WO2024201414A1 - Method of transforming a plant cell - Google Patents

Abstract

The present invention pertains generally to plant biotechnology. Aspects of the present disclosure relate to methods for plant transformation. More specifically, there is described a method for transforming sugarcane (Saccharum spp.). The present disclosure further relates to genetically altered sugarcane plants produced using these methods.

Description

METHOD OF TRANSFORMING A PLANT CELL

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/493,523, filed March 31, 2023, hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (207422000940seqlist.xml; Size: 15,225 bytes; and Date of Creation: March 26, 2024) are herein incorporated by reference in their entirety.

TECHNICAL FIELD

[0003] The present invention pertains generally to plant biotechnology. Aspects of the present disclosure relate to methods for plant transformation. More specifically, there is described a method for transforming sugarcane (Saccharum spp.). The present disclosure further relates to genetically altered sugarcane plants produced using these methods.

BACKGROUND OF THE INVENTION

[0004] There is a growing need to obtain new plant varieties to tackle the major problems in agriculture, such as pest control, susceptibility to diseases, resistance to adverse weather conditions, more productivity with less natural resources, among others. For the past decades, such problems have been the center of the conventional breeding programs that tried to obtain plants with desired characteristics to address such problems.

[0005] Although such conventional breeding methods are important for continuously supplying the market with new varieties of plants, there is now the need of combining them with vegetal biotechnology techniques (genetic engineering) to fulfill modem market’s needs, since part of the desired characteristics are not found in the genetic background of the varieties being crossed.

[0006] Due to the commercial success of incorporating desirable agronomic traits through genetic engineering (i.e., genetic transformation or gene editing) into several plant species in the past decades (soybean, corn, canola, beet and cotton, for example), the sugarcane industry has also gained interest in applying such techniques. Sugarcane (Saccharum spp.) is a grassy plant belonging to the botanic family Poaceae, originating from Southeast Asia, from the large central region of New Guinea and Indonesia (Daniels & Roach, 1987, Sugarcane improvement through breeding p. 7-84). It is one of the most important plant species cultivated in tropical and subtropical regions, with an area exceeding 23 million hectares distributed over 121 countries (FAO Statistical Yearbook 2012 p. 233). Alongside culinary importance (specially sugar production), sugarcane provides a source of biofuel in the form of ethanol, which has a global market of about 50 billion dollars. [0007] The economic and social importance of sugarcane stimulated, major research efforts are noted, aimed at defining better agricultural practices for cultivation and improved quality of the varieties cultivated. However, unlike in other crops such as maize and soybean, the introduction of transgenes (e.g. genetic transformation) and edition of genes cannot be done once in donor germplasm and then back-crossed into elite germplasm. Instead, each elite sugarcane germplasm cells must be invariably transformed or modified in order to develop new commercial cultivars. Thus, for the "spread" of the same trait in more than one germplasm, enabling gains in productivity for different varieties cultivated at different geographical regions, it is necessary to carry out a new genetic transformation/modification process.

[0008] Plant genetic engineering involves the transfer of nucleotide sequences or polypeptides of interest into plant cells in such a way that a agronomically superior progeny is produced by maintaining and stably expressing the sequences responsible for the desired trait and/or by stably maintain a desired phenotype (“heritable traits), either by introducing heterologous sequences and/or modifying endogenous sequences in the plant genome, or by modifying the expression pattern of genes and/or cellular function of interest without changes to the original DNA. Accordingly, one of the options is the use of in vitro cultivation techniques.

[0009] One of the in vitro cultivation techniques is somatic embryogenesis, which consists of the production of embryos from an isolated cell or a small group of cells which, by means of in vitro cultivation will give rise to somatic embryos and after to a plant, without the fusion of gametes (Jimenez. 2001. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Revista Brasileira de Fisiologia Vegetal, v. 13, p. 196-223).

[0010] Various types of explants have been used in the embryonic process in sugarcane. According to Lakshmanan el al. (2006. Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. Interspecific hybrids) leaf culture. Plant Cell Reports, v. 25, p. 1007- 1015), almost all plant tissues give rise to embryogenic calluses, but the younger leaves and developing inflorescences are very prolific and are preferred target tissues for fast production of embryogenic calluses.

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

[0012] The conversion of the somatic embryos in plants is the final phase of the process of somatic embryogenesis. Regeneration generally occurs in a medium devoid of growth regulators and in the presence of light (Genetic transformation of the euploid Saccharum officinarum via direct and indirect embryogenesis. Sugar tech, v. 12, p. 21- 25; Basnayake et al.. 2011. Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars. Plant Cell Reports, v. 30, p. 439-448), however, this process may be improved by using different regulators (Ali et al. 2008. An efficient protocol for large scale production of sugarcane through micropropagation. Pakistan Journal of Botany, v.40, p. 139-149; Nieves et al. 2008. Effect of exogenous arginine on sugarcane (Saccharum sp.) somatic embryogenesis, free polyamines and the contents of the soluble proteins and proline. Plant Cells, Tissue and Organ Culture, v. 95, p. 313-320; Wamaitha et al. 2010. Thidiazuron-induced rapid shoot regeneration via embryo-like structure formation from shoot tip-derived callus culture of sugarcane. Plant Biotechnology, v. 27, p. 365-368). However, such improvements are limited to few varieties and few laboratories managed to repeat these pioneer works in sugarcane tissue culture techniques.

[0013] Over the last decades, various scientific research have been carried out to develop efficient methods of genetic engineering of sugarcane. Different transformation techniques using electroporation, treatment with polyethylene glycol (PEG), microprojectile bombardment and Agrobacterium tumefaciens were used to introduce transgenes or modified genes in cells and cane calluses. However, there is still a need for improvements on handling and control of the in vitro culture conditions, considering the best age, type and stage of the embryonic culture to guarantee an efficient protocol for genetic engineering of this plant species. [0014] Additionally, some important sugarcane varieties exhibits recalcitrance to tissue culture and/or genetic manipulation, difficulties in tissue culture propagation, low rates of induction and regeneration of embryogenic calluses, and the impossibility of using the zygotic embryo as a target tissue in genetic transformation [(Anderson & Birch, 2012; Basnayake, Moyle, & Birch, 2011; Molinari et al., 2007)], which makes the development of a portfolio of genetically modified or edited varieties for this species even more challenging when comparing to other cereal crops.

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

[0016] Therefore, there is a clear need to develop cell genetic manipulation methods and approaches directed to sugarcane in order to satisfy the needs of a high-throughput commercial biotechnology pipeline of this species.

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

SUMMARY OF THE INVENTION

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

[0019] An aspect of the present disclosure includes methods of transforming a plant cell or plant tissue to include a trait of interest, said method including: (a) culturing a plant cell or plant tissue in vitro; (b) introducing a sequence of interest into the cell or tissue from step (a) thereby producing a transformed tissue or cell; (c) cultivating the cell or the tissue of step (b) in a culture medium for at least 15 to 60 days at 20°C to 35°C; (d) performing heat shock of the cell or tissue from step (c) for at least 1-3 days at 30°C to 45°C; and (e) regenerating the cell or tissue of (d), wherein the regenerated cell includes the trait of interest. In some embodiments of this aspect, wherein in step (c) the cell or tissue is maintained in the culture medium without manipulation or subculturing. Some embodiments of this aspect further include repeating steps (c) and (d) a second time to add a second selection step. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, step (b) further includes at least one of the additional steps of: (i) preparing Agrobacterium strains including the sequence of interest; (ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension of (i); (iii) co-cultivating the plant cell or tissue in a co-cultivation medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or (iv) cultivating the transformed plant cell and tissue in a rest medium including an agent (e.g. antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark. Some embodiments of this aspect further include step (f) allowing elongation of the regenerated plantlets from step (d). Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include screening the cells or the tissue between steps (b) and (c), screening the plantlets after step (e) or screening the plants after step (f) to identify the sequence introduced into the cells or tissues or the trait of interest. In some embodiments of this aspect, said sequence of interest includes at least one expression cassette including a nucleic acid that confers resistance to a selection agent, and wherein said selection agent is used to select the genetically altered plant cells and tissue at step (c) and/or after step (c). Some embodiments of this aspect further include selecting the genetically altered cells or tissues at step (c), selecting the genetically altered cells or tissues between steps (c) and (d), or selecting the genetically altered plantlets after step (e), optionally by using selectable markers. In some embodiments of this aspect, step (b) is achieved through Agrobacterium transformation, microprojectile bombardments, nanoparticle delivery, viral delivery, or a combination thereof. In some embodiments of this aspect, the polynucleotide includes a recombinase sequence under control of an inducible promoter and at least one polynucleotide sequence of interest, wherein both sequences are flanked by the recombination sites. In some embodiments of this aspect, the inducible promoter is selected from the group consisting of a stress-inducible promoter and a chemicalinducible promoter. Some embodiments of this aspect further include excising the polynucleotide sequences flanked by the recombination sites through the induction of expression of the site-specific recombinase by the conditions of the culturing at steps (c) and (d). Some embodiments of this aspect further include culturing the cells after step (c) in a culture medium including Abscisic Acid (ABA). In some embodiments of this aspect, the ABA is present at a concentration of 20 uM to 150 uM, preferably at a concentration of 50 uM to 100 uM. In some embodiments of this aspect, the culture medium further includes polyethylene glycol (PEG) in a range of 20 uM to 100 uM. In some embodiments of this aspect, the sequence of interest is selected from the group consisting of CRISPR machinery genes, selectable markers, herbicidal genes, silencing genes, dead nuclease genes, transcription factor genes, growth or development genes, morphogenes, reporter genes, insecticidal genes, DNA templates for homologous recombination, suppressor genes, agronomic trait genes, and a combination thereof. In some embodiments of this aspect, step (c) is performed in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days. In some embodiments of this aspect, step (c) is performed at 25°C to 30°C, more preferably 25°C to 29°C, particularly at 27°C. In some embodiments of this aspect, step (d) is performed at 35°C to 40°C, more preferably 35°C to 37°C, particularly at 35°C. In some embodiments of this aspect, step (d) is performed in 2 to 3 days, more preferably 3 days. In some embodiments of this aspect, the plant cell or plant tissue from step (a) is derived from the group consisting of embryo, callus, leaf disk, buds, axilliary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts, and any cells or tissues receptive to the introduction and uptake of a sequence. In some embodiments of this aspect, the plant cell or plant tissue from step (a) is a callus. In some embodiments of this aspect, transformation efficiency is increased by at least 5% as compared to a conventional method of transforming cells. In some embodiments of this aspect, transformation efficiency is increased by at least 10% - 30% as compared to a conventional method of transforming cells. In some embodiments of this aspect, the plant cell or plant tissue is derived from a sugarcane plant, plantlet, plant part, or plant tissue. [0020] Some aspects of the present disclosure relate to a plant, plant part, seed, or progeny plant including a sequence or a trait introduced by the method of any one of the preceding embodiments.

