Regeneration by protoplast callus grafting

Abstract

The present invention relates to a method for producing a shoot of a plant comprising a germline progenitor cell of a difficult-to-regenerate plant. The germline progenitor cell may be modified to contain a mutation in a sequence of interest. The present invention further relates to a plant obtainable by the method of the invention, wherein the plant preferably comprises at least the L2 meristem layer of the difficult-to-regenerate plant.

Description

[Technical Field]

[0001] The present invention relates to the field of molecular plant biology, in particular to the field of plant regeneration. The present invention relates to a method for improving the regeneration potential and / or regeneration efficiency of plants. [Background technology]

[0002] Because plant cells generally have limited regeneration potential, regeneration from single cells to whole plants is a bottleneck in many plant biotechnology workflows. Such workflows include protocols for targeted genome editing, clonal propagation of haploid or genetically complex plant material, stable transformant generation, and doubled haploid induction. While regeneration potential varies among different plant species, plant varieties, and plant tissue sources, the fraction of cells that are successfully regenerated into plants is usually quite small (Srinivasan et al., Planta 2007, 225:341-351). Plant species or varieties that fail to regenerate or regenerate inefficiently are considered difficult to regenerate.

[0003] Many biotechnology workflows, such as genome editing by programmed nucleases based on DNA-free protocols, involve protoplasts, and a major obstacle to these rapidly evolving technologies is the regeneration of whole plants from these single (edited) cells. Thus, there is a strong need in the art to improve plant regeneration efficiency, preferably to improve the regeneration efficiency of plants that are difficult to regenerate. In particular, there is a strong need to improve the regeneration efficiency of plants that carry heritable mutations. Summary of the Invention

[0004] The present invention can be summarized in the following embodiments.

[0005] Embodiment 1. A method for generating and selecting shoots of a plant, comprising: (a) the process of grafting callus between the scion and rootstock; (b) forming a graft joint between the callus and one of the scion and the rootstock to form a graft union; (c) generating a wound at or near at least one of the graft joints; (d) forming shoots at the wounded graft-healed site; (e) selecting the shoots formed in step (d), said shoots comprising cells derived from the callus of step (a); and optionally (f) growing plants from the selected shoots of step (e). A method comprising:

[0006] Embodiment 2. The method of embodiment 1, wherein in step (e), the selected shoots comprise germline progenitor cells derived from the callus of step (a), and optionally, the method further comprises steps (f) and (g) of obtaining seeds or progeny of the plant grown in step (f), preferably by sexual propagation, preferably by at least one of selfing and backcrossing.

[0007] Embodiment 3. The method of embodiment 1 or 2, further comprising steps (f) and (g) of obtaining progeny of the plant grown in step (f) by vegetative propagation.

[0008] Embodiment 4. The method according to any one of embodiments 1 to 3, wherein the callus in step (a) is from a first plant and the scion and / or rootstock is from a second plant.

[0009] Embodiment 5. The method of embodiment 4, wherein the scion and rootstock are from the same or similar plant.

[0010] Embodiment 6. The method according to any one of embodiments 1 to 5, wherein the wounding treatment in step (c) is removal of the shoot apical meristem by truncation.

[0011] Embodiment 7. The method of any one of embodiments 1 to 6, wherein in step (d), the axis of the (wounded) graft union is substantially perpendicular to the ground surface, and the root apical meristem is closer to the ground surface than the shoot apical meristem.

[0012] Embodiment 8. The step (d) of allowing shoot formation comprises: d1) allowing callus to form at or near the graft junction; and d2) growing shoots from the callus 8. The method of any one of embodiments 1 to 7, comprising:

[0013] Embodiment 9. The method of any one of embodiments 1 to 8, further comprising, prior to step (a), growing the callus of step (a) from protoplasts.

[0014] Embodiment 10. The method of embodiment 9, further comprising the step of introducing a transgene and / or mutation in a sequence of interest into protoplasts, wherein the selected shoots in step (e) comprise germline progenitor cells, or germline cells derived therefrom, containing the transgene and / or mutation.

[0015] Embodiment 11. The method of any one of embodiments 1 to 10, further comprising a step of introducing a transgene and / or mutation in a sequence of interest into cells located in the callus of step (a) and / or the shoot formed in step (d), wherein in step (e), the selected shoot comprises a germline progenitor cell, or a germline cell derived therefrom, containing the transgene and / or mutation.

[0016] Embodiment 12. The method of embodiment 10 or 11, comprising step (f), wherein the plant part of the plant grown in step (f) comprises a transgene and / or a mutation, and preferably, the plant part can be used for vegetative propagation.

[0017] Embodiment 13. The method of any one of embodiments 10 to 12, wherein the mutation is introduced by programmed genome editing, preferably using a site-specific endonuclease, preferably a CRISPR endonuclease.

[0018] Embodiment 14. A plant obtainable from the method according to any one of embodiments 10 to 13, i) germline progenitor cells and / or germline cells derived therefrom of the callus of step (a); and ii) Plant parts for vegetative propagation of the callus of step (a) and A plant, wherein the germline progenitor cells, germline cells and / or plant parts comprise the transgenes and / or mutations of embodiment 10 or 11.

[0019] Embodiment 15. A plant according to embodiment 14, comprising cells derived from the callus of step (a) and cells derived from the scion and / or rootstock of step (a).

[0020] definition Throughout the specification and claims, various terms are used in connection with the methods, compositions, uses, and other aspects of the present invention. Such terms are to be given their ordinary meaning within the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0021] It will be apparent to one skilled in the art that any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

[0022] It will be clear to one skilled in the art how to carry out the conventional techniques used in the methods of the present invention. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields is well known to those skilled in the art and is discussed, for example, in the following references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and regularly updated; and the series Methods in Enzymology, Academic Press, San Diego.

[0023] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. Thus, the indefinite article "a" or "an" typically means "at least one."

[0024] The term "and / or" refers to a situation in which one or more of the stated cases may occur alone or in combination with at least one of the stated cases, up to a maximum of all of the stated cases.

[0025] As used herein, the term "about" is used to express and take into account small variations. For example, the term can refer to ±(+ or -) 10% or less, e.g., ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. In addition, amounts, ratios, and other numerical values ​​are presented herein in range format. Such range formats are used for convenience and brevity and include numerical values ​​explicitly stated as range limits, but it should be understood that they should be flexibly interpreted as including any individual numerical value or subrange encompassed within the range, as if each numerical value and subrange were explicitly stated. For example, a ratio in the range of about 1 to about 200 includes the explicitly stated limits of about 1 and about 200, but should also be understood to include individual ratios such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50, about 20 to about 100, etc.

[0026] The term "comprising" is intended to be open-ended and inclusive, not exclusive. Specifically, this term and its variations mean that the specified features, steps, or components are included. These terms should not be interpreted to exclude the presence of other features, steps, or components.

[0027] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule consisting of a chain of amino acids, regardless of its specific mode of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "portion" of a protein can still be referred to as a "protein." An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial or plant host cell.

[0028] "Plant" refers to any whole plant or any part of a plant tissue or organ available from the plant (e.g., pollen, seed, root, leaf, flower, flower bud, anther, fruit, etc.), as well as any derivatives and progeny derived from such a plant by selfing or hybridization or apomixis. Non-limiting examples of plants include African eggplant, garlic, artichoke, asparagus, barley, sugar beet, bell pepper, bitter melon, ground cherry, bottle gourd, cabbage, canola, carrot, cassava, cauliflower, celery, chicory, kidney bean, corn salad, cotton, cucumber, eggplant, endive, fennel, gherkin, grapes, chili pepper, lettuce, corn, melon, rapeseed, okra, and parsley. Crop plants and cultivated plants include, for example, parsnip, pepino, chili pepper, potato, pumpkin, radish, rice, loofah, rocket, rye, snake gourd, sorghum, spinach, loofah, pumpkin, sugar beet, sugarcane, sunflower, tomatillo, tomato, tomato rootstock, Brassica vegetables, watermelon, wax gourd, wheat, and zucchini.

[0029] "Plant cells" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., originating from a plant, either isolated or within a tissue, organ, or organism. A plant cell may be part of a multicellular structure, such as, for example, a callus, a meristem, a plant organ, or an explant. A plant cell may be a meristematic cell, a somatic cell, and / or a germ cell.

[0030] A "scion" is a shoot of a plant, preferably a young shoot of a young plant, that has been cut for grafting. The scion preferably has a functional shoot apical meristem.

[0031] A "rootstock" is the stem of a plant, preferably a young plant, having a functional root system and preferably containing a functional root apical meristem.

[0032] "Similar conditions" for the cultivation of plants / plant cells means, inter alia, the use of similar temperature, humidity, nutrient and light conditions, as well as similar watering and light / dark cycles.

[0033] The terms "homology," "sequence identity," and the like are used interchangeably herein. Sequence identity is defined herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleotide (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as the case may be, as determined by the match between stretches of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The sequence identity / similarity percentage can be determined over the entire length of the sequences.

[0034] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are consistent across an alignment window of elements, e.g., nucleotides or amino acids. The "percent identity" for an aligned section of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences divided by the total number of elements in the reference sequence section, i.e., the entire reference sequence or a smaller, defined portion of the reference sequence. "Percent identity" is the percent identity multiplied by 100.

[0035] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms, depending on their lengths. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Thus, sequences can be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined herein) (e.g., when optimally aligned using default parameters with the programs GAP or BESTFIT). Percent sequence identity is preferably determined using the "BESTFIT" or "GAP" programs in the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). GAP uses the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, Journal of Molecular Biology 48:443-453, 1970) to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Global alignment is preferably used to determine sequence identity when two sequences are of similar length. Generally, the default GAP parameters are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins).For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA, or open-source software such as the programs "needle" (which uses the global Needleman-Wunsch algorithm) or "water" (which uses the local Smith-Waterman algorithm) in EmbossWIN, version 2.10.0, using the same parameters as GAP above or default settings (for both "needle" and "water," and for both protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; the default scoring matrices are Blossum62 for proteins and DNAFull for DNA). The gap extension penalty is 0.5; the default scoring matrices are Blossum62 for proteins and DNAFull for DNA). "BESTFIT" performs an optimal alignment of the best similarity interval between two sequences, inserting gaps to maximize the number of matches using the Smith-Waterman local homology algorithm (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981; Smith et al., Nucleic Acids Research 11:2205-2220, 1983). When sequences have substantially different overall lengths, local alignments, such as those using the Smith-Waterman algorithm, are preferred.

[0036] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Applied Math (1988) 48:1073. More particularly, preferred computer programs for determining sequence identity include the Basic Local Alignment Search Tool (BLAST) program, publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. Mol. Biol. 215:403-410 (1990); BLAST program version 2.0 and above allows the introduction of gaps (deletions and insertions) in the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0037] Alternatively, percentage similarity or identity may be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences described herein can also be used as "query sequences" to perform searches of public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0038] "Nucleic acid" or "polynucleotide," as used herein, can include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982), which is incorporated herein by reference in its entirety for all purposes). Any deoxyribonucleotide, ribonucleotide, or nucleic acid component, as well as any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, are contemplated. The polymer or oligomer may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or artificially or synthetically produced. In addition, the nucleic acid may be DNA (optionally cDNA) or RNA, or a mixture thereof, and may exist permanently or transiently in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0039] "Isolated nucleic acid" is used to refer to a nucleic acid that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial or plant cell. The nucleic acid and / or protein may be at least one of a recombinant, synthetic, or artificial nucleic acid and / or protein.

[0040] The terms "nucleic acid construct," "nucleic acid vector," "vector," and "expression construct" are used interchangeably herein and are defined herein as an artificially created nucleic acid molecule that results from the use of recombinant DNA technology. Thus, the terms "nucleic acid construct" and "nucleic acid vector" do not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (a portion of) a naturally occurring nucleic acid molecule.

[0041] The vector backbone can be, for example, a binary or super-binary vector (see, e.g., U.S. Pat. No. 5,591,616, U.S. Patent Application Publication No. 2002138879, and WO 95 / 06722), a co-integration vector, or a T-DNA vector into which the chimeric gene is integrated, or, if suitable transcriptional regulatory sequences are already present, into which only the desired nucleic acid sequence (e.g., coding sequence, antisense, or inverted repeat sequence) is integrated downstream of the transcriptional regulatory sequence, as known in the art and described elsewhere herein. Vectors can contain additional genetic elements to facilitate their use in molecular cloning, such as, for example, selectable markers, multiple cloning sites, etc.

[0042] The term "gene" refers to a DNA fragment comprising a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, operably linked to a suitable regulatory region (e.g., a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing a polyadenylation site.

[0043] "Gene expression" refers to the process by which a DNA region that is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into RNA that is biologically active, e.g., RNA that has the ability to be translated into a biologically active protein or peptide, or, e.g., regulatory non-coding RNA.

[0044] The term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked can mean that the linked DNA sequences are contiguous.

[0045] "Promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acids. A promoter fragment is preferably located upstream (5') of a gene's transcription initiation site in the direction of transcription, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase and a transcription initiation site. In addition, the promoter fragment may contain any other DNA sequence, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that functions to directly or indirectly regulate the amount of transcription from the promoter.

[0046] A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically regulated (e.g., by exogenous application of certain compounds) or developmentally regulated. A "tissue-specific" promoter is active only in particular types of tissues or cells.

[0047] Optionally, the term "promoter" may also include the 5' UTR region (5' untranslated region) (e.g., as used herein, a promoter may include one or more portions of a transcribed region upstream from the translation start codon, as that region may have a role in regulating transcription and / or translation).

[0048] "3'UTR" or "3' untranslated sequence" (often referred to as the 3' untranslated region or 3' end) refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, transcription termination sites and (in most, but not all, eukaryotic mRNAs) polyadenylation signals (e.g., AAUAAA or variants thereof). After transcription is terminated, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in transport of the mRNA to the cytoplasm (where translation takes place).

[0049] The term "cDNA" refers to complementary DNA. Complementary DNA is produced by reverse transcription of RNA into a complementary DNA sequence. Thus, a cDNA sequence corresponds to an RNA sequence expressed from a gene. Because RNA sequences expressed from the genome may be spliced, i.e., introns are removed from the pre-mRNA by splicing and exons are spliced ​​together before being translated into a protein in the cytoplasm, expression of a cDNA is understood to refer to expression of the mRNA encoding the cDNA. Thus, because a cDNA can encode only the complete open reading frame for a protein, consisting of spliced ​​exons, a cDNA sequence may not be identical to its corresponding genomic DNA sequence, whereas a genomic DNA sequence may contain exon sequences interspersed with intron sequences. Thus, genetic modification of a protein-encoding gene may involve not only modifying the protein-encoding sequence, but also mutating the intron sequences of the genomic DNA and / or other gene regulatory sequences of the gene.

