Regeneration of genetically modified plants

Abstract

The present invention relates to the field of plant breeding, particularly the generation of plants from cells and other tissues. More specifically, the present invention provides methods and means for improving plant regeneration, particularly from transformed or genetically modified plant cells.

Description

[Technical Field]

[0001] The present invention relates to the fields of plant breeding and biotechnology, particularly to the generation of plants from cells and other tissues. More specifically, the present invention provides methods and means for improving plant regeneration, particularly from transformed or genetically modified plant cells.

[0002] Plant breeding, the process of manipulating plant species to produce desired genotypes and phenotypes for specific purposes, has been practiced since the beginning of human civilization. With the development of genetic engineering, this agricultural field has undergone significant changes in recent decades. Various methods for plant genetic engineering have been developed. The choice of transformation method depends on many variables, primarily the plant species to be transformed, the purpose of the experiment, and the availability of the necessary equipment. Most plant transformation techniques require the use of explants with high regenerative potential as starting material. Furthermore, gene editing constitutes a new molecular biology method that can introduce specific modifications, such as insertions, deletions, or point mutations, or combinations thereof, into the genome of a plant. This requires, first of all, specific molecular equipment that possesses nuclease activity and, above all, can be directed to the target sequence to be modified with sufficient specificity to program and perform specific, site-directed mutagenesis. In recent years, specific genome editing has been developed in the field of plant biotechnology as an alternative to traditional breeding and transgenic strategies. However, currently available tools, such as meganucleases, zinc finger nucleases (ZFNs), "transcription activator-like effector nucleases" (TALENs) or CRISPR systems, have only been utilized to a limited extent in plant biotechnology due to their limited ability to regenerate the starting material of edited plants.

[0003] A wide variety of cells have the potential to develop into embryos, including haploid gametophyte cells, such as cells of pollen and embryo sacs (see Forster, BP, et al. (2007) Trends Plant Sci. 12:368-375 and Segui-Simarro, JM (2010) Bot. Rev. 76:377-404), as well as somatic cells derived from all three fundamental tissue layers of the plant (see Gaj, MD (2004) Plant Growth Regul. 43:27-47 or Rose, R., et al. (2010) "Developmental biology of somatic embryogenesis" in Plant Developmental Biology—Biotechnological Perspectives, Pua EC and Davey MR, Eds. (Berlin Heidelberg: Springer), pp. 3-26).

[0004] The ability to regenerate into a plant is often limited to a specific genotype in a plant species and is weakened in transformed and genetically modified plant cells and other precursor tissues. Even if the steps of transforming plant cells and genetically modifying them are successful, this does not necessarily mean that the desired plant will actually be obtained from the modified cells. It is assumed that the treatments applied to plant cells and other precursor tissues to achieve genetic modification will affect the development and regeneration of the plant. Therefore, the object of the present invention is to improve the effectiveness of previously known methods for generating transgenic and genetically modified plants and to support plant regeneration from modified plant cells and other plant precursors.

[0005] In the present invention, it has surprisingly been found that the Arabidopsis gene GRF5 (GROWTH-REGULATING FACTOR5) has a plant regeneration-promoting effect that has not been reported for this gene or its corresponding gene in the GRF gene family.

[0006] In Van der Knaap et al. (2000; "A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth", Plant physiology, 122(3), 695-704.), the authors identified and characterized the first member of the rice GRF gene family (OsGRF1), which is located in the intercalary meristem as a gene induced by gibberellic acid. Overexpression in Arabidopsis resulted in impaired stem growth, female sterility, and reduced male fertility. Application of gibberellic acid failed to restore the stem elongation defect in transformed plants, suggesting that OsGRF1 may be involved in GA-induced stem elongation. In 2003, Kim et al. ("The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis", The Plant Journal, 36(1), 94-104) characterized the GRF family in Arabidopsis and confirmed that its genes are expressed mainly in actively growing tissues. Analysis of null mutants and transgenic plants overexpressing AtGRF1 and AtGRF2 revealed that some members of the GRF family are involved in regulating cell expansion during leaf and cotyledon development. Furthermore, overexpressing plants showed delayed bolting time, revealing a possible role in flowering. In a study to determine the molecular mechanisms regulating cell proliferation in developing leaves, Rodriguez et al. found that miR396 antagonizes the expression pattern of its target GRF transcription factor in Arabidopsis thaliana ((2010), “Control of cell proliferation in Arabidopsis thaliana by microRNA miR396”, Development, 137(1), 103-112.).Thus, the balance between miR396 and GRFs controls the final cell number in leaves. Furthermore, the authors showed that GRFs, which target miR396, can control shoot meristem size.

[0007] In 2005, Horiguchi et al. ("The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana", The Plant Journal, 43(1), 68-78.) first characterized AtGRF5 and its interacting partner ANGUSTIFOLIA3 (AN3). Knockout mutants of atgrf5 and an3 developed narrow leaves due to reduced cell number, whereas overexpression lines of AtGRF5 and AN3 showed enhanced cell proliferation in leaf primordia. Kuijt et al. ((2014), "Interaction between the Growth-Regulating Factor and Knotted1-Like Homeobox Families of Transcription Factors", Plant Physiology, 164(4), 1952-1966.) showed that members of the GRF family function as players in a network that regulates the expression of Knotted1-Like Homebox (Knox) ​​genes, which are involved in restricting cell differentiation in the shoot meristem. AtGRF4, AtGRF5, and AtGRF6 can bind to the promoters of Knox genes and repress their expression. Arabidopsis seedlings overexpressing AtGRF4, AtGRF5, or AtGRF6 exhibit developmental abnormalities in the shoot meristem. A recent study by Vercruyssen et al. ((2015), “Growth regulating factor 5 stimulates Arabidopsis chloroplast division, photosynthesis, and leaf longevity”, Plant physiology, pp. 114) showed that Arabidopsis leaves overexpressing GRF5 exhibited a higher number of chloroplasts per cell, increased chlorophyll content, and delayed leaf senescence.

[0008] In summary, it is well known that the Arabidopsis gene AtGRF5 and other GRF genes play roles in leaf morphogenesis and stem development. Furthermore, GRF genes have been reported to function in flowering, seed, and root development, control plant growth under stress conditions, and regulate plant lifespan. However, during the course of their research, the inventors of the present invention surprisingly discovered another novel function of this gene family. GRF5 can promote plant regeneration, thereby enabling more efficient recovery of transgenic plants. The present invention enables improved regeneration from various tissues or cells (e.g., microspores), overcomes resistance to plant regeneration, particularly genotype-dependent regeneration, and improves the recovery of transgenic plants, for example, by coexpressing a gene of interest with GRF5, or by transiently coexpressing genome editing components with GRF5, as well as shortening the time required for generating and recovering transgenic lines. Therefore, a first aspect of the present invention is the use of a GRF5 polypeptide to improve the regeneration potential of plants.

[0009] Any reference below to a polypeptide or protein useful in the methods of the present invention shall be taken to mean a GRF5 polypeptide or GRF5 protein as defined herein. Any reference below to a nucleic acid or polynucleotide useful in the methods of the present invention (excluding a nucleic acid molecule optionally used as a target nucleotide sequence to be transformed or a repair template for modifying the genome of a plant) shall be taken to mean a nucleic acid or polynucleotide capable of encoding such a GRF5 polypeptide or GRF5 protein. In one embodiment, any reference to a polypeptide / protein or nucleic acid / polynucleotide useful in the methods of the present invention shall be understood to mean a protein or nucleic acid useful in the methods, constructs, expression cassettes, plant cells, plants, seeds, harvestable parts, and products of the present invention. The nucleic acid / polynucleotide, including mRNA(s), introduced into a plant cell or plant (and thus useful for performing the methods of the present invention) may be any nucleic acid / polynucleotide encoding a polypeptide / protein of the type described below, and may hereinafter also be referred to as a "GRF5 nucleic acid," "GRF5 polynucleotide," "GRF5 gene," "GRF5 mRNA," etc.

[0010] As defined herein, a "GRF5 polypeptide" or "GRF5 protein" refers to any transcription factor, preferably the 14-3-3-like protein GF14 upsilon, more preferably comprising the PFAM domain PF08880 (also known as the QLQ domain) and the PFAM domain PF08879 (also known as the WRC domain), and even more preferably comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the PFAM domains at or near the N-terminus of the GRF5 polypeptide, when analyzed using Interproscan software (www.ebi.ac.uk / interpro). and a PFAM domain PF08879 C-terminal to the PFAM domain PF08880 of the GRF5 polypeptide with a coverage of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, i.e. the matching amino acid stretch PF08879 is located in the translational direction after the matching amino acid stretch PF08880. Preferably, at least one of the matches has a coverage of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0011] In one embodiment, both matching amino acid stretches are located in the N-terminal half of the GRF5 polypeptide, preferably the matching amino acid stretch PFAM domain PF08880 is located in the N-terminal quarter of the GRF5 polypeptide. Preferably, PFAM domain PF08880 matches amino acid residues of the GRF5 polypeptide starting from residue 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably from residue 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, and PFAM domain PF08879 matches amino acid residues of the GRF5 polypeptide starting from residue 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21. and preferably matches amino acid residues starting from residues 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. Preferably, the distance between the starting amino acid residue of the matching amino acid stretch PFAM domain PF08880 and the starting amino acid residue of the matching amino acid stretch PFAM domain PF08879 is 60 to 82 amino acids within the GRF5 polypeptide, more preferably 60 to 75 amino acids within the GRF5 polypeptide, more preferably 61 to 75 amino acids, and even more preferably 62 to 73 amino acids.

[0012] In a further embodiment, the GRF5 polypeptide used herein comprises the index motif: [D]-[PL]-[E]-[P]-[G]-[R]-[C]-[R]-[R]-[T]-[D]-[G]-[K]-[K]-[W]-[R]-[C]-[SA]-[RK]-[ED]-[A]-[YH]-[P]-[D]-[S]-[K]-[Y]-[C]-[E]-[KR]-[H]-[M]-[H]-[R]-[G]-[RK]-[N]-[R], wherein the GRF5 polypeptide is allowable / tolerable to exhibit a maximum number of mismatches compared to the index motif of 3, i.e. Up to three mismatches may appear in the sequence alignment between each GRF5 polypeptide and the index motif, preferably up to two mismatches may appear compared to the index motif, i.e., up to two mismatches may appear in the sequence alignment between each GRF5 polypeptide and the index motif, more preferably up to one mismatch may appear compared to the index motif, i.e., only one mismatch may appear in the sequence alignment between each GRF5 polypeptide and the index motif, more preferably no mismatches compared to the index motif. In a particularly preferred embodiment, one of the mismatches or the sole mismatch is located at position 2 of the index motif, more preferably the mismatch is [A] rather than [P]. A mismatch means that an amino acid at a position according to the index motif has been substituted with a different amino acid, or has been deleted or shifted by the insertion of at least one additional amino acid. The letters of the index motif in square brackets indicate the amino acid residues (single-letter code), and the motif represents the order of amino acid residues in the N- to C-terminal direction as present in any GRF5 polypeptide of the present invention. Two letters within a single bracket represent alternatives. This motif was derived from a comprehensive comparison of GRF5 polypeptide sequences from 16 different plant species, including monocotyledonous and dicotyledonous plants, and distinguishes GRF5 polypeptides from other members of the GRF protein family, such as GRF1 (see Figure 5A).An exemplary motif analysis by sequence alignment / comparison to determine the number of mismatches is shown in Figure 5B. Preferably, the motif is located in the N-terminal half of the GRF5 polypeptide. More preferably, the motif is located in the N-terminal half of the GRF5 polypeptide and comprises a subregion of the matching amino acid stretch PFAM domain PF08879, i.e., the motif has a contiguous overlap with the matching amino acid stretch PFAM domain PF08879. Preferably, the index motif consists of any of the amino acid sequences SEQ ID NO:41 to SEQ ID NO:104 or SEQ ID NO:113 to SEQ ID NO:176. Correspondingly, the GRF5 polypeptide preferably comprises one contiguous motif consisting of any of the amino acid sequences SEQ ID NO:41 to SEQ ID NO:104 or SEQ ID NO:113 to SEQ ID NO:176.