[0021] A further aspect of the present disclosure relates to methods of increasing regeneration rate of plant cells or plant tissues including: (a) culturing a plant cell or plant tissue in vitro; (b) cultivating the cell or the tissue of step (a) in a culture medium for at least 15 to 60 days at 20°C to 35°C; (c) performing heat shock treatment of the cell or tissue from step (b) for at least 1-3 days at 30°C to 45°C in a culture medium; and (d) regenerating the cell or tissue of step (c). In some embodiments of this aspect, regeneration efficiency is increased by at least 5% as compared to a conventional method of regenerating cells and tissues. In some embodiments of this aspect, regeneration efficiency is increased by at least 50% - 100% as compared to a conventional method of regenerating cells and tissues. In some embodiments of this aspect, in step (b) the cell or tissue is maintained in the culture medium without manipulation or subculturing. Some embodiments of this aspect further include repeating steps (b) and (c) a second time to add a second selection step. In some embodiments of this aspect, step (b) further includes at least one of the additional steps of: (i) preparing Agrobacterium strains including the sequence of interest; (ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension of (i); (iii) co-cultivating the plant cell or tissue in a co-cultivation medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or (iv) cultivating the transformed plant cell and tissue in a rest medium including an agent (e.g. antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark. Some embodiments of this aspect further include step (e) allowing elongation of the regenerated plantlets from step (d). Some embodiments of this aspect further include culturing the cells after step (b) in a culture medium including Abscisic Acid (ABA). In some embodiments of this aspect, the Abscisic Acid (ABA) is present at a concentration of 20 uM to 150 uM, preferably at a concentration of 50 uM to 100 uM. In some embodiments of this aspect, the composition further includes polyethylene glycol (PEG) in a range of 20 uM to 100 uM. In some embodiments of this aspect, step (b) is performed in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days. In some embodiments of this aspect, step (b) is performed at 25°C to 30°C, more preferably 25°C to 29°C, particularly at 27°C. In some embodiments of this aspect, step (c) is performed at 35°C to 40°C, more preferably 35°C to 37°C, particularly at 35°C. In some embodiments of this aspect, step (c) is performed in 2 to 3 days, more preferably 3 days. In some embodiments of this aspect, the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disk, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts, and any cells or tissues receptive to the introduction and uptake of a sequence. In some embodiments of this aspect, the plant cell or plant tissue of step (a) is a callus. In some embodiments of this aspect, the plant cell or plant tissue is derived from a sugarcane plant, plantlet, plant part, or plant tissue.

[0022] Some aspects of the present disclosure relate to a plant, plant part, seed, or progeny plant including a sequence or a trait introduced by the method of any one of the preceding embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows expression cassettes for Agrobacterium transformation. The sequences of the expression cassettes are provided in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0024] FIGS. 2A-2B show representative images of the steps of the transformation protocol of the present disclosure. FIG. 2A shows plant tissue at regeneration step of the transformation protocol of the present invention. FIG. 2B shows plantlets at the elongation step of the transformation protocol of the present invention. The plant tissue and plantlets shown in FIGS. 2A-2B are of a sugarcane variety.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments. Unless defined otherwise, all the technical and scientific terms used herein have the same meaning as that understood by one skilled in the art to which the invention pertains. Unless indicated otherwise, all numbers expressing amounts, percentages and proportions, and other numerical figures used in the specification and in the claims, should be understood as being modified, in all cases, by the term “about”. So unless indicated otherwise, the numerical parameters shown in the specification and in the claims are approximations that may vary, depending on the properties to be obtained. [0026] The present disclosure provides a method for the efficient transformation of plants. More particularly, the method of the present invention is useful in agriculture for the transformation and regeneration of a monocotyledonous plant such as sugarcane.

[0027] The term “transformation” is used to refer the transfer of nucleotide or polypeptides sequences of interest into plant cells either transiently or stably, to introduce heterologous and/or modify endogenous sequences in the plant genome, or to modify the expression pattern of genes and/or cellular function of interest without changes to the original DNA. The term “transformation” includes but is not limited to nanotubes transformation, grafting, vortexing with silica fibers, microparticle/nanoparticle bombardment, Agrobacterium-mediated transformation, microinjection, polyethylene glycol (PEG) procedures, liposome-mediated DNA uptake, electroporation, nanoparticle delivery, among others.

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

[0029] The present disclosure relates to methods for producing these genetically altered plants, preferably sugarcane plants. In one embodiment of the present disclosure provides a genetically altered sugarcane plant including one or more transgenes (i.e., one or more heterologous genes) or one or more edited gene sequences in the sugarcane genome (i.e., one or more edited endogenous genes). In some embodiments, these methods use morphogenes to increase even more the transformation efficiency.

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

[0031] The terms “polynucleotide,” “nucleotide”, “nucleic acid” and “gene” are used interchangeably herein to refer to a polymer of nucleotide residues (DNA or RNA). The terms apply to nucleotide polymers in which one or more nucleotide residues is an artificial chemical analogue of a corresponding naturally occurring nucleotide, as well as to naturally occurring nucleotide polymers.

[0032] One embodiment of the present disclosure provides a method of transforming a plant cell or plant tissue to comprise a trait of interest, said method comprising: a. In vitro culturing a plant cell or plant tissue; b. introducing a sequence of interest in the cell or tissue from step (a) thereby producing a transformed tissue or cell thereof; c. cultivating the cell or the tissue of step (b) in a culture medium for at least 15 to 60 days at 20 to 35°C; d. performing heat shock treatment of the cell or tissue from step (c) for at least 1 to 3 days at 30 to 45°C; e. regenerating the cell or tissue of (d) comprising the trait of interest.

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

[0034] Any methodology known in the art to introduce a sequence of interest in a plant cell or tissue can be used in practicing the inventions disclosed herein. Any methodology known in the art to delete, insert or otherwise modify the cellular DNA (e.g., genomic DNA and organelle DNA) or the expression pattern of a DNA and/or a cellular function can be used in practicing the inventions disclosed herein. The term “introducing” in the context of inserting a nucleotide or polypeptide into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a polynucleotide/polypeptide into a cell. “Introducing” includes reference to the stable or transient transfer of a nucleotide or protein sequence to a plant cell or tissue, as well as the transfer or incorporation by cross-breeding. Therefore, “introduced” includes the incorporation into the genome of the cell (e.g. DNA of chromosome, plasmid, plastid, or mitochondria), converted into an autonomous replicon, or expressed transiently (e.g. Transfected mRNA). General molecular techniques used in the invention are provided, for example, by Sambrook et al. (eds.). 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, a disarmed Ti plasmid, containing a genetic construct for deletion or insertion of a target gene, in Agrobacterium tumefaciens can be used to transform a plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the present invention in combination with procedures described in the art. Ti-plasmid vectors each contain the gene between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Other types of vectors can also be used to transform the plant cell.

[0035] Recombinant DNA/RNA technology has enabled the isolation of genes and their stable insertion into a host genome or transient insertion and expression in the host cell. This technique can be defined as the controlled introduction of nucleic acids into a recipient genome, excluding introduction by fertilization. It is a controlled process where a defined DNA/RNA fragment is introduced into the host (or recipient) and can be integrated into it. The stable or transient insertion of these molecules into a host genome gives rise to an individual with a genome that is equal or substantially equal to the recipient (host) of the recombinant molecule, but with a new and particular feature. “Substantially equal” means a genome with more than 80%, preferably 85%, 90%, 95%, 98%, 99% or 100% of identity in relation to the recipient.