[0050] The term "regeneration" is defined herein as the formation of new tissues and / or new organs from a single plant cell, a group of cells, a callus, an explant, a tissue, or from an organ. Regeneration can include the formation of new plants from a single plant cell, or from, for example, a callus, an explant, a tissue, or an organ. Plant cells for regeneration may be undifferentiated plant cells. Preferred plant cells are protoplasts. The regeneration process can occur directly from parent tissue or indirectly, for example, via callus formation. The regeneration pathway may be somatic embryogenesis or organogenesis. Somatic embryogenesis is understood herein as the formation of a somatic embryo that can develop into a whole plant. Organogenesis is understood herein as the formation of a new organ from an (undifferentiated) cell. Organogenesis may be at least one of meristem formation, adventitious shoot formation, inflorescence formation, root formation, adventitious shoot elongation, and (subsequent) formation of a complete plant. Preferably, the regeneration is at least one of shoot regeneration, (ectopic) apical meristem formation, and root regeneration. Shoot regeneration as defined herein is de novo shoot formation. For example, the regeneration can be the regeneration of (inflorescence) shoots from (elongated) hypocotyl explants.

[0051] The term "normal growth conditions" is understood herein to mean the environment in which a plant grows, including, at a minimum, suitable temperature (i.e., between 0°C and 60°C), nutrients, a light-dark cycle, and watering.

[0052] The term "conditions permissive for regeneration" is understood herein as an environment in which plant cells or tissues can regenerate, preferably including normal growth conditions.

[0053] "Shoot organogenesis" is the regeneration pathway that occurs when cells, preferably cells from a callus or explant, form a de novo shoot apical meristem that develops into a leaf primordium and a leaf-bearing shoot. Because there is only one apical meristem, it is a monopolar structure, and no roots are formed at this stage. The vasculature of the shoot is often connected to the parent tissue. Only after the shoot is fully formed and elongated, e.g., after the callus or explant is removed, can root formation be induced in a separate root induction step on a different culture medium (Thorpe, TA (1993) In vitro Organogenesis and Somatic Embryogenesis: Physiological and Biochemical Aspects. In: Roubelakis-Angelakis KA, Van Thanh KT (eds) Morphogenesis in Plants. NATO ASI Series (Series A: Life Sciences), Vol. 253. Springer, Boston, MA).

[0054] Shoot organogenesis can occur spontaneously, i.e., without the exogenous addition of any plant growth regulators (PGRs). Shoot organogenesis can be induced by plant growth regulators, usually cytokinins at various concentrations, either alone or in combination with auxins, where the cytokinins preferably remain a component of the culture medium until the new shoot apical meristem and shoot have completed formation and are fully elongated, e.g., removed from the primary explant or callus. Preferably, the concentration of cytokinin exceeds the concentration of auxin for the induction of shoot formation.

[0055] "Somatic embryogenesis" leads to the formation of a bipolar structure resembling a zygotic embryo, which contains a root-shoot axis with a closed, independent vascular system. In other words, both the root and shoot primordia form simultaneously, without vascular communication with the underlying tissue (Dodds, JH and Roberts, LW (1985) Experiments in plant tissue culture. Cambridge University Press, Cambridge, UK). Somatic embryogenesis can be induced indirectly, for example, from callus or cell suspensions, or it can be induced directly on cells of an explant (Thorpe, supra). Somatic embryo formation passes through several distinct stages, from the globular embryo stage (a small, equal-diameter cell mass) through the cardiac embryo stage (a bilaterally symmetrical structure) to the torpedo embryo stage (elongation). The transition from a globular to a cardiac embryo is marked by the production of two cotyledons and the initiation of radicle development (Zimmerman, JL (1993) Somatic Embryogenesis: A Model for Early Development in Higher Plants. The Plant Cell 5:1411-1423; Von Arnold et al. (2002) Developmental pathways of somatic embryogenesis. Plant Cell, Tissue and Organ Culture 69:233-249). Finally, somatic embryos at the torpedo stage can develop into plantlets containing green cotyledons, an elongated hypocotyl, and a developed radicle with clearly differentiated root hairs in a process termed "germination" (similar to a zygotic embryo) or "conversion" or "maturation" (Von Arnold et al., supra). In direct or indirect induction of somatic embryogenesis, auxin is preferably used to induce embryogenic conditions in the callus at an early stage, but embryos form after subculture of the culture onto medium with or without reduced auxin levels. Auxins used to induce somatic embryos include, for example, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picloram, and dicamba.

[0056] The term "endogenous," when used in connection with a protein or nucleic acid in the context of the present invention, means that the protein or nucleic acid is still contained within the plant, i.e., is present in its natural environment. In most cases, an endogenous gene will be present in its normal genetic context in the plant.

[0057] "Plant hormones," "plant growth hormones," "plant growth regulators," or "phytohormones" are chemicals that affect the growth and / or development of plant cells and tissues. Plant growth regulators include five groups of chemicals: auxins, cytokinins, gibberellins, abscisic acid (ABA), and ethylene. In addition to these five major groups, two other chemical classes are often considered plant growth regulators: brassinosteroids and polyamines.

[0058] "Targeted mutagenesis" is mutagenesis that can be designed to modify specific nucleotides or nucleic acid sequences, including, but not limited to, oligonucleotide-directed mutagenesis, mutagenesis using RNA-guided endonucleases (e.g., CRISPR technology), meganuclease, TALEN, or zinc finger technology.

[0059] The term "sequence of interest" includes any genetic sequence preferably present in a cell, such as, but not limited to, a gene, a portion of a gene, or a non-coding sequence within or adjacent to a gene. A sequence of interest may be present in a chromosome, episome, organelle genome, such as a mitochondrial or chloroplast genome, or in genetic material that can exist independently of the body of genetic material, such as an infectious viral genome, a plasmid, an episome, or a transposon. A sequence of interest may be within the coding sequence of a gene, or within a transcribed non-coding sequence, such as a leader sequence, trailer sequence, or intron. The sequence of interest may be present in a double-stranded or single-stranded nucleic acid molecule. The nucleic acid sequence is preferably present in a double-stranded nucleic acid molecule. A sequence of interest may be any sequence within a nucleic acid, such as a gene, a gene complex, a locus, a pseudogene, a regulatory region, a highly repetitive region, a polymorphic region, or a portion thereof. A sequence of interest may also be a region containing a genetic or epigenetic variation that is indicative of a phenotype or disease. Preferably, the sequence of interest is a small or longer stretch of contiguous nucleotides (i.e., a polynucleotide) of double-stranded DNA, wherein said double-stranded DNA further comprises a sequence complementary to said sequence of interest on the complementary strand of said double-stranded DNA. The sequence of interest may be, or may be a portion of, a gene of interest, preferably an endogenous gene of interest. [Brief explanation of the drawings]

[0060] [Figure 1] Schematic representation of the present invention. [Figure 2] (A) Purple leaf of the RUBY tomato marker line. (B) Example of 11-week-old RUBY callus before grafting. (C) Example of grafted RUBY callus 7 days after grafting. (D) Ungrafted control callus after 2 weeks of culture. (E) Failed callus engraftment and (F) successful callus engraftment 3 weeks after the grafting procedure. [Figure 3](A) Brightfield image of a 2-week-old callus scion. (B) A callus scion like (A) several minutes after application of acid fuchsin (a red dye) to the root rootstock. Note the purple color change of the leaf due to uptake of acid fuchsin. (C) Brightfield image of a longitudinal section through the rootstock-callus graft area. (D) A section like (C) showing autofluorescence of chlorophyll and lignin as red and green signals, respectively. (E) Close-up of the indicated area in (D) showing the presence of whorl-shaped lignified callus cells. (F) Brightfield image of a second independent 2-week-old callus scion. (G) A callus scion like (F) several minutes after application of CFDA (a green dye) to the root rootstock. Note the green veins in the leaf due to uptake of CFDA molecules. (H) Close-up of the rootstock-callus-scion tissue showing localized CFDA signal (green signal to the left of the red asterisk) along the callus tissue. (I) Autofluorescence recording of (H) showing outlines of all tissues. (J) Shoot regeneration from the scion tissue (shoots are indicated by red arrows). [Figure 4] (A) Brightfield image of an early shoot regenerated from grafted RUBY marker callus tissue. (B) Autofluorescence recording of (A) showing the presence of phyllotactic patterning. (C) The same shoot 2 weeks after imaging in A and B. Green and red shoots were obtained from seedlings and callus, respectively. [Figure 5] (A) A 12-day-old intergraft of protoplast-derived Capsicum annuum cv. Maor callus between a C. baccatum seedling rootstock and scion. Capsicum annuum cv. Maor cells are labeled by GFP expression. (B) A leaf containing differentiated cells of both C. baccatum and Capsicum annuum cv. Maor genotypes. (C) An incomplete marginal chimeric shoot formed from the meristem associated with the leaf in (B). (D) A close-up of the shoot apex shown within the boxed area in (C), showing the presence of GFP-expressing Capsicum annuum cv. Maor cells. [Figure 6](A) Graft union of protoplast-derived tomato cv. Moneyberg callus between the F1 hybrid Solanum pennellii LA716 × Solanum lycopersicum LA3579 rootstock and scion. Protoplast-derived Moneyberg cells are labeled by GFP expression. (B) Sectioned chimeric leaf containing cells from both the F1 hybrid and Moneyberg genotype. (C) Chimeric shoot formed from the incomplete meristem from which the leaf in (B) was derived. DETAILED DESCRIPTION OF THE INVENTION

[0061] The inventors have discovered that, for example, grafting protoplast-derived callus between a scion and a rootstock, followed by inducing shoot regeneration, can result in shoots having the genotype of the callus. This has resulted in the effective regeneration of shoots from protoplast-derived callus of recalcitrant plants, which can then be directly regenerated into whole plants. Thus, the present method provides an efficient protocol for regenerating whole plants from callus derived from recalcitrant plant types, and also provides an efficient regeneration protocol for callus derived from more regenerative plant types. More specifically, the present method does not require the application of any (exogenously applied) hormones. Thus, the present method can be performed using hormone-free tissue culture media.

[0062] Thus, in a first aspect, there is provided a method for generating and selecting shoots of a plant, the method comprising: (a) the process of grafting callus between the scion and rootstock; (b) forming a graft joint between the callus and either the scion or the rootstock to form a graft union; (c) generating a wound at or near at least one of the graft joints; (d) forming shoots at the wounded graft-healed site; (e) selecting the shoots formed in step (d), said shoots comprising cells derived from the callus; and optionally (f) growing plants from the selected shoots of step (e). A method is provided that includes:

[0063] Preferably, the callus in step (a) is a protoplast-derived callus, which is understood to be a callus grown from a protoplast. Accordingly, step (a) may be preceded by a step of isolating protoplasts from a plant body, and an optional subsequent step of callus induction. Alternatively, the callus may be a wound-derived callus generated by wounding or truncating a plant body (i.e., removing all preformed shoot apical meristems), and an optional subsequent step of callus induction. For example, the callus formation in vitro and / or after grafting and wounding may occur spontaneously, i.e., in the absence of one or more exogenously supplied plant hormones. Alternatively, the callus formation may be induced and / or enhanced in the presence of one or more plant hormones.

[0064] Preferably, the graft joint formed in (b) is a bonded graft joint. A bonded graft joint should be understood as a joint that connects the rootstock or scion to the callus so as to transfer nutrients and water from the rootstock to the callus tissue. Intergrafting of the callus between the rootstock and scion should be understood herein as a process in which the callus is placed between the rootstock-explant and the scion-explant, thereby contacting the callus with either the rootstock or the scion. Subsequently, graft joints can be formed at the interface of the callus and on each of the two explants. The callus connects to both the rootstock and the scion through the de novo formation of one or more continuous vascular strands, thereby allowing, for example, nutrients, water, hormones, and other metabolites to flow through the callus, effectively creating the callus as an integral part of the grafted plant.

[0065] The graft union in step (a) can be carried out using any conventional method known to those skilled in the art. A callus grafted between a scion and a rootstock is herein understood as a callus in physical contact with at least a portion of the scion and a portion of the rootstock. Two junctions are then formed: one between the rootstock and the callus and one between the callus and the scion. The callus is preferably grafted along the hypocotyl or internode of the scion and the rootstock, preferably under sterile conditions. Preferably, the graft union results in the formation of vascular tissue in the callus, connecting the vascular tissue of the rootstock to the vascular tissue of the scion via the junction. The resulting overall structure of the rootstock, the grafted callus, and the scion is referred to as the graft union.

[0066] A rootstock can be prepared by removing the shoot containing the terminal bud before grafting, thereby giving a "truncated" plant, i.e., rootstock. Shoot removal is preferably performed by truncation at the hypocotyl or epicotyl, or at the internode. The scion may be a cotyledonary node. Preferably, the cotyledonary node is grafted onto a truncated hypocotyl rootstock with callus (substantially) placed between them (i.e., grafted) to form a graft union with two graft joints. A strip of callus may be formed at each of the two graft joints. Preferably, young plant material is used to prepare the scion and / or rootstock for grafting, and the young plant material is preferably seedling material 1 to 4 weeks or 1 to 3 weeks after sowing, preferably material about 2 weeks after sowing. Preferably, the young plant material used to prepare the scion and / or rootstock is seedling material having a width of 2-1 mm, 1.5-0.75 mm, 1-0.5 mm, or 0.5-0.25 mm. Preferably, the seedling material is used immediately after the first true leaves emerge. Preferably, the grafting process uses a suitable steel pin, preferably a sterilized steel pin, for aligning and fixing the rootstock, callus, and scion. Optionally, the steel pin is inserted into the center of the rootstock and scion (with the callus fixed therebetween).

[0067] Additionally or alternatively, ties, tape, bands, and / or clamps may be used around the graft union to hold the rootstock, callus, and scion together, and optionally, adhesive (glue, wax, or paste) may be used on the edges of the graft joint to secure them in place. Optionally, ties, tape, bands, and / or clamps may be used around the two grafting partners and callus to hold them together, and optionally, adhesive (glue, wax, or paste) may be used on the edges of the graft joint to secure them in place.

[0068] Preferably, at the graft union, the vascular tissues of the rootstock and the scion are connected to each other, allowing nutrients and water to move from the rootstock to the scion through the formation of vascular tissue in the graft callus. It should be understood that the callus and scion and / or rootstock in the method of the present invention are plants that naturally have the ability to form a graft union, optionally only under controlled experimental conditions. Preferably, the plant is a dicotyledonous plant. Optionally, the plant is a monocotyledonous plant. Preferably, the rootstock and / or scion used in the method of the present invention are derived from young plant material, such as young plant material obtained by in vitro micropropagation, young seedling material, or seed. Methods for grafting monocotyledonous plants are provided, for example, in WO 2020 / 099878 and WO 2020 / 099879, which are incorporated herein by reference. For use in the method of the present invention, known grafting methods are modified so that callus is placed between the scion and the rootstock or substantially between the scion and the rootstock.

