhaus et al, Theor. Appl. Genet. 75, “Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids”, 30-36 (1987)), silicon carbide fiber whisker technology (Kaeppler et al, Theor. Appl. Genet.84, “Silicon carbide fiber-mediated stable transformation of plant cells”, 560-566 (1992)), viral vector-mediated approaches (Gelvin, Nature Biotechnology 23, Viral-mediated plant transformation gets a boost, 684-685 (2005)) and particle bombardment (see, for example, Sood et al., 2011, Biologia Plantarum, 55, 1-15). For all the above methods, it is still mandatory to have explanted plant material, including suspension cells or calluses. Petition 870250016683, dated 28 / 02 / 2025, page 12 / 73 4 / 50 embryogenic, scutellar tissue, calluses of different types, including type 1 and type 2, type 3 organogenic calluses, protoplasts, immature embryos or green tissue that is then treated in vitro, that is, ex planta and not within the living plant. [6] An exemplary transformation process for maize is Agrobacterium-mediated transformation of immature embryos of defined genotypes through somatic embryogenesis. The process of regenerating a plant from the transformed material in vitro is, however, time-consuming, expensive, and the efficiency and parameters to be followed are strongly genotype-dependent. Furthermore, only some genotypes are suitable for genetic transformation, requiring backcrossing of the generated transgenic material with elite varieties, which is time-consuming and expensive. [7] US patent 6,603,061 B1 describes a method for transforming a maize plant cell or plant tissue using an Agrobacterium-mediated process by inoculating a transformable plant tissue or cell from a maize plant with Agrobacterium containing at least one genetic component capable of being transferred to the plant tissue or cell in an inoculation medium containing an effective amount of at least one antibiotic that inhibits or suppresses Agrobacterium growth. There are several publications in the scientific literature on Agrobacterium-mediated transformation of flowers, such as floral dip methodologies, routinely applied to Arabidopsis, but also to crops such as wheat (Clough and Bent, Plant J. 16, “Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana”, 735-743 (1998); Zale et al, Plant Cell Rep.28 “Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens”, 6:903-913 (2009)). These approaches, however, intrinsically require the complex regeneration of the transformed plant tissue or cell to obtain a genetically modified or transgenic plant. [8] Pace and Dupuis (Gene transfer to maize male reproductive structure by particle bombardment, 1993, Plant Cell reports, 12: 607-611.) describe a method for transforming maize tassel primordia using particle bombardment for transient expression of foreign genes. The method, according to Pace and Dupuis, relies on in vitro bombardment and all experiments are performed with explanted cells and no reproductive plant or plant material was or can be obtained from this approach. Pareddy and Greyson (“In vitro culture of immature tassels of an inbred field variety of Zea mays, cv. Oh43”, 1985, Plant Cell, Tissue Petition 870250016683, dated 28 / 02 / 2025, p. 13 / 73 5 / 50 Organ Cult. 5:119-128) published a method in which maize tassels (1 month old) of the OH43 genotype were dissected and bombarded in tassel maturation media. The bombardment was performed at 1350 psi with 1.6 μm gold particles, and the tassels were bombarded 4 times. The tassels cultivated in vitro matured but produced a low quantity of anthers compared to greenhouse plants. Transient expression in the tassels was evaluated. Most were detected in the outer glumes, only a few in the stamen primordia. After 1 month of in vitro culture, anthers were produced and tested for gus activity. Only 0.5% of all anthers showed gus activity, mainly in the anther vascular tissue. No pollen analyses were performed, and no pollinations were carried out. Therefore, it is still necessary to improve this method to obtain a sufficient quantity of transformed cells with a high transformation rate. [9] In wheat, a bombardment technology called micro-targeting was used to transform isolated immature wheat spikes (Leduc et al, Sex. Plant Reprod. 5 “Gene transfer to inflorescence and flower meristems using ballistic micro-targeting”, 119128 (1994)). These spikes were grown in an osmotic medium to increase transient expression. Even so, about 80% of the particles were directed to the L1 cell layer (the outer layer that will produce all the epidermal tissue of the spike) and approximately 20% to L2 cells (which will produce reproductive organs). The authors obtained multicellular sectors with transgenic expression 12 days after bombardment. These sectors were found in primordia of vegetative and reproductive organs such as anthers. They describe this methodology as a possible direct transformation of sporogenic tissues.However, there is no follow-up technology, especially no in-plant monitoring for other important crop plants.

[10] Bombarding plant cells with a nucleic acid of interest represents another process for transformation and has been known since the late 1980s (Taylor and Fauquet, DNA Cell Biol. 21, “Microparticle bombardment as a tool in plant science and agricultural biotechnology”, 12:963-977 (2002)) to manipulate the genome of (at that time) recalcitrant plants for transformation via Agrobacterium, including, inter alia, cereals (Klein et al, Nature 327, “High-speed microprojectiles for nucleic acid delivery into living cells”, 70-73 (1987); Taylor and Fauquet, DNA Cell Biol. 21, “Microparticle bombardment as a tool in plant science and agricultural biotechnology”, 12:963-977 (2002)). Transformation via bombardment Petition 870250016683, dated 28 / 02 / 2025, p. 14 / 73 6 / 50 of particles uses a metal microprojectile coated with the gene of interest, which is then fired into target cells using equipment known as biolistics (Sandford et al, Trends Biotechnol. 6, “The biolistic process”, 12:299-302 (1988)) at a speed high enough (~1500 km / h) to penetrate the cell wall of a target tissue, but not extreme enough to cause cell death. The precipitated nucleic acid or nucleic acid construct in at least one microprojectile is released into the cell after bombardment and integrated into the genome. Acceleration of microprojectiles is achieved by a high-voltage electrical discharge or compressed gas (helium). This technique has been progressively developed, for example, to insert more than one gene of interest into target cells (Chen et al, Theor. Appl. Genet.97, “Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolytic bombardment and the bar gene as a selection marker”, 1296-1306 (1998). Furthermore, several physical parameters correlated with biolytic equipment, such as pressure, macro and microcarrier displacement distance, and vacuum, must be optimized for successful transformation. Another crucial parameter is the target cell or tissue itself, as this target site must be easily accessible, as well as the nucleic acid construct of interest to be introduced.

[11] Although transformation methods based on biological approaches, such as Agrobacterium transformation or viral vector-mediated plant transformation, and methods based on physical delivery methods, such as microinjection or particle bombardment, have evolved as prominent techniques for introducing genetic material into a plant tissue or cell of interest in recent years, there are still several problems associated with this, hindering the broad approach for both transient and stable introduction of a construct of interest into any type of plant to be transformed.

[12] Helenius et al. (Gene delivery into intact plants using Helios™ Gene Gun, Plant Molecular Biology Reporter, 2000, 18 (3):287-288) describes particle bombardment as a physical method for transient transformation of gene constructs into intact plant tissue of model plants such as Arabidopsis and tobacco. However, there is no example of achieving stable transformation of the genetic construct or any guidance on how to use the technology for large-scale cultivation in plant organs other than leaves. Furthermore, the paper does not cite a method for directly getting a genetically modified seed into a modified plant, avoiding the need for cultivation. Petition 870250016683, dated 28 / 02 / 2025, page 15 / 73 7 / 50 in vitro or additional crossover steps, since the target tissue is not a meristematic tissue.

[13] In this way, this would represent a preferential targeting strategy to specifically transform the meristematic tissue of a plant of interest, since a targeted genetic manipulation performed on at least one meristematic cell provides the advantage of inheriting said manipulation to the progeny of cells not yet fully differentiated. For this purpose, however, a target meristematic structure must be adequately exposed to avoid destruction of the plant organism and / or the target structure of interest.

[14] In the case of cotton (Gossypium hirsutum), the most important source of natural fiber for the textile industry, approximately 78% of the global cultivated area is planted with genetically modified cultivars (ISAAA, International Service for the Acquisition of Agribiotech Applications, Brief 55-2019 Available at: isaaa.org / resources / publication / briefs / 55 / executivesummary / default.asp. The first reports of stable genetic transformation of cotton use protocols mediated by Agrobacterium tumefaciens and somatic embryogenesis tissue culture for the regeneration of transgenic plants (Firoozabady et al. 1987, “Transformation of cotton (Gossypium hirsutum) by Agrobacterium tumefaciens and regeneration of transgenic plants.” Plant Molecular Biology 10:105-115.; Umbeck et al. 1987, “Genetically modified cotton (Gossypium hirsutum) plants. Nature Biotechnology 5:263-266.”). Since then, a growing number of improved protocols with variations have been developed.The advantages of A. tumefaciens-mediated protocols are that they can be successfully used in a wide range of hosts and are well-established protocols for many plant species.

[15] Another well-known biotechnological method for obtaining genetically modified plants is the biolistic system, which involves the direct introduction of DNA molecules into the plant genome through a particle accelerator, which is used to bombard the target tissue with DNA coupled to metallic particles (Rech et al., 2008, “High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants.” Nature Protocols 3(3):410-418). This method offers interesting advantages in terms of plasticity and potential application in a wide range of plant species and tissues, but a high rate of multiple insertions of exogenous DNA fragments into the plant genome has been widely reported. Petition 870250016683, dated 28 / 02 / 2025, p. 16 / 73 8 / 50 reported, which can lead to transgene silencing over generations. (Keshavareddy et al., 2018, “Methods of plant transformation - A review”. International Journal of Current Microbiology and Applied Sciences 7(7):2656-2668”, Guo et al., 2018, “Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection.” Journal of Integrative Agriculture 17(10):2196-2203”; Yan et al., 2022, “Nanotechnology strategies for plant genetic engineering”. Advanced Materials 34(7):126). Most cotton transformation protocols developed to date rely on tissue culture for plant regeneration, which is a time-consuming and laborious process. In vitro cotton regeneration is the longest step in transformation protocols and is highly dependent on the plant genotype (Sakhanokho et al., 1998 “Tissue culture potential of diverse diploid and tetraploid cotton genotypes).Proceedings of the Beltwide Cotton Conference 1:590-593.”). Although plant regeneration is not a limitation in the genetic transformation of model cotton cultivars, such as Coker, many commercial cultivars exhibit high recalcitrance to existing regeneration protocols (Chakravarthy et al., 2014 “Current status of genetic engineering in cotton (Gossypium hirsutum L): An assessment.” Critical Reviews in Biotechnology 34(2):144-160.; Guo et al., 2018 “Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection.” Journal of Integrative Agriculture 17(10):2196-2203). This not only increases the time required to produce genetically modified plants but also significantly reduces the genetic base available for transformation. Furthermore, long in vitro cultivation periods cause a high incidence of abnormal cell formation and inconsistencies in the radicle development (Sakhanokho et al., 2001 “Induction of highly embryogenic calluses and plant regeneration in upland (Gossypium hirsutum L.) and pima (Gossypium barbadense L.).” Crop Science 41(4):1235-1240). Several improvements have been made to biolistic and Agrobacterium-mediated transformation using a wide variety of explants, including calluses induced from somatic or embryogenic tissues, shoot apex, embryogenic axis, among others. However, the average time to regenerate a genetically modified plant is still long, approximately seven months, with about 3% transformation efficiency (Zhu et al., 2006 “Transgene structures suggest that multiple mechanisms are involved in TDNA integration in plants.” Plant Science 171:308-322; Rech et al., 2008 “Highefficiency transformation by biolistics of common resin plants, common beans and cotton.” Nature Protocols 3(3):410-418; Liu et al., 2011 “Metallic engineering of. Petition 870250016683, dated 28 / 02 / 2025, p. 17 / 73 9 / 50 Artemisinin biosynthesis in Artemisia annua L.” Plant Cell Reports 30:689-694.; Ahmed et al., 2020 “An Agrobacteria-mediated genetic transformation using an embryonic axis in cotton (Gossypium hirsutum).” Russian Journal of Plant Physiology 67(3):581-587). Some protocols stand out for their combination of techniques, such as the one that uses microinjection and sonication to improve A. tumefaciens-mediated transformation, with transformation efficiency of up to 20% (Gurusaravanan et al., 2020 “An improved Agrobacterium-mediated transformation method for cotton (Gossypium hirsutum L. 'KC3') assisted by microinjection and sonication.” In Vitro Cellular & Developmental Biology 56: 111-121). Other methods combine A. tumefaciens-mediated transformation and biolistics with speed and simplicity, consisting of a transformation, co-culture, and regeneration workflow in a single step that does not last more than 3 months (Ribeiro et al., 2021 “Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods.” Planta 254(2):20). However, all these protocols require expensive reagents, specialized laboratory infrastructure, and well-trained technical skills.