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

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

[0038] Agrobacterium tumefaciens and A. rhizogenes are gram negative soil phytopathogenic bacteria belonging to the Rhizobiaceae family that cause diseases in dicotyledons, known as crown and hairy root galls, respectively. In this plant-pathogen interaction there is a process of natural gene transfer between the agrobacterium and the plant cell wherein fragments of bacterial DNA are transferred into the plant cell (T-DNA), integrating with the nuclear genome. In its natural form, the bacterium transfers T-DNA (“transferred DNA”), which is part of the bacterial plasmid called Ti (“tumor-inducing”) and integrates into the genome of infected plant cells. The T-DNA fragment that is transferred to the plant cell is comprised of genes involved in the constitutive biosynthesis of phytohormones (auxins and cytokinins), which alter the normal developmental program of infected tissue and cause tumor formation. In addition, it also contains oncogenes for the synthesis of sugars and amino acids called opines, which serve as carbon and nitrogen sources for bacteria (Oger et al. 1997). Repeated ends of 25 base pairs (bp) at the right and left edges delimit the T-DNA and are essential for its transfer. Phenolic compounds released by injured plant tissues activate specific regions (vir regions), initiating the process of transfer of T-DNA to the plant cell. Agrobacterium also has chromosomal (chv) genes that promote binding between bacterial and host cells, allowing the formation of the pore passage of the T-DNA-containing complex (Sheng & Citovsky. 1996).

[0039] Since the segment to be transferred is defined by its edges, any sequence flanked by the edges can be transferred to a plant by means of agrobacteria, making it possible to manipulate these sequences in order to transfer coding sequences of interest. The replacement or deletion of the coding regions of wild-type T-DNA (oncogenes) allows for the generation of non-oncogenic (disarmed) Agrobacterium strains, which can carry the sequences of interest. The modified T-DNA is able to transfer the sequences of interest to plants because the virulence genes (vir region) remain intact. [0040] Additionally, the Agrobacterium indirect transformation system allows for the transfer of artificial plasmid constructs to plants as long as the constructs contain such T- DNA edges, which enables the flexibility to use molecular tools and materials developed for other bacterial strains. These artificial plasmid constructs have promoters from different origins, as for example, plant promoters, viral promoters, bacterial and or chimeric promoters, besides genes that confer antibiotic resistance, herbicide resistance or tolerance or enzymatic activity (phosphomannose isomerase (PMI)/mannose (Man)) so these markers can be used for the selection of transformed cells or plants. These constructions also can contain auxiliary genes which interfere with relevant morphogenesis signaling pathways, enhancing the efficiency of the genetic transformation process and regeneration of vegetal tissues.

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

[0042] In one embodiment, the method of transforming a plant cell or plant tissue plant of the instant invention comprises the introduction of a sequence into a plant cell or tissue mediated by Agrobacterium strain (step b). A plant cell or plant tissue is placed in contact with an Agrobacterium strain. This is the inoculation phase and may be for at least about one minute up to about 12 hours, more preferably from about 5 minutes to about 2.5 hours, even more preferably from about 25 minutes to about 40 minutes at room temperature and with or without stirring. During or after the inoculation, it is possible to apply some treatments to assist the infection, such as, for example, vacuum infiltration and sonication of the solution of Agrobacterium. For example, in the vacuum infiltration, the tissue or the plant cell in contact with the bacterial suspension is subjected to a vacuum pressure, preferably from -300 mmHg to -1000 mmHg, more preferably from 400 mmHg to 800 mmHg, even more preferably from -500 mmHg to -700 mmHg, usually for a period of 1 to 10 minutes, more preferably from 1 to 7 minutes, even more preferably from 1 to 5 minutes. In another non-limitative example, the vacuum infiltration occurs in vacuum pressure of -700 mmHg for 5 minutes. Further in this inoculation phase, to improve the transformation efficiency, it is possible to incorporate additives such as acetosyringone and surfactants inside the suspension of Agrobacterium.

[0043] Optionally, in some embodiments, the cell or the plant tissue to be infected at step (b), before Agrobacterium inoculation, may be subjected to a temperature shock pretreatment, in which said tissue or cell is placed in a liquid plant culture medium such as Murashige and Skook, Gamborg's, Chu (Ne), Schenk and Hildebrand, and other known by those skilled in the art, pre-heated at the temperature in which the heat shock pretreatment will be conducted. The tissue or plant cell is then incubated in an incubator or water heating bath at a temperature above the temperature at which the inoculation will occur (for example, room temperature). So for example, the temperature of the temperature shock pre-treatment may occur at a temperature of about 30°C to about 55°C, preferably from about 35°C to about 50°C, even more preferably from about 40°C to 45°C, for a period from about 1 minute to about 60 minutes, about 1 minute to about 50 minutes, about 1 minute to about 40 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes. In another non-limitative example, the temperature shock treatment comprises placing and keeping the tissue or plant cell in a liquid plant culture medium pre-heated to a temperature of about 45 °C for about 5 minutes.

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

[0045] At step b), the period between the moment soon after inoculation (contact of the Agrobacterium with the plant tissue) to the moment when the bacteria is withdrawn or inactivated after the inoculation, the infected plant tissue or tissue is incubated on a support to enable the transfer of T-DNA of the Agrobacterium for the plant cells (“cocultivation phase”). In one embodiment, the co-cultivation of the plant tissue with Agrobacterium occurs on a culture medium, a filter paper or any other appropriate support.

[0046] The inoculated tissue may be co-cultivated for about 1 to 30 days, preferably from 1 to 20, more preferably from 1 to 10, and even more preferably, from 1 to 5 days. During the co-cultivation step, the temperature may be any suitable temperature for the target plant known in the art. Illustratively for sugarcane, the temperature may range from about 15°C to about 30°C, from about 16°C to about 29°C, from about 20°C to about 25°C, from about 21°C to about 24°C, or about 22°C to about 23°C. In some embodiments, the co-cultivation step occurs in the absence of light.

[0047] For purposes of this invention, “culture medium” refers to any media used in the art for supporting the viability and growth of a plant cell or tissue, or the growth of an entire plant, such as Murashige and Skook, Gamborg's, Chu (Ne), Schenk and Hildebrand, and other known by those skilled in the art. Such media commonly include defined components, but not limited to: macronutrients, providing nutritional sources of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium and iron; micronutrients, such as boron, molybdenum, manganese, cobalt, chlorine, iodine and zinc; carbohydrates, such as maltose, sorbitol and saccharide; phytohormones; vitamins; selection agents such as antibiotics or herbicides for selecting transformed cells or tissues; phenolic compounds (preferably those found in exudates of injury of plants, such as acetosyringone, sinapinic acid, syringic acid, ferulic acid, catechol, gallic acid, among others), antioxidants (for example, dithio treitol), and gelling agents. It may also include complex components not defined, such as casein hydrolyzate, coconut water, yeast extract and activated carbon.

[0048] In one aspect, the culture media of each step of the method of transformation of the present invention has particularities and may be any culture medium of plant tissues known in the art. Preferably, the culture media of the present invention are semi-solid and comprises a gelling agent. “Gelling agent” means any substance that increases the viscosity of a solution without substantially changing its properties, and include those gelling agents usually employed in plant tissue culture, such as agar, Agargel™, Phytablend™, Agargellan™, carrageenan and gellan gum (Gelzan™, Gelrite™, Phytagel™). [0049] Alternatively, direct nucleic acid/protein transfer can be used to directly introduce a molecule into a plant cell. One method of direct nucleic acid transfer is to bombard plant cells with a vector comprising DNA for insertion using a particle gun (particle-mediated biolistic transformation). Other methods for transformation of plant cells include protoplast transformation (optionally in the presence of polyethylene glycols (PEGs)); ultrasound treatment of plant tissues, cells, or protoplasts in a medium comprising the polynucleotide/polypeptide or the vector; microinjection of the polynucleotide/polypeptide or vector into plant material; microinjection, vacuum infiltration, sonication, use of silicon carbide, chemical transformation with PEG, electroporation of plant cells and the like. Disadvantages of direct transformation include challenges related to regeneration of plant tissue and the low transgene expression.

[0050] In addition, plant transformation can be performed by site direct insertion through homologous recombination mediated by nucleases (genome editing). In recent years, genome editing technology based on use of engineered or chimeric nucleases has enabling the generation of genetically modified organisms in a more precise and specific way. The introduction of exogenous or foreign genes occur by homologous recombination through introduction of a Homologous recombination template (HR) having the exogenous DNA linked to a DNA fragment homologous to the genome of the receptor organism. The tools available include the chimeric enzymatic system CRISPR(clustered, regularly interspaced, short palindromic repeats) - Cas, the Zinc finger nucleases (ZFN) and TAL effector nucleases (TALENs). Crispr-Cas systems are enzymatic systems including two main components: a endonuclease (Cas) and a guide- RNA (single-guide RNA - sgRNA; a guide to the specific cleavage site of Cas endonuclease). The guide RNA may also include two components: a Crispr RNA (crRNA) - a sequence of 17-20 mer complementary to specific DNA genomic sequences and, optionally, a tracr RNA. The specific cleavage performed by endonuclease and guide by the sgRNA is repair by homologous recombination, specifically inserting the exogenous DNA flanked by the homologous sequences to the cleavage site. The introduction of this enzymatic system to the cell could occur by several methods, including using plasmids, through direct or indirect transformation, or using carriers like proteins and other chemical agents. The expression of the system components may occur in a transient or stable manner, using the cellular machinery of the receptor organism or being used in a exogenous way, in vitro, delivering to the target cell or tissue all the components ready to use (endonucleases + sgRNA, in vitro transcribed and combined before cell delivery). The description presented herein is not exhaustive and should not limit the use of different variations, systems and methods of genome editing on scope of the present invention, known in the state of the art and even the ones not yet discovered. [0051] In another embodiment, the plant transformation also comprises delivery of genome editing reagents for modification of endogenous genes (knock-out, correction, overexpression, etc.) by base editing or template editing (HR or prime editing). According to this embodiment, editing reagents are delivery at step (b) by a plasmid containing a nuclease gene (e.g., Cas9 or Cpfl) and its crRNA. In another embodiment, the genome editing reagents are delivered using a ribonucleoprotein (RNP) complex. For editing mediated by homologous recombination (HR), a homologous template in the format of a plasmid is delivered in addition to Cas and crRNA. In a further embodiment, a homologous template in the format of dsDNA or ssDNA is delivered in addition to Cas and crRNA. The HR template may be delivered in the same plasmid or a separate plasmid as that of the genome editing reagent too. Genome editing reagents in plasmid(s) may be delivered by Agrobacterium transformation or particle bombardment. When RNP is used and/or when an HR template is used in a separate plasmid, particle bombardment may be used for delivery. A combination of both, plasmid and or RNP delivery methos is also provided, combining sequentially or at the same time different methods of plant cell or tissue transformation.