[0069] Preferably, step (b) of forming a graft union has a duration of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or about 1, 2, or 3 weeks, during culture under conditions suitable for said graft union to form as exemplified herein.

[0070] Step (d) preferably involves the formation of several shoots, i.e., two or more shoots. Accordingly, step (e) of the method of the present invention may include a step of selecting a shoot from the plurality of shoots formed in step (d), wherein the selected shoot comprises callus cells. "Derived from callus" is herein understood to mean originating from callus by regeneration and thus having substantially the same genotype as callus cells. "Cells derived from callus" may also be expressed as "callus cells." Preferably, at least a portion of the selected shoots consist of callus-derived cells. Preferably, step (e) of the method of the present invention may include a step of selecting a shoot from the plurality of shoots formed in step (d), wherein at least a portion of the selected shoots consist of callus cells. Preferably, the axis of the graft union in step (d) is perpendicular or substantially perpendicular to the soil surface, and the root apical meristem is closer to the soil surface than the shoot apical meristem. It is known in the art that gravity contributes to endogenous hormonal regulation associated with shoot formation. In positioning the graft union vertical or substantially vertical to the ground surface, the rootstock is preferably placed closest to the ground surface compared to the remaining elements of the callus and the (remaining) scion, and can contribute to the formation of a shoot at the wound made in step (c). Preferably, the scion and rootstock are scion and rootstock from seedlings, and the graft union is preferably placed on the medium plate so that the largest surface of the graft union is in contact with the medium. Since the graft union is substantially vertical to the ground surface, the medium plate is preferably placed in a substantially vertical position. Preferably, the medium is hormone-free.

[0071] Callus may be from a regenerable or difficult-to-regenerate plant. In one embodiment, the callus is from a difficult-to-regenerate plant, i.e., a plant that is unable to regenerate under normal growing conditions, or preferably, a plant that exhibits poor regeneration efficiency under conditions known in the art to be optimal for regeneration. Such optimal conditions may include, but are not limited to, the presence of exogenously supplied growth regulators. Within a species, there may be both difficult and regenerable cultivars, varieties, and / or accessions; typical, non-limiting examples of plants known in the art to be difficult to regenerate are pepper (Capsicum annuum), sugar beet (Beta vulgaris, more particularly sugar beet (Beta vulgaris subsp. vulgaris)), soybean (Gycine max), sunflower (Helianthus annuus), cotton (Gossipium hirsutum), hemp or cannabis (Cannabis sativa), strawberry (Fragaria x ananassa), hops (Humulus lupulus), melon (Cucumis melo), and cucumber (Cucumis sativus). Callus may be a plant that does not show any or very little regeneration, preferably under conditions optimal for regeneration, but may also be a plant for which regeneration efficiency can be (further) improved. Thus, and as exemplified herein, the callus may be, but is not limited to, that of a plant of the Solanaceae family. Similarly, the scion and / or rootstock may be, but is not limited to, that of a plant of the Solanaceae family. However, one skilled in the art will appreciate that the method of the present invention is applicable to any plant, plant cell, callus, or protoplast that may benefit from increased regeneration efficiency, depending on the circumstances. The callus of step (a) may be from the same or a different plant as the scion and / or rootstock of step (a). The callus of step (a) may be from a plant of the same or a different species as the scion and / or rootstock of step (a). The callus of step (a) may be from a plant of the same or a different genus as the scion and / or rootstock of step (a).Thus, the callus in step (a) may be derived from a plant from which the scion and / or rootstock in step (a) are derived. Optionally, the callus in step (a) is derived from a plant different from the plant from which the scion and / or rootstock are derived, although all plants from which the callus, scion, and rootstock in step (a) develop may be from the same variety, cultivar, species, genus section, and / or genus. Preferably, the cells of the callus in step (a) of the method of the present invention have a different genotype from the cells of the scion and / or rootstock in step (a) of the method of the present invention. Preferably, the callus in step (a) of the method of the present invention is from a plant from which genetic material, through traditional breeding methods, can be exchanged with the plant from which the scion and / or rootstock in step (a) of the method of the present invention was excised. Preferably, the cells of the callus are capable of sexually hybridizing with cells of the rootstock and / or scion.

[0072] In a preferred embodiment, the callus or cells thereof in step (a) of the method of the present invention is a plant that may exhibit a lower regeneration efficiency than the scion and / or rootstock plant or cells or callus thereof in step (a) of the method of the present invention under conditions suitable, preferably optimal, for the regeneration of the scion and / or rootstock plant or cells or callus thereof. Such suitable and / or optimal conditions include at least a suitable nutrient supply, optionally supplemented with hormones. Such conditions may further include a suitable and / or optimal temperature and / or light / dark regime. Preferably, such suitable and / or optimal conditions are applied in step (d) of the method of the present invention. Preferably, the callus of step (a) exhibits a lower regeneration efficiency when exposed to conditions similar to those applied in step (d) of the method of the present invention, except that the callus is not in contact with the rootstock and scion of step (a). These conditions are preferably conditions suitable for the regeneration of the scion and / or rootstock plant or cells or callus thereof. These conditions are preferably conditions suitable for wounded or decapitated plants of the scion and / or rootstock to induce shoots. Those skilled in the art are aware of conditions suitable for regenerating regenerable plant cells. Such conditions may be conditions under which the callus of step (a) normally (i.e., when not in contact with the scion and rootstock of step (a)) does not or hardly exhibits regeneration. Optionally, the callus of step (a) is from a plant belonging to a species that exhibits lower regeneration efficiency than the plants of the rootstock and / or scion of step (a) of the method of the present invention. Alternatively, the callus of step (a) may belong to the same plant species as the scion and / or rootstock, but the scion and / or rootstock may have been transformed and / or mutated to exhibit increased regeneration efficiency compared to the callus. For example, the scion and / or rootstock may include constructs and / or transgenes that increase regeneration efficiency, such as, but not limited to, those described in WO 2019 / 211296 and WO 2019 / 193143, which are incorporated herein by reference.

[0073] Alternatively, the callus in step (a) of the method of the present invention, or cells thereof, is from the scion and / or rootstock plant, or a plant that can exhibit increased regeneration efficiency compared to the scion and / or rootstock plant, or cells or callus thereof, in step (a) of the method of the present invention, under conditions that are suitable, preferably optimal, for the regeneration of the cells or callus. Such suitable and / or optimal conditions are preferably as defined herein. Optionally, the callus in step (a) is from a plant belonging to a species that exhibits increased regeneration efficiency compared to the rootstock and / or scion plant in step (a) of the method of the present invention. Alternatively, the scion and / or rootstock may be obtained from a plant belonging to the same plant species as the callus, but that has been transformed and / or mutated to exhibit reduced regeneration potential compared to the callus. For example, scions and / or rootstocks may be obtained from plants that are non-regenerable mutants in the goblet gene (Berger Y. et al. (2009) The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves. Development 136(5):823-832), preferably the plants are homozygous gob null mutants.

[0074] Preferably, step (d) of the method of the present invention is carried out under conditions suitable for regenerating plants of the scion and / or rootstock. Optionally, step (d) of the method of the present invention can be carried out under conditions known to those skilled in the art to be suitable for wounded plants of the scion and / or rootstock or for shoot induction of wounded scion and / or rootstock. Such conditions may be conditions under which the callus of step (a) normally (i.e., when not in contact with or grafted to the scion and rootstock of step (a)) exhibits little or no regeneration.

[0075] Preferably, the callus in step (a) is a protoplast-derived callus, and the protoplast may be a somatic cell. Optionally, the protoplast and cells of the callus derived therefrom contain a positive selection marker.

[0076] Preferably, the scion and / or rootstock plants (or cells thereof) of step (a) of the method of the present invention are capable of regenerating under normal growth conditions, preferably in the absence of exogenously supplied (e.g., chemical addition through human intervention) growth regulators such as auxins and / or cytokinins. In a preferred embodiment, said conditions are at least the minimum required conditions for the regeneration of the scion and / or rootstock plants. Optionally, said conditions are at least suitable conditions, and optionally optimal conditions for the regeneration of the scion and / or rootstock plants or plant cells. Preferably, under such conditions, the scion and / or rootstock plants are capable of forming new shoots on the multicellular tissue. Regeneration is preferably by organogenesis and / or somatic embryogenesis. Preferably, the (regenerable) scion and / or rootstock plants are capable of regenerating shoots after wounding or decapitation.

[0077] The scion and / or rootstock plant may be a naturally occurring regenerable plant, i.e., a plant with natural regeneration capabilities. Alternatively, the scion and / or rootstock may be genetically modified to increase regenerability. Examples of genes, transgenes, or constructs capable of increasing the regenerability or regenerability of a plant include, but are not limited to, the genes, transgenes, and constructs disclosed in WO 2019 / 211296 and WO 2019 / 193143, which are incorporated herein by reference. As a non-limiting example, the scion and / or rootstock may be modified to exhibit induced or increased expression of a histidine kinase selected from the group consisting of CHK4, CHK2, and CHK3, preferably as described in WO 2019 / 193143. Additionally or alternatively, the scion and / or rootstock plants may be modified to preferably transiently exhibit induced or increased expression of at least one transcription factor associated with regeneration, preferably a WUSCHEL-related homeobox protein (preferably WOX5, optionally AtWox5 of SEQ ID NO: 1), a PLETHORA protein (preferably PLT1, optionally AtPLT1 of SEQ ID NO: 2), and a WOUND INDUCED DEDIFFERENTIATION 1 protein (WIND1, optionally AtWIND1 of SEQ ID NO: 3), preferably both WOX5 and PLT1, even more preferably WOX5, PLT1 and WIND1, as described in WO 2019 / 211296. Preferably, the transcription factor is under the control of an inducible promoter, and regeneration is induced by exposing cells of the scion and / or rootstock to an agent that causes induction of the inducible promoter. Optionally, the scion and / or rootstock is transfected with a SHOOT REGENERATION-2 vector or a SHOOT REGENERATION vector, as described in WO 2019 / 211296.The vectors may be introduced by transient or stable transfection, and regeneration may be induced by exposing the scion and / or rootstock to at least one of dexamethasone and estradiol, preferably both, since the indicated regeneration-related transcription factors are under the control of promoters inducible through administration of these compounds (see WO 2019 / 211296 in this regard). Additionally or alternatively, the scion and / or rootstock may have a mutation in an endogenous gene that results in improved regeneration capacity and / or efficiency. Non-limiting examples are known in the art, such as ATHB15 mutants as described in Duclerq et al. (Plant biology, 2011, 13, p317-324), KCS1 mutants as described in Shang et al. (PNAS 2016, 113, 5101-5106), ARR mutants as described in Buechel et al. (European Journal of Cell Biology 2010, 89:279-284), and ATRXR2 mutants as described in Lee et al. (2021) Cell Reports 37, 1-13.

[0078] Optionally, cells of the scion and / or rootstock are modified to contain a negative selection marker.

[0079] The methods provided herein may include a step (g) of obtaining seeds and / or progeny of the plants grown in step (f), preferably by sexual and / or vegetative propagation, which is preferably at least one of selfing and backcrossing.

[0080] Preferably, in step (e) of the method of the present invention, the selected shoots containing cells of the callus contain germline progenitor cells derived from the callus. Preferably, the method further comprises steps (f) and (g) of obtaining seeds and / or plant progeny of the plant grown in step (f) by sexual propagation, optionally by selfing and / or backcrossing. In the above embodiment, the method of the present invention may also be expressed as a method for generating and selecting shoots of a plant, wherein the selected shoots contain germline progenitor cells of the callus, i.e., germline progenitor cells regenerated from the callus in step (a) of the method of the present invention. Germline progenitor cells are herein understood as cells that will become gametes upon terminal differentiation, or their clonal progeny. Therefore, the genotype of germline progenitor cells determines the genotype of the gametes, and any genomic modifications made in germline progenitor cells will be inherited by subsequent generations. Therefore, the transgene or mutation introduced into the germline progenitor cells is heritable, i.e., a heritable transgene or a heritable mutation. The L2 shoot meristem layer can determine the genotype of the gametes (see, for example, Filippis et al. Using a periclinal chimera to unravel layer-specific gene expression in plants, The Plant Journal, 2013, 75:1039-1049). Preferably, the selected shoots containing callus cells in step (e) contain germline progenitor cells of the callus. Preferably, the shoots selected in step (e) contain tissue derived from the callus of (a), and preferably, the tissue is (at least a portion of) the L2 shoot meristem layer.

[0081] The shoot selected in step (e) of the method of the present invention may further comprise at least one of an L1 and an L3 shoot meristem layer derived from the callus of step (a). Optionally, the shoot comprises the L1, L2, and L3 shoot meristem layers of the callus of step (a). Alternatively, the shoot selected in step (e) of the method of the present invention may comprise an L2 shoot meristem layer derived from or regenerated from the callus of step (a) and at least one of an L1 and an L3 shoot meristem layer derived from or regenerated from the scion and / or rootstock of step (a). Meristem layers derived from the scion and / or rootstock may be regenerated from the scion and / or rootstock of step (a) via callus formation.

[0082] Additionally or alternatively, in step (e) of the method of the invention, the selected shoots comprise cells of the callus, and optionally the method further comprises steps (f) and (g) of obtaining the progeny of the plant grown in step (f) by vegetative propagation. Thus, the present invention also relates to a method for generating and selecting shoots of a plant, wherein the shoots comprise cells that give rise to clonal propagation tissue and / or plant parts of the callus of step (a), i.e., plant parts regenerated from the clonal propagation tissue and / or callus of step (a) of the method of the invention.

[0083] Thus, preferably the method of the invention comprises a step (e) of selecting a shoot, wherein at least a portion of said shoot consists of cells of callus (i.e. regenerated from the callus of step (a)), and preferably said portion comprises: i) tissue containing germline progenitor cells; and ii) Clonal (vegetatively propagated) tissue and / or tissue containing cells that give rise to clonally (vegetatively propagated) plant parts. At least one of the following is true.

[0084] Clonal propagation tissue and / or plant part is understood herein to mean tissue and / or plant part that can be used for clonally propagating progeny, i.e., subsequent generations of plants. Such tissue and / or plant part may be, but is not limited to, tubers, bulbs, corms, bulblets, suckers, scions, crowns, scales, rhizomes, stem tips, shoots or cuttings, knob-like bases or cuttings, stolon, tuberous cuttings or eyes, (clonally propagated) seeds, etc. Thus, the genotype of the clonally propagated plant part or tissue (or the cells giving rise to it) determines the genotype of the progeny of the clonally propagated plant, and any genomic modifications made in this tissue or part (or the cells giving rise to it) can be inherited by subsequent generations. Thus, transgenes or mutations made in clonally propagated plant parts (or the cells giving rise to it) are heritable transgenes or mutations.