[0013] Examples of GRF5 polypeptides from various plant species useful in the methods of the present invention are further described below. Table 1 shows the location of PFAM domain PF08880 and PFAM domain PF08879 and their respective coverage in any of the presented GRF5 sequences.

[0014] [Table 1]

[0015] According to one aspect, the present invention provides a method for manufacturing a semiconductor device comprising: (a1) (i) at least one nucleotide sequence of interest; and (ii) introducing into the plant cell a polynucleotide encoding a GRF5 polypeptide, mRNA(s) encoding a GRF5 polypeptide, or an expression cassette comprising a GRF5 polypeptide, wherein (i) and (ii) can be introduced simultaneously or successively in any order (in parallel or sequentially); or (a2) introducing at least one nucleotide sequence of interest into a plant cell and simultaneously or sequentially inducing in said plant cell an enhanced level of expression of an endogenous gene encoding a GRF5 polypeptide; (b) optionally culturing the plant cell of (a1) or (a2) or a plant cell derived from the plant cell of (a1) or (a2), wherein in the plant cell a GRF5 polypeptide is expressed from an expression cassette, a GRF5 polypeptide is translated from introduced mRNA(s), a GRF5 polypeptide(s) is / are enhanced / increased in expression from an endogenous gene, or a GRF5 polypeptide(s) is / are present, preferably an expression cassette comprising a polynucleotide encoding a GRF5 polypeptide, mRNA(s) encoding a GRF5 polypeptide, or a GRF5 polypeptide(s) is / are present in an enhanced amount compared to that in a wild-type plant cell or plant cell not introduced according to step (a1), or an enhanced expression level of an endogenous gene encoding a GRF5 polypeptide is present according to step (a2); The present invention provides a method for transforming a plant cell, comprising:

[0016] The methods of the present invention result in modified or transformed plant cells that have improved regeneration potential due to the presence of GRF5 or the presence of enhanced amounts of GRF5, although it is preferred that the presence of GRF5 or the presence of enhanced amounts of GRF5 in the modified plant cells is transient.

[0017] Transformation of a plant cell refers to the introduction of a nucleic acid molecule into the plant cell in a manner that results in stable integration into the plant cell's genome or transient appearance within the plant cell, leading to constitutive, temporal, or specifically associated expression of the nucleic acid sequence, e.g., in specific tissue(s) or developmental stage(s). Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells are now routinely performed, and the selection of the most appropriate transformation technique will be determined by those skilled in the art. The choice of method will depend on the type of plant or genotype to be transformed. Those skilled in the art will recognize the suitability of a particular method for a given plant type or genotype. Suitable methods include, but are not limited to, electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG)-mediated transformation; viral transformation; microinjection of plant cells; microprojectile bombardment of plant cells; vacuum infiltration; and Agrobacterium-mediated transformation.

[0018] Step (a1)(i) or (a2) of introducing at least one nucleotide sequence of interest can be performed using any suitable method known in the art. Many methods are available for introducing nucleic acids of interest into plant cells. Exemplary vector-mediated methods for maize include Agrobacterium-mediated transformation, as described, for example, by Lindsay & Gallois, 1990, Journal of Experimental Botany and Kischenko et al., 2005, Cell Biology International for sugar beet, and Ishida et al., 2007, ("Agrobacterium-mediated transformation of maize," Nature protocols, 2(7), 1614-1621), and for wheat by PureWheat Technology from Japan Tobacco Inc. Other suitable techniques include particle bombardment and electroporation.

[0019] The nucleotide sequence of interest according to the present invention may be a DNA sequence or an RNA sequence, such as mRNA, siRNA, miRNA, etc. More specifically, the nucleotide sequence of interest encodes at least one phenotypic trait. Preferably, the phenotypic trait conferred by the DNA or RNA may be selected from the group consisting of resistance / tolerance to biotic stress, including pathogen resistance / tolerance (wherein the pathogen may be a virus, bacterium, fungus, or animal pathogen), resistance / tolerance to abiotic stress, including cold / freezing tolerance, drought stress resistance / tolerance, osmotic resistance / tolerance, heat stress resistance / tolerance, cold or frost stress resistance / tolerance, oxidative stress resistance / tolerance, heavy metal stress resistance / tolerance, salt stress or waterlogging resistance / tolerance, lodging resistance / tolerance, grain shattering resistance / tolerance, or resistance / tolerance to one or more herbicides, such as glyphosate, glufosinate, 2,4-D, dicamba, ALS inhibitors, etc. The at least one phenotypic trait of interest may also be selected from the group consisting of an increase in yield, an alteration in flowering time, an alteration in seed color, an alteration in endosperm composition, an alteration in nutrient content, or an alteration in metabolic engineering of a pathway of interest.

[0020] In the context of the present invention, GRF5 can be introduced as an expression cassette containing a polynucleotide encoding a GRF5 polypeptide, as an mRNA (including pre-mRNA or precursor mRNA) encoding a GRF5 polypeptide, or as a GRF5 polypeptide. Exemplary techniques for introducing nucleic acid molecules are described above. Alternatively, GRF5 can be provided in plant cells by activating the expression of an endogenous gene encoding a GRF5 polypeptide. This will lead to an enhanced expression level of the endogenous GRF5 gene, i.e., an enhanced amount of GRF5 polypeptide present or occurring in the plant cell. Activation of endogenous gene expression can be achieved by modifying the activity or structure of the promoter of the endogenous gene encoding the GRF5 polypeptide.For example, enhancer elements can be introduced into the promoter by gene editing; or enhancer elements regulating the promoter can be further strengthened, or silencer elements regulating the promoter can be weakened, for example, by targeted mutagenesis / modification; or modifications can be introduced into the epigenome associated with the enhancer by gene editing tools such as the CRISPR system (Hilton et al. (2015). Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature biotechnology, 33(5), 510-517); or synthetic transcription factors, for example, based on TALE activators or dCas9 activators, can be introduced into cells that can bind to target recognition sites on or near the promoter and activate transcription of the GRF5 gene (Cheng et al. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell research,23(10),1163); or the amount of microRNAs (miRNAs) in plant cells that regulate the expression of the GRF5 gene by post-transcriptional inhibition can be reduced, for example, by knockout (null mutant) or knockdown to increase the amount of GRF5 polypeptide translated in plant cells - for example, Rodriguez et al. (2010) (see above) identified the microRNA miR396 in Arabidopsis as a suitable target for antagonizing GRF5 expression and thus influencing the amount of GRF5 polypeptide in plant cells.

[0021] Depending on the plant species and cell type, different levels of gene or expression activation are required to ensure that appropriate amounts of GRF5 polypeptide are present in plant cells when regeneration occurs. Various techniques, such as qPCR, RT-PCR, Northern blot, or microarray, are available to those skilled in the art to measure the actual expression level of endogenous or introduced genes. Measurement of the expression level of the AtGRF5 gene introduced into a sugar beet plant (Beta vulgaris plant) is shown in Figure 4. These methods allow those skilled in the art to routinely adjust the expression level of the GRF5 gene to affect improved regeneration ability from various tissues or somatic and germ cells (e.g., microspores). In a preferred embodiment, the expression level of the endogenous gene encoding the GRF5 polypeptide in plant cells is increased by at least 2-, 3-, or 5-fold, preferably 10-, 25-, or 50-fold, and more preferably 100-, 200-, or 500-fold.

[0022] As further described above, induction of enhanced expression levels of endogenous genes in plant cells can be achieved by applying one or more activators or their precursors. These can be applied to the culture medium in which the plant cells are cultured and then actively or passively absorbed by the plant cells. Furthermore, one or more activators or their precursors can be directly introduced into plant cells by microinjection, electroporation, or biolistic bombardment. In addition to the synthetic transcriptional activators described above, numerous additional activators are known in the art that can be used to increase the expression levels of endogenous genes, particularly the endogenous GRF5 gene. Recently, the fields of chemical plant genetics and chemical plant biology have emerged, in which biological systems are treated with small molecules to specifically perturb cellular functions. Small molecules are commercially used in a variety of systems as drugs, herbicides, and fungicides, but in recent years they have also increasingly been used as tools for gene regulation. For example, chemical genetics involves the discovery of small-molecule effectors of various cellular functions through the screening of compound libraries (Dejonghe & Russinova (2017). Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology. Plant Physiology, pp-01805; Kawasumi, M., & Nghiem, P. (2007). Chemical genetics: elucidating biological systems with small-molecule compounds. Journal of Investigative Dermatology, 127(7), 1577-1584). Such small-molecule effectors suitable for activating the expression of target genes, such as GRF5, can be identified by chemical screening following various strategies (Dejonghe & Russinova, 2017).Comprehensive compound libraries are available, allowing for the easy screening of countless small molecules to identify effectors that can be used to activate the expression of genes such as GRF5. As mentioned above, another approach to enhancing the expression levels of endogenous genes such as GRF5 is the application of so-called synthetic transcription activators. These are typically engineered by fusing a recognition domain with at least one activation domain. The recognition domain can be derived from known systems such as zinc fingers, TAL effectors, or CRISPR; for example, fusing the VP-16 or VP-64 activation domains from herpes simplex virus to the recognition domain can result in increased transcription. Weaker activation domains, such as the AD of human NF-κB, offer a more diverse range of options for gene activation. Furthermore, as demonstrated on endogenous promoters, combinations of activators can be used to induce synergistic effects (Moore et al. (2014). "Transcription activator-like effectors: a toolkit for synthetic biology." ACS synthetic biology, 3(10), 708-716.; U.S. Patent Application Publication No. 2002 / 0046419; Lowder et al. (2017). "Multiplexed transcriptional activation or repression in plants using CRISPR-dCas9-based systems." Plant Gene Regulatory Networks: Methods and Protocols, 167-184.). Synthetic transcriptional activators can be delivered to plant cells or introduced into plant cells as precursors, i.e., DNA or RNA molecules encoding such artificial or synthetic transcriptional activators or domains thereof, or as inactive forms of transcriptional activators that are subsequently activated within the cell or in specific compartments of the cell.Finally, enhanced expression of the GRF gene can also be achieved by inactivating upstream negative regulators (i.e., miR396) or by creating mutant variants of the GRF gene that are resistant to such negative regulators.

[0023] Preferably, the GRF5 polypeptide of the present invention is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112, or 209, or an amino acid sequence having at least 70% identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112, or 209, preferably SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112, or 209. 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112 or 209, and preferably comprises an index motif described herein, particularly preferably an index motif consisting of any of amino acid sequences SEQ ID NO:41 to SEQ ID NO:104 or SEQ ID NO:113 to SEQ ID NO:176.For example, the GRF5 polypeptide encoded by the endogenous gene may be selected from the group consisting of SEQ ID NO:2 (Arabidopsis thaliana), SEQ ID NO:4 (sugar beet (Beta vulgaris)), SEQ ID NO:6 (maize (Zea mays)), SEQ ID NO:8 (bread wheat (Triticum aestivum)), SEQ ID NO:10 (Brassica napus), SEQ ID NO:12 (Brassica rapa), SEQ ID NO:14 (Brassica oleracea), SEQ ID NO:16 (radish (Raphanus sativus)), SEQ ID NO:18 (sorghum (Sorghum bicolor)), SEQ ID NO:20 (sunflower (Helianthus annuus)), SEQ ID NO:22 (potato (Solanum tuberosum)), SEQ ID NO:24 (barley (Hordeum vulgare)), SEQ ID NO:26 (rye (Secale cereale), SEQ ID NO:28 (soybean (Glycine max)), SEQ ID NO:30 (American cotton (Gossypium hirsutum)), SEQ ID NO:32 (rice (Oryza sativa)), SEQ ID NO:106 (soybean (Glycine max)), SEQ ID NO:108 (Brassica napus), SEQ ID NO:110 (sunflower (Helianthus annuus)), SEQ ID NO:112 (corn (Zea mays)), or SEQ ID NO:209 (corn (Zea mays)).