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

[0053] It is evident that the transformation of plants may involve the construction of an expression cassette or an expression vector that will act in a particular cell. Said expression cassette or vector may comprise a nucleotide sequence that includes a gene under the control of, or operably linked to, a regulatory element (for example, a promoter). The expression cassette or expression vector may contain one or more genes such as combinations of operably linked genes and regulatory elements. The vector may be a plasmid and can be used alone or in combination with other plasmids to provide transformed cells using transformation methods as provide by the present invention to incorporate the genetic sequences of interest or the characteristics into the plant cell or tissue.

[0054] Introduced genetic elements, whether in an expression vector or expression cassette, which result in the expression of an introduced gene, will typically comprises in the 5 ’-3’ transcription direction: a transcriptional and translational initiation region, a DNA sequence of interest, a functional transcriptional and translation termination region in plants. The transcription initiation region, the promoter, may be native or homolog or foreign or heterologous to the host., such region comprises usually a plant-expressible promoter. A “plant-expressible promoter” as used herein refers to a promoter that ensures expression of the genetic alteration(s) of the invention in a plant cell. Promoters suitable for plant expression may be isolated from plants or from other organisms. Several promoters have been isolated or developed including constitutive promoters, inducible promoters, and promoters that are responsive to tissue- specific abiotic stresses, tissue specific or cell specific, among others. Many of these promoters have intronic sequences described as relevant for proper gene expression. In a preferred aspect of the invention, promoters are constitutive promoters and may be selected from the non-limiting group consisting of CaMV 35S, CoYMV (Commelina yellow mottle virus), FMV 35S, ubiquitin (Ubi), Actin Rice Promoter (Act-1), Act -2, nopaline synthase promoter (NOS), octopine synthase promoter (OCS), com alcohol dehydrogenase promoter (Adh-1), PvUbil, among others. In one embodiment of the invention, the promoter is the Brachypodium distachyon ubiquitin gene promoter (BdUbilO). In one embodiment of the invention, the promoter is the Zea mays ubiquitin gene promoter (ZmUbil). Examples of promoters directing constitutive expression in plants are known in the art and include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic vims (CaMV), e.g., of isolates CM 1841 (Gardner et al, Nucleic Acids Res, (1981) 9, 2871- 2887), CabbB S (Franck et al, Cell (1980) 21, 285-294) and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., Plant Mol Biol, (1992) 18, 675-689), the gos2 promoter (de Pater et al, The Plant J (1992) 2, 834-844), the emu promoter (Last et al, Theor Appl Genet, (1990) 81, 581-588), actin promoters such as the promoter described by An et al. (The Plant J, (1996) 10, 107), the rice actin promoter described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. (Plant Mol Biol, (1998) 37, 1055-1067), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S4 or S7 promoter), an alcohol dehydrogenase promoter, e.g., pAdhlS (GenBank accession numbers X04049, X00581), and the TRT promoter and the TR2' promoter (the "TRT promoter" and "TR2' promoter", respectively) which drive the expression of the G and 2' genes, respectively, of the T DNA (Velten et al, EMBO J, (1984) 3, 2723 2730). Alternatively, a plant-expressible promoter can be a tissue- specific promoter, i.e., a promoter directing a higher level of expression in some cells or tissues of the plant. These plant promoters can be combined with enhancer elements, they can be combined with minimal promoter elements, or can include repeated elements to ensure the expression profile desired.

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

[0056] Terminator sequences are also contemplated on the expression cassette. Examples of suitable and functional plant polyadenylation signals include those from the Agrobacterium tumefaciens nopaline synthase gene (nos), pea proteinase inhibitor II gene rbcS (ribulose- 1,5-bisphosphate carboxylase small subunit), tobacco Lhcbl (tobacco chlorophyll a/b-binding proteins), heat shock protein (Hsp), CaMV 35S, octopine synthases, and alpha-tubulin genes among others. [0057] An introduced gene of the present invention can be inserted in host cell DNA so that the inserted gene part is upstream (i.e., 5') of suitable 3' end transcription regulation signals (e.g., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the gene in the plant cell genome. Preferred polyadenylation and transcript formation signals include those of the nopaline synthase gene (Depicker et al, J. Molec Appl Gen, (1982) 1, 561-573), the octopine synthase gene (Gielen et al, EMBO J, (1984) 3:835 845), the SCSV or the Malic enzyme terminators (Schunmann et al, Plant Funct Biol, (2003) 30:453-460), and the T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981 6998), which act as 3' untranslated DNA sequences in transformed plant cells. In some embodiments, one or more of the introduced genes are stably integrated into the nuclear genome. Stable integration is present when the nucleic acid sequence remains integrated into the nuclear genome and continues to be expressed (e.g, detectable mRNA transcript or protein is produced) throughout subsequent plant generations. Stable integration into and/or editing of the nuclear genome can be accomplished by any known method in the art (e.g., microparticle bombardment, Agrobacterium-mediated transformation, CRISPR/Cas9, electroporation of protoplasts, micro injection, etc.).

[0058] Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expressing genes in response to a specific signal, such as physical stimulus (e.g. Heat shock genes), light (e.g. ribulose-bis-phosphate carboxylase 1.5), hormones (e.g. glucocorticoid), antibiotic (e.g. tetracycline), metabolites and stress (e.g. drought). Other functional transcription and translation elements in plants can be used, such as, for example, untranslated 5’ leader sequences, 3’ transcription termination sequence and poly adenylate addition signal sequences.

[0059] The term recombinant or modified nucleic acids refers to polynucleotides which are made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.

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

[0061] Where a recombinant nucleic acid is intended for expression, cloning, or replication of a particular sequence, DNA constructs prepared for introduction into a host cell will typically comprise a replication system (e.g. vector) recognized by the host, including the intended DNA fragment encoding a desired polypeptide, and can also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Additionally, such constructs can include cellular localization signals (e.g., plasma membrane localization signals). In preferred embodiments, such DNA constructs are introduced into a host cell’s genomic DNA, chloroplast DNA or mitochondrial DNA.

[0062] In some embodiments, a non-integrated expression system can be used to induce expression of one or more introduced genes. Expression systems (expression vectors) can include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides can also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, cell wall, or be secreted from the cell. A non-integrated expression system allows transient expression, e.g., of morphogenes, so that heterologous sequences are only expressed during a limited time period. In some embodiments of the present disclosure, morphogenes are transiently expressed at one or more stages of the transformation process, and then the plant produced using the transformation process does not include the morphogene. [0063] Selectable markers useful in practicing the methodologies of the invention disclosed herein can be positive selectable markers. Typically, positive selection refers to the case in which a genetically altered cell can survive in the presence of a toxic substance only if the recombinant polynucleotide of interest is present within the cell. Negative selectable markers and screenable markers are also well known in the art and are contemplated by the present invention. One of skill in the art will recognize that any relevant markers available can be utilized in practicing the inventions disclosed herein. Thus, plant expression cassettes useful in practicing the invention can include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that enables transformed cells containing the marker to be either recovered via negative selection (that is, inhibiting the growth of cells that do not contain the selection marker gene) or via positive selection (that is, screening for the product produced by the genetic marker). Many of the genetic marker genes suitable for transforming plants are known and include, for example, genes that encode for enzymes which metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which may be sensitive to the inhibitor. Some methods of positive selection are known in the art. The gene selection marker may, accordingly, enable the selection of transformed cells while the growth of cells that do not contain the inserted DNA can be suppressed by the selection compound. The preference for one selection marker gene occurs at the discretion of the technician, but any one of the following selections markers can be used, as well as any other gene not listed here. Examples of selection markers include, but are not limited to, resistance or tolerance to kanamycin (e.g, nptll), hygromycin (HyG), bleomycin, G418, methotrexate, phosphinothricin (Bialaphos, Bar gene), imidazolinone, glyphosate (EPSPS), sulfonylureas and triazolopyrimidine herbicides, such as chlorosulforon, bromoxynil and dalapon, lethal genes, PMI, ALS gene , GUS, or fluorescent markers or reporter genes (e.g., GUS, GFP, CFP, YFP, RFP, dsRED, Td-Tomato, mNeonGreen, AmCyan, mCherry, Ruby, etc.).