[0085] The shoot, or at least one shoot, grown in step (d) and selected in step (e) of the method of the invention may be an adventitious shoot, or at least one adventitious shoot.

[0086] The callus in step (a) is contacted with the scion and rootstock to form a graft union. Optionally, the scion and rootstock are from the same plant or different plants. Alternatively, the scion and rootstock may be from similar plants, where similar plants are to be understood as plants of the same species, variety, or cross, and even more preferably, plants having substantially the same genotype. Preferably, the plants providing the scion and rootstock are obtained by vegetative propagation of the same plant and are therefore genetically identical. Optionally, the scion and rootstock are from two plants belonging to the same variety, cultivar, species, genus section, or genus. When the scion and rootstock are from the same or similar plants, in step (a) of the method of the present invention, the callus of a first plant may be contacted with the scion and rootstock of a second plant, where the first and second plants belong to different crosses, varieties, or species. Preferably, the first and second plants belong to the same genus or section. Alternatively, the callus can be derived from the same or similar plant as the scion and rootstock. For example, the callus in step (a) can be prepared from protoplasts isolated from leaves of a plant, and the plant (or a plant of the same variety, species, or hybrid) is later used to excise the rootstock and scion for grafting the callus in step (a). Alternatively, the callus in step (a) can be prepared from protoplasts isolated from leaves of a first plant, and the scion and rootstock are excised from a second plant, and the second plant is a plant of the same hybrid, species, or variety as the first plant.

[0087] If the scion is from a plant that is a different plant from the rootstock, in step (a) of the method of the present invention, the callus of the first plant may be contacted with the scion of the second plant and the rootstock of the third plant. Alternatively, the callus may originate from the same plant as the scion or the rootstock. Thus, in step (a) of the method of the present invention, the callus of the first plant may be contacted with the scion of the first plant and the rootstock of the second said plant, or in step (a) of the method of the present invention, the callus of the first plant may be contacted with the rootstock of the first plant and the scion of the second said plant. Preferably, the first, second and optional third plants belong to the same genus or section, preferably the same species, variety or hybrid, and even more preferably have substantially the same genotype.

[0088] In step (c), a wound is generated at or near at least one of the graft joints. This is performed after the formation of the graft joint in step (b). The wounding treatment can be performed by cutting, which induces the production of callus and adventitious shoots. Among these adventitious shoots, shoots comprising or consisting of cells of or derived from the callus of step (a) can appear spontaneously. The cells of or derived from the callus of step (a) can comprise derived germline progenitor cells and / or clonally propagated plant tissues and / or plant parts.

[0089] The wound is preferably made at the junction between the callus and the rootstock or scion at the graft union, i.e., at or near the graft joint formed between the callus and at least one of the rootstock or scion. Preferably, the wound is made at or near the graft joint formed between the callus and the scion. The wound is preferably made to the extent that substantially all of the scion is removed. More specifically, the cut is preferably made immediately above the junction between the callus and the scion, resulting in a thin layer of scion cells on the scion side of the callus-scion junction. Alternatively, or additionally, the wound may be made at or near the graft joint formed between the rootstock and callus, preferably immediately below the junction between the callus and the scion, resulting in a thin layer of rootstock cells on the rootstock side of the callus-scion junction. Thus, preferably, step (c) may comprise the step of making a wound at or near at least one of the graft joints, and allowing callus to form at the wounded graft joint.

[0090] The wound may be a complete cut, e.g., a transverse cut, separating the graft into two plant parts. Optionally, the wound (cut) does not completely separate the graft union into two plant parts, but is sufficient to initiate and / or stimulate callus production. Preferably, a shoot is grown from the callus, wherein the shoot may comprise or consist of tissue regenerated from the callus of step (a). Optionally, at least a portion of the shoot consists of cells regenerated from the callus. This particular method is preferably carried out under ambient conditions in a growth room or greenhouse.

[0091] Thus, optionally, the contacting in step (a) is carried out by grafting the callus onto a rootstock, and optionally grafting a scion onto said callus, and optionally forming two graft joints in step (b). The method further comprises step (c) of generating a wound at or near the very graft joint, preferably the graft joint between the scion and / or rootstock and the callus, allowing or inducing (further) callus to form at the (wounded) graft joint, and growing a shoot from said callus, said shoot comprising regenerated cells of the callus of step (a). Optionally, at least a portion of said shoot consists of regenerated cells of the callus of step (a).

[0092] Preferably, the method of the present invention allows the formation of at least one (adventitious) shoot comprising or consisting of tissue regenerated from the callus, preferably said tissue comprising germline progenitor cells and / or giving rise to clonally propagated tissue and / or clonally propagated plant part.

[0093] Step (d) is carried out under conditions suitable for shoot formation, optionally using conditions known by those skilled in the art to be suitable for shoot regeneration. Preferably, these conditions are at least the minimum requirements for shoot regeneration of the (wounded) plant from which the scion and / or rootstock are derived, and generally include at least the normal growth conditions of said plant. Preferably, step (d) includes callus formation prior to shoot formation. Step (d) may therefore include the substeps (d1) of allowing or inducing (further) callus formation at or near the wounded graft union; and (d2) of growing shoots from said callus, optionally with different culture conditions for (d1) and (d2). More specifically, step (d1) can be carried out under conditions suitable for at least the plant (preferably the plant from which the scion and / or rootstock are derived) to form a callus; and step (d2) can be carried out under conditions suitable for shoot formation from the callus of said plant. The (further) callus and shoots formed in steps (d1) and (d2) preferably comprise cells derived from the callus of step (a). Optionally, the (further) callus and shoots formed in steps (d1) and (d2) consist of cells derived from the callus of step (a). Those skilled in the art will recognize suitable conditions for callus and / or shoot regeneration. Preferably, in step (d), in addition to the cells of the callus of step (a), cells of the scion and / or rootstock also regenerate to form callus and / or shoots. Thus, optionally, in steps (d), (d1) and / or (d2), the callus and scion cells and / or rootstock cells are simultaneously regenerated.

[0094] Callus can be formed by (shoot) organogenesis or somatic embryogenesis prior to the regeneration process of step (d). The amount of callus formed can depend, for example, on the plant species used in the method of the present invention and / or the conditions used that allow shoot formation. For example, the callus formation in vitro and / or after grafting and wounding can occur spontaneously, i.e., in the absence of one or more exogenously supplied plant hormones. Similarly, shoot formation in step (d), e.g., after optional callus formation, can also occur spontaneously, i.e., in the absence of one or more exogenously supplied plant hormones. Alternatively, the callus formation can be induced and / or enhanced in the presence of one or more plant hormones. Alternatively, or in addition, shoot formation in step (d) can be induced and / or enhanced in the presence of one or more plant hormones.

[0095] In a particular embodiment, the formation of (further) callus between wounding step (c) and the formation of the shoot in step (d) of the method of the invention is minimal. This is particularly preferred when the callus of step (a) is from a plant that is less regenerable than the plant from which the scion and / or rootstock are excised, as this avoids cells of the scion and / or rootstock outcompeting cells of the callus of step (a). A minimal callus stage may therefore increase the likelihood of the development of a shoot comprising or consisting (at least in part) of cells of the callus of step (a).

[0096] To induce shoot regeneration in plant tissue, a combination of one or more plant hormones, preferably cytokinins and / or one or more auxins, may be used.

[0097] The cytokinins that can be used in the methods of the present invention can be adenine-type cytokinins or phenylurea-type cytokinins. Similarly, cytokinins can be naturally produced phytohormones or synthetic compounds. Adenine-type cytokinins can be phytohormones synthesized in at least one of roots, seeds, and fruits. In addition, the cambium and other actively dividing tissues can also synthesize cytokinins. Non-limiting examples of naturally occurring adenine-type cytokinins are zeatin and its metabolic precursor 2iP. Non-limiting examples of synthetic adenine-type cytokinins are kinetin and 6-benzylaminopurine (BAP). Substituted urea compounds, such as thidiazuron and CPPU, are not present in plants but can function as cytokinins in tissue culture. The adenine-type cytokinins can be selected from the group consisting of kinetin, zeatin, trans-zeatin, cis-zeatin, dihydrozeatin, 6-benzylaminopurine, 2iP, and combinations thereof. The phenylurea-type cytokinin may be diphenylurea or thidiazuron. It is known in the art that the type of cytokinin to be added depends on the type of plant cell, and those skilled in the art can easily select a suitable cytokinin as needed.

[0098] Alternatively, or in addition, the plant hormone may be an auxin. The auxin may be an endogenously synthesized auxin. The endogenously synthesized auxin may be selected from the group consisting of indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid. The auxin may be a synthetic auxin, such as an auxin analog. The synthetic auxin may be at least one of 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (α-NAA), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (trudon or picloram), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The auxin can be 1-naphthaleneacetic acid (NAA).

[0099] For shoot organogenesis by a combination of cytokinin and auxin, the ratio of cytokinin to auxin is preferably greater than 1 (Dodds, JH and Roberts, LW (1985) Experiments in plant tissue culture. Cambridge University Press, Cambridge, UK).

[0100] Optionally, in step (d), initial callus formation is stimulated (step d1), followed by shoot formation (step d2). Step (d1) may be performed using conditions that allow callus formation on the plant from which the scion and / or rootstock is derived (also referred to as "scion and / or rootstock plant"). Optionally, step (d1) is performed using minimal conditions that allow callus formation on the scion and / or rootstock plant. In a preferred embodiment, step (d1) is performed using conditions that are optimal for callus formation on the scion and / or rootstock plant. Step (d2) may be performed using conditions that allow shoot formation on the scion and / or rootstock plant. Optionally, step (d2) is performed using minimal conditions that allow shoot formation on the scion and / or rootstock plant. In a preferred embodiment, step (d2) is performed using conditions that are optimal for shoot formation on the scion and / or rootstock plant.

[0101] The method of the present invention may further comprise a step (f) of growing a plant from the shoot selected in step (e).

[0102] Optionally, in step (e) of the method of the present invention, shoots containing germline progenitor cells derived from the callus of step (a) are selected. Such shoots can give rise to plants containing germline progenitor cells and / or germline cells (e.g., gametes, egg cells, sperm cells) derived from the callus of step (a). The germline cells can form gametes for sexual reproduction. Such plants can then be used to produce seeds, wherein the seeds contain embryos, and wherein at least a portion of the genotype of the embryos is derived from the callus step (a) of the method of the present invention, and the seeds are optionally obtained by selfing or backcrossing.

[0103] Optionally, in step (e) of the method of the present invention, a shoot containing cells capable of giving rise to clonal propagation tissue or plant parts derived from the callus of step (a) of the method of the present invention is selected. Such shoots can give rise to plants containing plant parts derived from the callus of step (a) that can be used for clonal propagation. Such plant parts have the same or substantially the same genotype as the callus of step (a) of the method of the present invention.

[0104] The selected shoots may be substantially free of cells of the scion and / or rootstock of step (a). Such shoots may consist of cells derived from the callus of step (a) and can be used for the production of plants by (vegetative) propagation of said shoots, i.e., by growing whole plants from said shoots. The step of selecting shoots can be carried out using any conventional method known to those skilled in the art.

[0105] The selecting may comprise determining phenotypic traits and / or molecular markers that are present in cells of the callus of step (a) and / or that are present in the shoot meristem layer of the plant from which the callus was derived, but that are absent in cells of the scion and / or rootstock, and / or that are absent in the shoot meristem layer of the plant from which the scion and / or rootstock is excised. Alternatively, or in addition, the selecting may comprise determining phenotypic traits and / or molecular markers that are absent in cells of the plant from which the callus of step (a) was derived, and / or that are absent in the shoot meristem layer of said plant, but that are present in cells of the plant and / or that are present in the shoot meristem layer of the plant from which the scion and / or rootstock is excised. Preferably, the selecting may comprise determining phenotypic traits and / or molecular markers that are present in germline progenitor cells and / or clonally propagated plant parts of the plant from which the callus of step (a) was derived, but that are absent in germline progenitor cells and / or clonally propagated tissue and / or plant parts of a second plant. Alternatively, or in addition, the selection may comprise determining phenotypic characteristics and / or molecular markers that are not present in the germline progenitor cells and / or clonal propagation tissue and / or plant parts of the plant from which the scion and / or rootstock is excised, but are present in the germline progenitor cells and / or clonal propagation tissue and / or plant parts of the plant from which the scion and / or rootstock is excised. The molecular markers are preferably genomic sequences that are present either in the plant from which the callus in step (a) is derived or in the plant from which the scion and / or rootstock is excised.

[0106] Alternatively, or in addition, steps (e) and / or (f) may comprise bringing the (regenerated) shoot into contact with a compound that is toxic to the (plant) cells expressing the negative selection marker. In this embodiment, the negative selection marker may be expressed in cells of the plant from which the scion and / or rootstock are excised, and preferably, the negative selection marker is expressed in at least germline progenitor cells and / or clonal propagation tissues and / or plant parts of said plant. Optionally, the toxic selection marker is encoded by (the genome of) cells of the plant from which the scion and / or rootstock are excised, optionally under the control of an inducible promoter. Exposing the cells to a substance that activates the inducible promoter results in expression of the toxic selection marker and preferably kills these cells. Optionally, a precursor of the toxic selection marker is encoded by (the genome of) cells of the scion and / or rootstock. Exposing the cells to a substance that activates conversion of the precursor to the toxic component preferably kills these cells.

[0107] Alternatively, or in addition, steps (e) and / or (f) may comprise bringing the (regenerated) shoot into contact with a compound that is toxic to the (plant) cell but that can be converted to a non-toxic compound by expression of the positive selection marker. In this embodiment, the positive selection marker may be expressed in the shoot meristem layer of the plant from which the callus of step (a) is derived, and preferably the positive selection marker is expressed in at least germline progenitor cells and / or clonal propagation tissues and / or plant parts of said plant.

[0108] In a preferred embodiment, at least one or more germline progenitor cells and / or clonal propagation tissues and / or plant parts of the developed shoots selected in step (e) of the method of the present invention comprise a transgene or mutation in a sequence of interest. Preferably, at least one of the L1, L2 and / or L3 shoot meristem layers of the developed shoots comprises a transgene or mutation in a sequence of interest. Preferably, at least the L2 shoot meristem layer of the developed shoots comprises a transgene or mutation in a sequence of interest.