[0024] The exogenous (foreign) or endogenous polynucleotide encoding the GRF5 polypeptide of the present invention may be (i) a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 105, 107, 109, 111, 207, 208, or 210; (ii) a nucleotide sequence comprising a sequence that is at least 70%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, at least 98%, or at least 99% identical to a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 105, 107, 109, 111, 207, 208, or 210; (iii) a nucleotide sequence encoding a GRF5 polypeptide as defined above, or a nucleotide sequence encoding a polypeptide encoded by (i) and / or (ii) within the degeneracy of the genetic code; (iv) a nucleotide sequence complementary to the nucleotide sequence of (i), (ii), or (iii); and / or (v) a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of (iv); Includes.

[0025] Polynucleotides encoding GRF5 polypeptides, in particular polynucleotides encoding GRF5 polypeptides, are selected from the group consisting of SEQ ID NO: 1 (Arabidopsis thaliana), SEQ ID NO: 3 (sugar beet (Beta vulgaris)), SEQ ID NO: 5 (corn (Zea mays)), SEQ ID NO: 7 (bread wheat (Triticum aestivum)), SEQ ID NO: 9 (Brassica napus), SEQ ID NO: 11 (Brassica rapa), SEQ ID NO: 13 (Brassica oleracea), SEQ ID NO: 15 (radish (Raphanus sativus)), SEQ ID NO: 17 (sorghum (Sorghum bicolor)), SEQ ID NO: 19 (sunflower (Helianthus annuus)), SEQ ID NO: 21 (potato (Solanum tuberosum)), SEQ ID NO: 23 (barley (Hordeum vulgaris)), SEQ ID NO: 24 (barley (Hordeum vulgaris)), SEQ ID NO: 25 (barley (Hordeum vulgaris)), SEQ ID NO: 26 (barley (Hordeum vulgaris)), SEQ ID NO: 27 (barley (Hordeum vulgaris)), SEQ ID NO: 28 (barley (Hordeum vulgaris)), SEQ ID NO: 29 (barley (Hordeum vulgaris)), SEQ ID NO: 30 (barley (Hordeum vulgaris)), SEQ ID NO: 31 (barley (Hordeum vulgaris)), SEQ ID NO: 32 (barley (Hordeum vulgaris)), SEQ ID NO: 33 (barley (Hordeum vulgaris)), SEQ ID NO: 34 (barley (Hordeum vulgaris)), SEQ ID NO: 35 (barley ( vulgare), SEQ ID NO:25 (rye (Secale cereale)), SEQ ID NO:27 (soybean (Glycine max)), SEQ ID NO:29 (American cotton (Gossypium hirsutum)), SEQ ID NO:31 (rice (Oryza sativa)), SEQ ID NO:105 (soybean (Glycine max)), SEQ ID NO:107 (Brassica napus), SEQ ID NO:109 (sunflower (Helianthus annuus)), SEQ ID NO:111 (corn (Zea mays)), SEQ ID NO:207 (corn (Zea mays)), SEQ ID NO:207 (synthetic), SEQ ID NO:208 (synthetic) or SEQ ID NO:210 (synthetic).

[0026] For purposes of the present invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions with identical residues in two optimally aligned sequences divided by the number of positions compared (x100). Gaps, i.e., positions in the alignment where a residue is present in one sequence but not the other, are considered positions with non-identical residues. Alignment of two sequences is performed using the Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The above-mentioned computer-assisted sequence alignment can be conveniently performed using standard software programs, such as the NEEDLE program implemented in the European Molecular Biology Open Software Suite (EMBOSS), for example, version 6.3.1.2 (Trends in Genetics 16 (6), 276 (2000)), with default parameters, e.g., protein matrix = EBLOSUM62, gapopen = 10.0, and gapextend = 0.5.

[0027] The term "stringent conditions" or "hybridization under stringent conditions" refers to conditions under which nucleotide sequences that are sufficiently complementary to each other usually remain hybridized. These stringent conditions are known to those skilled in the art and are described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989) 6.3.1-6.3.6. Those skilled in the art know how to determine the necessary hybridization conditions, for example, based on Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989. In this regard, the term "hybridization conditions" refers not only to the actual conditions prevailing during the actual aggregation of nucleic acids, but also to the conditions prevailing during subsequent washing steps. Examples of stringent hybridization conditions include conditions under which only nucleic acid molecules with at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity undergo hybridization. Stringent hybridization conditions are, for example: 4xSSC at 65°C, followed by multiple washes with 0.1xSSC at 65°C for about 1 hour. As used herein, the term "stringent hybridization conditions" can also mean hybridization in 0.25M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA at 68°C for 16 hours, followed by two washes with 2xSSC and 0.1% SDS at 68°C. Preferably, hybridization is performed under stringent conditions.

[0028] The term "operably linked" means that the elements of the chimeric gene are linked to each other in such a way that their functions are coordinated and allow expression of the coding sequences, i.e., they are operably linked. For example, a promoter is operably linked to another nucleotide sequence if it is capable of effecting the transcription and ultimately expression of that other nucleotide sequence. Two protein encoding nucleotide sequences are functionally or operably linked to each other if they are connected in such a way that they can form a fusion protein of the first and second proteins or polypeptides.

[0029] A gene is said to be expressed when it leads to the formation of an expression product. An expression product refers to an intermediate or final product resulting from the transcription and optional translation of a nucleic acid, DNA or RNA, encoding such a product, such as a second nucleic acid described herein. During the transcription process, a DNA sequence under the control of a regulatory region, particularly a promoter, is transcribed into an RNA molecule. The RNA molecule may itself form an expression product, or it may be an intermediate product if it can be translated into a peptide or protein. If the RNA as the final product of gene expression can, for example, interact with another nucleic acid or protein, the gene is said to encode an RNA molecule as its expression product. Examples of RNA expression products include inhibitory RNA, such as sense RNA (cosuppression), antisense RNA, ribozyme, miRNA or siRNA, mRNA, rRNA, and tRNA. If the final product of gene expression is a protein or peptide, the gene is said to encode a protein as its expression product.

[0030] As used herein, nucleic acid (molecule) or nucleotide (sequence) or polynucleotide refers to both DNA and RNA. DNA also includes cDNA and genomic DNA. Nucleic acid molecules can be single-stranded or double-stranded, and can be chemically synthesized or produced by biological expression in vitro or in vivo.

[0031] Whenever the nucleotide sequence of an RNA molecule is defined by reference to the nucleotide sequence of a corresponding DNA molecule, it will be clear that thymine (T) in the nucleotide sequence should be substituted with uracil (U). It will be clear from the context of the application whether an RNA or DNA molecule is being referred to.

[0032] As used herein, "comprises" and the like should be interpreted as specifying the presence of the recited described feature, integer, step, or component, but not excluding the presence or addition of one or more features, integers, steps, or components, or groups thereof. Thus, for example, a nucleic acid or protein comprising a sequence of nucleotides or amino acids may contain more nucleotides or amino acids than those actually recited, i.e., may be embedded in a larger nucleic acid or protein. A chimeric gene comprising a functionally or structurally defined DNA region may contain additional DNA regions, etc.

[0033] GRF5 can provide plant cells or other plant precursor tissues with improved regenerative capabilities, which is particularly useful for genetically modified plant cells, especially those whose genomes have been edited.

[0034] According to a preferred embodiment of the present invention, step (a1) of introducing at least one nucleotide sequence of interest and GRF5 or the step of introducing at least one nucleotide sequence of interest and inducing an enhanced expression level of an endogenous gene encoding GRF5 results in transient transformation of the plant cell. In the context of the present invention, "transient transformation" means that the inserted sequence is not (stably) integrated into the genome of the plant cell. In another embodiment, stable transformation is performed, in which the nucleotide sequence of interest in steps (a1) and (a2) of the transforming method disclosed herein and / or the polynucleotide encoding the GRF5 polypeptide in step (a1) of the genome modifying method disclosed herein are inserted into the genome of the plant cell. According to a particularly preferred embodiment of the present invention, the nucleotide sequence of interest is stably transformed into the genome of the cell, and the nucleotide sequence encoding GRF5 is transiently transformed into the cell.

[0035] Genome modification of plant cells can preferably be achieved by means of double-stranded DNA break (DSB)-inducing enzymes that recognize predetermined sites within the genome of said cells.

[0036] Therefore, another embodiment of the present invention is (a1) introducing into a plant cell a polynucleotide encoding a GRF5 polypeptide, mRNA(s) (including pre-mRNA(s)) encoding a GRF5 polypeptide, or an expression cassette comprising GRF5 polypeptide(s); or (a2) inducing an enhanced expression level of an endogenous gene encoding a GRF5 polypeptide in a plant cell; (b) culturing the plant cell of (a1) or (a2) or a plant cell derived from the plant cell of (a1) or (a2), wherein in the plant cell, a GRF5 polypeptide is expressed from an expression cassette, a GRF5 polypeptide is translated from introduced mRNA(s), a GRF5 polypeptide(s) is / are enhanced / increased in expression from an endogenous gene, or a GRF5 polypeptide(s) is / are present, preferably under conditions in which a polynucleotide encoding a GRF5 polypeptide, a GRF5 polypeptide(s) encoding mRNA(s) or an expression cassette comprising a GRF5 polypeptide(s) is / are present in an enhanced amount compared to the amount in a wild-type plant cell or plant cell not introduced according to step (a1) or in which an enhanced expression level of an endogenous gene encoding a GRF5 polypeptide is present according to step (a2); (c) modifying the genome of the plant cell of (b) preferably using a double-stranded DNA break (DSB)-inducing enzyme that recognizes a predetermined site in the genome of said cell and, optionally, a repair nucleic acid molecule; wherein the modification of the genome at the predetermined site comprises: i. at least one nucleotide substitution; ii. a deletion of at least one nucleotide; iii. an insertion of at least one nucleotide; or iv. Any combination of i. to iii. is selected from Step (c) is carried out simultaneously with steps (a1) / (a2) and / or (b), before steps (a1) / (a2), between steps (a1) / (a2) and (b), or after step (b); A method for modifying the genome of a plant cell.

[0037] As used herein, a "double-stranded DNA break-inducing enzyme" or "DSBI enzyme" refers to an enzyme capable of inducing double-stranded DNA breaks at specific nucleotide sequences, referred to as "recognition sites." Double-stranded DNA break (DSB)-inducing enzymes can be selected from the group consisting of meganucleases, TAL effector nucleases, zinc finger nucleases, and CRISPR systems such as CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / Csm1, or CRISPR / MAD7. Rare-cutting endonucleases are DSBI enzymes that preferably have recognition sites of approximately 14 to 70 contiguous nucleotides and therefore cleave at very low frequencies, even within larger genomes such as most plant genomes. Homing endonucleases, also known as meganucleases, constitute a family of such rare-cutting endonucleases. These enzymes may be encoded by introns, independent genes, or intervening sequences and exhibit distinctive structural and functional characteristics that distinguish them from more classical restriction enzymes, usually bacterial type II restriction-modification systems. Their recognition sites have a general asymmetry, in contrast to the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns or inteins have been shown to facilitate homing of their respective genetic elements to intronless or inteinless sites in alleles. These nucleases replicate coding sequences by site-specific double-strand cleavage of intronless or inteinless alleles, creating recombination ends that are involved in the gene conversion process leading to the insertion of introns or intervening sequences at the DNA level. A list of other rare-cutting meganucleases and their respective recognition sites is provided in Table I of WO 03 / 004659 (pp. 17-20), incorporated herein by reference.