[0064] Some embodiments of this aspect further include selecting the genetically altered cells or tissue at step (c) or selecting between steps (c) and (d) or selecting the genetically altered plantlets after step (e), optionally by using selectable markers. These selectable markers may be nptll or EPSPS. In some embodiments of this aspect, step (b) is achieved through Agrobacterium transformation, microprojectile bombardments, nanoparticle delivery, viral delivery, or any other methods.

[0065] Optionally, in some embodiments, after the co-culture step (b-iii), the transformed cells can be subjected to a rest step. As used herein, “rest” refers to a step in which the plant cells, for example, embryogenic calluses, are incubated after the introduction of the sequence of interest by the infection mediated by Agrobacterium. The rest enables the preferred growth of a callus from transformed cells containing the sequence of interest and is usually carried out in the absence of selective pressure. The transformed plant tissue is subjected to a rest medium that typically includes an agent (e.g. antibiotic) that inhibits the growth of Agrobacterium. Said agents are known in the art and include cefotaxime, timetin, vancomycin, carbenicillin and the like. The concentrations of said agent will vary according to the standard for each antibiotic. A person skilled in the art will recognize that the concentration of the inhibitor agent of Agrobacterium may be optimized for a particular transformation protocol without undue experimentation .

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

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

[0068] The method provided herein further includes selecting the genetically altered plant cells or tissue comprising at least one copy of the gene sequence of interest (step c) or the protein of interest. “Select”, as used herein, means the situation in which a selective agent is used for the transformants, wherein said selective agent will enable the preferred growth of genetically altered plant cells or tissue. “Select” also means the step of the process wherein the genetically altered cells or tissues are maintained in an ideal culture condition for expression of the trait of interest in a manner that the trait can be used for selecting the genetically altered cells or tissues. As indicated above, any suitable selection marker, selection condition or selection method can be used. In some embodiments, an agent is also added to inhibit the growth of Agrobacterium. The selection may occur in conditions of light or dark, depending on the plant species being transformed, and on the genotype, for example. In the case of transformation of calluses, it is possible to maintain separate individual calluses to ensure that only one plant is regenerated per callus and, therefore, all the regenerated plants are derived from independent transformation events. In an embodiment the selection step is the step c) of the transformation method described herein. In a preferred embodiment, the selection step takes place in the dark. The selecting step is performed preferable in a sealed culture plate or vessel for at least 15 to 60 days at 20 °C to 35°C. In a preferred embodiment the selection step is performed during at least 20 to 45 days, more preferred for at least 21 to 42 days and even more preferred for 30 days. In an embodiment, the selection step is performed at 25 to 30°C. In a preferred embodiment, the selection step is performed at 25 to 29°C, particularly at 27°C. In an embodiment, the selection culture medium is a semi- solid or solid medium. In an embodiment, the selection medium is liquid, and the cells or tissue are cultivating on a solid support, wherein the solid support is a filter paper, a paper, a polymeric/nylon membrane, common petri dish, among others. The liquid selection medium is added to an amount enough to form a thin film over solid support or to moisten the filter paper or the membrane, without covering the genetically altered plant cells or tissue. In some embodiment, the selection medium is a dehydration medium comprising an osmotic agent, as for example high concentration of salts.

[0069] After selection phase (step (c)), the selected genetically altered cells or tissue must be submitted to a heat shock treatment to guarantee an efficient regeneration and the efficiency of the transformation method. The combination of steps (c) and (d) are crucial to the efficiency of the transformation method of the invention, promoting a high regeneration rate of genetically altered cells or tissue. The gain is even more prominent when observed for recalcitrant species or plant varieties.

[0070] By “heat shock”, the present invention refers to the controlled temperature variation that plant cell or tissue is submitted after or right after the selection phase (step (c)). Said controlled temperature variation can be achieved by any means and equipment provided that the plant cell or tissue is submitted from 1 to 5 days at 30 °C to 45°C. Preferred, the plant cell or tissue is submitted from 1 to 3 days at 30 °C to 45°C. More preferred, the heat shock is (step (c)) performed at 35 °C to 40°C, more preferably 35 to 37°C, particularly at 35°C. Additionally, the heat shock is performed in 2 to 3 days, more preferably 3 days. More preferred, the plant cell or tissue is submitted by 3 days at 35 °C. Optionally, the plant cell or tissue can be submitted first to a “cold shock”, incubating the plant cell or tissue form step c) at reduced temperature for less than 1 day to 3 days at 1 to 10°C. After the cold shock, the plant cell or tissue is submitted to the describes heat shock treatment at step d) of the invention.

[0071] By “transformation efficiency” or “transformation frequency” the present invention refers to a parameter that may be measured by the number of cells transformed and regenerated plants which are recovered under experiment conditions. For example, when calluses are used as start-up material for the transformation, the transformation frequency may be expressed as being the number of positive events obtained per gram of callus submitted to transformation.

[0072] In some embodiments of this aspect, transformation efficiency is increased by at least 5% as compared to a method of transforming sugarcane cells that does not use at least one morphogene nucleotide sequence. In some embodiments of this aspect, transformation is increased by 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 30%. In some embodiments of this aspect, transformation efficiency is increased by 50% to 100% as compared to a method of transforming sugarcane cells that does not use at least one morphogene nucleotide sequence.

[0073] In a specific embodiment, the present invention discloses a new transformation method in which a polynucleotide used in the transformation process is efficiently excised by a recombinase/extraction system activated through a thermal /stress stimulus, enabling the production of transformed events without undesirable integration of such polynucleotide sequences. The present invention involves methods for excising a polynucleotide of interest from plant cells or tissue by a heat shock treatment enabling excision without harming the regeneration rates. Thus, such transformation method comprises additionally at step b) the introduction of at least a polynucleotide sequence of interest in the cell or tissue from step (a) wherein said polynucleotide comprises a recombinase sequence under control of a inducible promoter and at least another polynucleotide, wherein both sequences are flanked by the recombination sites. In a preferred aspect, the inducible is selected from the group consisting of a stress-inducible promoter and a chemical- inducible promoter. The said method comprises excising the polynucleotide sequences flanked by the recombination sites through out the induction of expression of the site-specific recombinase by the conditions of the culturing at steps (c) and (d). In a preferred embodiment, the thermal inducible promoter is -rabl?.

[0074] In another embodiment of the invention, the recombinase gene present in the polynucleotide of interest can be any gene widely known by a person skilled in the art, such as, but not limited to LoxP/Cre, FLP/FRT, R-RS, Bxbl l, among others. The preferable aspect of this embodiment consists of using the LoxP/Cre recombinase system. Any inducible promoter known by the skilled person in the art could be used herein, with preference to those controlled by a thermal stimulus such as but limited to pHSP18.2, pHSP26, pHSP82, pHSP18, pRabl7, among others. In a preferable embodiment of the invention, the inducible promoter is pRabl7.

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

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

[0077] The polynucleotide of interest is flanked by the recombinase sites may be CRISPR machinery genes, prime editing machinery genes, selectable markers, herbicidal genes, silencing genes, dead nucleases genes, transcription factors, growth or development genes, antibody resistance gene, morphogens, reporter genes, among others. In a preferable embodiment of the invention, the second polynucleotide of interest is a selectable marker such as an antibody or herbicidal resistant gene or a growth or development stimulation gene such as a morphogene.

[0078] Despite being well known, the combination of a stress inducible promoter with a recombinase/extraction system is not yet mastered for transforming/modifying any monocot, specially for sugarcane varieties, since there is not any data showing that it is possible to proceed with an efficient excision with a heat shock step without harming the regeneration capacity of this species.

[0079] In one embodiment of the invention, after Heat Shock treatment (step (d)), the genetically altered plant cell or tissue is regenerate into a plantlet by cultivating the cells or tissue in a culture medium devoid of growth regulators and in the presence of light; and (e) growing the genetically altered plantlet into a genetically altered plant. When a calli culture is established at step (a) and submitted to the further steps of the transformation methods of the present invention, the conditions applied at selection step (c), in combination to the Heat shock treatment at step (d), synchronize the cells/tissue development to be responsive to the regeneration culture medium and conditions, rapidly convert the calli culture in plantlets.

[0080] Some embodiments of this aspect further include screening the sugarcane cells between steps (c) and (d), screening the plantlets after step (e), or screening the plants after step (f) to identify the modification introduced. Some embodiments of this aspect further include selection any means as disclosed herein. In some embodiments of this aspect, step (b) is achieved through Agrobacterium transformation, microprojectile bombardments, nanoparticle delivery, viral delivery, or a combination thereof.

[0081] As used herein, the term "regenerable plant or plant part” or “regenerated plant or plant part” or any other term referring to the regeneration process and results refers to plant cells or tissue in which a genetic alteration, such as transformation, has been performed as to a characteristic of interest, or is a plant or plant part, such as plant cells, which is descended from a plant or plant part that has been genetically altered.

[0082] Screening and molecular analysis of genetically altered plant, plant cell or tissue of the present invention can be performed during the selection step (step c and d) or further in the regeneration phase (step (e)), utilizing nucleic acid hybridization techniques. Hybridization procedures are useful for identifying polynucleotides, such as those modified using the techniques described herein, with sufficient homology to the subject regulatory sequences to be useful as taught herein. The particular hybridization techniques are not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied by one of skill in the art. Hybridization probes can be labeled with any appropriate label known to those of skill in the art. Hybridization conditions and washing conditions, for example temperature and salt concentration, can be altered to change the stringency of the detection threshold. See, e.g., Sambrook et al. (1989) vide infra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y., for further guidance on hybridization conditions.