[16] Independent tissue culture methods for cotton transformation have been developed and improved, most of them based on gamete transformation. The pollen tube pathway-mediated method uses the channels naturally formed by the style tissues after pollen germination, the pollen tube, to directly deliver the transgene into the plant ovary (Wang et al., 2019 “Transgenic cotton: Methods and protocols”, Methods in Molecular Biology, vol. 1902, Chapter 6, pp. 67-73). It was initially developed for cotton transformation and later used to transform other agricultural species (Zhou et al., 1983 “Introduction of exogenous DNA into cotton embryos.” Methods in Enzymology 101:433-481; Huang et al., 1999 “Introduction of exogenous DNA into cotton via pollen-tube pathway with GFP as reporter.” Chinese Science Bulletin 44(8)698-701).The pollen tube-mediated method can be performed by microinjecting exogenous DNA directly into the pollen tube or by inoculating A. tumefaciens cultures carrying a binary vector onto the stigmatic surface (TianZi et al., 2010 “Pistil drip following pollination: a simple in planta Agrobacterium-mediated transformation in cotton.” Biotechnology Letters 32(4):547-555; Bibi et al., 2013 “An efficient and highly reproducible approach for the selection of upland transgenic cotton produced by pollen tube pathway method.” Australian Journal of Crop Science 7(11):1714-1722). It is a genotype-independent transformation method that does not require expensive instruments or a large team to perform. Petition 870250016683, dated 28 / 02 / 2025, page 18 / 73 10 / 50 (Wang et al., 2019 “Transgenic cotton: Methods and protocols”, Methods in Molecular Biology, vol. 1902, Chapter 6, pp. 67-73). However, low rates of genetic transformation have been reported, ranging from 0.5% to 1% (Duncan, 2010 “Cotton transformation. In: Widholm JM, Lorz H, Nagata T (Eds), Biotechnology in agriculture and forestry.” Springer-Verlag Berlin Heidelberg 65:65-77).

[17] Particle-mediated gene delivery, combined with a plant transformation method, can be applied to overcome common limitations such as the cell wall barrier and nucleic acid degradation (Lv et al., 2020 “Nanoparticle-mediated gene transformation strategies for plant genetic engineering.” The Plant Journal 104:880-891). In this system, a wide range of nanomaterials can be used as DNA carriers, such as carbon dots and nanotubes, magnetic iron oxides, clay nanosheets, and organic polymers, including chitosan (polyglucosamine (1-4)-2-amino-β-D-glucose) and polyethyleneimine hydrochloride (PEI) (Yan et al., 2022 “Nanotechnology strategies for plant genetic engineering.” Advanced Materials 34(7):1-26; Vasquez et al., 2023 “Simultaneous silencing of juvenile hormone metabolism genes through RNAi interrupts metamorphosis in the cotton boll weevil.” Frontiers in Molecular Biosciences 10:1073721).Chitosan is widely used in nanotechnology approaches due to its biocompatibility and non-toxicity to plants, humans, and the environment. It is obtained by the deacetylation of chitin, an abundant carbohydrate present in the exoskeleton of arthropods (Mohammed et al., 2017 “An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics 9(4):53”; Malerba and Cerana, 2019 “Recent applications of chitin- and chitosan-based polymers in plants.” Polymers 11:839). Meanwhile, PEI is a cationic polymer known to possess cytotoxic properties (Kafil et al., 2011 “Cytotoxic impacts of linear and branched polyethyleneimine nanostructures in A431 cells.” Bioimpacts 1(1):23-30). It facilitates the integration of DNA in fertilization and improves the efficiency of plant transformation (Firoozi et al., 2018 “Enhancement of the transfection efficiency of DNA into Crocus sativus L. cells via PEI nanoparticles.(Journal of Integrative Agriculture 17(8):1768-1778). The first study of plant transformation mediated by nanoparticles used mesoporous silica nanoparticles to deliver DNA to cells (Torney et al., 2007 “Mesoporous silica nanoparticles deliver DNA and chemicals into plants.” Nature Nanotechnology 2:295300). The authors induced transient expression of a marker gene in tobacco protoplasts and also in intact leaf cells using a system of... Petition 870250016683, dated 28 / 02 / 2025, page 19 / 73 11 / 50 gene gun. A nanoparticle-mediated method called pollen magnetofection has been developed for the stable transformation of cotton, making intelligent use of pollen anatomy, which has openings where the pollen wall is reduced or absent. In this method, DNA coupled to a magnetic metallic polymer is delivered to pollen grains in the presence of a magnetic field. Magnetofected pollen grains are then used for the artificial pollination of cotton flowers to produce transgenic seeds directly, without the need for in vitro culture and plant regeneration steps (Zhao et al., 2017 “Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers.” Nature Plants 3:956-964). Although it is a simple and cell culture-free methodology, the pollen magnetofection system has shown reduced transformation efficiencies, ranging from 0.18 to 1%, and has not yet been reproduced in other plant species (Vejlupkova et al., 2020 “No evidence for transient transformation via pollen tube magnetofection in several monocot species.” Nature Plants 6(11):1323-1324).

[18] Therefore, there remains a need for a plant transformation methodology with a high rate of efficiency and reduced cost and time compared with the methodologies available in the state of the art. SUMMARY OF THE INVENTION

[19] The present invention demonstrates that, surprisingly, it was possible to obtain high plant transformation efficiency rates by administering positively charged particles containing exogenous DNA in planta, into a floral structure that is a pollen tube passage, wherein said administration is carried out at a floral development stage prior to the formation of the pollen tube in said floral structure. This method eliminates the need for in vitro tissue culture, reducing the costs associated with materials and personnel training, as well as the method execution time relative to methods involving in vitro tissue culture. Furthermore, the method of the present invention results in a higher rate of genetic transformation than other in planta transformation methods.

[20] The main object of the present invention is, therefore, to provide methods for the in planta transformation of plants that are suitable for both the stable and transient integration / introduction of a genetic construct of interest. In addition, the present invention provides methods for producing a genetically modified plant based on an in planta approach to transform a plant. Petition 870250016683, dated 28 / 02 / 2025, page 20 / 73 12 / 50 Finally, it was an object to obtain a plant or plant progeny derived from plants produced by the methods according to the present invention, or plant cells or plant material, genome or its derivatives, or to provide and use a plant transformed by the methods according to the present invention for the production of a genetically modified plant material or plant.

[21] The main objective was achieved by providing a method by which gametes are transformed with exogenous DNA, in theory, when the pollen tube grows through a floral structure in which positively charged particles containing the exogenous DNA have been administered, so that the seeds produced from said floral bud contain the genetic modification of interest.

[22] In a first embodiment, a method is provided for the in planta transformation of plants with exogenous DNA comprising administering positively charged particles complexed with the exogenous DNA into a floral structure that is a passage for the pollen tube, wherein said administration is carried out at a stage of floral development prior to the formation of the pollen tube in said floral structure.

[23] Em uma concretização preferential, a planta é uma planta de cultivation. In a preferred embodiment, the crop plant is a plant of a genus selected from the group consisting of Triticum spp., Oryza spp., Zea spp., Hordeum spp., Avena spp., Secale spp., Malus spp., Pyrus spp., Prunus spp., Citrus spp., Vaccinium spp., Rubus spp., Fragaria spp., Citrullus spp., Solanum spp., Cucumis spp., Cucurbita spp., Capsicum spp., Solanum spp., Phaseolus spp., Abelmoschus spp., Helianthus spp., Brassica spp., Glycine spp., Olea spp., Sesamum spp., Carthamus spp., Prunus spp., Carya spp., Corylus spp., Anacardium spp., Juglans spp., Pisum spp., Lens spp., Cicer spp., Ipomoea spp., Solanum spp., Vanilla spp., Crocus spp., Gossypium spp., Coffea spp., Theobroma spp., Saccharum spp., and Linum species.In a more preferred embodiment, a cultivated plant is a plant selected from the group consisting of Wheat (Triticum aestivum), Rice (Oryza sativa), Maize (Zea mays), Barley (Hordeum vulgare), Oats (Avena sativa), Rye (Secale cereale), Apple (Malus domestica), Pear (Pyrus spp.), Peach (Prunus persica), Plum (Prunus domestica), Cherry (Prunus avium and Prunus cerasus), Orange (Citrus sinensis), Lemon (Citrus limon), Blueberry (Vaccinium spp), Raspberry (Rubus idaeus), Strawberry (Fragaria ananassa), Watermelon (Citrullus lanatus), Tomato (Solanum lycopersicum), Cucumber (Cucumis sativa). Petition 870250016683, dated 28 / 02 / 2025, p. 21 / 73 13 / 50 Pumpkin (Cucurbita spp.), Pepper (Capsicum spp.), Eggplant (Solanum melongena), Green bean (Phaseolus vulgaris), Okra (Abelmoschus esculentus), Sunflower (Helianthus annuus), Rapeseed / Canola (Brassica napus), Soybean (Glycine max), Olive (Olea europaea), Sesame (Sesamum indicum), Safflower (Carthamus tinctorius), Almond (Prunus dulcis), Pecan (Carya illinoinensis), Hazelnut (Corylus avellana), Cashew (Anacardium occidentale), Walnut (Juglans regia), Pea (Pisum sativum), Lentil (Lens culinaris), Chickpea (Cicer arietinum), Sweet potato (Ipomoea batatas), Potato (Solanum tuberosum), Vanilla (Vanilla planifolia), Saffron (Crocus sativus), Cotton (Gossypium spp), Coffee (Coffea arabica and Coffea canephora), Cocoa (Theobroma cacao), Sugarcane (Saccharum spp.) and Flax (Linum usitatissimum). More preferably, the cultivated plant is a cotton plant (Gossypium hirsutum).

[24] In a preferred embodiment, the floral structure that is the passage of the pollen tube is an unpollinated floral bud or a pre-anthesis bud. Preferably, the floral structure is selected from the group consisting of the androecium and gynoecium of an unpollinated floral bud or a pre-anthesis bud.

[25] In a preferred embodiment, the positively charged particles containing the exogenous DNA are in the form of a solution of exogenous DNA complexed with a cationic polymer. Preferably, the cationic polymer is selected from the group consisting of chitosan and polyethyleneimine hydrochloride (PEI).

[26] In a preferred embodiment, positively charged particles complexed with exogenous DNA are administered by injection with a syringe or microsyringe.

[27] In a preferred embodiment, the solution containing exogenous DNA comprises from about 1 ng to about 300 ng of exogenous DNA, preferably from about 75 ng to about 150 ng.

[28] In a preferred embodiment, exogenous DNA encodes one or more selected from the group consisting of a transgene of interest, an RNAi and a genome editing protein.

[29] The present invention further provides a method for producing a genetically modified plant comprising transforming a plant by the method according to the present invention with exogenous DNA. Preferably, the DNA Petition 870250016683, dated 28 / 02 / 2025, page 22 / 73 14 / 50 exogenous encodes one or more selected components from the group, consisting of a transgene of interest, an RNAi, and a genome-editing protein.

[30] The present invention further provides a genetically modified plant produced by the method as defined in any of the preceding claims or descended therefrom, seeds, plant parts as well as the genome of said plant. In a preferred embodiment, the genetically modified plant is a transgenic plant.

[31] Additional embodiments and aspects of the present invention may be derived from the detailed description below, the drawings and also the attached set of claims. BRIEF DESCRIPTION OF THE FIGURES

[32] Figure 1 (Fig. 1) shows an evaluation of the cotton flower bud region being microinjected with the biocomplex solution. In this evaluation, a test solution with bromophenol blue dye was used. a) External view of an 8 mm cotton flower bud. b) Longitudinal section of a cotton flower bud. c) Microinjection of the androecium of floral buds, e) Longitudinal section of a floral bud after microinjection of the androecium with bromophenol blue dye. e) Microinjection of the gynoecium of floral buds, f) Longitudinal section of a floral bud after microinjection of the gynoecium with bromophenol blue dye. Scale bar 5 mm.

[33] Figure 2 (Fig. 2) presents the analysis of the size of the biocomplexes by DLS, using formulations obtained from the complexation between DNA and the polymers of chitosan and polyethyleneimine hydrochloride (PEI), and their respective standard deviations.

[34] Figure 3 (Fig. 3) presents a schematic representation of the minimal expression gene cassettes used for genetic transformation of cotton. The binary vector pCAMBIA 3300 was used as a scaffold for DNA construction. The pCAMBIA Cry10A / Bar vector (10,000 bp) is used for the overexpression of the entomotoxic protein CrylOA, regulated by the cotton flower bud-specific FS1 promoter and the t-nos terminator. This vector also contains the bar gene regulated by the CAMV-35S-2x promoter and the t-nos terminator. Petition 870250016683, dated 28 / 02 / 2025, page 23 / 73 15 / 50 B- pCAMBIA uidA / ahas vector (20,000 bp), for the overexpression of the GUS protein, regulated by the CAMV-35S-2x promoter and the t-nos terminator. This vector also contains the ahas gene regulated by the CAMV-35S-2x promoter and the t-nos terminator.

[35] Figure 4 (Fig. 4) shows the result of selecting cotton plants transformed with the pCAMBIA Cry10A / Bar Vector according to the method described in the present invention. GM seeds in the T0 generation were sown and selected by spraying with the herbicide glufosinate ammonium. In the figure, it is possible to observe plants obtained from the treatment with chitosan-based DNA biocomplexes and PEI. After 15 days, the herbicide-resistant transformed plants were selected to proceed to the characterization and generation advancement steps.