[0038] Furthermore, methods are available for designing custom-tailored rare-cutting endonucleases that recognize essentially any target nucleotide sequence. Briefly, chimeric restriction enzymes can be prepared using hybrids between zinc finger domains designed to recognize specific nucleotide sequences and nonspecific DNA cleavage domains from natural restriction enzymes, such as FokI. Such methods are described, for example, in WO 03 / 080809, WO 94 / 18313, or WO 95 / 09233, and in Isalan et al. (2001). "A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter." Nature biotechnology, 19(7), 656; Liu et al. (1997). "Design of polydactyl zinc-finger proteins for unique addressing within complex genomes." Proceedings of the National Academy of Sciences, 94(11), 5525-5530.

[0039] Another example of a custom-designed endonuclease is the TALE nuclease (TALEN), which is based on a transcription activator-like effector (TALE) from the bacterial genus Xanthomonas fused to the catalytic domain of a nuclease (e.g., FokI or its variants). The DNA-binding specificity of these TALEs is defined by the repeating variable dinucleotides (RVDs) of tandemly arranged 34 / 35 amino acid repeat units, with each RVD specifically recognizing a single nucleotide in the target DNA. The repeat units can be assembled to recognize essentially any target sequence and fused to the catalytic domain of a nuclease to create a sequence-specific endonuclease (e.g., Boch et al. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326(5959), 1509-1512; Moscou & Bogdanove (2009). A simple cipher governs DNA recognition by TAL). effectors. Science, 326(5959), 1501-1501; and WO 2010 / 079430, WO 2011 / 072246, WO 2011 / 154393, WO 2011 / 146121, WO 2012 / 001527, WO 2012 / 093833, WO 2012 / 104729, WO 2012 / 138927, WO 2012 / 138939). WO 2012 / 138927 further describes monomeric (compact) TALENs and TALEs with various catalytic domains and combinations thereof.

[0040] In recent years, a new type of customizable endonuclease system, the so-called CRISPR / Cas system, has been described. In nature, CRISPR systems represent molecular complexes containing at least one small, individual non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease, such as the Cpf1 nuclease, which can generate specific DNA double-strand breaks (Zetsche et al., "Cpf1 Is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System," Cell, 163, pp. 1–13, October 2015). Currently, CRISPR systems are classified into two classes, including five types of CRISPR systems: type II systems, which use Cas9 as an effector, and type V systems, which use Cpf1 as an effector molecule (Makarova et al., Nature Rev. Microbiol., 2015). Artificial CRISPR systems utilize synthetic non-coding RNAs and CRISPR nucleases, and / or modified CRISPR nucleases, optionally modified to function as nickases or lacking any nuclease function, in combination with at least one synthetic or artificial guide RNA or gRNA that combines the functions of crRNA and / or tracrRNA (Makarova et al., 2015, see above). CRISPR / Cas-mediated immune responses in natural systems require CRISPR-RNA (crRNA), and the maturation of this guide RNA, which controls the specific activation of CRISPR nucleases, differs significantly among the various CRISPR systems characterized to date. First, an invading DNA fragment, also known as a spacer, is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. Type II CRISPR systems encode the Cas9 nuclease as the key enzyme for the interference step, and these systems contain both a crRNA and a transactivating RNA (tracrRNA) as guide motifs.These hybridize to form double-stranded (ds)RNA regions that can be recognized by RNAse III and cleaved to form mature crRNA. These then associate with Cas molecules to specifically direct the nuclease to the target nucleic acid region. Recombinant gRNA molecules can contain both a variable DNA recognition region and a Cas interaction region, allowing them to be specifically designed regardless of the specific target nucleic acid and desired Cas nuclease. As an additional safety mechanism, PAMs (protospacer adjacent motifs) must be present in the target nucleic acid region; these are DNA sequences that directly follow the Cas9 / RNA complex recognition DNA. The PAM sequence of Cas9 from Streptococcus pyogenes has been described as "NGG" or "NAG" (standard IUPAC nucleotide code) (Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science 2012, 337:816-821). The PAM sequence of Cas9 from Staphylococcus aureus is "NNGRRT" or "NNGRR(N)." Additional variant CRISPR / Cas9 systems are known. Thus, Neisseria meningitidis Cas9 cleaves the PAM sequence NNNNGATT. Streptococcus thermophilus Cas9 cleaves the PAM sequence NNAGAAW. Recently, an additional PAM motif, NNNNRYAC, has been described for the Campylobacter CRISPR system (WO 2016 / 021973).For the Cpf1 nuclease, the Cpf1-crRNA complex without tracrRNA efficiently recognizes and cleaves target DNA generated by short T-rich PAMs, in contrast to the generally G-rich PAMs recognized by the Cas9 system (Zetsche et al., supra). Furthermore, specific single-strand breaks can be obtained by using engineered CRISPR polypeptides. Highly specific double-strand breaks in DNA can also be induced by double-nicking using Cas nickases in combination with various recombinant gRNAs. Furthermore, the specificity of DNA binding and therefore DNA cleavage can be optimized by using two gRNAs. Meanwhile, additional CRISPR effectors, such as the CasX and CasY effectors originally described in bacteria, are available and represent additional effectors that can be used for genome engineering purposes (Burstein et al., "New CRISPR-Cas systems from uncultivated microbes," Nature, 2017, 542, 237-241).

[0041] The cleavage site of a DSBI enzyme is related to the precise location on DNA where a double-stranded DNA break is induced. The cleavage site may or may not be contained within (overlapping with) the DSBI enzyme's recognition site; therefore, the cleavage site of a DSBI enzyme is said to be located at or near the recognition site. The recognition site of a DSBI enzyme, sometimes referred to as the binding site, is a nucleotide sequence that is specifically recognized by the DSBI enzyme and determines its binding specificity. For example, a TALEN or ZNF monomer has a recognition site determined by its RVD or ZF repeats, respectively, but its cleavage site, determined by its nuclease domain (e.g., FokI), is usually located outside the recognition site. In the case of a dimeric TALEN or ZFN, the cleavage site is located between the two recognition / binding sites of each monomer; this intervening DNA region where cleavage occurs is called the spacer region.

[0042] One skilled in the art can select a DSBI enzyme that recognizes a recognition site and induce a DSB at or near a preselected / predetermined cleavage site, or engineer such a DSBI enzyme. Alternatively, the DSBI enzyme recognition site can be introduced into a target genome using any conventional transformation method or by mating with an organism that has the DSBI enzyme recognition site in its genome, and then any desired DNA can be introduced at or near the cleavage site of the DSBI enzyme.

[0043] In a particularly preferred aspect of this embodiment, a repair nucleic acid molecule is additionally introduced into plant cells.As used herein, " repair nucleic acid molecule " refers to a single-stranded or double-stranded DNA molecule or RNA molecule that is used as a template for modifying genomic DNA at a preselected site near or at the cleavage site.As used herein, "using as a template for modifying genomic DNA" means that the repair nucleic acid molecule is copied or integrated at a preselected site by homologous recombination between the flanking region(s) and the corresponding homologous region(s) in the target genome adjacent to the preselected site, optionally combined with non-homologous end joining (NHEJ) at one of the two ends of the repair nucleic acid molecule (for example, when there is only one flanking region).Integration by homologous recombination allows the repair nucleic acid molecule to be accurately integrated into the target genome at the nucleotide level, and NHEJ can cause small insertions / deletions at the junction between the repair nucleic acid molecule and genomic DNA.

[0044] As used herein, "genome modification" means that the genome has been changed by at least one nucleotide. This can occur by substitution of at least one nucleotide and / or deletion of at least one nucleotide and / or insertion of at least one nucleotide, as long as it results in an overall change of at least one nucleotide compared to the nucleotide sequence of the preselected genome target site before modification, thereby allowing the identification of the modification by techniques such as sequencing or PCR analysis, as those skilled in the art will be familiar with.

[0045] As used herein, a "preselected site," "predetermined site," or "predefined site" refers to a specific nucleotide sequence within a genome (e.g., a nuclear genome or a chloroplast genome) at which insertion, substitution, and / or deletion of one or more nucleotides is desired. This may be, for example, an endogenous locus or a specific nucleotide sequence within or linked to previously introduced foreign DNA or a transgene. The preselected site may be (downstream of) a specific nucleotide position at which one or more nucleotides are intended to be inserted. The preselected site may also include the sequence of one or more nucleotides to be exchanged (substituted) or deleted.

[0046] As used in the context of this application, the term "about" means + / - 10% of the recited value, preferably + / - 5% of the recited value. For example, about 100 nucleotides (nt) shall be understood as a value between 90 and 110 nt, preferably between 95 and 105 nt.

[0047] As used herein, a "flanking region" refers to a region of a repair nucleic acid molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a DNA region adjacent to (i.e., upstream or downstream of) a preselected site. It will be apparent that the length and percentage sequence identity of the flanking region should be selected to allow homologous recombination between the flanking region and its corresponding DNA region upstream or downstream of the preselected site. One or more DNA regions adjacent to a preselected site that are homologous to one or more flanking DNA regions of a repair nucleic acid molecule are also referred to as one or more homologous regions in genomic DNA.

[0048] To ensure sufficient homology for recombination, the flanking DNA regions of the repair nucleic acid molecule may vary in length, preferably at least about 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 40 nt, or 50 nt. However, the flanking regions may be as long as practical (e.g., up to about 100-150 kb, such as in a complete bacterial artificial chromosome (BAC)). Preferably, the flanking regions will be about 50 nt to about 2,000 nt, e.g., about 100 nt, 200 nt, 500 nt, or 1,000 nt. Furthermore, the regions flanking the target DNA need not be identical to the homologous region (the DNA region flanking the preselected site) but may share about 80% to about 100% sequence identity, preferably about 95% to about 100% sequence identity, with the DNA region flanking the preselected site. The longer the flanking region, the less stringent the homology requirement. Furthermore, to achieve exchange of the target DNA sequence at the preselected site without altering the DNA sequence of the flanking DNA sequences, it is preferable that the flanking DNA sequences are identical to the upstream and downstream DNA regions adjacent to the preselected site.

[0049] As used herein, "upstream" refers to a position on a nucleic acid molecule that is closer to the 5' end of said nucleic acid molecule. Similarly, the term "downstream" refers to a position on a nucleic acid molecule that is closer to the 3' end of said nucleic acid molecule. For the avoidance of doubt, nucleic acid molecules and their sequences are typically represented in a 5' to 3' direction (left to right).

[0050] To target a sequence modification at a preselected site, the flanking regions must be selected such that the 3' end of the upstream flanking region and / or the 5' end of the downstream flanking region align with the ends of the preselected site. Thus, the 3' end of the upstream flanking region determines the 5' end of the predefined site, and the 5' end of the downstream flanking region determines the 3' end of the predefined site.

[0051] As used herein, the preselected site being outside or distant from the cleavage (and / or recognition) site means that the site at which genome modification is intended (the preselected site) does not contain the cleavage and / or recognition site of the DSBI enzyme, i.e., the preselected site does not overlap with the cleavage (and / or recognition) site. Thus, outside / distant in this context means upstream or downstream of the cleavage (and / or recognition) site.

[0052] The modified plant cells that have been transformed or gene-edited according to the methods of the present invention, and optionally have a modified genome, can be regenerated into intact (fertile) plants. The presence of additional GRF5 in the plant cells significantly improves their regeneration ability. Therefore, in a preferred embodiment of the present invention, each transformation of a plant cell or modification of the genome of a plant cell is followed by a step of regenerating a plant. Thus, the present invention provides: (a) transforming a plant cell as described above; (b) regenerating a plant from the plant cell of (a) or a plant cell derived from the plant cell of (a), the plant cell comprising at least one plant cell comprising at least one nucleotide sequence of interest as a transgene; The present invention provides a method for producing a transgenic plant comprising:

[0053] Furthermore, the present invention also provides (a) modifying the genome of a plant cell as described above; (b) regenerating a plant from the plant cell of (a) or a plant cell derived from the plant cell of (a) comprising the genome modified or modified plant cell in at least one cell; The present invention provides a method for producing a genetically modified plant, comprising:

[0054] Regeneration techniques rely on the manipulation of certain plant hormones in tissue culture growth media and may also rely on biocide and / or herbicide markers, which can be introduced along with the desired nucleotide sequence of interest. Plant regeneration from cultured protoplasts is described in Evans et al., "Protoplast Isolation and Culture," Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and "Binding, Regeneration of Plants," Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, protoplasts, immature or mature embryos, embryonic tissue, meristems, organs, or parts thereof. Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones (a process known as tissue culture). Once whole plants have developed and seeds have been produced, evaluation of the progeny can begin.