[0083] Similarly, screening can be performed using polypeptide-based techniques including enzyme-linked immunosorbent assays (ELISAs), fluorescence detection (if a fluorescent marker was used), or Western blots. One of skill in the art will recognize that any polypeptide-based techniques available can be utilized in screening the inventions disclosed herein.

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

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

[0086] Nucleic acids and proteins of the present disclosure can also encompass homologues of the specifically disclosed sequences. Homology (e.g., sequence identity) can be 50%-100%. In some instances, such homology is greater than 80%, greater than 85%, greater than 90%, or greater than 95%. The degree of homology or identity needed for any intended use of the sequence(s) is readily identified by one of skill in the art. As used herein percent sequence identity of two nucleic acids is determined using an algorithm known in the art, such as that disclosed by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See www.ncbi.nih.gov.

[0087] Preferred host cells or explants are plant cells or tissue. The plant cells may be derived from plants including com (e.g., maize, Zea mays), barley (e.g., Hordeum vulgar ), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), oat (e.g., Avena sativa), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), rye (e.g., Secale cereale, Secale cereanuni), sugarcane (e.g., Saccharum sp.), setaria (e.g., Setaria italica, Setaria viridis). Brachypodium sp., sorghum (e.g., Sorghum bicolor), teff (e.g., Eragrostis tef), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), wheat (e.g., common wheat, spelt, durum, einkom, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), switchgrass (e.g., Panicum virgatum), Brassica sp., or tobacco (e.g., Nicotiana benthamiana, Nicotiana tabacum). The plant cells may also be derived from other monocot and dicot plant species. Preferably, the plant cells are from monocot species, specially sugarcane (e.g., Saccharum spp.). The methods of this disclosure may be particularly suited for transformation of recalcitrant species.

[0088] Sugarcane plants of the present disclosure include species and hybrids in the genus Saccharum, e.g., Saccharum officinarum, Saccharum sinense, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, Saccharum spp., Saccharum spp. hybrid, S. edule, S. aegyptiacum, S. esculentum, S. aenicol, S. arundinaceum, S. bengalense, S. biflorum, S.ciliare, S. cylindricum, S. elephantinum, S. exaltatum, S.f allax, S. floridulum, S. giganteum, S. japonicum, S. koenigii, S. laguroides, S. munja, S. narenga, S. paniceum, S. pophyrocoma, S. purpuratum, S. ravennae, S. roseum, S. sanguineum, S. sara, S. chinense, S. tinctorium, S. versicolor, S. violaceum. Even more preferably, these are interspecific hybrids produced by cross-breeding commercial species and varieties thereof. Methods of the present disclosure are genotype-independent.

[0089] Plant cells and tissues may be derived from tissue types including embryo, callus, leaf disk, buds, axilliary buds, section or fragment of plant parts, leaf blade, stem, stem apex, leaf sheath, internodes, petioles, flower stalks, root, inflorescence and other explants. Suitably, the explant is a segment, a slice or section of tissue. Plant cells can be differentiated or undifferentiated (e.g. callus, undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf sneath, leaf cells, root cells, phloem cells and pollen). Plant cells include, without limitation, cells from seeds, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristem, somatic meristems, organogenic callus, protoplasts, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, cotyledons, immature cotyledons, embryonic axes, meristematic regions, callus tissue, cells from leaves, cells from stems, cells from roots, cells from shoots, gametophytes, sporophytes, pollen and microspores. Plant cells further include various forms of cells in culture (e.g., single cells, protoplasts, embryos, and callus tissue), wherein the protoplasts or cells are produced from a plant part selected from the group of leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo, or meristematic cell. Genetically altered plant cells, in the present context, are those which have been modified to contain a nucleic or protein molecule, contain one or more deleted or otherwise non-functional genes normally present and functional in the host cell, or a different pattern of expression of a characteristic (such as, an altered cellular function without genome modification, e.g epigenetic variations). The nucleic acid(s) and/or proteins can be introduced by any means known to the art which is appropriate for the particular type of cell, including without limitation, transformation by Agrobacterium or Bombardment, lipofection, electroporation or any other methodology known by those skilled in the art.

[0090] More preferably, the plant cell or tissue from step (b) of the method of the present invention is a callus. More preferably, the embryogenic callus is of the type II or III. Embryogenic calluses can be formed from any suitable tissue of a plant, preferably from a sugarcane plant. The culture of tissues in sugarcane is well known and follows a conventional production model of calluses and regeneration of plants initially described by Ho & Vasil. 1983. Protoplasma, 118:169-180; Brisibe et al. 1993. Plant Science, 89:85-92, and further by Falco et al. 1996. R. Bras. Fisiol. Veg., 8(2):93-97. Preferably, it uses an immature tissue to initiate the callus, such as sugarcane heart or meristems. [0091] Type I, II and III calluses can be initiated from tissues including, but not limited to, immature embryos, apex meristems, axillary meristems, microspores and others. Those cells capable of proliferating as calluses are also target cells for plant transformation. Target cells can also be somatic cells, which are those cells that, during normal development of the plant, do not contribute to reproductive processes thereof. Meristem cells (that is, capable of continuous cell division and characterized by a undifferentiated cytological appearance, normally found at growing points as root tips, axillary meristems, shoot apices, side buds and others) may represent another type of target cell. Due to the undifferentiated state and capacity for differentiation and totipotency, a single transformed meristem cell has the potential to regenerate a whole transformed plant.

[0092] Suitable cell cultures can be initiated from various types of explants. For example, for varieties of sugarcane, explants can be obtained from suitable plant tissue, including sett or sugarcane heart (set of young and curled sheets containing apical meristem), leaf blade, axillary buds, stem, stem apex, leaf sheath, internodes, petioles, flower stalks, seeds, roots or inflorescence. Suitably, the explant is a segment, a slice or section of tissue. More preferably, the explant is a section of the apical sugarcane heart portion of sugarcane saplings. The explants can be obtained from plants grown in vitro, in greenhouses or in the field. Preferably, the plant age is less than about 24 months, less than about 23 months, less than about 22 months, less than about 21 months, less than about 20 months, less than about 19 months, less than about 18 months, less than about 17 months, less than about 16 months, less than about 15 months, less than about 14 months, less than about 13 months, less than about 12 months, less than about 11 months, less than about 10 months, less than about 9 months, less than about 8 months, less than about 7 months, less than about 6 months, less than about 5 months, less than about 4 months, less than about 3 months, less than about 2 months or less than about 1 month. Preferably, the plant age is preferably about 24-12 months, more preferably about 12-8 months, even more preferably about 6-2 months. Said tissue culture is generally initiated from sterile pieces of a plant, such as outlined above. Many explant characteristics are known to affect the efficiency of initiation of the culture, however, it is considered that generally young, faster-growing tissues, or a tissue in an earlier stage of development, are more efficient. Explants cultivated in appropriate media may give rise to an unorganized mass of dividing cells (calluses) that may, in culture, be maintained more or less undefine as long as periodic subcultures are carried out in a fresh culture medium.

[0093] As used herein, “plant” refers both to the entire plant, a plant tissue, a plant part (such as embryo), a plant cell, or a group of plant cells. More preferably, the plants are monocot, and even more preferably, are those used as food or energy generation, such as rice, maize, wheat, barley, millet, sorghum, rye, triticale, sugarcane and other species such as Erianthus, Miscanthus, Narenga, Sclerostachya, and Brachypodium. Included are all the genera of the Bambusoideae subfamilies (e.g., the genus Bambusa), Andropogonoideae (e.g. genus Saccharum, Sorghum and Zea), Arundineae (e.g. genus Phragmites'), Oryzoideae (e.g. genus Oryza), Panicoideae (e.g. genera Panicum, Pennisetum and Setaria), Pooideae (Festuciadeae) (e.g. genera Poa, Festuca, Lolium, Trisetum, Agrostis, Phleum, Dactylis, Alopecurus, Avena, Triticum, Secale, and Hordeum). More specifically, a plant that may be transformed according to the present invention is sugarcane.

[0094] By means of “genetic alteration”, a plant, preferably a sugarcane plant or plant cell, may be modified to exhibit improved or superior agronomic characteristics, in relation to the non-transformed plants of the same genotype. For example, transgenic plants can be modified so as to express genes having resistance to diseases and insects, having tolerance to herbicides, which confer nutritional value, increase in the content of sucrose, of fibers, influence in the plant growth, tolerance to abiotic stresses, increased production of biomass, modification of content (composition/content) of lignin, sterility, among others.

[0095] When appropriate, the sequence of interest to be transferred to a plant may be modified to optimize the expression. For example, a sequence may be modified to improve expression in a monocot plant, more preferably, in sugarcane. Methods for synthetic optimization are available in the technique, for example, US 5,380,831; US 5,436,391 and Murray, et al. 1989. Nucleic Acids Res. 17:477-498. The preferred codons of the target plant can be determined from higher frequency codons in the target plants of interest. Other modifications can be made in order to increase the gene expression in the target plant, including, for example, the elimination of spurious polyadenylation signals, of exon-intron splice signals, of similar transposons repetitions, among others. The G-C content of the sequence may be adjusted to average levels to a given target plant, calculated having as reference the known genes expressed in the target plant. Further, the sequence may be modified so as to prevent hairpin structures in the mRNA.