[0109] The transgene or mutation may be present in or cells derived from the callus in step (a) of the method of the present invention, and optionally in or cells derived from the scion and / or rootstock of the second plant in step (a) of the method of the present invention. Preferably, the transgene or mutation is present in all or substantially all cells of the callus in step (a) of the method of the present invention. Preferably, the transgene or mutation is present at least in germline progenitor cells and / or clonal propagation tissue and / or plant parts of the developed shoot selected in step (e) of the method of the present invention. Optionally, the transgene or mutation is present in all or substantially all cells of the developed shoot selected in step (e), wherein said cells are cells regenerated from the callus in step (a) of the method of the present invention. Preferably, at least the L2 shoot meristem layer of the developed shoot contains the transgene or mutation in a sequence of interest, wherein at least the L2 shoot meristem layer is regenerated from the callus in step (a). Seeds subsequently produced from such shoots may contain the transgene or mutation, preferably within the embryo of said seed.

[0110] Thus, preferably, the method of the invention comprises the step of introducing a transgene or mutation in a sequence of interest into one or more cells of the callus of step (a). Alternatively, or in addition, the method of the invention (further) comprises the step of introducing a transgene or mutation in a sequence of interest into one or more cells originating from the callus of step (a) and present in the shoot formed in step (d), and optionally in the callus formed in step (d1).

[0111] The transgene or mutation may be introduced into one or more cells of the callus in step (a) before contacting the callus with the scion and rootstock in step (a). Optionally, the transgene or mutation may be introduced into protoplasts that are subsequently expressed in callus for use in step (a). Alternatively, the transgene or mutation may be introduced into one or at least a portion of the callus in step (a) and / or into wounded plant tissue from which callus is subsequently formed for use in step (a). Similarly, the transgene or mutation may be introduced into one or more cells of a plant, and cells of the plant carrying the transgene or mutation may be induced to form callus for use in step (a).

[0112] Alternatively, or in addition, the transgene or mutation may be introduced into cells of the callus of step (a) after contacting the callus with at least one of the scion or rootstock of step (a). The transgene or mutation is preferably introduced before shoot formation. Thus, preferably, the step of introducing the mutation occurs before step (d) or (d2) of the method as defined herein, but may occur during or after step (c) or (d1). Optionally, the transgene or mutation may be introduced into one or more cells of at least one of the scion or rootstock of step (a).

[0113] Optionally, the graft union is first wounded before introducing the transgene or mutation into at least one or more cells of or derived from the callus of step (a). Optionally, the graft union is first cut or "wounded" before introducing the transgene or mutation into at least one or more cells of or derived from the callus of step (a). Optionally, (further) callus formation is first induced by wounding before introducing the transgene or mutation into at least one or more cells of or derived from the callus of step (a).

[0114] Preferably, the shoot selected in step (e) of the method of the present invention contains a transgene or mutation in a sequence of interest in cells regenerated from the callus in step (a) of the method of the present invention, i.e., a transgene or mutation separate from the genotype of the callus. The shoot preferably contains a transgene or mutation in at least one of the L1, L2, and L3 shoot meristem layers regenerated from the callus in step (a). In other words, at least one of the L1, L2, and L3 shoot meristem layers of the shoot selected in (e) has the genotype of the callus in step (a) separate from the transgene or mutation. Preferably, at least germline progenitor cells and / or clonal propagation tissue and / or plant parts of the selected shoot are regenerated from the callus in step (a) of the method of the present invention (i.e., have the genotype of the callus in step (a)) and contain a transgene or mutation in a gene of interest. Thus, a preferred method of the invention is a method of generating and selecting shoots of a plant, wherein the shoots comprise germline progenitor cells and / or cells that give rise to clonally propagated tissue and / or plant parts derived from the callus of step (a), and wherein one or more of the germline progenitor cells and / or clonally propagated plant tissue and / or plant parts comprise a transgene or mutation in a sequence of interest. Preferably, all or substantially all of the germline progenitor cells and / or clonally propagated tissue and / or plant parts comprise a transgene or mutation in a sequence of interest.

[0115] The transgene or mutation may be present in at least the L2 shoot meristem layer. Thus, a preferred method of the present invention is a method of generating and selecting shoots of a plant, the shoots comprising an L2 shoot meristem layer derived from the callus of step (a), and wherein one or more cells of the L2 shoot meristem layer contain a transgene or mutation in a sequence of interest. Preferably, all or substantially all cells of at least the L2 shoot meristem layer contain a transgene or mutation in a sequence of interest. Optionally, the transgene or mutation is present in all or substantially all cells of the generated shoot selected in step (c), wherein said cells are cells regenerated from the callus of step (a) of the method of the present invention. Optionally, the shoots selected in step (e) have substantially the same genotype as the cells of the callus of step (a), preferably containing the transgene or mutation.

[0116] Introducing a transgene or mutation in a sequence of interest in the methods of the present invention preferably results in one or more improved phenotypic characteristics, including, but not limited to, increased yield, disease resistance, agronomic traits, abiotic traits, protein composition, oil composition, starch composition, insect resistance, fertility, silage, and morphological traits.

[0117] The transgene may be introduced by stable or transgenic transfection using any method known to those skilled in the art for transfecting plants, plant parts, callus, plant cells or protoplasts.

[0118] Mutations herein should be understood as either modifications of the nucleotide sequence of a gene, preferably the genetic code (insertion, deletion, or substitution of one or more nucleotides, or chromosomal translocation), or epigenetic modifications such as methylation changes. Mutations may be introduced by random mutagenesis or targeted mutagenesis, the latter also referred to as programmed genome editing. Random mutagenesis may be, but is not limited to, chemical mutagenesis and gamma irradiation. Non-limiting examples of chemical mutagenesis include, but are not limited to, EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), NaN3 (sodium azide), ENU (N-ethyl-N-nitrosourea), AzaC (azacytidine), and NQO (4-nitroquinoline 1-oxide). Optionally, a mutagenesis system such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both of which are incorporated herein by reference) can be used to create mutations in cells of the recalcitrant plant. TILLING uses traditional chemical mutagenesis (e.g., EMS mutagenesis) followed by high-throughput screening for mutations. In this way, TILLING can be used to obtain plants, seeds, and tissues containing genes with one or more desired mutations. Preferably, plants, seeds, and tissues containing genes with one or more desired mutations can be obtained using KeyPoint® Breeding as described in WO 2007 / 037678, which is incorporated herein by reference.

[0119] Targeted mutagenesis or programmed genome editing is mutagenesis that can be designed to modify specific nucleotides or nucleic acid sequences, including but not limited to oligo-directed mutagenesis, RNA-guided endonucleases (e.g., CRISPR technology), TALEN, meganuclease, or zinc finger technology.

[0120] Preferably, targeted mutagenesis is introduced by site-specific protein, preferably site-specific endonuclease.Site-specific endonuclease is preferably at least one of CRISPR protein complexed with guide RNA, TALEN, zinc finger protein, meganuclease and Argonaute complex.Preferably, site-specific endonuclease is CRISPR protein complexed with guide RNA.

[0121] The CRISPR protein that is part of the CRISPR protein complex used in the methods of the present invention is preferably at least one of a CRISPR endonuclease, a CRISPR nickase, and a CRISPR deaminase. Preferably, the CRISPR protein is a CRISPR endonuclease.

[0122] CRISPR protein can be any suitable CRISPR protein known in the art. Optionally, CRISPR protein comprises a nuclear localization signal (NLS) that directs CRISPR protein to the nucleus of plant cells. Any known nuclear localization signal can be suitable for use in the present invention. Preferred nuclear localization signals include, but are not limited to, the NLS of SV40 large T antigen, MEDPTMAPKKKRKV (SEQ ID NO: 4), and the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 5).

[0123] CRISPR endonuclease comprises nuclease domain and at least one domain that interacts with guide RNA.When CRISPR protein complex is combined with guide RNA, guide RNA guides CRISPR protein complex to specific nucleic acid sequence.Guide RNA interacts with CRISPR endonuclease and target specific nucleic acid sequence, so that when CRISPR endonuclease is guided by guide sequence to the site that contains target nucleic acid sequence, it can introduce double-strand break at target site.

[0124] If the CRISPR protein is a CRISPR endonuclease, both domains of the nuclease are catalytically active and the protein is capable of introducing a double-stranded break at the target site. If the CRISPR protein is a CRISPR nickase, one domain of the nuclease is catalytically active and one domain is catalytically inactive and the protein is capable of introducing a single-stranded break at the target site.

[0125] Those skilled in the art are well aware of how to design a guide RNA such that, when combined with a CRISPR endonuclease or CRISPR nickase, it achieves the introduction of a single- or double-stranded break at a predetermined site in a nucleic acid molecule.

[0126] CRISPR proteins can be broadly classified into six major types (I-VI) based on the content and sequence of their core elements, which are further subdivided into subtypes (Makarova et al., 2011, Nat Rev Microbiol 9:467-77 and Wright et al., 2016, Cell 164(1-2):29-44). In general, the two key elements of a CRISPR protein complex are the CRISPR protein and the guide RNA.

[0127] Type II CRISPR protein complexes contain a single protein (approximately 160 kDa) with the signature Cas9 protein, which is capable of specifically cleaving double-stranded DNA. Cas9 proteins typically contain two nuclease domains: a RuvC-like nuclease domain near the amino terminus and an HNH (or McrA-like) nuclease domain near the center of the protein. Each nuclease domain of the Cas9 protein is specialized for cleaving one strand of the double helix (Jinek et al., 2012, Science 337(6096):816-821). The Cas9 protein is an example of a CAS protein in the type II CRISPR-CAS protein complex, which forms an endonuclease when combined with a crRNA and a second RNA called a trans-activating crRNA (tracrRNA). The crRNA and tracrRNA function together as guide RNAs. The CRISPR protein complex then introduces a DNA double-strand break (DSB) at the location in the genome defined by the crRNA. Jinek et al. (2012, Science 337:816-820) demonstrated that a single-stranded chimeric guide RNA (defined herein as "sgRNA" or "single guide RNA") made by fusing the essential portions of crRNA and tracrRNA was able to form a functional CRISPR protein complex in combination with the Cas9 protein.

[0128] A type V CRISPR protein complex, Clustered Regularly Interspaced Short Palindromic Repeats 1 (CRISPR / Cpf1), from the genera Prevotella and Francisella has been described. The Cpf1 gene encodes an endonuclease that associates with CRISPR loci and targets DNA using crRNA. Cpf1 is a smaller endonuclease than Cas9, potentially overcoming some of the limitations of the CRISPR-Cas9 system. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, which utilizes a T-rich protospacer-adjacent motif. Cpf1 cleaves DNA with sticky-end DNA double-strand breaks (Zetsche et al. (2015) Cell 163(3):759-771). The type V CRISPR protein system preferably includes at least one of Cpf1, C2c1, and C2c3.

[0129] The CRISPR protein complex used in the present invention can comprise any CRISPR protein as defined herein above.Preferably, CRISPR protein is type II CRISPR protein, preferably type II CRISPR endonuclease, for example Cas9 (for example, the protein of SEQ ID NO: 6, or the protein of SEQ ID NO: 5, encoded by SEQ ID NO: 7) or type V CRISPR protein, preferably type V CRISPR endonuclease, for example Cpf1 (for example, the protein of SEQ ID NO: 9, encoded by SEQ ID NO: 10) or Mad7 (for example, the protein of SEQ ID NO: 11 or 12), or the protein derived therefrom, and preferably has at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with said protein throughout its entire length.

[0130] Preferably, the CRISPR protein is a type II CRISPR endonuclease, preferably a Cas9 endonuclease.

[0131] Those skilled in the art know how to find and prepare CRISPR proteins for use in the methods of the present invention. Numerous reports on their design and use are available in the prior art. For example, see the review by Haeussler et al. (J Genet Genomics. (2016) 43(5): 239-50. doi: 10.1016 / j.jgg.2016.04.008.) or the review by Lee et al. (Plant Biotechnology Journal (2016) 14(2) 448-462) on the design of guide RNAs and their use in combination with CAS proteins (originally obtained from Streptococcus pyogenes).

[0132] Generally, CRISPR endonucleases such as Cas9 contain two catalytically active nuclease domains. For example, Cas9 protein can contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to create a double-strand break in DNA, since both domains break a single strand (Jinek et al., Science, 337:816-821).

[0133] A dead CRISPR endonuclease includes modifications such that none of the nuclease domains exhibit cleavage activity. A CRISPR nickase can be a mutant of a CRISPR endonuclease in which one of the nuclease domains is mutated so that it is no longer functional (i.e., there is no nuclease activity). One example is an SpCas9 mutant with either the D10A or H840A mutation.

[0134] The CRISPR protein may comprise or consist of the entire Type II or Type V CRISPR protein, or a variant or functional fragment thereof, preferably such fragment binds to a guide RNA and retains at least partial endonuclease activity.

[0135] Preferably, the CRISPR protein used in the methods of the present invention is a Cas9 protein. The Cas9 protein is expressed in the bacteria Streptococcus pyogenes (SpCas9; NCBI reference sequence NC_017053.1; UniProtKB-Q99ZW2), Geobacillus thermodenitrificans (UniProtKB-A0A178TEJ9), Corynebacterium ulcerans (NCBI references: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI references: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI reference: NC_021284.1), and Prevotella intermedia. intermedia (NCBI Reference: NC_017861.1); Spiroplasma taiwanense (NCBI Reference: NC_021846.1); Streptococcus iniae (NCBI Reference: NC_021314.1); Belliella baltica (NCBI Reference: NC_018010.1); Psychroflexus torquis (NCBI Reference: NC_018721.1); Streptococcus thermophilus (NCBI Reference: YP_820832.1); Listeria innocua (NCBI Reference: NP_472073.1); Campylobacter jejuni jejuni (NCBI Reference: YP_002344900.1); or Neisseria meningitidis (NCBI Reference: YP_002342100.1).These include Cas9 mutants with inactive HNH or RuvC domains homologous to SpCas9, such as SpCas9_D10A or SpCas9_H840A, or Cas9s that have been made into nickases by having equivalent substitutions at positions corresponding to D10 or H840 of the SpCas9 protein.

[0136] The CRISPR protein used in the method of the present invention may be or be derived from Cpf1, such as Cpf1 from Acidaminococcus sp. UniProtKB-U2UMQ6. The mutant may be a Cpf1 nickase with an inactive RuvC or NUC domain, where the RuvC or NUC domain no longer has nuclease activity. Those skilled in the art are well aware of techniques available in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, which allow for inactive nucleases, such as inactive RuvC or NUC domains. An example of a Cpf1 nickase with an inactive NUC domain is Cpf1 R1226A (see Gao et al. Cell Research (2016) 26:901-913, Yamano et al. Cell (2016) 165(4):949-962). In this mutant, there is an arginine to alanine (R1226A) conversion in the NUC domain, which renders the NUC domain inactive.