[36] Figure 5 (Fig. 5) shows the detection of transgenes in transformed cotton plants using PCR assays. a) Amplification of Cry10Aa gene (257 bp) in T0, T1, and T2 transgenic plants from treatments 6 (DNA + polyethyleneimine hydrochloride) and treatment 10 (DNA + chitosan), visualized by electrophoresis on 1% (w / v) agarose gel. M: DNA molecular weight; WT: non-transgenic wild-type control; NC: negative control; PC: positive control (plasmid). In this evaluation, the presence of electrophoretic bands is indicative of positivity.

[37] Figure 6 (Fig. 6) shows the immunoenzymatic detection and quantification of the Cry10Aa protein by indirect ELISA in floral buds of non-transgenic (NT) and transgenic cotton plants (generations T0, T1, and T2) resulting from the transformation of the pCAMBIA Cry10A / Bar vector using the method described: A) treatment 6 (DNA + polyethyleneimine hydrochloride) and B) treatment 10 (DNA + chitosan).

[38] Figure 7 (Fig. 7) presents the qualitative evaluation of β-glucuronidase (GUS) enzyme activity by means of a histological assay in GM cotton plants obtained by transformation of the pCAMBIA uidA / ahas vector using the method described in treatment 10 (DNA + chitosan), in the present invention. Histochemical staining of β-glucuronidase (GUS) activity in leaf and root tissues of transgenic cotton lines (event 1 to 5, generation T0) overexpressing the uidA / GUS gene compared to wild-type control plants (WT). DEFINITIONS Petition 870250016683, dated 28 / 02 / 2025, page 24 / 73 16 / 50

[39] As used herein, the term in planta or in planta transformation means that the actual transformation process, i.e., the introduction of a genetic construct, according to the present invention, is achieved by transforming a plant, plant cell or plant material that is still connected with the living plant from which it is derived. Optionally, said in planta transformation may be conducted with a plant, plant cell, plant tissue or plant material that has been specifically prepared or exposed to a transformation process while still connected with the living plant. The said term is thus used to distinguish the respective methods from ex planta in vitro transformation methods, in which a plant, plant cell, plant tissue or plant material is first dissected from its natural environment to be subsequently transformed in vitro or ex vivo / ex planta.The terms “transform” or “transformation” or transformed, in this context, therefore encompass any form of biological, chemical or physical introduction of at least one genetic construct or molecule by means of, for example, transformation, transfection, (micro)injection, biolytic bombardment, viral infection and the like.

[40] The term cultivated plant, as used in accordance with the present invention, implies any species of plant intentionally cultivated by humans for agricultural or ornamental purposes. These purposes may include the production of food and beverages, compounds or compositions for therapeutic cosmetic purposes, raw materials, biofuels, for reforestation and for ornamental purposes, among others.

[41] The term culture-free in vitro, as used in accordance with the present invention, therefore implies that no in vitro culture is required for the respective process step. This implies that the respective process occurs directly in planta and not in a plant cell, tissue, organ, or explanted or dissected material. The present invention, in certain embodiments, may comprise in vitro steps, for example, analytical steps to analyze the stable integration or transient introduction of a genetic construct of interest according to the present invention. In vitro steps may also occur to cultivate a specific transformed plant material, if desired, depending on the desired plant, plant cell, plant tissue, organ, or material to be obtained.Whenever the term "free from in vitro culture" is used, the respective transformation method or production process, or a product that can be obtained from it, can occur entirely in planta. Petition 870250016683, dated 28 / 02 / 2025, p. 25 / 73 17 / 50 without requiring an in vitro culture step for propagation of a cell, tissue, or plant material.

[42] A CRISPR system, as used herein, refers to any characterized CRISPR system. Currently, five types (IV) of CRISPR systems have been described (Barrangou et al., 2007, Science, 315(5819):1709-12.; Brouns et al., 2008, Science, 321(5891):960-4; Marraffini and Sontheimer, 2008, Science, 322(5909):1843-5; Makarova et al., Nature Rev. Microbiol., 13, 722-736, 2015), wherein each system comprises a cluster of CRISPR-associated genes (cas or others, e.g., cpf) and a corresponding CRISPR array. These characteristic CRISPR arrays consist of repetitive sequences (direct repeats) in which short fragments of non-repetitive sequences are interspersed (called spacers), where the spacer elements originate from short fragments of exogenous genera (protospacer).CRISPR arrays are subsequently transcribed to generate short CRISPR RNAs (crRNAs), in which the crRNAs direct Cas proteins or other effector nucleases of a CRISPR system to the respective target nucleic acid to be cleaved, while directing operations via Watson-Crick base pairing. Type I and III CRISPR systems use complexes created from Cas proteins and crRNAs to mediate the recognition and subsequent cleavage of target nucleic acid sequences (Wiedenheft et al., 2011, Nature, 477 (7365): 486-9). In contrast, Type II CRISPR systems, in their natural environment, act on the target DNA through an orchestrated action of the RNA-guided Cas9 nuclease in conjunction with two non-coding RNAs, a crRNA and a transactivation RNA (tracrRNA) (Garneau et al. 2010, Sapranauskas et al., 2011, Nucleic Acids Res., 39(21):9275-82; Deltcheva et al., 2011, Nature, 471(7340):602-7). A possible Type IV CRISPR system has also been proposed (Makraova et al, Biol.Direct 6, “Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems, 38 (2011)). Furthermore, a Type V CRISPR system or CRISPR / Cpf1 system has recently been described (Zetsche et al., Cpf1 is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, pp. 113, October 2015; Makarova et al., Nature Rev. Microbiol., 2015, supra). In contrast to the Cas9 nuclease of a Type II CRISPR system, Cpf1 recognizes T-rich PAM sequences and cuts the target DNA in a way that results in so-called sticky ends, while the native Cas9 protein leaves the so-called blunt end to cut. Like Cas9 nucleases, Cpf1 also contains a domain. Petition 870250016683, dated 28 / 02 / 2025, page 26 / 73 18 / 50 of the RuvC type endonuclease, however, the second HND endonuclease domain present in Cas9 does not have Cpf1 (Makarova & Koonin, Methods Mol. Biol. 1311, “Annotation and classification of CRISPR-Cas systems”, 47-75 (2015)).

[43] A CRISPR system for use in accordance with the methods of the present invention is a recombinant CRISPR system using a CRISPR nuclease or a variant, for example, a variant creating a nickase or nuclease-deficient endonuclease, or a catalytically active fragment of a CRISPR nuclease that acts in conjunction with an artificial gRNA or guide RNA.

[44] The terms guide RNA and gRNA are used interchangeably here. The gRNA can combine the function of a crRNA and / or a tracrRNA. Depending on the assay of interest, at least one gRNA, but also more gRNAs, can be used to target at least one CRISPR nuclease or a variant thereof.

[45] The term construct, especially genetic construct or recombinant construct (used interchangeably herein), as used herein, refers to a construct comprising, inter alia, plasmids or plasmid vectors, cosmids, artificial bacterial or yeast chromosomes (YACs and BACs), phagomids, bacterial phage-based vectors, an expression cassette, isolated single-strand or double-strand nucleic acid sequences comprising DNA and RNA sequences or amino acid sequences, viral vectors, including modified viruses, and a combination or mixture thereof, for introduction or transformation, transfection or transduction into a cell or plant, plant cell, tissue, organ or target material in accordance with the present invention.A recombinant construct, according to the present invention, may comprise an effector domain, either in the form of a nucleic acid or an amino acid sequence, wherein an effector domain represents a molecule that can exert an effect on a target cell and includes a transgene, a single-stranded or double-stranded RNA molecule, including a guide RNA, a miRNA or an siRNA, or an amino acid sequence, including, inter alia, an enzyme or a catalytically active fragment thereof, a binding protein, an antibody, a transcription factor, a nuclease, preferably a site-specific nuclease, and the like. Furthermore, the recombinant construct may comprise regulatory sequences and / or localization sequences. The recombinant construct may be integrated into a vector, including a plasmid vector, and / or may be present in isolation from a vector structure, for example. Petition 870250016683, dated 28 / 02 / 2025, page 27 / 73 19 / 50 example, in the form of a polypeptide sequence or as a single-stranded or double-stranded nucleic acid connected to a non-vector. After its introduction, for example, by transformation, the genetic construct may persist extrachromosomally, i.e., not integrated into the genome of the target cell, for example, in the form of single-stranded or double-stranded DNA, double-stranded or single-stranded RNA, or as an amino acid sequence. Alternatively, the genetic construct, or parts thereof, according to the present invention, may be stably integrated into the genome of a target cell, including the nuclear genome or other genetic elements of a target cell, including the genome of plastids such as mitochondria or chloroplasts. The term plasmid vector, as used in this connection, refers to a genetic construct originally obtained from a plasmid.A plasmid generally refers to a circular, autonomously replicating extrachromosomal element in the form of a double-stranded nucleic acid sequence. In the field of genetic engineering, these plasmids are routinely subjected to specific modifications, inserting, for example, genes encoding resistance to an antibiotic or herbicide, a gene encoding a target nucleic acid sequence, a localization sequence, a regulatory sequence, a tagging sequence, a marker gene, including an antibiotic marker or a fluorescent marker, and the like. The structural components of the original plasmid, such as the origin of replication, are maintained. According to certain embodiments of the present invention, the localization sequence may comprise a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrial localization sequence, or a chloroplast localization sequence.The aforementioned localization sequences are available to those skilled in the art of plant biotechnology. A variety of plasmid vectors for use in different target cells of interest are commercially available, and their modification is known to those versed in the respective field.

[46] A fertile plant is a plant that can produce viable and self-fertile male and female gametes. This self-fertile plant can produce offspring without the contribution of any other plant of a gamete and the genetic material contained therein. A plant that is not self-fertile means a plant that cannot produce viable male or female gametes, or both, that are otherwise capable of fertilization. As used herein, a plant Petition 870250016683, dated 28 / 02 / 2025, page 28 / 73 A 20 / 50 male sterile plant is one that does not produce viable male gametes or gametes otherwise capable of fertilization. As used herein, a female sterile plant is one that does not produce viable female gametes or gametes otherwise capable of fertilization. It is recognized that male sterile and female sterile plants can be female fertile and male fertile, respectively. It is further recognized that a male fertile (but female sterile) plant can produce viable progeny when crossed with a female fertile plant and that a female fertile (but male sterile) plant can produce viable progeny when crossed with a male fertile plant.

[47] The term genetically modified or genetically manipulated or genetically engineered is used in a broad sense herein and means any modification of a nucleic acid sequence or an amino acid sequence, of a target cell, tissue, organ or organism, which is carried out by human intervention, directly or indirectly, to influence the endogenous genetic material or the transcriptome or proteinome of a target cell, tissue, organ or organism to intentionally modify it so that it differs from its state found without human intervention. Human intervention may occur in vitro or in vivo / in planta, or both.Other modifications may include, for example, one or more point mutations, for example, for targeted protein engineering or codon optimization, deletions and one or more insertions or deletions of at least one nucleic acid or amino acid molecule (including homologous recombination), modification of an amino acid or nucleic acid sequence, or a combination thereof. The terms should also include a nucleic acid molecule or an amino acid molecule or a host cell or organism, including a plant or plant material that is similar to a comparable sequence, organism or material as occurring in nature, but which has been constructed by at least one intentional manipulation step. In certain embodiments of the present invention, a “genetically modified” plant may be a transgenic plant.

[48] ​​Targeted genetic manipulation, as used herein, is the result of genetic manipulation that is carried out in a targeted manner, that is, at a specific position in a target cell and under specific appropriate circumstances to achieve a desired effect in at least one cell, preferably a plant cell, to be manipulated. Petition 870250016683, dated 28 / 02 / 2025, page 29 / 73 21 / 50

[49] The term “RNA”, as used herein, refers to its meaning as generally accepted in the art. The term generally refers to a molecule comprising at least one ribofuranoside residue, such as a ribonucleotide. The term ribonucleotide means a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. The term refers to a double-stranded RNA, a single-stranded RNA, an isolated RNA, such as a partially purified RNA, an essentially pure RNA, a synthetic RNA, a recombinantly produced RNA, or an altered RNA that differs from a natural RNA. - RNA occurring by the addition, deletion, substitution and / or alteration of one or more nucleotides contained therein. Such alterations may include the addition of non-nucleotide material, for example, at one or more non-terminal nucleotides of an RNA molecule.As such, the nucleotides in the single-stranded RNA molecules of the invention may comprise non-standard nucleotides, such as unnaturally occurring nucleotides, chemically synthesized and / or modified nucleotides, or deoxynucleotides. The altered RNA is referred to as “modified RNA” or “RNA analog”.