[0055] According to the present invention, it is possible to improve not only the regeneration capacity of transformed or genetically modified plant cells, but also the regeneration capacity of other types of susceptible cells that have poor regeneration capacity. In particular, the production of haploid plant embryos from precursors such as immature male gametophytes or microspores can be improved by GRF5.

[0056] Therefore, another aspect of the present invention is (a1) introducing into immature male gametophytes or microspores a polynucleotide encoding a GRF5 polypeptide, mRNA(s) encoding a GRF5 polypeptide, or an expression cassette comprising GRF5 polypeptide(s); or (a2) inducing enhanced expression levels of an endogenous gene encoding a GRF5 polypeptide in immature male gametophytes or microspores; (b) culturing the immature male gametophyte or microspore of (a), wherein the GRF5 polypeptide is expressed from an expression cassette, the GRF5 polypeptide is translated from introduced mRNA(s), the GRF5 polypeptide is enhanced / increased in expression from an endogenous gene, or the GRF5 polypeptide(s) is / are present in the immature male gametophyte or microspore, preferably under conditions in which a polynucleotide encoding a GRF5 polypeptide, mRNA(s) encoding a GRF5 polypeptide, or an expression cassette comprising a GRF5 polypeptide is present in an enhanced amount compared to the amount in a wild-type plant cell or plant cell not introduced according to step (a1) or in which the expression level of an endogenous gene encoding a GRF5 polypeptide is enhanced according to step (a2); (c) selecting haploid plant embryos derived from the immature male gametophytes or microspores of step (b); A method for producing a haploid plant embryo, comprising:

[0057] The present invention also includes methods for producing haploid seedlings, comprising exposing haploid plant material to a polynucleotide encoding a GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or an expression cassette containing one or more GRF5 polypeptides to produce haploid embryos, and then converting (i.e., germinating) the haploid embryos into seedlings. Accordingly, the present invention includes methods for producing haploid plants, comprising growing seedlings produced according to the aforementioned methods. The present invention also provides methods for producing double haploid plants, comprising culturing haploid plant material in the presence of GRF5 for a period of time to stimulate or allow spontaneous chromosome doubling, and growing the double haploid plant material into seedlings, plantlets, or plants. In some embodiments, haploid embryo formation and chromosome doubling can occur substantially simultaneously. In other embodiments, there can be a time delay between haploid embryo formation and chromosome doubling. In the unlikely event that the growth of haploid seedlings, plants, or plantlets is not accompanied by spontaneous chromosome doubling events, a chemical chromosome doubling agent, such as colchicine, can be used. Many procedures require contacting plant cells with colchicine, an antimicrotubule agent or antimicrotubule herbicide, such as pronamide, nitrous oxide, or any mitotic inhibitor. This results in homozygous doubled haploid cells. When colchicine is used, its concentration in the medium is typically 0.01% to 0.2%. The colchicine concentration range can be approximately 400 to 600 mg / L.

[0058] When the microspores are exposed to GRF5 polypeptide(s), callus forms, which undergoes organogenesis to form embryos. Thus, the present invention includes methods for producing haploid plant callus, comprising exposing an immature male gametophyte or microspore to GRF5 polypeptide(s), or introducing into the immature male gametophyte or microspore a polynucleotide encoding a GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or an expression cassette comprising GRF5 polypeptide(s).

[0059] The immature male gametophytes or microspores are preferably exposed to GRF5 during the culture step for a time sufficient to induce haploid embryogenesis. The required time period may depend on the plant species, all of which can be readily ascertained by one skilled in the art. A preferred range for GRF5 exposure is from about 1 to about 20 hours, more preferably from about 2 to about 20 hours.

[0060] In one embodiment of the present invention, physical stress is applied to haploid plant material prior to the introduction of a polynucleotide encoding a GRF5 polypeptide, an mRNA encoding a GRF5 polypeptide, or an expression cassette containing (multiple) GRF5 polypeptides. The physical stress may be, for example, temperature, darkness, light, ionizing radiation, starvation, or osmotic stress. The light may be full-spectrum sunlight, or one or more frequencies selected from the visible, infrared, or UV spectrum. The stress may be continuous or intermittent (periodic); regular or random over time.

[0061] The present invention is applicable to all plant species, whether monocotyledonous or dicotyledonous. Preferably, plants that may be subjected to the methods and uses of the present invention are from the genus Hordeum, Sorghum, Saccharum, Zea, Setaria, Oryza, Triticum, Secale, Triticale, Malus, Brachypodium, Euonymus, and the like. Aegilops, Daucus, Beta, Eucalyptus, Nicotiana, Solanum, Coffea, Vitis, Erythrantha, Genlisea, Cucumis, Marus, Arabidopsis, Curcuma Crucihimalaya, Cardamine, Lepidium, Capsella, Olimarabidopsis, Arabis, Brassica, Eruca, Raphanus, Citrus, Jatropha, Poplar More preferably, the plant is a plant of a genus selected from the group consisting of: Populus, Medicago, Cicer, Cajanus, Phaseolus, Glycine, Gossypium, Astragalus, Lotus, Torenia, Allium, or Helianthus. More preferably, the plant is a plant of a genus selected from the group consisting of barley (Hordeum vulgare), Hordeum bulbusom, sorghum (Sorghum bicolor), Saccharum officinarium, maize species (Zea mays),spp., Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, wheat (Triticum aestivum), Triticum durum, rye (Secale cereale), Triticale, Malus domestica, wheatgrass (Brachypodium distachyon), Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, beet species (Beta vulgaris) spp.), Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrantha guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalayahimalaica, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Shepherd's purse (Capsella bursa pastoris), Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Radish (Raphanus sativus), Brassica juncacea, Brassica nigra, Arugula (Eruca vesicaria subsp. sativa), Orange (Citrus sinensis), Jatropha curcas), black cottonwood (Populus trichocarpa), alfalfa (Medicago truncatula), Cicer yamashitae (Cicer yamashitae), Cicer bijugum (Cicer bijugum), chickpea (Cicer arietinum), Cicer reticulatum (Cicer reticulatum), Cicer judaicum (Cicer judaicum), Cajanus cajanifolius (Cajanus scarabaeoides), kidney bean (Phaseolus vulgaris), soybean (Glycine max), cotton (Gossypium sp.), astragalus sinicus, lotus grass (Lotus japonicas), melon grass (Torenia fournieri), onion (Allium cepa), leeks (Allium fistulosum), garlic (Allium sativum), sunflowers (Helianthus annuus), Jerusalem artichokes (Helianthus tuberosus) and / or chives (AlliumParticularly preferred are sugar beet (Beta vulgaris), corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), sunflower (Helianthus annuus), potato (Solanum tuberosum), sorghum (Sorghum bicolor), Brassica rapa, rapeseed (Brassica napus), Brassica juncacea, Brassica oleracea, radish (Raphanus sativus), Oryza sativa, soybean (Glycine max) and / or cotton (Gossypium sp.).

[0062] Suitable plant cells according to the invention are in particular cells of callus tissue, preferably friable callus, meristematic tissue, reproductive tissue (eg microspores) or embryonic tissue, as well as protoplasts.

[0063] The plant part or parts may be attached to or separate from the whole intact plant, and such plant parts include, but are not limited to, plant organs, tissues and cells, preferably seeds.

[0064] The subject of the present invention is also a plant obtained or obtainable by the above-mentioned method.Accordingly, one embodiment of the present invention is a transgenic plant obtained or obtainable by the above-mentioned method of transforming a plant cell and regenerating a plant from said cell, as well as its progeny or parts, wherein the progeny or parts contain at least one nucleotide sequence of interest as a transgene.Another embodiment of the present invention is a genetically modified transgenic plant obtained or obtainable by the above-mentioned method of modifying the genome of a plant cell and regenerating a plant from said cell, as well as its progeny or parts, wherein the progeny or parts contain modifications in the genome introduced by the above-mentioned modification method.

[0065] Another subject of the present invention is plant cells or seeds derived from the above-mentioned transgenic or genetically modified plants. Such plant cells preferably contain a polynucleotide encoding a GRF5 polypeptide and a double-stranded DNA break (DSB)-inducing enzyme, which preferably recognizes a predetermined site in the genome of the cell and, optionally, a repair nucleic acid molecule, transiently or stably integrated therein. The polynucleotide encoding the GRF5 polypeptide is preferably operably linked to an appropriate regulatory sequence so that the plant cell can express the GRF5 polypeptide. Regulatory sequence refers, for example, to a "promoter," a nucleotide sequence typically located upstream (5') of the coding sequence, which controls the expression of the coding sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. A "constitutive promoter" refers to a promoter that constantly drives gene expression in almost all tissues. Examples of constitutive promoters include the CaMV 35S promoter, the dual CaMV 35S promoter (70S promoter), the nopaline synthase (nos) promoter, the BdEF1 promoter, or ubiquitin promoters such as PcUbi4 or ZmUbi1. A "regulated promoter" refers to a promoter that induces gene expression in a temporally and / or spatially regulated manner, rather than constitutively, and includes both tissue-specific and inducible promoters. It includes natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. Different promoters can induce gene expression in different tissue or cell types, at different developmental stages, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered and are well known to those skilled in the art. A "tissue-specific promoter" refers to a regulated promoter that does not express in all plant cells, but only in one or more cell types of a specific organ (e.g., leaves or seeds), a specific tissue (e.g., embryos or cotyledons), or a specific cell type (e.g., leaf parenchyma or seed storage cells).These also include promoters that are temporally regulated (e.g., during fruit ripening in developing seeds or fruits, in fully differentiated leaves, or at the onset of senescence (e.g., early or late embryogenesis). An "inducible promoter" refers to a regulated promoter that can be turned on in one or more cell types by an external stimulus (e.g., a chemical, light, hormone, stress, or pathogen). Examples of inducible promoters include promoters inducible by ecdysone, dexamethasone, and ethanol. Such promoters are well known to those skilled in the art (e.g., Samalova et al. (2005). pOp6 / LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. The Plant Journal, 41(6), 919-935; Gatz & Lenk (1998). Promoters that respond to chemical inducers. Trends in Plant Science, 3(9), 352-358.).

[0066] A further subject of the present invention is a haploid plant embryo obtained or obtainable by the method of the invention.

[0067] Another subject of the present invention is a catalytic cell comprising a transiently or stably integrated polynucleotide encoding a GRF5 polypeptide, a double-stranded DNA break (DSB)-inducing enzyme that preferably recognizes a predetermined site in the genome of said cell, and optionally a repair nucleic acid molecule, wherein preferably the polynucleotide encoding the GRF5 polypeptide is operably linked to a suitable regulatory sequence so that the plant cell can express the GRF5 polypeptide. Such a plant cell can be obtained by carrying out the above-mentioned method for modifying the genome of a plant cell.

[0068] A further aspect of the present invention is the use of a polynucleotide encoding a GRF5 polypeptide, an mRNA encoding a GRF5 polypeptide, a GRF5 polypeptide, or an activator of expression of an endogenous gene encoding a GRF5 polypeptide in a method for transforming plant cells, preferably in a transformation method as described above, in a method for modifying the genome of a plant cell, preferably in a method for modifying the genome as described above, in a method for producing an embryo of a plant or haploid plant, preferably in a method for producing an embryo of a plant or haploid plant as described above, or in a method for regenerating a plant, preferably in a method for regenerating a plant as described above.