[0137] The CRISPR protein used in the methods of the present invention may be or be derived from CRISPR-CasΦ, a nuclease that is approximately half the size of Cas9. CRISPR-CasΦ uses a single crRNA for nucleic acid targeting and cleavage, as described, for example, in Pausch et al. (CRISPR-CasΦ from huge phages is a hypercompact genome editor, Science (2020); 369(6501):333-337).

[0138] The methods of the present invention may use active, partially active, or dead CRISPR proteins to guide fused functional domains, as detailed herein, to specific sites in DNA as determined by, for example, a guide RNA.

[0139] Therefore, the CRISPR protein may be fused to a functional domain. Optionally, the functional domain is for epigenetic modification, such as a histone modification domain. The epigenetic modification domain may be selected from the group consisting of methyltransferase, demethylase, deacetylase, methylase, deacetylase, deoxygenase, glycosylase, and acetylase (Cano-Rodriguez et al., Curr Genet Med Rep (2016) 4:170-179). The methyltransferase may be selected from the group consisting of G9a, Suv39h1, DNMT3, PRDM9, and Dot1L. The demethylase may be LSD1. The deacetylase may be SIRT6 or SIRT3. The methylase may be at least one of KYP, TgSET8, and NUE. The deacetylase may be selected from the group consisting of HDAC8, RPD3, Sir2a, and Sin3a. The deoxygenase may be at least one of TET1, TET2, and TET3, preferably TET1cd (Gallego-Bartolome J et al., Proc Natl Acad Sci U S A. (2018); 115(9):E2125-E2134). The glycosylase may be TDG. The acetylase may be p300.

[0140] Optionally, the functional domain is a deaminase selected from the group consisting of apolipoprotein B mRNA editing complex (APOBEC) deaminase family, activation-induced cytosine deaminase (AID), ACF1 / ASE deaminase, adenine deaminase, and ADAT family deaminase, or a functional fragment thereof. Alternatively, or in addition, the deaminase or functional fragment thereof may be ADAR1 or ADAR2, or a variant thereof.

[0141] The apolipoprotein B mRNA editing complex (APOBEC) cytosine deaminase enzyme family includes 11 proteins that function to induce mutagenesis in a controlled and beneficial manner. Preferably, the APOBEC deaminase is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. Preferably, the APOBEC family cytosine deaminase is activation-induced cytosine (or cytidine) deaminase (AID) or apolipoprotein B editing complex 3 (APOBEC3). Preferably, the deaminase domain is fused to a CRISPR protein, an APOBEC1 family deaminase.

[0142] Another exemplary preferred type of deaminase domain that can be fused to a CRISPR system nuclease is adenine or adenosine deaminase, such as the ADAT adenine deaminase family. Furthermore, the adenine deaminase may preferably be TadA or a variant thereof, as described in Gaudelli et al., 2017 (Gaudelli et al. 2017 Nature 551:464-471). Furthermore, the CRISPR system nuclease may be fused to an adenine deaminase domain derived from, for example, ADAR1 or ADAR2. The deaminase domain of the present invention may comprise or consist of the entire deaminase protein or a catalytically active fragment thereof. Preferably, the deaminase domain has deaminase activity. Optionally, the CRISPR protein is further fused to a UDG inhibitor (UGI) domain.

[0143] The CRISPR protein used in the method of the present invention is complexed with a guide RNA molecule, and the guide RNA molecule guides the CRISPR protein to a specific location in the genome of the plant cell to achieve targeted genome modification. Preferably, the plant cell is a cell of a plant that is difficult to regenerate. Optionally, the plant cell is a cell that gives rise to a germline or germline progenitor cell and / or a clonal propagation tissue and / or plant part of the plant that is difficult to regenerate.

[0144] A complex comprising a CRISPR protein and a guide RNA may also be annotated as a ribonucleoprotein complex.

[0145] The guide RNA molecule directs the complex to a predetermined target site, also known as a protospacer sequence, within the double-stranded nucleic acid molecule. The guide RNA molecule preferably comprises a sequence for targeting the CRISPR protein complex to a protospacer sequence that is near, at, or within the target sequence in the genome of the plant cell. The guide RNA may be a single guide (sg) RNA or a combination of crRNA and tracrRNA (e.g., for Cas9) or crRNA alone (e.g., for Cpf1 and CasΦ).

[0146] The CRISPR protein complex used in the methods of the present invention may thus comprise a guide RNA molecule, wherein the guide RNA molecule comprises a combination of crRNA and tracrRNA, and wherein the CRISPR protein is preferably Cas9. The crRNA and tracrRNA are preferably combined to form an sgRNA (single guide RNA). Alternatively, the CRISPR protein complex used in the methods of the present invention may comprise a guide RNA molecule, wherein the guide RNA molecule comprises crRNA, and wherein the CRISPR protein is preferably Cpf1 or CasΦ.

[0147] The guide RNA molecules used in the methods of the present invention may comprise a sequence capable of hybridizing to or adjacent to a sequence of interest, preferably a sequence of interest as defined herein. The guide RNA molecule may comprise a nucleotide sequence that is perfectly complementary to a sequence in the sequence of interest, i.e., the sequence of interest comprises a protospacer sequence. Alternatively, or in addition, the guide RNA molecules used in the methods of the present invention may comprise a sequence capable of hybridizing to or adjacent to the complement of the sequence of interest.

[0148] The portion of the crRNA complementary to the protospacer sequence is designed to have sufficient complementarity with the protospacer sequence to hybridize with the protospacer sequence and direct sequence-specific binding of the complexed CRISPR protein. The protospacer sequence is preferably adjacent to a protospacer adjacent motif (PAM) sequence, which can interact with the CRISPR protein of the RNA-guided CRISPR protein complex. For example, if the CRISPR protein is S. pyogenes Cas9, the PAM sequence is preferably 5'-NGG-3' (where N can be any one of T, G, A, or C).

[0149] Those skilled in the art have the ability to engineer crRNA to target any desired sequence, preferably by engineering the sequence to be at least partially complementary to, and thus hybridize to, any desired protospacer sequence. Preferably, the complementarity between a portion of the crRNA sequence and its corresponding protospacer sequence is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% when optimally aligned using a suitable alignment algorithm. The portion of the crRNA sequence complementary to the protospacer sequence may be at least about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some preferred embodiments, the sequence complementary to the sequence of interest is less than about 75, 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. Preferably, the length of the sequence complementary to the sequence of interest is at least 17 nucleotides. Preferably, the complementary crRNA sequence is about 10-30 nucleotides in length, about 17-25 nucleotides in length, or about 15-21 nucleotides in length. Preferably, the portion of the crRNA complementary to the protospacer sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably 20 or 21 nucleotides, preferably 20 nucleotides.

[0150] Molecules suitable as crRNAs and tracrRNAs are well known in the art (see, e.g., WO 2013142578 and Jinek et al., Science (2012) 337, 816-821). The guide RNA molecules crRNA and tracrRNA can be linked together to form a single-guide (sg) RNA. The crRNA and tracrRNA can be linked, preferably covalently, using any conventional method known in the art. Covalent linkages between crRNAs and tracrRNAs are described, for example, in Jinek et al. (supra) and WO 13 / 176772, which are incorporated herein by reference. The crRNA and tracrRNA can be covalently linked, for example, using linker nucleotides or via direct covalent linkage between the 3' end of the crRNA and the 5' end of the tracrRNA.

[0151] Preferably, the method of the present invention uses at least one CRISPR protein complex comprising a CRISPR nuclease and a guide RNA.However, those skilled in the art will readily understand that the method of the present invention can use additional CRISPR protein complexes, for example, by using at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different guide RNAs.These different guide RNAs can be designed to target and bind to the same sequence of interest.Alternatively, different guide RNAs can direct the CRISPR protein complex to different genes of interest.

[0152] A transgene or mutation in a sequence of interest may be introduced before, during, and / or after callus formation. The transgene or mutation is preferably introduced before shoot formation begins. Preferably, the transgene or mutation is present in at least the germline progenitor cells and / or clonal propagation tissue and / or plant part of the shoot formed in step (d) of the method of the present invention. Preferably, the transgene or mutation is present in at least the germline progenitor cells of the shoot selected in step (e) and / or in cells that will give rise to the clonal propagation tissue and / or plant part. Preferably, the transgene or mutation is present in all the germline progenitor cells and / or all the clonal propagation tissue and / or plant part of the shoot formed in step (d). Preferably, the transgene or mutation is present in at least one cell of the L2 shoot meristem layer of the shoot formed in step (d) of the method of the present invention. Preferably, the transgene or mutation is present in all cells of the L2 shoot meristem layer of the shoot formed in step (d). The transgene or mutation may also be present in other cells, such as cells of the L1 and L3 shoot meristem layers. Optionally, all cells of the shoot formed in step (d) of the method of the invention contain the transgene or mutation in the sequence of interest.

[0153] The transgene or mutation in the sequence of interest may be introduced into cells of the plant from which the callus of step (a) is derived and / or cells of the plant from which the scion and / or rootstock of step (a) is excised. Preferably, the transgene or mutation in the sequence of interest is introduced into at least cells of the callus of step (a) and / or cells of the scion and / or rootstock of step (a).

[0154] Mutation can be introduced by transfecting plant cell with site-specific endonuclease, preferably CRISPR endonuclease.Transgene can be introduced by transfecting target transgene into plant cell.Transfection of plant cell can be carried out by any conventional means known to those skilled in the art.

[0155] "Transfection" or "transformation" is herein understood to mean the delivery of a transgene and / or site-specific endonuclease protein or a nucleic acid molecule encoding the transgene and / or site-specific endonuclease into a plant cell. The nucleic acid molecule may be DNA or RNA encoding the transgene and / or site-specific nuclease. Optionally, the transgene and / or site-specific endonuclease is introduced by transfection of (pre)mRNA. Transfection may further include the delivery of a guide RNA or a nucleic acid molecule encoding the guide RNA that is (will be) associated with the site-specific endonuclease into the plant cell. Optionally, the site-specific endonuclease is delivered as a CRISPR endonuclease complex comprising the CRISPR endonuclease complexed with the guide RNA. Alternatively, or in addition, the CRISPR endonuclease and guide RNA are delivered to the plant cell and form a complex within the cell. Alternatively, or in addition, the CRISPR endonuclease is expressed from the transfected nucleic acid and forms a complex with the optionally expressed guide RNA within the cell.

[0156] Preferably, the transgene and / or site-specific endonuclease, or the nucleic acid encoding it, can be introduced into the cells of the difficult-to-regenerate plant as a protein, or in the case of a CRISPR endonuclease, as a protein-guide RNA complex (also called a ribonucleoprotein complex) using any conventional means known to those skilled in the art. Non-limiting examples of transfection include, but are not limited to, viral infection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whisker technology, Agrobacterium-mediated transformation, etc. The choice of method generally depends on the type of cell to be transformed and the context in which the transformation is performed (i.e., in vitro, ex vivo, or in vivo; protein transfection or nucleic acid transfection).

[0157] Transfection methods based on the soil bacterium Agrobacterium tumefaciens can be particularly useful for introducing nucleic acid molecules into plant cells. Methods for cocultivating Agrobacterium with cultured plant cells or wounded tissues, such as leaf tissue, root explants, lower cotyledons, stem segments, or tubers, are well known in the art. See, for example, Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993). Microprojectile-mediated transformation can also be used to transfect plant cells. This method, first described by Klein et al. (Nature 327:70-73 (1987)), relies on microprojectiles, such as gold or tungsten, coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine, or polyethylene glycol. Microprojectile particles are accelerated to high speeds using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules, Calif.) and into angiosperm tissue.

[0158] The nucleic acid encoding the transgene and / or site-specific endonuclease, and optionally the guide RNA, may be introduced into the plant in a manner that allows the nucleic acid to enter the plant cell, for example, by in vivo or ex vivo protocols. "In vivo" means that the nucleic acid is administered to the living plant, for example, by infiltration. "Ex vivo" means that cells or explants are modified outside the plant, and then such cells or organs are regenerated into shoots of the plant.

[0159] A number of vectors suitable for the transformation of plant cells and / or the establishment of transgenic plants have been described, including those described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al., (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Examples include Agrobacterium tumefaciens-mediated transformation, as well as methods disclosed, for example, by Herrera-Estrella et al. (1983) Nature 303:209, Bevan (1984) Nucl Acid Res. 12:8711-8721, and Klee (1985) Bio / Technology 3:637-642. Conventional methods for transforming plant cells include, but are not limited to, biolistic bombardment, polyethylene glycol transformation, and microinjection (e.g., Danieli et al. Nat. Biotechnol 16:345-348, 1998; Staub et al. Nat. Biotechnol 18:333-338, 2000; O'Neill et al. Plant J. 3:729-738, 1993; Knoblauch et al. Nat. Biotechnol 17:906-909; U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,576,198; WO 95 / 16783; and Boynton et al., Methods in Enzymology 217:510-536 (1993), Svab et al., Proc. Natl. Acad. Sci. USA 90:913-917 (1993), and McBride et al., Proc. Natl. Acad. Sci. USA 91:7301-7305 (1994).

[0160] Preferably, a transgene is introduced into the cells of the callus in step (a) or the plant that gives rise to the callus, and / or a mutation is present in a sequence of interest in the cells of the callus in step (a) or the plant that gives rise to the callus. The cells are preferably transfected with at least one of a transgene, a CRISPR endonuclease, and / or one guide RNA. Preferably, the CRISPR endonuclease and guide RNA form a ribonucleoprotein complex that is transfected into the cells of the callus in step (a) or the plant that gives rise to the callus. Preferably, the cells are protoplasts. Preferably, the protoplasts are transfected with the transgene protein and / or the CRISPR guide RNA ribonucleoprotein complex using polyethylene glycol transformation, as described, for example, in WO 2017 / 222370 or WO 2020 / 089448 (which are incorporated herein by reference). The cells may be cells in a single cell suspension, cells present in a protoplast, callus or slice, and / or cells present in a plant, preferably present at the graft union, preferably present at the graft union or junction of steps (b) and (c) of the methods provided herein.

[0161] Alternatively, or in addition, cells of the callus or of the plant from which the callus develops may be transfected with a nucleic acid molecule encoding a transgene and / or at least one site-specific endonuclease and / or at least one guide RNA. Optionally, the cells are protoplasts. Optionally, the protoplasts are transfected with one or more plasmids encoding the transgene and / or CRISPR endonuclease and guide RNA using polyethylene glycol transformation, such as described in WO 2018 / 115390 and WO 2020 / 011985 (which are incorporated herein by reference).