[50] As used herein, the phrase RNA interference (also referred to herein as RNAi) refers to its meaning as generally accepted in the art. The term generally refers to the biological process of inhibiting, decreasing, or downregulating gene expression in a cell, and which is mediated by short interfering nucleic acid molecules (e.g., siRNAs, miRNAs, shRNAs), see, for example, Zamore and Haley, 2005, Science 309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526; Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429; Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al., International Publication PCT No. WO 00 / 44895; Zernicka-Goetz et al., International Publication PCT No. WO 01 / 36646; Fire, International Publication PCT No. WO 99 / 32619; Plaetinck et al., International Publication PCT No. WO 00 / 01846; Mello and Fire, International Publication PCT No. WO 01 / 29058; Deschamps-Depaillette, International Publication PCT No.WO 99 / 07409; and Li et al., PCT International Publication No. WO 00 / 44914; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagner and Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA 8:842-850; Reinhart et al., 2002, Gene & Dev. 16:1616-1626; (and Reinhart & Bartel, 2002, Science 297:1831). Furthermore, the term “RNA interference” (or “RNAi”) is intended to be equivalent to other terms used to describe interference. Petition 870250016683, dated 28 / 02 / 2025, page 30 / 73 22 / 50 of sequence-specific RNA, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, the single-stranded RNA molecules of the invention can be used to epigenetically silence genes either at the post-transcriptional or pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by single-stranded RNA molecules of the invention may result from modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science 303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237).In another non-limiting example, the modulation of gene expression by single-stranded RNA molecules of the invention may result from RNA cleavage (both coding and non-coding RNA) via RISC, or via translational inhibition, as is known in the art, or the modulation may result from transcriptional inhibition (see, for example, Janowski et al., 2005, Nature Chemical Biology 1:216-222).

[51] The term transgenic, as used in accordance with the present description, refers to a plant, plant cell, tissue, organ or material comprising a gene or a genetic construct, comprising a transgene, which has been transferred to the plant, plant cell, tissue, organ or material by natural means or by means of genetic engineering techniques from another organism. The term transgene comprises a nucleic acid sequence, including DNA or RNA, or an amino acid sequence, or a combination or mixture thereof. Therefore, the term transgene is not restricted to a sequence commonly identified as a gene, i.e., a protein-coding sequence. It may also refer, for example, to a DNA or RNA sequence that does not code for protein.Therefore, the term transgenic generally implies that the respective amino acid or nucleic acid sequence is not naturally present in the respective target cell, including a plant, plant cell, tissue, organ, or material. The terms transgene or transgenic, as used herein, refer to a nucleic acid sequence or an amino acid sequence that is extracted from the genome of an organism or produced synthetically and then introduced into another organism, stably or transiently, by artificial molecular biology, genetics, and similar techniques.

[52] The term plant or plant cell, as used herein, refers to a plant organism, a plant organ, undifferentiated plant tissues and Petition 870250016683, dated 28 / 02 / 2025, p. 31 / 73 23 / 50 differentiated, plant cells, seeds and derivatives and their progeny. Plant cells include, without limitation, for example, seed cells, mature and immature embryos, meristematic tissues, seedlings, callus tissues in different states of differentiation, leaves, flowers, roots, buds, gametophytes, sporophytes, pollen and microspores, protoplasts, macroalgae and microalgae. The different plant cells can be haploid, diploid or multiploid.

[53] Plant material, as used herein, refers to any material that can be obtained from a plant during any stage of development. Plant material may be obtained in planta or from an in vitro culture of the plant or of a plant organ or tissue thereof. The term also includes plant cells, tissues and organs, as well as developed plant structures, as well as subcellular components such as nucleic acids, polypeptides and all plant chemical substances or metabolites that can be found within a plant cell or compartment and / or that can be produced by the plant, or that can be obtained from an extract of any plant cell, tissue or plant at any stage of development. The term also includes a derivative of plant material, for example, a protoplast, derived from at least one plant cell composed of plant material.The term, therefore, also includes meristematic cells or a meristematic tissue of a plant.

[54] The term transient introduction, as used herein, refers to the transient introduction of at least one recombinant construct, according to the present invention, into a target plant structure, for example, a plant cell, wherein the at least one recombinant construct is introduced under suitable reaction conditions, such that no integration of the at least one recombinant construct into the endogenous nucleic acid material of a target plant structure, i.e., the plant genome as a whole, occurs, such that the at least one recombinant construct is not integrated into the endogenous DNA of the target cell. As a consequence, in the case of transient introduction, the introduced genetic construct will not be inherited by progeny of the target plant structure, for example, the plant cell.At least one genetic or recombinant construct, or the products resulting from its transcription or translation, are present only temporarily, that is, transiently, in constitutive or inducible form, and thus can only be active in the target cell to exert its effect for a limited time. Therefore, at least one genetic or recombinant construct introduced through... Petition 870250016683, dated 28 / 02 / 2025, page 32 / 73 24 / 50 Transient introduction will not be inherited by the progeny of a cell. The effect that a transiently introduced recombinant construct will cause may, however, potentially be inherited by the progeny of the target cell.

[55] The term stable integration or stably integrated, as used herein, refers to the stable integration of at least one genetic or recombinant construct according to the present invention. Integration may occur in the nuclear genome or in any other extranuclear genomic material within a plant compartment of interest, for example, a mitochondrion. At least one stably integrated recombinant construct will therefore be heritable to the progeny of a target cell thus modified.Depending on the nature of the genetic construct, all or part of the genetic construct will be stably integrated, as the genetic construct may include several regions of interest comprising a target region to be stably integrated, in addition to other regions necessary for the transport, delivery, maintenance, and correct localization of the genetic construct within a plant cell, which regions, however, will not themselves be integrated, but serve as cargo for the region of interest to be fully integrated, as is known to those skilled in the art. The stable integration of at least one genetic construct into a transformed gamete according to the present invention will consequently lead to the inheritance of the genomic region to the embryo formed by the fertilization of said gamete.

[56] The term particle bombardment, as used herein, also referred to as biolistic transfection or microparticle-mediated gene transfer, refers to a physical delivery method for transferring a coated nanoparticle or microparticle comprising a nucleic acid or genetic construct of interest into a target tissue or cell. The micro or nanoparticle functions as a projectile and is fired at the target structure of interest under high pressure using a suitable device, usually called a biolistic device. Transformation via particle bombardment uses a metal microprojectile coated with the gene of interest, which is then fired into target cells using equipment known as a biolistic device (Sandford et al., 1987) at a speed high enough (~1500 km / h) to penetrate the cell wall of a target tissue, but not powerful enough to cause cell death.For protoplasts, which have their cell wall completely removed, the conditions are logically different. The precipitated nucleic acid or genetic construct is at least one microprojectile. Petition 870250016683, dated 28 / 02 / 2025, page 33 / 73 25 / 50 is released into the cell after bombardment and integrated into the genome. Microprojectile acceleration is achieved by a high-voltage electrical discharge or compressed gas (helium). Regarding the metal particles used, it is mandatory that they be non-toxic, non-reactive, and have a diameter smaller than that of the target cell. The most commonly used are gold or tungsten. Much publicly available information from manufacturers and providers of biolistics and associated systems regarding general use is available.

[57] The term derivative or descendant or progeny, as used herein in the context of a plant or plant cell or plant material, according to the present application, refers to the offspring of such plant, plant cell or plant material resulting from natural reproductive propagation, including sexual and asexual propagation. It is well known to those skilled in the art that such propagation can lead to the introduction of mutations into the genome of an organism resulting from natural phenomena that result in offspring or progeny that are genomically different from the plant, plant cell or plant material of origin, yet still belong to the same genus / species and possess the same characteristics as the recombinant host cell of origin. Such derivatives or descendants or progenies resulting from natural phenomena during reproduction or regeneration are therefore encompassed by the present invention.

[58] The term vector, as used herein, refers to a means of transport for administering a genetic or recombinant construct, according to the present invention, into a target cell, tissue, organ or plant. A vector, therefore, comprises nucleic acid sequences, optionally comprising sequences such as regulatory sequences or localization sequences for delivery, directly or indirectly, to a target cell of interest or to a target plant structure in the desired cellular compartment of a plant. A vector may also be used to introduce an amino acid sequence into a target cell or target structure. Generally, a vector, as used herein, may be a plasmid vector.The term direct introduction implies that the target cell or desired target structure containing a nucleic acid sequence to be modified, according to the present invention, is directly transformed or transduced or transfected into the specific target cell of interest, where the material delivered with the vector will exert its effect. The term indirect introduction implies that the introduction is achieved into a structure, for example, leaf cells or plant organ or tissue cells, which do not represent the actual target cell or structure of interest to be transformed, but rather these. Petition 870250016683, dated 28 / 02 / 2025, page 34 / 73 26 / 50 structures serve as the basis for systemic dissemination and transfer of the vector, preferably comprising a genetic construct, according to the present invention, to the actual plant target structure, for example, a gamete. In the case of the term vector being used in the context of transfection of amino acid sequences into a target cell, the term vector implies suitable agents for peptide or protein transfection, such as, for example, mixtures of ionic lipids. In the context of nucleic acid delivery, the term vector may imply not only plasmid vectors but also suitable carrier materials that can serve as the basis for the delivery of nucleic acid or amino acid sequences to a target cell of interest, for example, by means of particle bombardment. Said carrier material comprises, inter alia, gold or tungsten particles. Finally,The term vector also implies the use of viral vectors for the introduction of at least one genetic construct, according to the present invention, such as, for example, modified viruses, for example, derived from the following virus strains: Corn Streak Virus (MSV), Barley Streak Mosaic Virus (BSMV), Bromegrass Mosaic Virus (BMV, registration numbers: RNA1: X58456; RNA2: X58457; RNA3: X58458), Corn Streak Virus (MSpV), Corn Streak Virus (MYDV), Corn Yellow Dwarf Virus (MYDV), Corn Dwarf Mosaic Virus (MDMV), positive-strand RNA viruses of the Benyviridae family, for example, Beetroot Necrotic Yellow Vein Virus (registration numbers: RNA1: NC_003514; RNA2: NC_003515; RNA3: NC_003516; RNA4: NC_003517) or of the Bromoviridae family, for example, viruses of the genus Alfalfa mosaic virus (registration numbers: RNA1: NC_001495; RNA2: NC_002024; RNA3: NC_002025) or of the genus Bromovirus, for example, BMV (above),or of the genus Cucumovirus, for example, Cucumber Mosaic Virus (registration numbers: RNA1: NC_002034; RNA2: NC_002035; RNA3: NC_001440), or of the genus Oleavirus, dsDNA viruses of the family Caulimoviridae, particularly of the family Badnavirus or Caulimovirus, for example, different banana streak viruses (for example, registration numbers: NC_007002, NC_015507, NC_006955 or NC_003381) or cauliflower mosaic virus (registration number: NC_001497), or viruses of the genus Cavemovirus, Petuvirus, Rosadnavirus, Solendovirus, Soymovirus or Tungrovirus, positive-strand RNA viruses of the family Closteroviridae, for example, of the genus Ampelovirus, Crinivirus, for example, infectious lettuce chlorosis virus (registration numbers: RNA1: NC_003617; RNA2: NC_003618) or tomato chlorosis virus (registration numbers: RNA1: NC_007340; RNA2: NC_007341), Closterovirus, for example, Virus, Petition 870250016683, dated 28 / 02 / 2025, p. 35 / 73 27 / 50 Beetroot yellow (registration number: NC_001598), or Velarivirus, single-stranded DNA virus (+ / -) of the Geminiviridae family, for example, virus of the Becurtovirus family, Begomovirus, for example, Bean golden yellow mosaic virus, Tobacco curly bud virus, Tobacco leaf mottled curl virus, Tomato chlorotic mottle virus, Tomato leaf dwarf virus, Tomato golden mosaic virus, Tomato curly leaf virus, Tomato mottle virus, Tomato yellow spot virus, or Geminiviridae of the genus Curtovirus, for example, Beetroot curly bud virus, or Geminiviridae of the genus Topocuvirus, Turncurtvirus or Mastrevirus, maize necrosis virus zB (above), Tobacco yellow dwarf virus, Wheat dwarf virus, positive-strand RNA virus of the family Luteoviridae, for example, of the genus Luteovirus, for example, barley yellow dwarf virus-PAV (registration number: NC_004750), or of the genus Polerovirus, for example, potato leafroll virus (registration number: NC_001747), single-stranded DNA viruses of the family Nanoviridae, comprising the genus Nanovirus or Babuvirus, double-stranded RNA viruses of the family Partiviridae, comprising inter alia the families Alphapartitivirus, Betapartitivirus or Deltapartitivirus, viroids of the family Pospiviroidae, positive-strand RNA viruses of the family Potyviridae, for example, comprising the genera Brambyvirus, Bymovirus, Ipomovirus, Macluravirus, Poacevirus, for example, Triticum mosaic virus (registration number: NC_012799), or Potyviridae of the genus Potyvirus, for example, beet mosaic virus (registration number: NC_005304), maize dwarf mosaic virus (registration number: NC_003377), potato Y virus (registration number: NC_001616), or Zea mosaic virus (registration number: NC_018833), or Potyviridae of the genus Tritimovirus, for example, bromegrass necrosis mosaic virus (registration number: NC_003501) or wheat necrosis mosaic virus (registration number: NC_001886), single-stranded RNA viruses of the family Pseudoviridae, for example, of the genus Pseudovirus or Sirevirus, double-stranded RNA viruses of the family Reoviridae, for example,Rice dwarf virus (registration numbers: RNA1: NC_003773; RNA2: NC_003774; RNA3: NC_003772; RNA4: NC_003761; RNA5: NC_003762; RNA6: NC_003763; RNA7: NC_003760; RNA8: NC_003764; RNA9: NC_003765; RNA10: NC_003766; RNA11: NC_003767; RNA12: NC_003768), positive-strand RNA viruses of the family Tombusviridae, for example, comprising the genera Alphanecrovirus, Aureusvirus, Petition 870250016683, dated 28 / 02 / 2025, page 36 / 73 28 / 50 Betanecrovirus, Carmovirus, Dianthovirus, Gallantivirus, Macanavirus, Machlomovirus, Panicovirus, Tombusvirus, Umbravirus or Zeavirus, for example, maize necrotic necrosis virus (registration number: NC_007729), or positive-strand RNA viruses of the family Virgaviridae, for example, viruses of the genus Furovirus, Hordeivirus, for example, barley banded mosaic virus (registration numbers: RNA1: NC_003469; RNA2: NC_003481; RNA3: NC_003478), or of the genus Pecluvirus, Pomovirus, Tobamovirus or Tobravirus, for example, tobacco bell virus (registration numbers: RNA1: NC_003805; RNA2: NC_003811), as well as negative-strain RNA viruses of the order Mononegavirales, particularly of the family Rhabdoviridae, for example, viruses of barley yellow striated mosaic virus (registration number: KM213865) or lettuce yellow necrotic virus (registration number / specimen: NC_007642 / AJ867584), positive-strand RNA viruses of the order Picornavirales, particularly of the family Secoviridae,e.g., of the genus Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus, Torradovirus or Waikavirus, positive-strand RNA viruses of the order Tymovirales, particularly of the family Alphaflexiviridae, e.g., viruses of the genus Allexivirus, Lolavirus, Mandarivirus or Potexvirus, Tymovirales, particularly of the family Betaflexiviridae, e.g., viruses of the genus Capillovirus, Carlavirus, Citrivirus, Foveavirus, Tepovirus or Vitivirus, positive-strand RNA viruses of the order Tymovirales, particularly of the family Tymoviridae, e.g. viruses of the order Maculavirus, Marafivirus or Tymovirus.