[0069] Unless otherwise specified in the examples, all recombinant DNA techniques are performed according to the standard protocols described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY, and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Standard materials and methods for plant molecular processes are described in R.D.Cray's Plant Molecular Biology Labfax (1993) (co-published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK). Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, and Brown (1998) Molecular Biology Labfax, Second Edition, Academic Press (UK), Volumes 1 and 2. Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0070] All patents, patent applications, and publications or public information (including internet publications) referenced or cited herein are incorporated by reference in their entirety.

[0071] The present invention will be further described with reference to the following figures and examples described herein, however, it should be understood that the present invention is not limited to such examples. [Brief explanation of the drawings]

[0072] [Figure 1] Figure 1 shows the total number of developing shoots at each selection step (S1–S4). Data obtained from five independent transformation experiments performed with either the control construct pZFN-tdT-nptII or pZFN-nptII-GRF5 (compare Table 3). [Figure 2] Shoot regeneration at the start of selection step 3 (S3) of 14 independently transformed calli in a control experiment (A) and in an experiment performed with pZFN-nptII-GRF5 (B). Arrows indicate developing shoots. Experiments were performed according to the method in 2.1. [Figure 3] Shoot regeneration in sugar beet calli 10 days after bullet transduction with pZFN-nptII-GRF5 (A) and the control plasmid pABM70S-TurboYFP (B). Calli were transfected with the pUbi4-tDT plasmid to control transient expression levels via red fluorescence. The experiment was performed according to the method in 2.2. Three independently transfected calli are shown. [Figure 4] FIG. 1 shows the expression levels of GRF5 in 11 independent sugar beet transgenic events. [Figure 5A] FIG. 1 shows a sequence comparison of GRF5 polypeptide sequences from 16 different plant species and the deduced GRF5-specific indicator motif (SEQ ID NO: 177); partial sequences of the GRF5 polypeptide sequences shown are set forth in SEQ ID NOs: 178-206. [Figure 5B]

[0023] Figure 1 shows motif analysis by sequence alignment / comparison of complete protein sequences from 16 different plant species, allowing up to 10 mismatches. The "Start" and "Stop" columns indicate the start and end positions of the GRF5 polypeptide sequence fragment corresponding to the index motif; the "Mismatch" column indicates the number of mismatches between each GRF5 polypeptide and the index motif. The partial sequences of the GRF5 polypeptide sequences shown are set forth in SEQ ID NOs: 178-184 and 186-206. AtGRF1_AT2G22840.1 (sequence number 33); AtGRF2_AT4G37740.1 (sequence number 34); AtGRF3_AT2G36400.1 (sequence number 35); AtGRF4_AT3G52910.1 (sequence number 36); AtGRF6_AT2G06200.1 (sequence number 37); AtGRF7_AT5G53660.1 (sequence number 38); AtGRF8_AT4G24150.1 (sequence number 39); AtGRF9_AT2G45480.1 (sequence number 40). [Figure 6] FIG. 1 shows the transformation frequencies in sugar beet obtained with the control construct 70S::tdT, a construct overexpressing AtGRF5 (cDNA—SEQ ID NO: 1; amino acid sequence—SEQ ID NO: 2), and BvGRF5, an orthologue of sugar beet GRF5 (synthetic DNA—SEQ ID NO: 207; amino acid sequence—SEQ ID NO: 4). [Figure 7] Number of transgenic shoots per inoculated sugar beet callus obtained in transformation experiments using constructs overexpressing either tdTomato (tDT) (control) or AtGRF5 (cDNA - SEQ ID NO: 1; amino acid sequence - SEQ ID NO: 2). [Figure 8] Figure 1 shows the average number of transgenic shoots per sugar beet callus obtained from transformation experiments using either the control construct 70S-tDT (control) or a construct overexpressing AtGRF5 (70S-GRF5). Two resistant genotypes, A and B, were used. Genotype A exhibits a very high level of resistance to shoot regeneration, while genotype B exhibits a milder resistance. [Figure 9] Figure 1 shows a callus regeneration experiment of transgenic lines overexpressing AtGRF5 in sugar beet. Callus formation frequency (A) and shoot regeneration frequency (B) were scored using callus induction and shoot regeneration media as described in Kischenko et al., 2005 (see also Example 1). A total of five independent events overexpressing AtGRF5 were assayed (AtGRF5-36, AtGRF5-41, AtGRF5-14, AtGRF5-94, and AtGRF5-50). Nontransgenic sugar beet shoots (WT1 and WT2) and transgenic shoots overexpressing the tDT reporter protein (tDT-event) served as controls. AtGRF5 expression levels were determined by qRT-PCR of samples isolated from the GRF5 transgenic events used in the assay (C). [Figure 10] Figure 1 shows the transformation frequency in maize using either the control construct 70S::tDT, or a construct overexpressing AtGRF5 (cDNA - SEQ ID NO: 1; amino acid sequence - SEQ ID NO: 2), or two GRF5 maize orthologs [ZmGRF5 version A (synthetic DNA - SEQ ID NO: 208; amino acid sequence - SEQ ID NO: 1) and ZmGRF5 version B (synthetic DNA - SEQ ID NO: 210; amino acid sequence - SEQ ID NO: 112)]. Transformation efficiency was calculated as the number of transgenic events divided by the number of immature embryos inoculated. [Figure 11] Figure 1 shows shoot development and DsRed fluorescence on soybean explants 21 days after transformation. Representative photographs of explants from the DsRed control, AtGRF5, and GmGRF5 plants show (A) shoot development at the primary node and (B) DsRed fluorescence in the shoot. The DsRed photograph is a composite of multiple photographs and is roughly aligned for orientation. [Figure 12] The formation of "good" shoot outgrowth continued to increase rapidly from days 16 to 22 of selection. To emphasize the rapid shoot outgrowth during selection of explants transformed with the three constructs, the percentage of explants was scored at two time points. [Figure 13] Figure 1 shows shoot formation in soybean explants transformed with or without GRF5 variants. Photographs taken from the top and side of one replicate are shown for the three treatments (constructs). Explants transformed with AtGRF5 (middle panel) and GmGRF5 (bottom panel) show more pronounced shoot outgrowth compared to the DsRed control (top panel). [Figure 14] Box plots are shown visualizing the significant increase in transgenic shoot regeneration in explants transformed with either AtGRF5 or GmGRF5 at 16 and 22 days of selection. [Figure 15] Figure 1 shows shoot development and DsRed fluorescence on canola explants 21 days after transformation. Representative photographs of explants from the DsRed control, AtGRF5, and BnGRF5 show (A) callus development on hypocotyl sections and (B) DsRed fluorescence on the explants. [Figure 16] Boxplot depicting the percentage of explants expressing DsRed across five Brassica napus experiments transformed with AtGRF5, BnGRF5, and the DsRed control. The percentage of explants expressing DsRed is significantly higher with AtGRF5 and BnGRF5 compared to the control construct, increasing by nearly three-fold on average. [Figure 17] Figure 1 shows co-transformation of sugar beet callus with the control constructs 70S-tDT and 70S-AtGRF5. In the co-transformation experiments, callus was inoculated with a 1:1 mixture of Agrobacterium strains containing each construct individually.

[0073] Example 1. Experiments with sugar beet (Beta vulgaris) Preparation of binary plasmids: The binary vector pZFN-nptII-GRF5 was constructed using standard cloning procedures. The cDNA encoding GRF5 (At3g13960) was cloned into the T-DNA of this vector between a dual CaMV35S promoter and a nopaline synthase (NOS) terminator to ensure high ectopic expression levels of the GRF5 protein. The T-DNA also contains the neomycin phosphotransferase II (nptII) gene, which confers resistance to a range of aminoglycoside antibiotics, such as kanamycin or paromomycin, and was used for selection of transgenic plant cells and tissues. The nptII gene is flanked by the NOS promoter and pAG7 terminator. The backbone of the binary vector contains the colE1 and pVS1 origins for plasmid replication in E. coli and Agrobacterium tumefaciens, respectively; and the aadA gene, which confers streptomycin / spectinomycin resistance for bacterial selection. The pZFN-nptII-GRF5 plasmid was transformed into the AGL-1 Agrobacterium strain using standard procedures.

[0074] Transformation of micropropagated shoots: Sugar beet shoots were transformed in different ways: a) Agrobacterium-mediated transformation based on Kischenko et al., 2005 Cell Biology International: 1. Micropropagated shoots of genotype S706 were used as starting material. Shoots were grown in MS salts supplemented with 30 g / L sucrose and 0.25 mg / L benzyladenine (BAP). 2. To induce friable callus, the seeds were cultured in MS salts containing 15 g / l sucrose and 2 mg / l BAP at approximately 30°C for several weeks. 3. Friable callus was harvested. 4. Agrobacterium AGL-1 containing the vector pZFN-nptII-GRF5 was grown in an appropriate medium supplemented with the appropriate antibiotic. 5. Callus was inoculated with an Agrobacterium suspension. Co-cultivation of callus tissue with Agrobacterium was carried out for at least 2 days in a medium containing 440 mg / L CaCl2·2H2O, 170 mg / L KH2PO4, 1900 mg / L KNO3, 370 mg / L MgSO4, 1650 mg / L NH4NO3, 2 mg / L BAP, 40 μg / L acetosyringone, 20 g / L sucrose, and 2 g / L glucose. 6. The calli were subcultured onto MS salts supplemented with 30 g / l sucrose, 1 mg / l GA3, 1 mg / l TDZ and 500 mg / l timentin and incubated in the dark for about 1 week. 7. For selection of transgenic cells, the calli were transferred to step 6 medium supplemented with 100 mg / l paromycin and incubated in the light for several weeks. 8. Transgenic calli were selected and subcultured several times on the same medium and conditions. 9. Regenerated shoots were isolated and propagated in MS salts containing 30 g / l sucrose, 0.25 mg / l BAP and 100 mg / l kanamycin. 10. The green shoots were transferred to MS salts supplemented with 30 g / l sucrose, 0.1 mg / l BAP, 250 mg / l timentin and 200 mg / l paromycin to finish the selection stage and were propagated periodically in this medium several times. 11. Leaves were isolated from green growing shoots for DNA extraction and PCR analysis to confirm the putative transformed lines. 12. The selected shoots were rooted in MS salts supplemented with 0.5 mg / l IBA, 100 mg / l cefotaxime and 10 mg / l PPT and transferred to greenhouse for seed production.

[0075] b) Particle gun method for sugar beet callus Friable callus was prepared as previously described in the methods section 1.2.1. 2. Osmotic treatment was carried out for several hours. 3. Preparation of gold particles and DNA coating were performed using standard procedures as described in the PDS-1000 / He instruction manual. Plasmids pZFN-nptII-GRF5 and pUbi4-tDT (containing a red fluorescent reporter) were co-precipitated with gold particles. As a control, gold particles were coated with pUbi4tDT and pABM70STurboYFP (containing a yellow fluorescent reporter). 4. Calli were bullet-transduced using a PDS-1000 / He unit (Bio-Rad) with 30 ng of gold particles coated with 500 ng of DNA per shot. 5. To assess the effect of transient expression of GRF5 on shoot regeneration frequency, the transfected calli were incubated in MS salts supplemented with 30 g / L sucrose, 1 mg / L GA3, and 1 mg / L thidiazuron for approximately 2 weeks in light. Shoot number was scored using a standard binocular.

[0076] result: Agrobacterium tumefaciens-mediated transformation of calli derived from micropropagated shoots of sugar beet with the construct pZFN-nptII-GRF5 significantly increased the number of regenerated shoots at the end of the selection step (Table 2). The construct pZFN-tdT-nptII (control), which contains a red fluorescent reporter, was used as a control. Five independent experiments show an increase in the average transformation frequency from 5.0% to 33.9%. Furthermore, the number of PCR-confirmed transgenic events increased from an average of 4.8 events per transformation experiment to 25.4 events.