[0162] Preferably, the codon sequence of the transgene and / or site-specific endonuclease is optimized for expression in plant cells. The nucleic acid molecule encoding at least one transgene and / or site-specific endonuclease and / or at least one guide RNA is preferably contained in a nucleic acid vector. The nucleic acid vector is preferably a vector for transient expression of the transgene and / or site-specific endonuclease and / or guide RNA. Alternatively, the nucleic acid vector is a vector for stable expression of the transgene and / or site-specific endonuclease and / or guide RNA. Optionally, at least one, and optionally all, cells of the callus in step a) of the method of the present invention contain a transgene integrated into its genome that encodes a gene of interest and / or a programmable endonuclease, preferably a CRISPR endonuclease, wherein the transgene and / or programmable endonuclease may be stably expressed, or wherein expression of the transgene and / or programmable endonuclease is under the control of an inducible or tissue-specific promoter.

[0163] The transgene and / or site-specific endonuclease, and optionally at least one guide RNA, may thus be transcribed from an expression cassette contained in the vector. The vector backbone may, for example, be a plasmid into which the expression cassette is integrated, or, if suitable transcriptional regulatory sequences are already present (e.g., an (inducible) promoter), into which only the desired nucleotide sequence (e.g., a sequence encoding the transgene and / or site-specific endonuclease) is integrated downstream of the transcriptional regulatory sequences.

[0164] The vectors used in the methods of the present invention may further comprise genetic elements that facilitate their use in molecular cloning, such as, for example, selectable markers, multiple cloning sites, etc. The vector backbone may be, for example, a binary or super-binary vector (see, e.g., U.S. Pat. No. 5,591,616, U.S. Patent Application Publication No. 2002138879, and WO 95 / 06722), a cointegration vector, or a T-DNA vector, as known in the art.

[0165] The vectors used in the methods of the present invention are preferably particularly suitable for introducing expression of a transgene and / or a site-specific endonuclease and optionally one or more guide RNAs into plant cells, where the plant cells are preferably plant cells of the callus of step (a) or of the plant giving rise to said callus. Preferred expression vectors are naked DNA, DNA complexes or viral vectors.

[0166] Preferred naked DNA is a linear or circular nucleic acid molecule, such as a plasmid. A plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. The DNA complex may be a DNA molecule coupled to any carrier suitable for delivering DNA to cells. Preferred carriers are selected from the group consisting of lipoplexes, liposomes, polymersomes, polyplexes, PEG, dendrimers, inorganic nanoparticles, virosomes, and cell-penetrating peptides.

[0167] The vector used in the method of the present invention is preferably a viral expression vector. The viral vector can be a DNA virus or an RNA virus. The viral vector can be or be based on a tobamovirus, tobravirus, potexvirus, geminivirus, alphamovirus, cucumovirus, potyvirus, tombusvirus, hordivirus, or nucleorhabdovirus.

[0168] The tobamovirus viral vector can be at least one of tobacco mosaic virus (TMV) and sun hemp mosaic virus (SHMV). The tobravirus viral vector can be tobacco rattle virus (TRV). The potexvirus viral vector can be at least one of potato virus X (PVX) and papaya mosaic potexvirus (PapMV). The geminivirus viral vector can be comovirus cowpea mosaic virus (CPMV). Further examples of suitable geminivirus viral vectors can include cabbage leaf curl virus, tomato golden mosaic virus, bean yellow dwarf virus, African cassava mosaic virus, wheat dwarf virus, Miscanthus streak mastre virus, tobacco yellow dwarf virus, tomato yellow leaf curl virus, bean golden mosaic virus, beet curly top virus, maize streak virus, and tomato pseudocurly top virus. The alphamovirus can be alfalfa mosaic virus (AMV). The cucumovirus may be cucumber mosaic virus (CMV). The potyvirus may be plum pox virus (PPV). The tombusvirus may be tomato bushy stunt virus (TBSV). The hordeivirus may be wheat stripe mosaic virus. The nucleorhabdovirus may be sonchus yellow net virus (SYNV) (see, e.g., Hefferon K, Plant Virus Expression Vectors: A Powerhouse for Global Health, Biomedicines). 2017, 5(3):44 and Lico et al, Viral vectors for production of recombinant proteins in plants, J Cell Physiol, 2008;216(2):366-77).

[0169] Preferably, the viral vector is selected from the group consisting of tobacco rattle virus (TRV), tobacco mosaic virus (TMV), sonchus yellow net virus (SYNV), and potato virus X (PVX). Preferably, the viral vector is at least one of tobacco rattle virus (TRV), tobacco mosaic virus (TMV), and sonchus yellow net virus (SYNV).

[0170] The viral vector used in the methods of the present invention may contain a gene deletion to increase the packaging capacity of the virus. Preferably, the virus contains a deletion of the gene encoding the coat protein (CP). A preferred viral vector containing a coat protein deletion is a tobamovirus or a tobravirus. Preferably, the viral vector containing a coat protein deletion is a tobamovirus, preferably a tobacco mosaic virus (TMV). A preferred viral vector is a TMV RNA-based overexpression vector (TRBO), for example, as described by Lindbo (TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector, Plant Physiol, 2007;145(4):1232-40). The viral vector may be a self-replicating RNA vector, for example, as described in WO 2018 / 226972, which is incorporated herein by reference.

[0171] The vector, preferably a viral vector, may initially be contained in an Agrobacterium to introduce the viral vector into plant cells of the plant. After infection, the Agrobacterium expresses the viral vector in the plant cells. The viral vector can replicate and infect surrounding plant cells. The viral vector may be modified, for example, by deletion of a coat protein to prevent systemic spread of the virus.

[0172] The transfected callus of step (a), or cells of the plant giving rise to said callus, will preferably develop into tissue that is part of a newly formed shoot, wherein the tissue includes one or more germline progenitor cells and / or clonal propagation tissue and / or one or more cells that will give rise to a plant part. The transfected cells may be primary transfected cells, or, for example, secondary or subsequently transfected cells. As a non-limiting example, cells of the callus of step (a), or the plant giving rise to said callus, may be transfected with a vector expressing a transgene and / or a site-specific endonuclease, such as an Agrobacterium and / or viral vector. Viruses produced in such initially infected cells can spread and infect the (regenerated) callus or the plant giving rise to the callus, i.e., a secondary infection.

[0173] For example, one or more cells of the second plant may be infected with Agrobacterium containing a viral vector expressing a transgene and / or a site-specific endonuclease. At the graft union, the produced virus may be transferred to cells of the callus in step (a) or to the plant from which the callus will develop. Subsequent infection with the viral vector results in expression of the transgene and / or site-specific endonuclease in one or more cells of the callus in step (a) or in the plant from which the callus will develop. The site-specific endonuclease introduces a mutation into the sequence of interest in the one or more cells, and upon shoot formation, this mutation will be present in the resulting shoot. The transgene may be integrated into the genome during shoot formation, and the transgene may be present in the resulting shoot.

[0174] As noted above, the method of the present invention may further comprise step (f) of growing a plant from the shoot selected in step (e). Optionally, particularly when root regeneration is troublesome, step (f) may comprise grafting the selected shoot onto a suitable rootstock. The plant grown in step (f) preferably comprises at least one inflorescence for reproduction, i.e., for producing seeds and / or progeny plants.

[0175] More specifically, the present invention provides a method for generating a plant, the method comprising steps (a), (b), (c), (d), (e), and (f) as defined herein, and further comprising generating a plant from the shoot, wherein preferably the plant comprises at least one inflorescence. Optionally, the generated plant is free of, or substantially free of, cells of or derived from the scion and / or rootstock of step (a). In other words, optionally, the generated plant is a non-chimeric plant having the same or substantially the same genotype as the callus of step (a) of the method of the present invention. Thus, the generated plant may also belong to the same species and variety as the callus of step (a). When the method of the present invention involves the introduction of a mutation and / or transgene, preferably at least one, and optionally all, of the cells of the generated plant also contain the mutation and / or transgene. Therefore, "substantially the same genotype" as used herein should be understood to mean the same genotype, even if it contains a mutation and / or transgene that may be introduced using the method of the present invention.

[0176] Additionally or alternatively, the germline cells, preferably gametes, of the generated plant may have the same or substantially the same genotype as the gametes of the plant from which the callus of step (a) was derived, optionally including mutations and / or transgenes introduced into the cells of the callus of step (a) as further detailed herein. Optionally, the plant may be a chimeric plant further comprising cells or tissue layers of or derived from the scion and / or rootstock of step (a). Optionally, the plant may be a chimeric plant further comprising cells or tissue layers of a regenerable plant. Optionally, the plant is used to produce seeds and / or progeny by crossing, selfing, and / or, in the case of germline progenitor cells of an apomictic genotype, apomictic propagation (i.e., apomictic reproduction). Optionally, the plant is pollinated and / or the pollen is used to pollinate another plant or the same plant (selfing). Optionally, the plant is used to produce tissue and / or plant parts for clonal propagation as defined herein, and optionally the tissue and / or plant parts are isolated and used for clonal or vegetative propagation. Accordingly, the present invention also provides a method of producing a plant or seed, comprising steps (a), (b), (c), (d) and (e) as defined herein, and further comprising generating a plant by vegetative or clonal propagation from the shoot selected in step (e), wherein preferably the plant comprises at least one inflorescence; and optionally producing seeds and / or progeny plants of the generated plant by sexual or apomictic reproduction.

[0177] Thus, the method of the present invention is a method for producing a plant or a seed, comprising: (a) the process of grafting callus between the scion and rootstock; (b) forming a graft joint between the callus and either the scion or the rootstock to form a graft union; (c) generating a wound at or near at least one of the graft joints; (d) forming shoots at the wounded graft-healed site; (e) selecting the shoots formed in step (d), wherein said shoots comprise cells derived from the callus of step (a); (f) growing plants from the shoots selected in step (e); and (g) producing seeds and / or progeny from the plant of step (f); The method may include:

[0178] Where the method of the invention involves the introduction of a mutation and / or transgene, the seeds and / or progeny of the resulting plant may be selected to carry the mutation and / or transgene. The seeds (or embryos of the seeds) produced may optionally have the same or substantially the same genotype as the progeny of the plant from which the callus in step (a) was isolated or part of, except for the mutation and / or transgene introduced.

[0179] Optionally, step (d) of regenerating shoots comprises the formation of a callus prior to shoot regeneration. Thus, the method of the present invention may be a method of producing a plant, wherein the plant comprises germline progenitor cells and / or tissues and / or plant parts for clonal propagation, and wherein the method comprises: (a) the process of grafting callus between the scion and rootstock; (b) forming a graft joint between the callus and either the scion or the rootstock to form a graft union; (c) generating a wound at or near at least one of the graft joints; (d) forming shoots at the wounded graft-healed site; (e) selecting the shoots formed in step (d) that contain germline progenitor cells and / or cells that give rise to tissues and / or plant parts for clonal propagation, derived from the callus of step (a); and (f) growing a plant body from the shoot selected in step (e); Includes.

[0180] Optionally, a plurality of seeds and / or progeny plants are produced, and the method further comprises the step of selecting at least one seed and / or progeny plant, preferably after genotyping and / or evaluating the presence of a mutation and / or transgene that may have been introduced in at least one or substantially all cells of the callus of step (a) of the method of the present invention as detailed herein. The seeds and / or progeny plants may be genotyped to evaluate whether the plant has the same or substantially the same genotype as the cells of the callus of step (a). The seeds may be germinated and allowed to develop into plants.

[0181] Optionally, the callus cells of step (a) of the method of the invention are cells with ploidy abnormalities and may be haploid. Thus, the method of the invention may be a method for propagating haploid plant material. The method may further comprise screening plants and / or seeds for ploidy level for the production of callus of step (a).

[0182] Optionally, during steps (b), (c), and / or (d), the genome may be doubled spontaneously or chemically, resulting in the development of at least one shoot comprising or consisting of doubled haploid cells. In such cases, the genotype of the developed shoot may differ from the cells of the callus in step (a) in that the genome has been doubled. Thus, the method of the present invention may be a method of producing doubled haploid plant material, and may include a step of screening the regenerated plants and / or seeds for ploidy level.

[0183] In another embodiment, the selected shoots of step (e) are not isolated, but an inflorescence can be grown on the shoot developed in steps (d), (e) and optionally (f) of the method of the invention, wherein the shoot optionally includes further shoots. The inflorescence can be used for sexual or apomictic reproduction. The inflorescence can be pollinated, or pollen from the inflorescence can be used to pollinate another plant or the same plant (i.e., the inflorescence is self-pollinating).

[0184] As noted herein, the method includes injecting at least one or substantially all of the cells of the callus of step (a) or the cells originating therefrom in the regenerated shoot of step (d). (i) a transgene; or (ii) mutations in the sequence of interest; The method may further comprise the step of introducing:

[0185] Preferably, the sequence of interest is an endogenous sequence of interest. In methods involving the introduction of a transgene or a mutation, preferably the step of introducing the transgene or mutation occurs before step (d), even more preferably before step (a).

[0186] Additionally or alternatively, in the method, at least the germline progenitor cells of the shoot regenerated in step (d) and / or the clonal propagation tissue and / or the cells giving rise to the plant part comprise a transgene or a mutation. Optionally, the mutation is introduced by programmed genome editing, preferably using a site-specific endonuclease, preferably a CRISPR endonuclease.

[0187] Optionally, the cells of the callus in step (a) of the method of the invention are (highly) heterogeneous, and the method of the invention is a method for propagating heterologous plant material. Optionally, the cells of the callus in step (a) are sterile, and the method of the invention is a method for propagating sterile plant material.

[0188] Plants can be grown from the shoots selected in step (d) of the methods of the present invention using any conventional culture conditions known to those skilled in the art. These culture conditions may depend on the plant produced by the methods of the present invention, and those skilled in the art will know how to adjust these conditions to create an optimal environment for growing plants produced by the methods of the present invention. The plants grown in step (f) may contain a transgene or mutation in a sequence of interest as defined herein.

[0189] The method of the present invention may further comprise a step (g) of producing or obtaining progeny of the plant grown in step (f). The progeny may be produced, for example, by sexual propagation, i.e., through the production of seeds by fusion of pollen and eggs. Preferably, at least one of the pollen and eggs originates from the plant produced in step (f). When the method comprises the introduction of a transgene or mutation in a sequence of interest as defined herein, preferably at least one of the pollen and eggs contains the transgene or mutation in the sequence of interest. Optionally, both the pollen and eggs originate from the plant grown in step (f). Preferably, the pollen and eggs contain the same transgene and / or mutation in the sequence of interest. Alternatively, the progeny is obtained by asexual (vegetative) propagation of the plant grown in step (f). Preferably, in such an embodiment, the transgene and / or mutation in the sequence of interest is present in tissues and / or plant parts that will form the next generation by clonal propagation.