[59] Other suitable vectors may be bacterial vectors, such as, for example, Agrobacterium spp., such as, for example, Agrobacterium tumefaciens. Finally, the term vector also implies suitable transport agents for the introduction of linear nucleic acid sequences (single or double stranded) into a target cell. The usual production processing and the vectors used, according to the present invention, can be carried out by a person skilled in the art with knowledge of the present invention.

[60] The term target region, target site, target structure, target construct, target nucleic acid or target cell / tissue / organism, as used herein, refers to a target that may be any genomic region within a target cell, but also refers to extrachromosomal DNA or RNA, including mRNA, a target cell or organism, or any structural element within a target cell, where modification or manipulation is desired. The term target region, target site, structure Petition 870250016683, dated 28 / 02 / 2025, page 37 / 73 29 / 50 target, target construct, target nucleic acid or target cell / tissue / organism is therefore not restricted to genomic regions that encode a gene, that is, a region that encodes the information to be transcribed into an mRNA.

[61] Complementarity or complementary, as used herein, describes the relationship between two nucleic acid regions of DNA or RNA. Defined by the nucleobases of DNA or RNA, two nucleic acid regions can hybridize with each other according to the lock-and-key model. For this purpose, the Watson-Crick base pairing principles have adenine and thymine / uracil, as well as guanine and cytosine, respectively, as complementary bases. In addition, non-Watson-Crick pairing, such as reverse Watson-Crick, Hoogsteen, reverse Hoogsteen and Wobble pairing, are also understood by the term complementary, as used herein, provided that the respective base pairs can form hydrogen bonds with each other, i.e., two different nucleic acid chains can hybridize with each other based on said complementarity.

[62] The term regulatory sequence, as used herein, refers to a nucleic acid or amino acid sequence that can direct the transcription and / or translation and / or modification of a nucleic acid sequence of interest. Regulatory sequences may comprise cis-acting or trans-acting sequences. Exemplary regulatory sequences include promoters, enhancers, terminators, operators, transcription factors, transcription factor binding sites, introns, and the like.

[63] A promoter refers to a DNA sequence capable of controlling the expression of a coding sequence, i.e., a gene or part thereof, or of a functional RNA, i.e., an RNA that is active without being translated, for example, a miRNA, a siRNA, an IncRNA, an inverted repeat RNA or a loop-forming RNA. The promoter sequence consists of proximal and distal elements in relation to the regulated sequence, the latter often being referred to as enhancers. Promoters can have broad-spectrum activity, but they can also have tissue-stage specific activity. For example, they can be active in root cells, seed cells and meristematic cells, etc. A promoter can be constitutively active or it can be inducible. Induction can be stimulated by a variety of environmental conditions and stimuli. There are strong promoters that can allow high transcription of Petition 870250016683, dated 28 / 02 / 2025, p. 38 / 73 30 / 50 regulated sequence, and weak promoters. Generally, promoters are highly regulated. A promoter of the present invention may include an endogenous promoter natively present in a cell, or an artificial, synthetic (chimeric) or transgenic promoter, even of another species, or an artificial or chimeric promoter, that is, a promoter that does not occur naturally in nature in this composition and is composed of different promoting elements.

[64] The term terminator, as used herein, refers to DNA sequences located downstream, i.e., in the 3' direction, of a coding sequence and may include a polyadenylation signal and other sequences, i.e., other sequences that encode regulatory signals that are capable of affecting mRNA processing and / or gene expression. The polyadenylation signal is generally characterized by the addition of poly-A nucleotides at the 3' end of an mRNA precursor.

[65] The term nucleic acid (molecule) or nucleic acid construct or nucleic acid sequence, as used herein, refers to natural or synthetic ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequences, which may optionally comprise synthetic nucleic acid analogs. A nucleic acid, according to the present invention, may optionally be codon-optimized. Codon optimization implies that the use of codons from a DNA or RNA is tailored to a cell or organism of interest to improve the transcription rate of said recombinant nucleic acid in the cell or organism of interest.The expert in the field is well aware of the fact that a target nucleic acid can be modified at a position due to codon degeneracy, while this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into account the use of species-specific codons of a target organism or cell.

[66] The terms protein, polypeptide and enzyme are used interchangeably herein. The term amino acid or amino acid sequence or amino acid molecule includes any naturally occurring or chemically synthesized protein, peptide, polypeptide and enzyme or a modified protein, peptide, polypeptide and enzyme, wherein the term modified includes any chemical or enzymatic modification of the protein, peptide, polypeptide and enzyme.

[67] The term “cationic polymers”, as used herein, refers to cationic polymers of natural or synthetic origin. Examples of cationic polymers include Petition 870250016683, dated 28 / 02 / 2025, page 39 / 73 31 / 50 of natural origin, not limited to, chitosan (polyglucosamine (1-4)-2-amino-e-D-glucose), cationic gelatin, cationic cellulose, cationic dextran, among others. Examples of synthetic cationic polymers include Poly(2N,N-dimethylaminoethyl methacrylate), Poly-L-lysine, the different forms of Polyamidoamines, and the different forms of polyethyleneimine hydrochloride (PEI).

[68] The term homologous recombination, as used herein, refers to a process such as occurs naturally in all organisms. This requires homologous double-stranded DNA segments. Homologous therefore means that there is a high degree of similarity between two nucleotide sequences. In naturally occurring double-strand breaks, the damage can thus be repaired by homologous recombination and the information about the undamaged chromatid in an organism's genome can be used as a template. According to the present invention, a precise and targeted double-strand break or notch during the course of the genome editing method performed in planta can be inserted into a target region of nucleic acid of interest and homologous recombination can then be used to repair the resulting break.

[69] Homology-directed repair (HDR) denotes a cellular mechanism for repairing single- and double-strand DNA breaks. Thus, HDR includes elements of homologous recombination, as well as the so-called single-strand annealing (SSA) (Dear Michael et al, Annu.Rev.Biochem.79. 181-211, 2010). The most common form of HDR in a cell is homologous recombination; this type of repair also requires the highest sequence homology between donor and acceptor DNA. Other forms of HDR include single-strand annealing (SSA). SSA is non-conservative and occurs naturally between direct repeats of > 30 bp, resulting in deletions. HDR in notches, i.e., in single-strand breaks, occurs through a different mechanism than HDR in double-strand breaks (Davis and Maizels PNAS, 2014 E924-32).According to the present invention, both double-strand and single-strand break-inducing endonucleases, for example, CRISPR nucleases or variants or their catalytically active fragments modified to act as a nickase endonuclease, are proposed. The term HDR or homologous recombination, therefore, refers to the repair of single-strand and double-strand break(s) using specifically a suitable repair mechanism. Petition 870250016683, dated 28 / 02 / 2025, page 40 / 73 32 / 50

[70] Herbicide resistance or herbicide tolerance, as used herein, refers to the ability of a plant or plant cell to develop resistance or tolerance to a particular herbicide or pesticide. This property is commonly transmitted by introducing at least one protein, or a functional RNA, which has been introduced into a plant cell, or whose protein or RNA can be obtained by targeted modification of an endogenous gene or non-coding sequence.

[71] The term genome, as used herein, refers to the sum of the genetic material of a cell, comprising the coding non-coding genes present in the cell of an organism or a virus or an organelle of an organism. The term thus includes the nuclear genome (if present) as well as extranuclear genomic material. DETAILED DESCRIPTION

[72] The present invention provides a method whereby the male gamete is transformed with exogenous DNA when the pollen tube grows through a floral structure into which positively charged particles containing the exogenous DNA have been administered, so that the seeds produced from said floral bud contain the genetic modification of interest.

[73] In a first embodiment, a method is provided for the in planta transformation of plants with exogenous DNA comprising administering positively charged particles containing the exogenous DNA into a floral structure that is a passage for the pollen tube, wherein said administration is carried out at a stage of floral development prior to the formation of the pollen tube in said floral structure.

[74] In a preferred embodiment, the plant is a cultivated plant. In a preferred embodiment, the crop plant is a plant of a genus selected from the group consisting of Triticum spp., Oryza spp., Zea spp., Hordeum spp., Avena spp., Secale spp., Malus spp., Pyrus spp., Prunus spp., Citrus spp., Vaccinium spp., Rubus spp., Fragaria spp., Citrullus spp., Solanum spp., Cucumis spp., Cucurbita spp., Capsicum spp., Solanum spp., Phaseolus spp., Abelmoschus spp., Helianthus spp., Brassica spp., Glycine spp., Olea spp., Sesamum spp., Carthamus spp., Prunus spp., Carya spp., Corylus spp., Anacardium spp., Juglans spp., Pisum spp., Lens spp., Cicer spp., Ipomoea spp., Solanum spp., Vanilla spp., Crocus spp., Gossypium spp., Coffea Petition 870250016683, dated 28 / 02 / 2025, page 41 / 73 33 / 50 spp., Theobroma spp., Saccharum spp., and Linum spp. In a more preferred embodiment, a cultivated plant is a plant selected from the group consisting of Wheat (Triticum aestivum), Rice (Oryza sativa), Maize (Zea mays), Barley (Hordeum vulgare), Oats (Avena sativa), Rye (Secale cereale), Apple (Malus domestica), Pear (Pyrus spp.), Peach (Prunus persica), Plum (Prunus domestica), Cherry (Prunus avium and Prunus cerasus), Orange (Citrus sinensis), Lemon (Citrus limon), Blueberry (Vaccinium spp.), Raspberry (Rubus idaeus), Strawberry (Fragaria ananassa), Watermelon (Citrullus lanatus), Tomato (Solanum lycopersicum), Cucumber (Cucumis sativa), Pumpkin (Cucurbita spp.)Pepper (Capsicum spp), Eggplant (Solanum melongena), Green beans (Phaseolus vulgaris), Okra (Abelmoschus esculentus), Sunflower (Helianthus annuus), Rapeseed / Canola (Brassica napus), Soybeans (Glycine max), Olives (Olea europaea), Sesame (Sesamum indicum), Safflower (Carthamus tinctorius), Almonds (Prunus dulcis), Pecans (Carya illinoinensis), Hazelnuts (Corylus avellana), Cashews (Anacardium occidentale), Walnuts (Juglans regia), Peas (Pisum sativum), Lentils (Lens culinaris), Chickpeas (Cicer arietinum), Sweet potato (Ipomoea batatas), Potato (Solanum tuberosum), Vanilla (Vanilla planifolia), Saffron (Crocus sativus), Cotton (Gossypium spp), Coffee (Coffea arabica and Coffea canephora), Cocoa (Theobroma cacao), Sugarcane (Saccharum spp.) and Flax (Linum usitatissimum). More preferably, the cultivated plant is a cotton plant (Gossypium hirsutum).