[0077] [Table 2]

[0078] In callus transformation experiments, shoot regeneration was also measured at each selection step. Quantification of the amount of shoot regeneration at each selection step revealed that overexpression of GRF5 in sugar beet callus promoted shoot organogenesis (Table 3, Figures 1 and 2). In a separate callus transformation experiment, shoot regeneration was measured for two resistant sugar beet genotypes (Figure 8). Transformation with the construct pZFN-tdT-nptII (control) resulted in no transgenic shoots in genotype A and an average of only 0.3 shoots per callus in genotype B. Using AtGRF5 overexpression under the 70S promoter, the average number of transgenic shoots increased from 0 to 1.67 in genotype A and from 0.3 to 4.82 in genotype B. This impressively demonstrates the potential of AtGRF5 and opens up the possibility of genotype-independent action using standard transformation methods.

[0079] Further experiments demonstrated that the use of a construct for overexpressing AtGRF5 in sugar beet callus (Beta vulgaris calli) could increase the number of transgenic events per inoculated callus by ninefold (Figure 9). As a control, the construct pZFN-tdT-nptII (control), which contained a red fluorescent reporter, was used. The GRF gene and reporter gene were under the control of a constitutive 70S promoter (a double-enhanced constitutive 35S promoter derived from cauliflower mosaic virus).

[0080] The expression level of GRF5 was determined in 11 randomly selected independent transgenic events in sugar beet. Expression analysis was performed using primers that bind to the 3'-UTR of the NOS terminator. High levels of GRF5 expression were detected in all transgenic events analyzed (see Figure 4).

[0081] [Table 3]

[0082] In further callus regeneration experiments, five different transgenic events overexpressing AtGRF5 in sugar beet were analyzed. Two nontransgenic sugar beet lines (WT1 and WT2) served as controls, and a transgenic line overexpressing the tdT reporter protein served as controls. Figure 9A shows that the average callus formation frequency in the control lines and transgenic AtGRF5 events was similar and possibly slightly enhanced in the transgenic events. Figure 9B shows the average number of transgenic shoots. Four of the five AtGRF5 events showed a significant increase in shoot formation compared with the two controls. Figure 9C reveals that the favorable effect on shoot formation is related to the expression level of AtGRF5 in the transgenic events.

[0083] In cotransformation experiments, the control constructs 70S-tDT and 70S-AtGRF5 were stably integrated into the genome of sugar beet callus cells. In these cotransformation experiments, callus was inoculated with a 1:1 mixture of Agrobacterium strains containing each construct individually. The average cotransformation frequency was 31.5%. As shown in Figure 17, the number of red-fluorescent (tDT-positive) events was approximately 8-fold higher in the cotransformation experiments than in the single-transformation experiments.

[0084] Particle bombardment transformation using the method described in 2.2 demonstrated a significant increase in transformation frequency, even with transient (over)expression of GRF5. The bombardment-transfected calli exhibited enhanced regeneration ability. Figure 3 shows that calli biolistically transformed with the construct pZFN-nptII-GRF5 exhibited an increased number of regenerated shoots compared to calli produced by the control construct.

[0085] In additional callus transformation experiments, the transformation frequency in sugar beet was determined using the construct pZFN-nptII-AtGRF5 compared with the same construct, but also expressing a GRF homologue from sugar beet (Beta vulgaris) (BvGRF5). As a control, the construct pZFN-tdT-nptII (control), containing a red fluorescent reporter, was used. The GRF gene and reporter gene were under the control of a constitutive 70S promoter. The experiment shown in Figure 6 was stopped during the early selection phase. This explains why the transformation efficiency of the tDT construct was 0%. We were able to achieve an average transformation frequency of 70% with the AtGRF5 construct and 32.5% with the BvGRF5 construct.

[0086] 2. Oryza sativa experiment Constructs used in binary plasmids: A binary vector was constructed using standard cloning procedures. Within the T-DNA of this vector, a cDNA encoding GRF5 (At3g13960) was cloned between an appropriate promoter and terminator to ensure sufficient ectopic expression levels of the GRF5 protein in rice (single construct). The T-DNA also contained a GFP gene, which was used for the selection of transgenic plant cells and tissues. Furthermore, a binary vector carrying an additional gene of interest under the control of a promoter and terminator was constructed (double (stack) construct), in addition to the cDNA encoding GRF5 (At3g13960) containing an appropriate promoter and terminator to ensure sufficient ectopic expression levels of the GRF5 protein in rice.

[0087] Seed sterilization and sowing: Wild-type green seeds were incubated in 70% ethanol and shaken for approximately 1 minute. After removing the ethanol, the seeds were washed once with sterile mQ water. Next, 30 ml of 6% sodium hypochlorite solution was added, and the seeds were shaken for 40–60 minutes. After removing the sodium hypochlorite solution, the seeds were washed 3–5 times with sterile mQ water.

[0088] After sterilization (0-3 hours), the seeds were dried on sterile filter paper and placed on the surface of induction medium R001. Incubation was carried out at 32°C under continuous light (3000 lux) for 6 days.

[0089] Transformation and co-cultivation: For explant preparation, expanded embryos (calli derived from the scutellum) from wild-type seeds suitable for transformation were selected and transferred to liquid infection medium (R002) containing Agrobacterium tumefaciens transformed with the plasmids introduced into plant cells for 1.5 min, then transferred to co-cultivation plates (R003). The plates were incubated in the dark at 25°C for 3 days.

[0090] Selection of resistant tissues: Three days after co-cultivation, the calli were removed from the seeds, washed several times with sterile mQ water, washed once with sterile mQ water containing 250 mg / L cefotaxime, and transferred to R004 selection medium. Incubation was carried out at 32°C under continuous light (3000 lux) for 2 weeks.

[0091] Microcallus isolation and regeneration: Using sterile forceps, the microcallus was transferred to R005 and incubated at 32°C under continuous light (3000 lux) for one week. The GFP, used as a selection marker, was then confirmed in the dark. Healthy GFP-positive callus was transferred to R006 and further incubated at 32°C under continuous light (3000 lux) for one week. For continuous regeneration, the callus was transferred to R007 and incubated at 32°C under continuous light (3000 lux) for three weeks. Healthy seedlings were extracted using sterile forceps and transferred to R008. They were incubated at 32°C under continuous light (3000 lux) for two weeks, after which the seedlings were brought into the greenhouse.

[0092] [Table 4-1] [Table 4-2] [Table 4-3]

[0093] result: Agrobacterium tumefaciens-mediated transformation of immature rice embryos with different constructs containing GRF5 leads to a significant increase in the average transformation frequency. As a control or comparison, the transformation efficiency observed for 28 randomly selected other constructs without GRF5 was used, which showed an average transformation efficiency of 63%. With the "single" construct, transformation efficiency increased on average from 63% to 78%, and with the "double" construct, it could be increased from 63% to 84% (Table 5).

[0094] [Table 5]

[0095] 3. Corn (Zea mays) experiment Constructs used in the Binar plasmid: Binary vectors were prepared using standard cloning procedures. A) cDNA encoding AtGRF5 (SEQ ID NO: 1), b) synthetic DNA encoding ZmGRF5 (version A) (SEQ ID NO: 208), and c) synthetic DNA encoding ZmGRF5 (version B) (SEQ ID NO: 210) were cloned into the T-DNA of these vectors between an appropriate promoter (e.g., the BdEF1 promoter) and terminator to ensure sufficient ectopic expression levels of the GRF5 protein in maize. As a control, a construct containing a tDT reporter gene under the control of a 70S promoter with a ZmUbi intron was used.

[0096] Transformation: Agrobacterium tumefaciens-mediated transformation has been performed by standard methods to transform monocotyledonous plants by using the scutellum of immature embryos (e.g., WO 95 / 067222).

[0097] result: As shown in Figure 10, Agrobacterium tumefaciens-mediated transformation of immature embryos with constructs containing GRFs derived from Arabidopsis thaliana led to a significant increase in the average transformation frequency from 8% to 14%. In contrast, using both versions of ZmGRF5 derived from different maize (Zea mays) genotypes resulted in a more robust increase in transformation efficiency. ZmGRF5 version A increased transformation efficiency from 8% to 52%, and ZmGRF5 version B increased transformation efficiency from 8% to 48%.

[0098] 4. Glycine max (soybean) experiment Soybean transformation: Transformation of Glycine max (soybean) was performed using Agrobacterium rhizogenes to deliver T-DNA to axillary meristem cells of epicotyls located at the primary node of soybean seedlings of the cultivar "Jake" (Olhoft PM, Bernal LM, Grist LB, Hill DS, Mankin SL, Shen Y., Kalogerakis M., Wiley H., Toren E., Song H.-S., Hillebrand H., and Jones T. 2007, A novel Agrobacterium rhizogenes-mediated transformation method for soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings, In Vitro Cell. Dev. Biol. - Plant 43:536-549; U.S. Patent Application Publication No. 2014237688, WO 2006024509, WO 2005121345).

[0099] Preparation of binary plasmids: Three binary plasmids were constructed using standard cloning procedures. The first binary plasmid was a control plasmid (referred to as the DsRed control) used in the experiments and served as the base vector for the other vectors. This plasmid contained within the T-DNA the DsRed gene, which was used for phenotypic scoring of transgenic plant cells and tissues, and the AtAHAS gene, which was used for preferential selection of transgenic cells. Both genes were cloned with appropriate promoters and terminators to ensure sufficient ectopic expression levels for the purposes described above. The second binary plasmid contained a base vector with a cDNA encoding AtGRF5 (SEQ ID NO: 2) cloned between an appropriate promoter and terminator to ensure sufficient ectopic expression levels of the GRF5 protein in soybean. The third binary plasmid contained a base vector with a cDNA encoding GmGRF5 (SEQ ID NO: 106) cloned between an appropriate promoter and terminator to ensure sufficient ectopic expression levels of the GRF5 protein in soybean.

[0100] Seed sterilization and germination: Soybean seeds of the "Jake" cultivar were sterilized in a chamber using chlorine gas generated by adding 3.5 ml of 12 N HCl to 100 ml of bleach. After sterilization, approximately 65 seeds were placed on PlantCons™ solid germination medium (1x B5 salts and vitamins, 2% sucrose, 0.8% Noble agar (A5431 Sigma-Aldrich®); pH 5.8). The seedlings were grown at 26°C under a light (150 μm) exposure. -2 s -2 ) for 7 days and used as explant material for transformation.

[0101] Preparation of Agrobacterium: Agrobacterium rhizogenes (WO 2006024509) was transformed with one of the following vectors: (1) pSUPER:DsRed and pPcUBI:AHAS selectable marker as a control, or the control vector plus either (2) pPcUbi-AtGRF5 or (3) pPcUBI-GmGRF5. A. rhizogenes was grown and inoculated into 50 ml of liquid inoculation medium (1 / 10 th B5 salts (G768 Phytotech, 3% sucrose, 20 mM MES, 1x Gamborg Vitamins, 200 μM acetosyringone, 1.44 μM gibberellic acid, 5.0 μM kinetin; pH 5.4) were added to a Falcon tube at OD 400. 600 The Agrobacterium suspension was then resuspended until the pH reached 1.5. The Agrobacterium suspension was then placed into a deep Petri dish for harvesting the prepared explants.

[0102] Explant preparation and transformation: Seedling explants were prepared from 8-day-old seedlings by removing the root and most of the hypocotyl, one cotyledon, the axillary bud tissue outgrowth of the cotyledonary node, and the epicotyl above the primary node, including all preformed leaves. After explant preparation, approximately 45-50 explants were incubated with the Agrobacterium suspension in a Petri dish for 30 minutes. The explants were then placed in co-cultivation medium (1 / 10 th The seeds were placed on moist filter paper in a Petri dish containing B5 salts (G768 Phytotech), 3% sucrose, 20 mM MES, 0.5% Noble agar (A5431 Sigma-Aldrich®), 1x Gamborg vitamins, 200 μM acetosyringone, 1.44 μM gibberellic acid, 5.0 μM kinetin, 4.1 mM L-cysteine, 0.5 mM dithiothreitol, 0.5 mM sodium thiosulfate; pH 5.4) and sealed in a container at room temperature for 5 days.