[0190] The present invention also relates to a plant obtainable by the method of the present invention, preferably obtainable in step (f) by the method of the present invention. The plant may be a chimeric plant comprising cells of the same or substantially the same genotype from the callus of step (a) and cells or tissues of the same or substantially the same genotype from the scion and / or rootstock of step (a). Preferably, the plant comprises germline or germline progenitor cells and / or clonal propagation tissue and / or plant parts of the plant from which the callus of step (a) is derived. Preferably, the plant comprises the L2 shoot meristem layer of said plant. Optionally, the plant is a perichimera and / or plant comprising a transgene or mutation in a sequence of interest. Thus, the plant may be a non-natural plant, an artificially created plant, a mutant plant, and / or a transformed plant.

[0191] In one aspect, the present invention thus relates to periclinal chimeras obtainable from the method of the present invention, preferably obtainable from step (d) as defined herein. A "periclinal chimera" is a chimera in which one or more entire cell (tissue) layers L1, L2, and / or L3 are genetically distinct from another cell layer. In the case of periclinal chimeras, the single tissue layer itself is homogenous and not chimeric. Periclinal chimeras are the most stable form of chimera and produce distinctive, valuable plant phenotypes. These plants produce axillary buds with the same apical configuration as the terminal meristem from which they originated. Periclinal chimeras can therefore be propagated by vegetative propagation to maintain their chimeric layer configuration.

[0192] The periclinal chimeric plant obtainable from the method of the present invention preferably comprises at least one shoot meristem layer derived from the callus of step (a) and at least one shoot meristem layer derived from the scion and / or rootstock of step (a). Preferably, at least one of the L1, L2 and L3 shoot meristem layers is derived from the callus of step (a). The shoot meristem layer not derived from the callus of step (a) is preferably derived from the scion and / or rootstock of step (a). Preferably, the L2 shoot meristem layer of the periclinal chimera is derived from the callus of step (a) and at least one of the L1 and L3 shoot meristem layers is derived from the second plant.

[0193] The L2 meristem layer and the L1 and L3 shoot meristem layers of the peripheral plant may be from the same genus or from different genera. Preferably, the L2 meristem layer and the L1 and L3 shoot meristem layers of the peripheral plant are from the same genus. As a non-limiting example, the L1, L2, and L3 shoot meristem layers are from the genus Solanum or Capsicum. For example, the L2 shoot meristem layer may be from a pepper (Capsicum annuum) plant, and at least one of the L1 and L3 shoot meristem layers may be from a yellow pepper (Capsicum baccatum) plant. Similarly, the L2 shoot meristem layer may be from a potato (Solanum tuberosum) plant, and at least one of the L1 and L3 shoot meristem layers may be from a tomato (Solanum lycopersicum) plant.

[0194] The periclinal chimera may further comprise a transgene or mutation in a sequence of interest. The mutation is preferably present at least in the germline or germline progenitor cells of the callus and / or clonal propagation tissue and / or plant part of step (a). Preferably, the transgene or mutation is present in cells located in at least one of the L1, L2, and L3 shoot meristem layers of the periclinal chimera. Preferably, the transgene or mutation is present in cells located in at least the L2 shoot meristem layer of the periclinal chimera.

[0195] Peripheral chimeras produced by the methods of the present invention may find applications such as, but not limited to, those identified in WO 2018 / 115395 and / or WO 2018 / 115396, which are incorporated herein by reference.

[0196] In a further aspect, the present invention relates to a plant obtainable from the method of the present invention, the plant comprising a transgene and / or a mutation in a sequence of interest. The plant may therefore be a transgenic plant and / or a mutant plant. The plant may also be an artificially created plant. Preferably, the transgene or mutation in the sequence of interest is located in the germline or germline progenitor cells and / or clonal propagation tissue and / or plant part of the plant from which the callus of step (a) was derived. Thus, the plant preferably comprises at least the germline or germline progenitor cells of the callus of step (a) and / or the clonal propagation tissue and / or plant part of the callus of step (a), and preferably comprises a transgene or mutation in the sequence of interest. Preferably, the plant of the present invention is not obtained by or is not obtained solely by an essentially biological process. The plant of the present invention preferably differs from a naturally occurring plant at least in that it comprises at least one transgene or mutation in a sequence of interest. The transgene or mutation in the sequence of interest is preferably located in at least the germline or germline progenitor cells and / or clonal propagation tissues and / or plant parts of the plant. The transgene or mutation in the sequence of interest is preferably located in at least the L2 shoot meristem layer. The transgene or mutation in the sequence of interest is preferably present in at least one of the pollen and ovum of the plant.

[0197] The plant preferably comprises at least germline or germline progenitor cells and / or clonal propagation tissue and / or plant parts derived from the callus of step (a). The plant preferably comprises at least an L2 shoot meristem layer derived from the callus of step (a). The plant obtainable from the method of the present invention is preferably a plant having substantially the same genotype as the plant from which the callus of step (a) was derived, preferably comprising a transgene or mutation in the sequence of interest.

[0198] The present invention further relates to progeny or seeds from plants or perichamera as defined herein. The progeny may be produced by sexual or asexual (vegetative) propagation. The progeny preferably contain a transgene or mutation in the sequence of interest as defined herein. The seed coat may have a different genotype from the embryo. Preferably, the genotype of the coat is from the scion and / or rootstock of step (a) and the genotype of the embryo is from the callus of step (a).

[0199] The present invention also relates to a plant part or plant product derived from a plant obtained from the method of the present invention, preferably step (e), (f), or (g) of the method of the present invention. Optionally, the plant part or plant product is characterized in that it contains genetic material originating from both the callus of step (a) and the rootstock and / or scion. Preferably, the plant part or plant product contains cells, tissue, or genetic material derived from the callus of step (a). Optionally, the plant part or plant product is free of, or substantially free of, cells, tissue, or genetic material derived from the scion and / or rootstock of step (a). Optionally, the plant part or plant product consists of cells, tissue, or plant material characterized in that it contains the genotype of the callus of step (a). Optionally, the plant part or plant product is characterized in that it contains a transgene or a mutation in a sequence of interest. Such genetic material may be genomic DNA or a fragment of genomic DNA. Such genetic material may be mitochondrial DNA or a fragment of mitochondrial DNA. Such genetic material may be chloroplast DNA or fragments of chloroplast DNA.

[0200] The plant part may be propagating or non-propagating material.

[0201] All patents and references cited herein are hereby incorporated by reference in their entirety. [Example]

[0202] Example 1 Protoplasts from leaves of Solanum lycopersicum cultivar Garden Pearl, stably transformed to express the purple RUBY marker (He et al. Horticulture research 2020,7(1):152 doi:10.1038 / s41438-020-00390-1), were cultured on callus induction medium for 11 weeks to generate purple callus (Figure 2A, B). The RUBY callus was grafted onto a scion and rootstock of an F1 hybrid of Solanum pennellii accession LA716 and Solanum lycopersicum accession LA3579, a highly renewable tomato genotype containing the semidominant marker xa (Figure 2C). The semidominant phenotypic marker xa, when present in the heterozygous state at L2 and / or L3, causes yellow leaves (Szymkowiak and Sussex, Plant Cell 1992, 4:1089-1100). In successful scions, callus tissue remained viable, as indicated by the presence of ruby ​​color and the formation of a continuous callus mass within the graft union (Figure 2F). In contrast, ungrafted control callus died after 2 weeks of culture on hormone-free medium (Figure 2D). A similar response was observed in unsuccessful grafting attempts, where callus tissue rapidly browned and withered (Figure 2E).

[0203] Successfully grafted callus tissue remained viable for 7 weeks when cultured on near-vertical plates with 25 mL of MS10 agar. This suggests the formation of rootstock / scion connectivity as a critical requirement for rootstock-scion water / nutrient exchange. The three lines of evidence together suggest that a certain level of connectivity is established after application of the grafting procedure.

[0204] First, when the acidic fuchsin solution was applied to the rootstock root system, it rapidly accumulated in the scion (Figure 3A, B). Consistent with the presence of conductive callus tissue types, whorled lignified callus cells were observed in the callus grafted between the rootstock and the scion (Figure 3C-E). Whorled lignin deposition is a unique feature of vascular xylem cells.

[0205] Second, similar results were obtained when the experiment was repeated using the cell-permeable, amine-reactive green fluorophore 5-(and-6)-carboxyfluorescein diacetic acid (CFDA) as a tracer molecule (Fig. 3F, G). Notably, the pattern of CFDA within the callus tissue was localized to the central region of the grafted callus tissue (Fig. 3H, I).

[0206] Third, shoot regeneration was observed from the scion tissue, indicating extended viability of the scion tissue (see below and Figures 2F and 3J). Thus, the grafted callus tissue established rootstock / scion connectivity.

[0207] Two weeks after grafting, the callus scion was truncated at the callus-scion interface to induce naturally occurring tomato shoot regeneration (Fig. 2C, cut site indicated by red dotted line). The truncated scion was transferred to a greenhouse where the rootstock / scion tissue naturally regenerated shoots under ex vitro conditions (Figs. 2F, 3J). Notably, the callus scion tissue formed organs in parallel with shoot regeneration (Figs. 4A, B). Importantly, the initially observed organs were able to develop into functional shoots (Fig. 4C). This demonstrates effective regeneration during callus grafting.

[0208] Example 2 Maor pepper (Capsicum annuum) is known to be a difficult-to-regenerate plant, failing to regenerate using any conventional tissue or protoplast culture and regeneration procedures. As previously described (WO 2019 / 211296, Example 2), stable transgenic Capsicum annuum cultivar Maor plants were generated by transformation with construct pKG11052. Construct pKG11052 contains the following promoter-transgene expression cassette: CaMV 35S-XVE CaMV 35S-GVG XVE-inducible promoter -WIND1 GVG-inducible promoter-PLT1 GVG-inducible promoter - WOX5 CaMV 35S-erGFP

[0209] Protoplasts from leaves of pepper cultivar Maor, stably transfected with a GFP marker, were cultured on callus induction medium for 7 weeks to allow callus development. Maor GFP-expressing callus was grafted between the rootstock and scion of a 9-day-old wild-type C. baccatum seedling (Figure 5A). Wound healing was allowed for 12 days (cultured on near-vertical plates containing 25 mL of MS10 agar), after which the graft union was truncated at the callus-scion graft junction and maintained on near-vertical plates containing 25 mL of MS10 agar. Spontaneous regeneration of C. baccatum genotypes on the truncated surface occurred without the addition of any hormones. Two weeks after decapitation, incomplete meristem leaves containing differentiated cells from both C. baccatum and Capsicum annuum cv. Maor genotypes had formed (Fig. 5B). The chimeric nature was maintained during developmental growth, leading to the development of shoots containing shoot meristems containing Capsicum annuum cv. Maor cells (Fig. 5C, D).

[0210] Example 3 Stable transgenic tomato cv. Moneyberg plants containing construct pKG11052 (as described in Example 2) were generated. Protoplasts from leaves expressing the GFP marker were cultured on callus induction medium for 6 weeks to allow callus to develop. GFP-expressing callus was grafted between the rootstock and scion of an F1 hybrid of a highly regenerable tomato genotype, Solanum lycopersicum LA3579 × Solanum pennellii LA716 (Figure 6A). Graft healing was allowed for 10 days (cultured on a nearly vertical plate with 25 mL of MS10 agar), after which the graft union was truncated at the callus-scion graft junction. The F1 hybrid genotype spontaneously regenerated from the truncated surface cultured on a nearly vertical plate with 25 mL of MS10 agar, without any hormone application. Two weeks after decapitation, a segmented chimeric leaf containing differentiated cells from both the F1 hybrid and the protoplast-derived Moneyberg genotype had formed (Fig. 6B). The incomplete marginal meristem associated with this leaf gave rise to a chimeric shoot and shoot meristem containing protoplast-derived cells from the Moneyberg genotype (Fig. 6C).

Claims

1. 1. A method for generating and selecting shoots of a plant, comprising: (a) grafting the callus between the scion and the rootstock; (b) forming a graft joint between the callus and each of the scion and the rootstock to form a graft union; (c) creating a wound at or near at least one of the graft joints; (d) forming a shoot at the wounded graft hemisphere; (e) selecting the shoots formed in step (d), wherein said shoots comprise cells derived from said callus of step (a); and optionally (f) growing a plant from the selected shoot of step (e). A method comprising:

2. 2. The method of claim 1, wherein in step (e), the selected shoots comprise germline progenitor cells derived from the callus of step (a), and optionally the method further comprises steps (f) and (g) of obtaining seeds or progeny of the plants grown in step (f), preferably by sexual propagation, preferably by at least one of selfing and backcrossing.

3. The method according to claim 1 or 2, further comprising steps (f) and (g) of obtaining progeny of the plant grown in step (f) by vegetative propagation.

4. The method according to any one of claims 1 to 3, wherein the callus in step (a) is from a first plant and the scion and / or rootstock is from a second plant.

5. 5. The method of claim 4, wherein the scion and the rootstock are from the same or similar plant.

6. The method according to any one of claims 1 to 5, wherein the wounding treatment in step (c) is the removal of the shoot apical meristem by truncation.

7. 7. The method according to any one of claims 1 to 6, wherein in step (d), the axis of the wounded graft union is substantially perpendicular to the ground surface, and the root apical meristem is closer to the ground surface than the shoot apical meristem.

8. Step (d) of allowing shoot formation d1) allowing callus to form at or near the graft union; and d2) Growing shoots from the callus The method according to any one of claims 1 to 7, comprising:

9. 9. The method of any one of claims 1 to 8, further comprising, prior to step (a), growing the callus of step (a) from protoplasts.

10. 10. The method of claim 9, further comprising the step of introducing a transgene and / or a mutation in a sequence of interest into the protoplasts, wherein the selected shoots in step (e) comprise germline progenitor cells, or germline cells derived therefrom, containing the transgene and / or the mutation.

11. 11. The method of any one of claims 1 to 10, further comprising the step of introducing a transgene and / or a mutation in a sequence of interest into cells located in the callus of step (a) and / or the shoot formed in step (d), wherein in step (e) the selected shoot comprises a germline progenitor cell, or a germline cell derived therefrom, containing the transgene and / or the mutation.

12. 12. The method according to claim 10 or 11, comprising step (f), wherein the plant part of the plant grown in step (f) comprises the transgene and / or the mutation, and preferably the plant part can be used for vegetative propagation.

13. 13. The method of any one of claims 10 to 12, wherein the mutation is introduced by programmed genome editing, preferably using a site-specific endonuclease, preferably a CRISPR endonuclease.

14. A plant obtainable from the method according to any one of claims 10 to 13, i) germline progenitor cells and / or germline cells derived therefrom of the callus of step (a); and ii) Plant parts for vegetative propagation of the callus of step (a) and A plant body, wherein the germline progenitor cells, germline cells and / or plant parts contain the transgene and / or mutation described in claim 10 or 11.

15. 15. The plant of claim 14, comprising cells derived from the callus of step (a) and cells derived from the scion and / or rootstock of step (a).

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