[75] In a preferred embodiment, the floral structure that is the passage of the pollen tube is an unpollinated floral bud or a pre-anthesis bud. Preferably, the floral structure is selected from the group consisting of the androecium and gynoecium of an unpollinated floral bud or a pre-anthesis bud.

[76] In a preferred embodiment, the positively charged particles containing the exogenous DNA are in the form of a solution of exogenous DNA complexed with a cationic polymer. Preferably, the cationic polymer is selected from the group consisting of chitosan and polyethyleneimine hydrochloride (PEI).

[77] In a preferred embodiment, the positively charged particles containing the exogenous DNA are administered by injection with a syringe. Optionally, the method further comprises producing at least one hole in the flower bud before administering the positively charged particles containing the exogenous DNA. In a preferred embodiment, the hole is produced using the needle of an injection syringe. Petition 870250016683, dated 28 / 02 / 2025, page 42 / 73 34 / 50

[78] In a preferred embodiment, the solution containing exogenous DNA comprises from about 1 ng to about 300 ng of exogenous DNA, preferably from about 75 ng to about 150 ng.

[79] In a preferred embodiment, exogenous DNA encodes one or more selected from the group consisting of a transgene of interest, an RNAi and a genome editing protein.

[80] The present invention further provides a method for producing a genetically modified plant comprising transforming a plant by the method according to the present invention with exogenous DNA. Preferably, the exogenous DNA encodes one or more selected from the group consisting of a transgene of interest, an RNAi and a genome editing protein.

[81] The present invention further provides a genetically modified plant produced by the method as defined in any of the preceding claims or descended therefrom, seeds, plant parts as well as the genome of said plant. In a preferred embodiment, the genetically modified plant is a transgenic plant.

[82] Plant genetic transformation has become routine in genetic improvement programs, especially those of agricultural commodities in which advanced biotechnological tools are widely applied to improve agronomic characteristics (Basso et al, Front. Plant Sci. 15, “Insights into genetic and molecular elements for transgenic crop development”, 11:509 (2020)). Given this growing demand for genetically modified crops containing new characteristics, the supply of optimized cotton transformation protocols is also required to obtain greater cotton transformation efficiencies in a fast and economical way (Ribeiro et al, Plant Biotechnol. J. 15, “Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil” 997-1009 (2017); Ribeiro et al, Planta 254, “Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods, 2:20 (2021)).Furthermore, the recalcitrance of cotton makes in vitro tissue culture inefficient, dependent on the genotype or explant, and its genetic transformation is not a trivial activity (Ribeiro et al, Planta 254, “Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods, 2:20 (2021)). The methods and uses provided by the present invention and the plant products that may... Petition 870250016683, dated 28 / 02 / 2025, p. 43 / 73 35 / 50 being obtained from them offers a superior alternative to current transformation protocols.

[83] According to the methods of the present invention, the genetic transformation of cotton is carried out by means of a tissue culture-independent method combined with DNA microinjection directly into floral organs. This method allows the generation of genetically modified cotton plants in a shorter time relative to other available techniques with a high-efficiency transformation rate, without the need for in vitro tissue culture, expensive laboratory facilities or skilled labor.

[84] As an example, cotton flowers are hermaphrodites, containing both male and female organs in the same structure. Pollen grains and ovules are formed in cotton flower buds with a diameter of 6-8 mm (Moura et al, Planta 252, “Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton floral development”, 71 (2020)). On the day of anthesis, the flower is white and turns pink after pollination, which occurs a few hours after anthesis. Approximately 5 days after anthesis (dpa) the petals dry and fall from the plant, exposing the developing cotton boll (Ritchie et al., 2007 “Cotton growth and development.” The University of Georgia, Bulletin 1252:1-5.).

[85] As shown in Table 1, the transformation methods, according to the present invention, should be carried out in the floral bud stage, in cotton plants, preferably using floral buds with a diameter of 6-8 mm, in which the transformation efficiency and number of transformants are superior in relation to other stages of floral development.

[86] The methods, according to the present invention, provide a further significant improvement over current strategies for obtaining genetically modified organisms.

[87] In one embodiment, effector nucleases, for example, homing endonuclease, meganucleases, zinc nucleases, transcription activator-type effector nucleases (TALENs) or CRISPR nucleases, for example, Cas nucleases or Cpf1 nucleases, or their catalytically active variants or fragments, alone or in combination as fusions in at least one genetic construct or introduced via protein transfection, can be inserted in planta together with other regulatory sequences and / or effector molecules. The effector molecules can be composed of Petition 870250016683, dated 28 / 02 / 2025, page 44 / 73 36 / 50 DNA, RNA, or synthetic molecules, or a combination thereof, can associate, for example, with CRISPR nuclease and / or gRNA. Effector molecules can be DNA-binding or DNA-modifying enzymes, or coding sequences, therefore, sequences that mediate epigenetic modifications.

[88] In one embodiment, according to any aspect of the present invention, at least one CRISPR polypeptide, or the nucleotide sequence encoding it, is selected independently from the group consisting of a Cas polypeptide from Streptococcus spp., including Streptococcus pyogenes, Streptococcus thermophiles, Staphylococcus aureus or Neisseria spp., including Neisseria meningitides, Corynebacterium, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Roseburia, Parvibaculum, Nitratifractor, Mycoplasma and Campylobacter, including Campylobacter jejuni, or the CRISPR polypeptide, or the nucleotide sequence encoding it, is selected from a Cpf1 polypeptide derived from an archaebacterium or bacterium, including a Cpf1 polypeptide from Acidaminococcus spp., including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., including Lachnospiraceae bacterium ND2006, Francisella spp., including Francisella novicida U112, Eubacterium eligens, Prevotella spp. or Porphyromonas spp., or their variants and / or functional fragments and / or combinations thereof, including nickase endonucleases, or a CRISPR polypeptide without endonucleolytic activity, or a CRISPR polypeptide fusion protein, wherein one CRISPR polypeptide portion may be fused to another CRISPR polypeptide portion or to any effector domain of polypeptide, DNA or RNA.

[89] Furthermore, according to the present invention, at least one DNA repair template can be transformed in planta together with a genome editing tool of interest. The use of such a repair template is of particular interest in the case where an endonuclease or at least a nickase endonuclease is used to introduce a specific double-strand break or multiple nicks at a genomic target site of interest. By providing a suitable repair template that assists homology-directed repair, and the introduction of a modification of interest, the genome editing event can be controlled even more precisely. A double-strand DNA break in vivo is generally repaired by natural mechanisms in plant cells: either by non-homologous end joining (NHEJ) or by homologous recombination (HR, also known as Petition 870250016683, dated 28 / 02 / 2025, page 45 / 73 37 / 50 homology-directed repair (HDR)). Furthermore, in plants, so-called alternative end-jointing (AEJ) pathways have been described (Charbonnel C, Allain E, Gallego ME, White CI (2011) Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis. DNA Reapir (Amst) 10:611-619). Because NHEJ is highly error-prone, there is a great need to provide repair mechanisms that allow for greater accuracy.

[90] In the context of the present invention, in one embodiment, a repair model for HDR is described that can optionally be introduced into the target cell together with or at a separate time from an endoclease, for example, a CRISPR construct, and / or at least one additional effector domain, in order to induce a specific HDR mechanism and thereby introduce specific sequence changes at the site of a double-strand break. In this sense, specific edits, knock-ins, gene swaps or specific DNA damage repair can be performed to avoid an unwanted mutation at the DNA break site.A knock-in can mean the specific insertion of at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, at least eight nucleotides, at least nine nucleotides, at least ten nucleotides, at least fifteen nucleotides, at least twenty nucleotides, at least fifty nucleotides, at least one hundred nucleotides, at least two hundred nucleotides, at least five hundred nucleotides, or at least one thousand nucleotides into the target nucleic acid in the plant cell. An edit can mean the specific exchange of at least one nucleotide for another nucleotide. Furthermore, an edition can also be obtained through two, three, four or more swaps, or a combination of insertions and swaps.Insertion(s) means the specific insertion of at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, at least eight nucleotides, at least nine nucleotides, at least ten nucleotides, at least fifteen nucleotides, at least twenty nucleotides, at least fifty nucleotides, at least one hundred nucleotides, at least two hundred nucleotides, at least five hundred nucleotides, at least one thousand nucleotides, at least two hundred nucleotides, at least five hundred nucleotides, at least one thousand nucleotides, at least two thousand nucleotides, at least five thousand nucleotides, at least one hundred thousand nucleotides, or at least one five thousand nucleotides into the acid. The target nucleic acid sequence of the plant cell. A nucleic acid sequence such as an insertion can be all or part of a transcription factor binding site sequence, a sequence. Petition 870250016683, dated 28 / 02 / 2025, page 46 / 73 38 / 50 regulatory, a polypeptide coding sequence, an intron sequence, a non-coding RNA sequence (e.g., IncRNA), an expression cassette, another non-coding sequence with various uses, such as the introduction of a replication marker at a desired location, or an RNAi construct. Additionally, a knock-in can also be triggered by the deletion of sequence sections that disrupt gene functionality (e.g., the removal of transposon insertions). The repair template can be introduced into the target plant structure of interest as a single-stranded or double-stranded nucleic acid or a combination thereof.

[91] The term functional integration or functionally integrated, as used herein, refers to the integration of a genetic construct of interest into at least one cell that enables transcription and / or translation and / or catalytic activity and / or binding activity, including the binding of one nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within at least one cell. Where relevant, functional integration occurs in a particular cellular compartment of at least one cell, including the nucleus, cytosol, mitochondria, chloroplast, vacuole, membrane, cell wall, and the like.Therefore, the term functional integration – in contrast to the term stable integration detailed above – implies that the genetic construct of interest is introduced into at least one cell, where the genetic construct exerts its effect within or on at least that cell into which it was introduced.Depending on the nature of the genetic construct to be introduced, the said effect may naturally vary and include, alone or in combination, inter alia, the transcription of a DNA encoded by the genetic construct into a ribonucleic acid, the translation of an RNA into an amino acid sequence, the activity of an RNA molecule within a cell, comprising the activity of a guide RNA or a miRNA or siRNA for use in RNA interference, and / or a binding activity, including the binding of one nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within at least one cell, including the integration of a sequence delivered via a vector or genetic construct, transiently or stably. The said effect may also comprise the catalytic activity of an amino acid sequence representing an enzyme or a catalytically active portion thereof in at least one cell.The aforementioned effect was achieved after the. Petition 870250016683, dated 28 / 02 / 2025, p. 47 / 73 39 / 50 The functional integration of the genetic construct, according to the present invention, may depend on the presence of regulatory sequences or localization sequences that are comprised by the genetic construct of interest, as is known to those skilled in the art. Since the present invention comprises both embodiments directed toward the stable integration of a genetic construct of interest, as well as the transient introduction of a genetic construct of interest into at least one cell, the effect achieved by the functional integration of the genetic construct may vary further depending on whether the mode of introduction is stable or transient.Stable integration, as detailed above, is based on the integration of the genetic construct into the genome of at least one cell, including the nuclear genome as well as the genome of other plant compartments, whereas transient introduction implies that functional integration means the introduction of the genetic construct of interest into at least one cell followed by its transcription and / or translation and / or catalytic activity and / or binding activity in the cell without the need for integration into the cell's genome.

[92] If a CRISPR system is used as part of a genetic construct according to the present invention, the nuclease component, or a catalytically active variant or fragment thereof, and the gRNA component, and optionally other components such as effector domains or an artificial repair template, can be introduced into a target cell of interest stably or transiently. Furthermore, the different components can be introduced into the same or different genetic constructs. The different CRISPR components can thus be transformed in planta simultaneously or subsequently. For example, the CRISPR nuclease, for example, a Cas nuclease or a Cpf1 nuclease, or a catalytically active variant or fragment thereof, can be stably incorporated into at least one cell to generate a first plant.The aforementioned first plant, in the next generation, can then be transformed in planta, with the additional CRISPR component comprising at least one gRNA in a transient manner. In another embodiment, the CRISPR components are transformed in planta, which allows for the rapid and precise generation of a genome editing event of interest. In one embodiment, the components can be provided in / as the same genetic construct or the components can be provided in / as different genetic constructs, simultaneously or subsequently. As the CRISPR nuclease component as a protein will be more stable than the gRNA component, it can be. Petition 870250016683, dated 28 / 02 / 2025, page 48 / 73 40 / 50 desirable transformation in planta in such a way that, firstly, a stable level of a CRISPR nuclease is achieved, comprising necessary localization signals, for example, nuclear or plastid localization signals, before a gRNA is provided, either in the form of RNA or as a genetic construct of DNA that can be transcribed into active RNA.