[0103] Shoot development and selection: After 5 days, explants were transferred to selective medium (1x B5 salts and vitamins (G398 Phytotech), 3% sucrose, 3 mM MES, 1 μM 6-benzylaminopurine, 5 μM kinetin, 250 mg / L timentin STK, 3 μM imazapyr, 0.8% Noble agar (A5431 Sigma-Aldrich®); pH 5.6), five per plate, and incubated at 26°C. Explants showed significant growth from the axillary meristems of the primary node 16 days after selection and were first scored for shoot development (regeneration). After 22 days of selection (the end of selection), explants were removed from the solid medium and placed on Oasis® medium. They were then scored for (1) the quality of shoot formation and (2) DsRed fluorescence of shoots developing from the primary node.

[0104] Experimental Design and Results: One experiment was conducted by four researchers using three constructs (Table 6). Soybean primary node axillary meristems were transformed with the DsRed control, AtGRF5, and GmGRF5. A total of 524 seedling explants were transformed and scored 16 days after selection (21 days after transformation) and 22 days after selection (27 days after transformation). Shoots 16 days after selection showed rapid growth at the primary node, consisting of a small, compact shoot pad combined with larger, elongated shoots (Figure 11). The regeneration rate at the target tissue, the primary node [(number of explants with shoots) / total explants × 100], was 80–100% for all constructs and was fixed for 16 days of selection. Explant shoots continued to grow rapidly between days 16 and 22. At both time points, explants were subjectively scored for the presence of healthy, elongated shoot growth with a morphology predictive of successful formation of rooted transgenic plants ("good") (Figure 12). There was an increase in "good" shoot formation in explants transformed with all three constructs across both time points, particularly in explants transformed with AtGRF5 and GmGRF5. Across the four replicates, explants transformed with either form of GRF5 tended to exhibit greater shoot formation (larger shoots, more elongated shoots) compared to the DsRed control (Figure 13).

[0105] To obtain an early measure of transformation efficiency, primary nodes resulting from transgenic cells expressing DsRed protein were scored for the presence or absence of DsRed-fluorescent shoots at 16 and 22 days of selection. DsRed expression was significantly stronger in constructs containing AtGRF5 or GmGRF5 than in the DsRed control at both time points, but was even stronger at 16 days of selection (Figure 11). Explants characterized as having "good" shoot morphology generally had shoots with DsRed expression (Figure 11). The percentage of explants with DsRed-expressing shoots was significantly higher (α = 0.05) in explants transformed with either AtGRF5 or GmGRF5 compared with explants transformed with the DsRed control at both time points (Table 6; Figure 14). Of the explants transformed with the DsRed control construct, 54.5% displayed shoots expressing DsRed at 22 days after selection, compared with 70.2% for AtGRF5 and 74.6% for GmGRF5 (Table 6). The data indicate that at 27 days after transformation, soybean explants transformed with either AtGRF5 or GmGRF5 regenerated significantly more transgenic shoots at the primary node than explants not transformed with either form of GRF5.

[0106] [Table 6]

[0107] 5. Experiments with Brassica napus (canola) Canola transformation: Transformation of Brassica napus was performed using Agrobacterium rhizogenes to deliver T-DNA into hypocotyl segments of B. napus seedlings of the genotype BNS3.

[0108] Seed sterilization and germination: Seeds were surface sterilized by placing 200-300 seeds in 70% ethanol in a 50 mL Falcon tube for 2 minutes. After gentle shaking for 2 minutes, the ethanol was removed and 40-50 mL of 30% Clorox bleach was added with one drop of Tween. The seeds were incubated for 10 minutes with occasional mixing. The liquid was removed, and the seeds were then rinsed three times with sterile water. The seeds were then placed in germination medium (1 / 2x MS salts / vitamins, 10 g / L) in a PlantCon box. -1 Sucrose, Phyto Agar 7g -1 The boxes were placed in a chamber at 23°C in the dark for 4 to 5 days.

[0109] Preparation of Agrobacterium: Agrobacterium rhizogenes was transformed with one of the following vectors: (1) pSUPER:DsRed and PcUBI:AHAS selectable marker as a control, or the control vector plus either (2) PcUbi-AtGRF5 or (3) PcUBI-GmGRF5 (SEQ ID NO: 108). A. rhizogenes was grown at OD 600 The culture was grown until the β-glucan content reached 1.0, and then inoculated with liquid inoculation medium (1x MS salts / vitamins, 30g / -1 The Agrobacterium suspension was diluted to 0.1 with sucrose (pH 5.8) and then placed in a dish for harvesting the prepared hypocotyl explants.

[0110] Explant preparation and transformation: Hypocotyl explants were prepared from 4- to 5-day-old etiolated seedlings by removing them from the germination box and placing them on sterile filter paper moistened with Agrobacterium-free infection medium to keep the seedlings swollen. After removing the roots, cotyledons, and epicotyls, hypocotyl segments 7- to 10-mm long were prepared. After cutting, the explants were immersed in an Agrobacterium suspension, blotted onto dry sterile filter paper, and finally plated in co-cultivation medium (1x MS salts / vitamins, 30 g / ml). -1Sucrose, 0.6gl -1 MES, 18gl -1 Mannitol, 7gl -1 Phyto Agar, 1mgl -1 2,4-D, 100mgl -1 Acetosyringone, 200mgl -1 After plating approximately 50 explants, the plates were sealed with micropore tape and incubated at 23°C under a 16-hour light / 8-hour dark photoperiod for 3 days.

[0111] Callus development and shoot initiation: After 3 days, all explants were transferred to recovery medium (1x MS salts / vitamins, 30g l -1 Sucrose, 0.6gl -1 MES, 18gl -1 Mannitol, 7gl -1 Phyto Agar, 1mgl -1 2,4-D, 300 mg / l -1 The explants were transferred to selective medium #1 (1x MS salts / vitamins, 30g l -1 Sucrose, 0.5gl -1 MES, 7gl -1 Phyto Agar, 3mgl -1 BAP, 0.1 mg / l -1 NAA, 0.1 mg / l -1 GA3, 2.5mgl -1 AgNO3, 100nM Imazethapyr, 300mgl -1 After 1 week, the explants were transferred to Selection Medium 2 (1x MS salts / vitamins, 30g l -1 Sucrose, 0.5gl -1 MES, 7gl -1 Phyto Agar, 0.5mgl -1 BAP, 0.1 mg / l -1 GA3, 2.5mgl-1 AgNO3, 100nM Imazethapyr, 300mgl -1 The solution was transferred to timentin (pH 5.8).

[0112] Experimental Design and Results: Five experiments were conducted over time, involving three researchers, with at least one replicate per researcher (Canola-1 in the table). In Experiment 1, Brassica napus hypocotyls were transformed with the DsRed control and BnGRF5 constructs (except for Experiment 1); in other experiments, explants were transformed with the DsRed control, AtGRF5, and BnGRF5 constructs. A total of 3,156 explants were inoculated and scored for DsRed fluorescence 16–22 days later. At this time, explants rapidly developed callus, particularly at the cut ends of the hypocotyls. Explants were scored for the presence or absence of DsRed fluorescence, regardless of the size, intensity, and frequency of single explants. The frequency of DsRed-expressing sectors is an early indicator of transformation efficiency for a treatment.

[0113] DsRed expression was significantly stronger with constructs containing AtGRF5 or BnGRF5 than with the DsRed control (Figure 15). The percentage of explants with DsRed-expressing shoots was significantly higher in explants transformed with either AtGRF5 or BnGRF5 compared with explants transformed with the DsRed control (Table 7; Figure 16). Of the explants transformed with the DsRed control construct, 20.1% showed shoots expressing DsRed, compared with 56.6% for AtGRF5 and 58.5% for BnGRF5. The data show that 21 days after transformation, B. napus explants transformed with either AtGRF5 or BnGRF5 had nearly three times as many transgenic sectors as explants not transformed with either form of GRF5 (statistically significant α = 0.05). No significant differences between investigators and experiments were detected across treatments (constructs).

[0114] Table 7

Claims

1. A method for increasing the number of shoots regenerated from plant callus, comprising: (a) i. at least one nucleotide sequence of interest; and ii. An expression cassette comprising a polynucleotide encoding a GRF5 polypeptide, an mRNA encoding a GRF5 polypeptide, or a GRF5 polypeptide into a plant callus simultaneously or sequentially; (b) culturing the plant callus of (a) under conditions in which the GRF5 polypeptide is expressed from the expression cassette, translated from the introduced mRNA, or in the presence of the GRF5 polypeptide in the plant callus, and regenerating a shoot from the plant callus; (c) regenerating a plant from the shoot of (b) comprising at least one cell containing at least one nucleotide sequence of interest as a transgene; Including, the nucleotide sequence of interest encodes at least one phenotypic trait selected from the group consisting of resistance / tolerance to biotic stress, including pathogen resistance / tolerance, resistance / tolerance to abiotic stress, including cold / freezing tolerance, drought stress resistance / tolerance, osmotic resistance / tolerance, heat stress resistance / tolerance, cold or frost stress resistance / tolerance, oxidative stress resistance / tolerance, heavy metal stress resistance / tolerance, salt stress or waterlogging resistance / tolerance, lodging resistance / tolerance, grain shattering resistance / tolerance, resistance / tolerance to one or more herbicides, and alteration of further agronomic traits of interest, including increased yield, altered flowering time, altered seed color, altered endosperm composition, altered nutrient content, or altered metabolic engineering of a pathway of interest; The GRF5 polypeptide is (i) comprising an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112, or 209; method.

2. The method of claim 1, wherein the GRF5 polypeptide comprises a PFAM domain PF08880 and a PFAM domain PF08879, preferably wherein the PFAM domain PF08880 has at least a 90% coverage match at or near the N-terminus of the GRF5 polypeptide, and the PFAM domain PF08879 has at least a 90% coverage match at a position C-terminal to the PFAM domain PF08880 of the GRF5 polypeptide.

3. The method of claim 2, wherein both matching amino acid stretches are located in the N-terminal half of the GRF5 polypeptide, and preferably the matching amino acid stretch PFAM domain PF08880 is located in the N-terminal quarter of the GRF5 polypeptide.

4. 4. The method of any one of claims 1 to 3, wherein the GRF5 polypeptide comprises the motif: [D]-[PL]-[E]-[P]-[G]-[R]-[C]-[R]-[R]-[T]-[D]-[G]-[K]-[K]-[W]-[R]-[C]-[SA]-[RK]-[ED]-[A]-[YH]-[P]-[D]-[S]-[K]-[Y]-[C]-[E]-[KR]-[H]-[M]-[H]-[R]-[G]-[RK]-[N]-[R] (SEQ ID NO: 177), preferably wherein the motif comprises a subregion of the matching amino acid stretch PFAM domain PF08879.

5. the polynucleotide encoding the GRF5 polypeptide is (i) a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 105, 107, 109, 111, 207, 208, or 210; (ii) a nucleotide sequence comprising a sequence that is at least 70% identical to the nucleotide sequence of (i); (iii) a nucleotide sequence encoding, within the degeneracy of the genetic code, a polypeptide encoded by (i) or (ii); (iv) a nucleotide sequence complementary to the nucleotide sequence of (i), (ii), or (iii); or (v) a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of (iv); 5. The method of claim 1, comprising:

6. The method described in any one of claims 1 to 5, wherein introducing the expression cassette containing a polynucleotide encoding a GRF5 polypeptide into a plant callus results in stable integration into the genome of the plant callus, or introducing the expression cassette containing a polynucleotide encoding a GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or a GRF5 polypeptide into a plant callus results in transient expression of the GRF5 polypeptide within the plant callus or its descendant cells.

7. 7. The method of claim 1, wherein the polynucleotide encoding the GRF5 polypeptide is operably linked to at least one regulatory sequence suitable for expression of the GRF5 polypeptide in the plant callus.

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