Methods for producing organogenic calluses and compositions, systems, and methods related thereto

Abstract

Described herein are methods of producing organogenic calluses, as well as compositions, systems, and methods related thereto.

Description

[0001] Statement Regarding Electronic Submission of Sequence Listings

[0002] A sequence listing in XML text format, titled 1499-94WO_ST26.xml, of size 175,004 bytes, was generated on March 13, 2023 and submitted with this application, and is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0003] The present invention relates to methods of generating organogenic callus and compositions, systems and methods related thereto. Background Art

[0004] Outcrossing plant species may be difficult to regenerate in tissue culture because the regeneration method may not be reproducible or may produce plants that are different from the original species. For example, the outcrossing species sweet cherry (Prunus cerasus) is often referred to as a recalcitrant species because it is difficult to produce plants that are identical to the original species during plant regeneration using tissue culture methods and during genetic modification. Publications demonstrating organogenesis of sweet cherry (e.g., obtaining buds directly from somatic tissues such as leaves, petioles, and stems) generally report low regeneration rates or reported results that cannot be reproduced by other institutions. See, for example, De March, G., et al., (1993) Plant cell, tissue and organ culture, 34(2), 209-215; Garin, E., et al., (1997) Plant Cell, Tissue and Organ Culture, 48(2), 83-91; and Reidiboym-Talleux, L., et al., (1998) Plant Cell, Tissue and Organ Culture, 55(3), 199-209.

[0005] Organogenesis callus generated from plant tissue is a developmental stage in which plant cells occur, and the callus is a disordered cell mass and these cells can be differentiated into other tissues or cell types, such as embryos or buds. Organogenesis callus can produce somatic embryos, and somatic embryos can produce plants after the stages with clear characteristics such as globular (cell sphere) stage, followed by the heart-shaped stage, the torpedo-shaped stage and the cotyledon stage. The original cell that produces organogenesis callus is a cell from a tissue (for example, leaf or stem tissue) that does not usually participate in embryogenesis. For sweet cherry, somatic embryos are usually formed by immature cotyledons, and cotyledons are immature leaves present in seeds. Because sweet cherry is a kind of outcrossing species, and its seed may not be identical with the original species, the plant produced by the somatic embryo in the cotyledon source may not be identical with excellent cultivars.

[0006] Therefore, for outcrossing species, new methods are needed to cultivate and regenerate plants. Summary of the invention

[0007] One aspect of the present invention relates to a method for producing organogenic callus, the method comprising: wounding a plant or plant part comprising stem tissue including a node to provide wounded tissue (e.g., cut tissue); culturing the wounded tissue on a culture medium comprising thidiazuron (TDZ) at a concentration of at least about 5 mg / L of culture medium, thereby producing organogenic callus.

[0008] Another aspect of the present invention relates to a method of propagating plants from organogenic callus, the method comprising: culturing the organogenic callus of the present invention on a culture medium (eg, a regeneration medium) in the presence of light.

[0009] Yet another aspect of the present invention relates to a method for transforming an organogenic callus, the method comprising: contacting the organogenic callus of the present invention with a bacterial cell (eg, Agrobacterium), thereby transforming the organogenic callus with the bacterial cell to provide a transformed organogenic callus.

[0010] Another aspect of the invention relates to a method of modifying a target nucleic acid, the method comprising: contacting the organogenic callus of the invention with a bacterial cell (e.g., Agrobacterium), wherein the organogenic callus comprises the target nucleic acid and wherein the bacterial cell comprises and / or encodes an editing system, and the editing system modifies the target nucleic acid, thereby providing a modified organogenic callus.

[0011] Yet another aspect of the present invention relates to a method for propagating plants from organogenic callus, the method comprising: culturing the transformed organogenic callus of the present invention or the modified organogenic callus of the present invention on a culture medium (eg, a regeneration medium) in the presence of light.

[0012] Another aspect of the invention relates to a composition (e.g., a culture medium) comprising thidiazuron (TDZ) in an amount of at least about 5 mg / L of the composition (e.g., about 5, 6, 7, 8, 9, or 10 mg / L to about 11, 12, 13, 14, or 15 mg / L of the composition); a sugar (e.g., sucrose) in an amount of about 10, 11, 12, 13, 14, 14, 16, 17, 18, or 19 g / L to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L of the composition; and optionally an antibiotic (e.g., cefoxatime) in an amount of about 100, 150, or 200 mg / L to about 250, 300, 350, or 400 mg / L of the composition.