[0096] In some embodiments of this aspect, step (b) is achieved through Agrobacterium transformation, microprojectile bombardments, nanoparticle delivery, viral delivery or any other transformation method as described herein. In some embodiments of this aspect, the at least one nucleotide sequence introduced encodes a nucleotide or protein sequence capable to exhibit a characteristic selected from the group consisting of expressing a fluorescent protein (e.g., GFP, CFP, dsRED, etc.), herbicide resistance or tolerance (e.g., CP4-EPSPS, BAR, ALS, etc.), Agronomic trait, and a disease/pest resistance or tolerance protein (e.g., BT, Cry, VIP, etc.), or a morphogene. In some embodiments, the agronomic trait includes a biomass trait, a sucrose trait, a flowering trait, and/or an aluminum tolerance trait.

[0097] Morphogene are genes that have been functionally demonstrated to improve somatic embryogenesis and transformation. Any combination of morphogenes may be used in the methods of the present disclosure. Thus, in some embodiments of this aspect, at least one morphogene is introduced in step (b). In some embodiments of this aspect, one nucleotide sequence, other than a morphogene, is introduced in step (b). In some embodiments of this aspect, the at least one nucleotide sequence is introduced at the same time as the morphogene. In some embodiments of this aspect, the at least one morphogene sequence is introduced before the at least one nucleotide sequence. In some embodiments of this aspect, the at least one morphogene sequence is introduced after the at least one nucleotide sequence. In some embodiments of this aspect, the morphogene and the nucleotide sequence are introduced by separate vectors. In one aspect, the vector includes a promoter operably linked to the at least one nucleotide sequence. In some embodiments of this aspect, the promoters are selected from the group of a constitutive promoter, an inducible promoter, or a tissue- specific or cell-type-specific promoter. In some embodiments of this aspect, the morphogene and the nucleotide sequence are introduced by the same vectors in different expression cassettes. [0098] In some embodiments of this aspect, the introduction of the at least one morphogene sequence is transient. In some embodiments of this aspect, the genetically altered plant of step (d) does not include the at least one morphogene sequence. In a specific embodiment, the one morphogene sequence is excised by a recombinase/extraction system activated through a thermal stimulus, according to the method described by the present invention, enabling the production of transformed events without integration of such polynucleotide sequences.

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

[0100] In some aspects, the transformation method of the present invention further comprises the step (f) for elongation of the regenerated plantlets from step (d). The elongation step comprises cultivating the plantlets from step (e) in a culture medium and maintain them for 14-21 days in the light at 27°C± 2°C. Optionally, this cultivation cycle can be repeat for additional 14-21 days in the light at 27°C± 2°C.

[0101] After elongation step, the plants were submitted to molecular and morphological analyses to confirm the incorporation of the trait of interested.

[0102] In some aspect, the present disclosure relates to a seed, plant part, or plant tissue from the genetically altered plant of any of the above embodiments. In some embodiments, the plant part is selected from the group of leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo, or meristematic cell. Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, shoots, leaves, pollen, seeds.

[0103] In some aspects, the present disclosure relates to a pollen grain or an ovule of the genetically altered sugarcane plant of any of the above embodiments. [0104] In some aspects, the present disclosure relates to a protoplast from the genetically altered sugarcane plant of any of the above embodiments.

[0105] In some aspects, the present disclosure relates to a tissue culture produced from protoplasts or cells from the genetically altered plant of any of the above embodiments.

[0106] In one aspect of the invention is provided a method of increasing regeneration of plant cells or plant tissues comprising, a) In vitro culturing a plant cell or plant tissue; b) cultivating the cell or the tissue of step (a) in a culture medium for at least 15 to 60 days at 20 to 35°C; c) performing heat shock treatment of the cell or tissue from step (b) for at least 1-3 days at 30 to 45°C in a culture medium; d) regenerating the cell or tissue of (c).

[0107] The method as disclose herein increase the regeneration efficiency by at least 5% as compared to a conventional method of regenerating cells and tissues. Preferred the said method increase regeneration efficiency by at least 50% - 100% as compared to a conventional method of regenerating cells and tissues.

[0108] Having generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

EXAMPLES

[0109] The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such a description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0110] The combination of selection step and heat shock treatment as described by the instant invention increase significantly the regeneration rate of genetically altered cells or tissues, improving the efficiency of the transformation method. To demonstrate the present invention, a recalcitrant sugarcane variety was submitted to Agrobacterium genetic transformation using a conventional protocol (Control) and the Invention Method. [0111] Three experiments were conducted with the recalcitrant sugarcane variety using a heat shock of 3 days at 35°C (step d) after a selection phase of 21 days without culture medium exchange or cell manipulation at 27°C.

[0112] The plasmid used in these examples containing a herbicide tolerance gene (expression cassette 3, SEQ ID NO: 3) and a insecticidal gene (expression cassette 4, SEQ ID NO: 4) as the traits to be incorporate in the plant cell and, a recombinase gene (moCRE, expression cassette 2, SEQ ID NO: 2) in combination with a selection marker gene (nptll, cassette 1, SEQ ID NO: 1) both flanked by recombination sites (LoxP) and planned to be excised during steps (c) and (d) of the transformation method.

1. Expression cassette 1: pBdUbil0::LoxP::nptII::T-35S

2. Expression cassette 2: P-rabl7::moCRE::T-NOS::LoxP

3. Expression cassette 3: CTP2::Herbicide gene::T35S

4. Expression cassette 4: P-2X35S::L-CAB::I-OsActl "insecticidal gene::T-35S.

[0113] The cassettes flanked by recombination sites (LoxP) were included to exemplify the possibility of using recombinase excision systems driven by thermal/stress inducible promoters (as rab-17) with efficiency, even for a recalcitrant variety, without affecting the viability of transformed cells and increasing the regeneration rate.

Example 1: Plant Material: explant for transformation (step a)

[0114] Tissue culture is normally used for transforming plants by generating cells that are potentially transformable. Maintenance of tissue cultures requires the use of culture media (mixture of nutrients and phytoregulators for growth and maintenance of cells in vitro) and controlled environmental conditions. The tissue-explant chosen to exemplify the present invention is the embryogenic callus of sugarcane.

[0115] To obtain the embryogenic calluses, young, curled leaves (heart) of sugarcane, developed in the field or greenhouse for 3-12 months, were collected for isolation of the initial explants.

[0116] After surface disinfection, cross sections about 0.05-5mm thick were cut from the region above the meristem under aseptic conditions. The sections were placed on the surface of the SCIM culture medium (Table 1). The cultures were kept in the dark at a temperature of 26°C± 2°C, and subcultivated every 15 days, for three to five cycles of 7- 28 days.

Example 2: Preparation of the Agrobacterium and Infection of the calluses (step b - i and b-ii) [0117] The culture of Agrobacterium, comprising the strain EHA105 (Hood et al. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Research, v. 2, p. 208-218), comprising the plasmid with the expression cassettes as described below (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; FIG. 1), was initiated from a glycerol stock kept at -80°C in solid LB plus appropriate antibiotics. This culture was kept in the dark at 28°C for two to three days. The suspension of Agrobacterium to infect the plant material was prepared by resuspending the culture in a liquid medium * MS plus 200pM of acetosyringone, adjusting to a final ODeoo of 0.1-1.0.

[0118] The calluses were directly transferred to the suspension of Agrobacterium, where they remained for 30 minutes, in the dark under constant stirring at 50 rpm.

[0119] After this period, the calluses were separated from the Agrobacterium and the excess suspension was removed by drying on sheets of filter paper.

[0120] Alternatively, prior to infection, the calluses can be subjected to a treatment in a * MS liquid medium at about 45°C for about 5 minutes.

[0121] Another optional treatment is the submission of the infected plant material for about 5 minutes at vacuum pressure from about -700 mmHg.

Example 3: Co-cultivation and rest of the calluses (step b-iii e b-iv)

[0122] This step was carried out in a liquid or solid SCIM culture medium (Table 1) with 7; 14; 21; 28; 35; 42 or 49g/L of Agargel™, weighting between 0.5-10g of callus per plate (100 x 20mm). The co-cultivation was carried out for a period of 1-5 days at a temperature of 22 °C in the dark.

[0123] After co-cultivation, the calluses were transferred to the DT resting medium (Table 1) plus Timentim® bacteriostatic in a concentration of 200mg/L in order to control undesirable growth of the Agrobacterium. The rest period was 5-14 days at 26°C in the dark.

Example 4: Selection of genetically altered cells or tissues (step c) and Heat Shock treatment (step d).

[0124] The calluses were transferred to the SGT selection medium (Tablet), supplemented with 200mg/L of Timentim® + 50mg/L of the geneticin selective agent. The calluses remained in this condition for 21 days at 26°C± 2°C in the dark (Selection 1, optional step) and after, was transferred to a new SGT selection medium and remained for additional 21 days at 26°C ± 2°C in the dark without any other culture replacement or cell manipulation.

[0125] After Selection step, the culture plates submitted to the method of the present invention were incubated in Bio-Oxygen Demand chamber (BOD) (Thermolab Scientific Equipments) at approximately 35°C± 2°C for 3 days (heat shock treatment - step d). The heat shock can be done with structures still in selection medium or after changing it for Regeneration medium (Table 1). The plates were sealed with micropore or plastic film, according to the profile of the material.

[0126] In the control treatment, after Selection step, the calluses were transferred to the RG1 regeneration medium.

Table 1: Culture media recipes.