[93] In certain embodiments, transformation according to methods of the present invention may comprise the introduction of an additional effector domain, including an effector domain for epigenetic modulation, or the introduction of a DNA repair template. DNA repair templates are highly suitable for precision genome editing when used in combination with a site-specific endonuclease or nickase endonuclease, as providing the repair template can help provide a direct homology-targeted repair event.The term targeted homology repair, according to the present invention, comprises any type of alteration that can be introduced by the repair template sequence, according to the present application, which may independently include sequence insertions, edits of at least one sequence position, deletions or rearrangements, the preferred strategy for genome editing approaches in higher eukaryotes currently being insertions or edits, as these strategies allow targeted knock-in or knock-out of a sequence of interest within a target DNA sequence or a site-specific modification of at least one sequence.

[94] In one embodiment, according to this aspect, the target nucleic acid sequence, where the functional integration of the genetic construct of interest occurs, is nuclear genomic DNA. In another embodiment, according to this aspect, the target nucleic acid sequence, in which the functional integration of the genetic construct of interest is mediated, is a mitochondrion or plastid DNA, wherein the genetic construct comprises a localization sequence that mediates the localization of the genetic construct in the respective plant compartment of interest, for example, a mitochondrion or a chloroplast. In another embodiment, functional integration refers to the delivery of a genetic construct of interest into at least one cell, for example, the cytosol or another plant compartment, without stable integration occurring. Petition 870250016683, dated 28 / 02 / 2025, page 49 / 73 41 / 50

[95] In one embodiment, according to the above and in accordance with the additional aspects of the present invention, the transformation is carried out without in vitro culture.

[96] In another modality, in planta transformation, the cell, tissue, organ or material that is transformed is explanted and further cultivated in vitro.

[97] As detailed above, the methods according to the invention provide the advantage that both the transformation and further development of at least one transformed cell can occur in planta, avoiding the need for complex in vitro culture steps for the regeneration of a plant or plant material therefrom. In certain embodiments, however, it may be appropriate to explant or dissect a cell, tissue, organ or plant material for further culture, screening or testing depending on the specific needs. Several methods for the in vitro culture of a cell, tissue, organ or plant material are available to those skilled in the art.

[98] In another embodiment, functional integration of the genetic construct of interest is performed as (a) a stable integration such that the integrated genetic construct of interest, or a part thereof, is heritable to a zygote formed from the gamete comprising the genetic construct of interest, or (b) a transient introduction permitting targeted genetic manipulation through the genetic construct of interest or its products, wherein the targeted genetic manipulation, but not the genetic construct of interest, or a part thereof, is heritable to a zygote formed from the gamete comprising the genetic construct of interest, from which the progeny comprising the targeted genetic manipulation is developed.

[99] The methods according to the present invention are highly suitable for achieving controlled and efficient trait development in a plant, for example, for introducing or modifying marker genes or for conferring certain desired characteristics to a plant in a short period of time.

[100] Particularly with regard to the targeted development of positive traits in plants, including resistance, especially against pests and environmental influences such as cold, drought, salinity, increased yield, herbicide resistance, there is a need to provide a reliable method for the selective activation and deactivation or modification of genomic material, as well as the Petition 870250016683, dated 28 / 02 / 2025, pp. 50 / 73 42 / 50 RNA silencing within a short period of time without in vitro cultivation or intensive crossing steps.

[101] In a further embodiment, an in planta transformation method is thus provided for the production of a plant, plant material or plant cell, wherein a plant is transformed with at least one gRNA, at least one CRISPR nuclease or its catalytically active fragment and / or an effector domain and at least one DNA repair template, wherein a targeted modification of at least one nucleic acid target region of interest is obtained, either by modification of an endogenous sequence or by insertion of a heterologous, i.e., non-endogenous sequence to which the targeted modification relates, or wherein the heterologous sequence comprises a gene that is selected from a reporter gene, a selectable marker, a resistance gene mediating resistance to a disease, a gene mediating resistance to herbicides, a gene mediating resistance to insects or nematodes, a gene that is involved in carbohydrate metabolism,a gene that is involved in fatty acid metabolism, a gene that is involved in amino acid metabolism, a gene that is involved in plant development, a gene that is involved in the regulation of plant growth, a gene that is involved in improving the yield of a plant material of interest, a gene that is involved in mediating drought resistance, a gene that participates in the transmission of cold resistance, a gene involved in the transmission of heat resistance, a gene involved in the transmission of resistance to a salt or salts or a specific salt concentration, or a gene that encodes a functional RNA, wherein the functional RNA is selected from the group consisting of a miRNA, an siRNA or other RNA that can form an inverted repeat structure, for example, a ddRNAi construct encoding both a sense and antisense strand and a connecting loop.

[102] In one embodiment, new molecular markers can be developed. In relevant crop plants, there is the potential to increase the efficiency of targeted plant crossing through marker-assisted selection (MAS). Genetic marker alleles, or alternatively, the quantitative trait controlling alleles (QTLs) described above are used for this purpose in order to identify plants or plant material, or a plant cell, that contains a desired genotype at one or more loci / locations. It is assumed that they can inherit the desired genotype along with a desired phenotype to the offspring. The alleles of Petition 870250016683, dated 28 / 02 / 2025, pp. 51 / 73 43 / 50 Genetic markers (or QTL alleles) can also be used to identify plants having a desired genotype at a locus or at several unlinked or linked loci, for example, a haplotype, from which it is assumed they can transmit to their offspring the desired genotype along with a desired phenotype. For the purpose of marker-assisted selection, the term marker as used here therefore refers to both markers and QTL loci. Once it has been determined that a desired phenotype and a polymorphic chromosomal locus, for example, a marker locus or a QTL, segregate together, it is possible to use these polymorphic loci to select alleles corresponding to a desired phenotype. This procedure is called marker-assisted selection (MAS). To this end, a nucleic acid sequence corresponding to the marker nucleic acid is detected in a biological sample of interest from a plant to be analyzed.This detection can be in the form of hybridization of a nucleic acid probe to a label, for example, using allele-specific hybridization, Southern blot analysis, in situ hybridization of Northern blot analysis, primer hybridization followed by PCR amplification of a marker region, or similar, by any combination thereof. A variety of methods for marker detection are known to those skilled in the art. After the presence or absence of a specific marker in the biological sample of interest, comprising at least one plant cell, the corresponding plant is selected and can be used for selective breeding to obtain progenies.

[103] In yet another aspect, according to the present invention, a plant produced by the production methods of the second or third aspect of the present invention is provided, or plant cells, plant material or derivatives thereof, or an offspring thereof.

[104] The present invention is further described with reference to the following non-limiting examples. EXAMPLES OF CONCRETIZATIONS OF THE INVENTION Example 1:

[105] Establishing the appropriate floral stage for genetic modification. To identify the most suitable floral stage and organ for genetic transformation, DNA microinjections were tested in flowers at three developmental stages. Petition 870250016683, dated 28 / 02 / 2025, pp. 52 / 73 44 / 50 (floral bud, white flower and pink flower) and in floral organs of the androecium and gynoecium (Table 1).

[106] Microcomplexation of DNA in microparticles with the cationic polymers chitosan and polyethyleneimine hydrochloride (PEI) was also tested.

[107] Genetic construction. The minimal expression cassettes used for the genetic transformation of cotton contained the sequences of the selectable marker genes resistance to bialaphos (bar) or acetohydroxyacid synthase (ahas) under the control of the CaMV 35S promoter and the T-Nos terminator, which confer tolerance to the herbicides glufosinate ammonium and imazapyr, respectively. Furthermore, the coding sequence of the Cry10Aa gene (Bacillus thuringiensis toxin) driven by the Flower Specific 1 (FS1) promoter confers flower-specific expression (Artico et al., 2014 Isolation and characterization of three new promoters from Gossypium hirsutum that show high activity in reproductive tissues. Plant Molecular Biology Reporter 32:630-643) and resistance to CBW (Ribeiro et al., 2017 “Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil."Plant Biotechnology Journal 15:997-1009) (Figure 3a), or the uidA / GUS gene driven by the CaMV 35S promoter (Figure 3b) was also present. Minimal expression cassettes were chemically synthesized and assembled into the pCAMBIA 3300 binary vector by Epoch Life Science (Missouri, TX, USA). The DNA plasmid was transfected into an OmniMAX Escherichia coli strain and purified using the QIAGEN Plasmid Maxi Kit (QIAGEN, Hilden, Germany). Then, 20 μg of plasmid DNA was digested with Bam HI restriction enzyme to release the minimal expression cassettes. The released minimal expression cassettes were recovered by electroelution from a 1% agarose gel, as described by Sambrook and Russel (2001). After trapping the DNA in a cellophane membrane, the eluate was treated with Chloroform / isoamyl alcohol (24:1) and centrifuged at 6,000g for 5 minutes. The aqueous phase was transferred to a new tube and the DNA was precipitated with 3M sodium acetate (pH 5.0) and isopropanol.After precipitation at -20°C overnight, the DNA was recovered by centrifugation at 13,000g for 10 min, rinsed with 75% ethanol, air-dried at room temperature, and suspended in TE buffer.

[108] Formulation of microparticles for DNA complexation and particle analysis. The cationic polymers chitosan (chitosan polymer, 95% deacetylation) and PEI (Sigma-Aldrich, St. Louis, Missouri, USA) were used in Petition 870250016683, dated 28 / 02 / 2025, pp. 53 / 73 45 / 50 assembly of microparticles for flower microinjections. All formulations were prepared by gently complexing one microgram of polymer and one microgram of purified minimal expression cassette DNA FS1::Cry10Aa, at a final concentration of 10 micrograms per mL. In addition, concentrations of purified minimal expression cassette DNA 35S::uidA / GUS biocomplexes of 10 or 20 ng^L were also used. Measurements of microparticle hydrodynamic diameters (HD), Zeta potentials, and polydispersity indices (Pdl) were performed using a ZetaSizer Nano ZS instrument (Malvern, Worcestershire, UK) coupled with a helium-neon laser (4 mW) operated at 633 nm for dynamic light scattering (DLS technique) to determine HD and Pdl, and electrophoretic mobility to determine the Zeta potential. For each formulation, the samples were diluted 1:20 with deionized water and analyzed in triplicate.The number of steps in automatic mode and the scattering angle were set to 173° at 25°C. The collected data were analyzed using ZetaSizer v10.11.1 software. DNA molecules ranging from 10,000 bp to 20,000 bp were used for the transformation, with the potential for DNA containing more than 20 K bp to be used for the transformation described in this document (Figure 3).

[109] Microinjection of cotton floral structures. Three-month-old plants of the BRS372 cultivar of G. hirsutum, maintained in a greenhouse, were used. Flowers at three developmental stages were selected for microinjection, as described by Moura et al. 2020 “Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.” Plant 252:71. (i) floral buds with 6-8 mm diameter, (ii) unpollinated white flowers at 0 days post anthesis (dpa), and (iii) pollinated pink flowers at 2 dpa. Microinjections were performed in the gynoecium (lower part of the reproductive structure, at the level of the ovule) and, for floral buds, also in the androecium (upper part of the reproductive structure, near the stamens) (Figure 1a to 1f, and Figure 7). A 100 μL Hamilton Microliter 701 microsyringe (Hamilton Co, Reno, NV, USA) was used for the microinjection.Microinjections into the androecium were performed directly at the level of the stamens. Previous tests were conducted by injecting a blue dye (0.25% bromophenol blue, 0.25% xylene cyanol, and 1 mM EDTA) into the androecium and gynoecium of flower buds. After 30 min, the flower buds were sectioned in half to verify the internal location of the injected solution. Subsequently, experiments were performed by injecting 10 μL of DNA biocomplexed by... Petition 870250016683, dated 28 / 02 / 2025, pp. 54 / 73 46 / 50 organ, flower or flower bud, following the formulations, stages of floral development and floral organs described in Table 1.