Example 5: Regeneration of genetically altered plants (step e)

[0127] After selection step and/or Heat shock treatment, calluses were transferred to the RG1 regeneration medium, supplemented with 200mg/L of Timentim® + 30mg/L of geneticin, and cultivated in a photoperiod of 15 hours at 4,000 lux at 27°C± 2°C for 14 to 21 days. After 14-21 days, the calluses showing the formation of seedlings (FIGS. 2A- 2B) were transferred to the RG2 medium, supplemented with 200mg/L of Timentim® + 30mg/L of geneticin and maintained for additional 14-21 days in a photoperiod of 15 hours at 4,000 lux at 27°C± 2°C.

[0128] It is observed that the application of said heat shock in combination with the conditions applied to the selection step (c) is able to increase in 4.6X the regeneration rate of the genetically altered plant cell or tissue (Table 2).

Table 2. Regeneration rate.

[0129] This indicates that the combination of a selection step as defined by the present invention followed by a heat shock treatment not only did not affect the viability of transformed sugarcane cells, as it is necessary to achieve an increase regeneration rate.

[0130] When the plants reached an average height of five centimeters, they were transferred to the elongation medium.

Example 6: Elongation step (step f)

[0131] When the plants reached an average height of five centimeters, they were transferred to the elongation medium and maintained for 14-21 days in the light at 27°C± 2°C (Elongation 1). After, the plants were sub- cultured at the same culture medium and maintained for additional 14-21 days in the light at 27°C± 2°C (Elongation 2, optional). After elongation step, the plants were submitted to molecular and morphological analyses to confirm the incorporation of the trait of interested.

Example 7: Thermal and ABA-mediated excision by Cre-LOX recombinase system [0132] Part of the calluses submitted to the Heat Shock step (Example 4) were transferred to a regeneration medium containing ABA (RG1 plus ABA) with concentrations in the approximate range 25 to lOOuM and maintaining for a period ranging from 24 to 72 hours in a dark room at 27 ± 2 °C. Subsequently, the calluses were transferred to a new regeneration medium without ABA and in the presence of light and submitted to the conditions describe at regeneration step (Example 5).

[0133] Three analyses were carried out based on such methodology with three repetitions with 40 callus clusters each treatment/replication, totaling 120 clusters per treatment. The excision treatments were:

• Treatment 1: 3 days of Heat shock treatment in BOD;

• Treatment 2: Treatment 1 plus 50uM ABA for 24 hours; and

• Treatment 3: Treatment 1 plus lOOuM ABA for 24 hours. [0134] After regeneration step, a total of 1.163 plants were analyzed. It was found that the application of ABA in the excision phase for 24 hours was efficient to increase the percentage of excised plant in 15,6% and 17,5% (50 and lOOuM of ABA, respectively) compared to the Method without ABA.

Table 3: Excision Percentage of Plants Under Different Excision Treatments.

Example 8: ABA/PEG-mediated excision by Cre-Lox recombinase system

[0135] In order to verify the effect of ABA 50uM plus PEG 50uM for 3 days in the excision rates, two repetitions were performed with 20 calli clusters each. The excision treatments were:

• Treatment 1: 3 days of Heat shock treatment in BOD;

• Treatment 2: Treatment 1 plus 50uM ABA + 50g/L PEG for 72 hours

[0136] The results showed that adding PEG to the ABA treatment is also efficient for the excision mediated by recombinase moCre. It was observed the same range of increase as ABA treatment alone in the excision rate in comparison to the Treatment 1 (15,6%).

[0137] Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

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

2. The method of claim 1, wherein in step (c) the cell or tissue is maintained in the culture medium without manipulation or subculturing, and/or further comprising repeating steps (c) and (d) a second time to add a second selection step.

3. The method of claim 1 or claim 2, wherein step (b) further comprises at least one of the additional steps of: i) preparing Agrobacterium strains comprising the sequence of interest; ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension of (i); iii) co-cultivating the plant cell or tissue in a co-cultivation medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium; or iv) cultivating the transformed plant cell and tissue in a rest medium comprising an agent (e.g. antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark.

4. The method of claim 1, further comprising step (f) allowing elongation of the regenerated plantlets from step (d).

5. The method of any one of claims 1-4, further comprising

(i) screening the cells or the tissue between steps (b) and (c), screening the plantlets after step (e) or screening the plants after step (f) to identify the sequence introduced into the cells or tissues or the trait of interest; and/or (ii) selecting the genetically altered cells or tissues at step (c), selecting the genetically altered cells or tissues between steps (c) and (d), or selecting the genetically altered plantlets after step (e), optionally by using selectable markers.

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

7. The method of claim 1, wherein step (b) is achieved through Agrobacterium transformation, microprojectile bombardments, nanoparticle delivery, viral delivery, or a combination thereof.

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

9. The method of claim 8, further comprising excising the polynucleotide sequences flanked by the recombination sites through the induction of expression of the sitespecific recombinase by the conditions of the culturing at steps (c) and (d); and/or further comprising culturing the cells after step (c) in a culture medium comprising Abscisic Acid (ABA); optionally wherein the ABA is present at a concentration of 20 uM to 150 uM, preferably at a concentration of 50 uM to 100 uM; optionally wherein the culture medium further comprises polyethylene glycol (PEG) in a range of 20 uM to 100 uM.

10. The method of claim 1, wherein the sequence of interest is selected from the group consisting of CRISPR machinery genes, selectable markers, herbicidal genes, silencing genes, dead nuclease genes, transcription factor genes, growth or development genes, morphogenes, reporter genes, insecticidal genes, DNA templates for homologous recombination, suppressor genes, agronomic trait genes, and a combination thereof.

11. The method of claim 1, wherein:

(i) step (c) is performed in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days; (ii) step (c) is performed at 25°C to 30°C, more preferably 25°C to 29°C, particularly at 27°C;

(iii) step (d) is performed at 35°C to 40°C, more preferably 35°C to 37°C, particularly at 35°C; and/or

(iv) wherein step (d) is performed in 2 to 3 days, more preferably 3 days.

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

13. The method of claim 1, wherein transformation efficiency is increased by at least 5% as compared to a conventional method of transforming cells, or wherein transformation efficiency is increased by at least 10% - 30% as compared to a conventional method of transforming cells.

14. The method of claim 1, wherein the plant cell or plant tissue is derived from a sugarcane plant, plantlet, plant part, or plant tissue.

15. A plant, plant part, seed, or progeny plant comprising a sequence or a trait introduced by the method of any one of the claims 1 - 14.

16. A method of increasing regeneration rate of plant cells or plant tissues comprising: a) culturing a plant cell or plant tissue in vitro; b) cultivating the cell or the tissue of step (a) in a culture medium for at least 15 to 60 days at 20°C to 35°C; c) performing heat shock treatment of the cell or tissue from step (b) for at least 1-3 days at 30°C to 45°C in a culture medium; and d) regenerating the cell or tissue of step (c).

17. The method of claim 16, wherein regeneration efficiency is increased by at least 5% as compared to a conventional method of regenerating cells and tissues; or wherein regeneration efficiency is increased by at least 50% - 100% as compared to a conventional method of regenerating cells and tissues.

18. The method of claim 16 or claim 17, wherein in step (b) the cell or tissue is maintained in the culture medium without manipulation or subculturing; and/or further comprising repeating steps (b) and (c) a second time to add a second selection step.

19. The method of any one of claims 16-18, wherein step (b) further comprises at least one of the additional steps of: i) preparing Agrobacterium strains comprising the sequence of interest; ii) inoculating the plant cell or plant tissue with the Agrobacterium strain suspension of (i); iii) co-cultivating the plant cell or tissue in a co-cultivation medium capable of supporting the growth of the plant cell or tissue and inhibiting the growth of Agrobacterium', or iv) cultivating the transformed plant cell and tissue in a rest medium comprising an agent (e.g. antibiotic) that inhibits the growth of Agrobacterium for 1 to 30 days in the dark.

20. The method of claim 16, further comprising step (e) allowing elongation of the regenerated plantlets from step (d).

21. The method of claim 16, further comprising culturing the cells after step (b) in a culture medium comprising Abscisic Acid (ABA); optionally wherein the Abscisic Acid (ABA) is present at a concentration of 20 uM to 150 uM, preferably at a concentration of 50 uM to 100 uM; optionally wherein the composition further comprises polyethylene glycol (PEG) in a range of 20 uM to 100 uM.

22. The method of claim 16, wherein:

(i) step (b) is performed in 20 to 45 days, more preferably in 21 to 42 days, particularly in 30 days;

(ii) step (b) is performed at 25°C to 30°C, more preferably 25°C to 29°C, particularly at 27°C;

(iii) step (c) is performed at 35°C to 40°C, more preferably 35°C to 37°C, particularly at 35°C; and/or

(iv) step (c) is performed in 2 to 3 days, more preferably 3 days.

23. The method of claim 16, wherein the plant cell or plant tissue of step (a) is derived from the group consisting of embryo, callus, leaf disk, buds, axillary buds, internodes, root, inflorescence, cotyledon, embryonic axis, suspension culture cells, protoplasts, phloem cells, pollen, leaf disc cells, callus cells, protoplast cells, sections or fragments of plant parts, and any cells or tissues receptive to the introduction and uptake of a sequence.

24. The method of claim 16, wherein the plant cell or plant tissue is derived from a sugarcane plant, plantlet, plant part, or plant tissue.

25. A plant, plant part, seed, or progeny plant comprising a sequence or a trait introduced by the method of any one of the claims 16-24.