[110] Characterization of transgenic cotton plants. Approximately 55 days after microinjection, cotton bolls developed from treated flowers were collected. Cotton seeds were manually delinted and sown in germination trays filled with soil mixed with commercial substrate (Vivatto, São Paulo, SP, Brazil) in a 1:1 ratio (w:w). A glufosinate ammonium herbicide solution (200 mg / L) was sprayed once the plants were 30 days old, and surviving plants were evaluated by PCR. Genomic DNA from glufosinate-resistant plants was purified using the DNeasy Plant Maxi kit (QIAGEN) following the manufacturer's instructions. DNA concentration was determined with a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). DNA integrity was verified by electrophoresis on a 1% agarose gel stained with ethidium bromide.The insertion of T-DNA was confirmed by PCR amplification using specific primers for bar and Cry10Aa. Reactions were performed with 40 ng of genomic DNA and the QIAGEN Multiplex PCR kit (QIAGEN) according to the manufacturer's instructions. Gene amplification was performed using 95°C for 15 minutes; 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 min; and 72°C for 10 min. Untransformed (NT) plant DNA and 20 ng of plasmid DNA were used in negative and positive control reactions, respectively. PCR results were evaluated by electrophoresis on a 1% agarose gel stained with ethidium bromide. Transgenic cotton plants were transplanted into pots, maintained in a greenhouse, and self-fertilized to generate segregating Ti lines.The same procedures for collecting cotton bolls, seed sowing, and plant genotyping using PCR assays described above were followed to obtain segregating plants of Ti and T2 generations. As a result of transformation treatments using DNA complexed with cationic polymers microapplied to floral structures, the best treatments that resulted in the highest transformation rates were the administration of PEI and DNA biocomplexes, as well as chitosan and DNA biocomplexes, to the gynoecium of 6-8 mm floral buds, resulting in survival rates after treatment with glufosinate ammonium herbicide of 60% and 42%, respectively, and regarding PCR test positivity, the rates were 17.04% and 17.97%, respectively (Table 1). Intermediate rates (>3% and...) were also found. Petition 870250016683, dated 28 / 02 / 2025, pp. 55 / 73 47 / 50 <15%) when DNA and cationic polymer biocomplexes were applied to other locations on the flower bud and at other stages of floral structure development. In another transformation-independent experiment, microapplication of a DNA and chitosan biocomplex to cotton flower buds in different amounts of DNA (100 and 200 ng) showed survival rates above 20% after treatment with glufosinate ammonium herbicide.

[111] Detection of transgenic proteins by indirect ELISA. Transgenic plants of To, Ti, and T2 generations obtained from treatments 6 and 10 were also screened by detecting the Cry10Aa protein in flower buds using indirect ELISA. Total protein extraction was performed as described by Ribeiro et al. 2017 (Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil. Plant Biotechnol. J. 15:997-1009) using 80 mg of leaf discs from fully expanded young leaves and flower buds pulverized in a mortar with liquid nitrogen, for Cry10Aa protein, respectively. Total protein extracts were quantified using Bradford's reagent as described by Bradford 1976 (A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 254:248-254).The amount of target protein was determined by spectrophotometry at 495 nm based on standard curves of the Cry10Aa protein. Indirect ELISA assays were performed using a polyclonal antibody produced in rabbits against the Cry10Aa protein (peptide “VSSDSKIVKGPGHT”; GenScript, New Jersey, USA), as described by Ribeiro et al. 2017 (Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil. Plant Biotechnol. J. 15:997-1009).

[112] Transgene copy number estimation. The transgene copy number was estimated in transgenic cotton plants of To, Ti, and T2 generations for treatments 6 and 10 using real-time PCR assay with the 2-ΔΔα method (Yang et al., 2012 “Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-ΔΔ0ϊ method.” African Journal of Biotechnology 11(23):6226-6233). The pBSK-cry10Aa-ubc1 plasmid containing a fragment of the G. hirsutum ubiquitin C gene (GhUbc1) and a fragment of the Cry10Aa transgene was used as a reference. The absolute quantity of each of these genes was determined in reference to a standard curve, plotting the cycle limit (Ct) values ​​relative to the log-transformed concentration of ten-fold serial dilutions (101, 102, 103, 104 and 105) of the pBSK-cry10Aa-ubc1 plasmid, using specific primers. Petition 870250016683, dated 28 / 02 / 2025, pp. 56 / 73 48 / 50 of the target. Real-time PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with 5 μL of SYBR Green mix (Cellco Biotech, São Carlos, SP, Brazil), 20 ng of DNA, and 0.2 μM of each primer in a final volume of 10 μL. Plants obtained using the described method and characterized to determine the number of transgene copies per haploid genome were able to be identified with up to two copies per haploid genome.

[113] Histochemical assay. The accumulation of uidA / GUS protein and enzymatic activity in transgenic cotton plants were determined by a histochemical assay. Leaf and root tissue sections were immersed in 1 mL of X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) solution [1 mg / mL of X-Gluc in 1% (v / v) N,N-dimethylformamide, 0.1 M phosphate buffer (40 mM KH2DEPOIS4 and 60 mM K2HPO4, pH 7.0), 50 μM potassium ferricyanide, 50 μM potassium ferrocyanide, 0.1% (v / v) Triton X-100] and incubated at 37°C in the dark for 16 h. Chlorophyll pigments from plant tissues were removed by extraction with ethanol, and the activity of the GUS (glucuronidase) enzyme was observed and photographed under a magnifying glass. GM plants generated from the microapplication of biocomplexes showed a strong signal of deposition of the blue reaction product, catalyzed by the beta-glucuronidase enzyme, indicating that the evaluated plants were transformed with the pCAMBIA uidA / ahas exogenous DNA construct.

[114] Table 1. List of treatments used for the application of DNA-associated biocomplexes in floral structures of cotton plants. The different types of chitosan- or PEI-based biocomplexes are described, as well as the application site at different stages of floral development (androecium or gynoecium, closed floral bud, open white flower and in the pink-colored flower, already fertilized). The respective transformation efficiencies when using the FS1::Cry10Aa minimal expression cassette are described. Petition 870250016683, dated 28 / 02 / 2025, pp. 57 / 73 49 / 50 Treatment Formulation Floral Stage Target Floral Organ Number of Microinjected Cotton Capsules Number of Seeds Harvested After Microinjection Number of Glufosinate-Resistant T0 Plants Number of PCR-Positive T0 Plants Transformation Efficiency (%) 1 DNA + H2O Floral Bud Androecium 5 96 17 9 9.37 2 DNA + H2O Floral Bud Gynoecium 5 22 1 1 4.54 3 DNA + H2O White Flower Gynoecium 5 0 0 0 0 4 DNA + H2O Pink Flower Gynoecium 5 17 1 0 0 5 DNA + PEI Floral Bud Androecium 5 50 19 3 6 6 DNA + PEI Floral Bud Gynoecium 5 88 53 15 17.04 7 DNA + PEI White Flower Gynoecium 5 8 0 0 0 8 DNA + PEI Pink Flower Gynoecium 5 0 0 0 0 9 DNA + chitosan Floral bud Androecium 5 81 8 3 3.7 10 DNA + chitosan Floral bud Gynoecium 5 89 38 16 17.97 11 DNA + chitosan White flower Gynoecium 5 0 0 0 0 12 DNA + chitosan Pink flower Gynoecium 5 12 3 3 25 Petition 870250016683, dated 28 / 02 / 2025, pp. 58 / 73 50 / 50

[115] Table 2. Inheritance and copy number of transgenes (FS1::Cry10Aa minimum expression cassette) in GM cotton plants in generations T1 and T2. Transgene segregation ratios were obtained by PCR detection of the bar gene in plants from treatments 6 (DNA + PEI) and 10 (DNA + chitosan). Copy number estimates were obtained by qPCR analysis of the Cry10Aa gene. Treatment Generation T1 Generation T2 Total number of plants Number of GM plants Segregation (%) Number of transgene copies1 Total number of plants Number of GM plants Segregation (%) Number of transgene copies1 6 (DNA + PEI) 85 46 54.11 2 28 19 67.85 2 10 (DNA + chitosan) 16 8 50 2 42 32 76.19 2 1 The same number of transgene copies was obtained for all GM plants tested. PEI: polyethyl enimine hydrochloride. Petition 870250016683, dated 28 / 02 / 2025, pp. 59 / 73

Claims

1 / 4 CLAIMS 1. A method for the in planta transformation of a plant with exogenous DNA, characterized in that it comprises administering positively charged particles containing the exogenous DNA into a floral structure that is a pollen tube passage, wherein said administration is carried out at a stage of floral development prior to the formation of the pollen tube in said floral structure.

2. Method according to claim 1, characterized in that the plant is a cultivated plant.

3. Method according to claim 2, characterized in that the crop plant is a plant of a genus selected from the group consisting of Triticum spp., Oryza spp., Zea spp., Hordeum spp., Avena spp., Secale spp., Malus spp., Pyrus spp., Prunus spp., Citrus spp., Vaccinium spp., Rubus spp., Fragaria spp., Citrullus spp., Solanum spp., Cucumis spp., Cucurbita spp., Capsicum spp., Solanum spp., Phaseolus spp., Abelmoschus spp., Helianthus spp., Brassica spp., Glycine spp., Olea spp., Sesamum spp., Carthamus spp., Prunus spp., Carya spp., Corylus spp., Anacardium spp., Juglans spp., Pisum spp., Lens spp., Cicer spp., Ipomoea spp., Solanum spp., Vanilla spp., Crocus spp., Gossypium spp., Coffea spp., Theobroma spp., Saccharum spp., and Linum spp.

4. Method according to claim 3, characterized in that the cultivation plant is a plant selected from the group consisting of Wheat (Triticum aestivum), Rice (Oryza sativa), Corn (Zea mays), Barley (Hordeum vulgare), Oats (Avena sativa), Rye (Secale cereale), Apple (Malus domestica), Pear (Pyrus spp.), Peach (Prunus persica), Plum (Prunus domestica), Cherry (Prunus avium and Prunus cerasus), Orange (Citrus sinensis), Lemon (Citrus limon), Blueberry (Vaccinium spp.), Raspberry (Rubus idaeus), Strawberry (Fragaria ananassa), Watermelon (Citrullus lanatus), Tomato (Solanum lycopersicum), Cucumber (Cucumis sativa), Pumpkin (Cucurbita spp.Pepper (Capsicum spp), Eggplant (Solanum melongena), Green beans (Phaseolus vulgaris), Okra (Abelmoschus esculentus), Sunflower (Helianthus annuus), Rapeseed / Canola (Brassica napus), Soybeans (Glycine max), Olives (Olea europaea), Sesame (Sesamum indicum), Safflower (Carthamus tinctorius), Almonds (Prunus dulcis), Pecans (Carya illinoinensis), Hazelnuts (Corylus avellana), Cashews (Anacardium occidentale), Walnuts (Juglans regia), Peas (Pisum sativum), Lentils (Lens culinaris), Chickpeas (Cicer arietinum), Sweet potato (Ipomoea batatas), Potato (Solanum tuberosum), Vanilla (Vanilla planifolia), Saffron (Crocus sativus), Cotton (Gossypium spp), Coffee (Coffea arabica and Coffea canephora), Cocoa (Theobroma cacao), Sugarcane (Saccharum spp.) and Linen (Linum usitatissimum).

5. A method according to any one of claims 1 to 4, characterized in that the floral structure through which the pollen tube passes is selected from the group consisting of the androecium and gynoecium of the floral bud.

6. Method according to claim 5, characterized in that the floral structure through which the pollen tube passes is the gynoecium of a floral bud.

7. Method according to claim 5, characterized in that the floral structure through which the pollen tube passes is the androecium of a floral bud.

8. Method according to claim 5, characterized in that the floral structure is an unpollinated floral bud or a pre-anthesis bud.

9. Method according to claim 4, characterized in that the plant is cotton (Gossypium hirsutum).

10. Method according to claim 8, characterized in that the floral structure that is the passage of the pollen tube is a floral bud with about 0.5 mm to about 8 mm in diameter, preferably about 6 mm to about 8 mm in diameter.

11. Method according to claim 1, characterized in that the positively charged particles containing the exogenous DNA are in the form of a solution of exogenous DNA complexed with a cationic polymer, or a mixture of cationic polymers.

12. Method according to claim 11, characterized in that the cationic polymer is selected from the group consisting of chitosan (polyglucosamine (1-4)-2-amino-β-D-glucose), cationic gelatin, cationic cellulose, cationic dextran, Poly(2-N,N-dimethylaminoethyl methacrylate), Poly-L-lysine, the different forms of Polyamidoamines and the different forms of polyethyleneimine hydrochloride (PEI).

13. Method according to any one of claims 8-12, characterized in that the solution containing exogenous DNA comprises from about 1 ng to about 10000[5][6] ng of exogenous DNA, preferably from about 75 ng to about 150 ng, complexed or not with cationic polymers.

14. Method according to claim 1, characterized in that the exogenous DNA encodes one or more selected genes from the group consisting of a transgene for the expression of one or more proteins of interest, the expression of a gene encoding a dsRNA, and a genome-editing protein.

15. A method for producing a genetically modified plant, characterized in that it comprises transforming a plant by the method as defined in any of the preceding claims with exogenous DNA.

16. Method according to claim 18, characterized in that the exogenous DNA encodes one or more selected from the group consisting of a transgene of interest, an RNAi and / or a genome editing protein. Petition 870250016683, dated 28 / 02 / 2025, pp. 70 / 73 4 / 4 17. Genome of a genetically modified plant, characterized in that it is produced by the method as defined in any of the preceding claims.

18. Genetically modified plant: a plant part or seed thereof, characterized in that it is produced by the method as defined in any of the preceding claims or is descended from it.

19. Method according to claim 1, Genome according to claim 17, or plant, plant part or seed thereof according to claim 18, characterized in that the genetically modified plant is a transgenic plant. Petition 870250016683, dated 28 / 02 / 2025, pp. 71 / 73

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