[0013] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated into different embodiments, although not specifically described therewith. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to change any initially filed claim and / or to submit any new claim accordingly, including the right to be able to amend any initially filed claim to be subordinate to and / or incorporate any features of any other claim, although the claim was not initially made in this manner. These and other objects and / or aspects of the invention will be explained in detail in the specification set forth below. Those of ordinary skill in the art will understand further features, advantages and details of the invention from reading the accompanying drawings and the subsequent detailed description of the preferred embodiments, and such description is merely an illustration of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a diagrammatic representation of a chopping blade according to some embodiments of the present invention.

[0015] Figure 2 is another illustration of a chopping blade according to some embodiments of the present invention. DETAILED DESCRIPTION

[0016] The present invention is now described more fully below, wherein embodiments of the present invention are described. However, the present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein; on the contrary, these embodiments are provided to make this disclosure comprehensive and complete and to fully convey the scope of the present invention to those skilled in the art.

[0017] The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. As used in the description of the present invention and the appended claims, the singular forms "a", "an" and "the" are intended to include plural forms as well, unless the context clearly indicates otherwise.

[0018] Unless otherwise defined, all terms used herein (including technical and scientific terms) will have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. It should also be understood that those terms such as those defined in common dictionaries should be interpreted as having the same meaning as their meaning in the context of the present application and the related art, and unless explicitly defined as such herein, should not be interpreted in an idealized or overly formal sense. The terms used herein in the description of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference as a whole. In the case of a conflict of terms, this specification shall prevail.

[0019] Additionally, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as no combinations when interpreted as an alternative ("or").

[0020] Unless the context indicates otherwise, it is expressly intended that the various features of the invention described herein may be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. For example, if the specification states that a certain compound comprises components A, B, and C, it is expressly intended that any one or combination of A, B, or C may be omitted and disclaimed individually or in any combination.

[0021] As used herein, the conjunction "consisting essentially of" (and grammatical variations) should be interpreted to cover the recited materials or steps "and those which do not materially affect the basic and novel characteristics of the claimed invention." See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original); see also MPEP §2111.03. Therefore, as used herein, the term "consisting essentially of" should not be interpreted as equivalent to "comprising."

[0022] As used herein, the terms “comprise”, “comprises” and “comprising” specify the presence of stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0023] As used herein in reference to a measurable value such as an amount or concentration, the term "about" is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, "about X" (where X is a measurable value) is intended to include X as well as ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X.

[0024] ±0.5% or even ±0.1% variation.A range of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0025] Unless otherwise indicated herein, recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range of 10 to 15 is disclosed, 11, 12, 13, and 14 are also disclosed.

[0026] As used herein, the terms "increase," "increasing," "enhance," "enhancing," and "improve," "improving" (and grammatical variations thereof) describe an improvement such as by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to another measurable property or quantity (e.g., a control value).

[0027] As used herein, the terms "reduce," "reduced," "reducing," and "reduction" (and grammatical variations thereof) describe a reduction such as, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% compared to another measurable property or amount (e.g., a control value). In some embodiments, reduction can result in no or substantially no (i.e., a negligible amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0028] As used herein, "editing system" refers to any site-specific (e.g., sequence-specific) nucleic acid editing system now known or later developed, which can introduce modifications (e.g., mutations) in a nucleic acid in a target-specific manner. For example, an editing system (e.g., a site-specific and / or sequence-specific editing system) may include, but is not limited to, a CRISPR-Cas editing system, a meganuclease editing system, a zinc finger nuclease (ZFN) editing system, a transcription activator-like effector nuclease (TALEN) editing system, a base editing system, and / or a guide editing system, each of which may include one or more polypeptides and / or one or more polynucleotides, which, when present and / or expressed together (e.g., as a system) in a composition and / or cell, can modify a target nucleic acid in a sequence-specific manner (e.g., mutate a target nucleic acid). In some embodiments, an editing system (e.g., a site-specific and / or sequence-specific editing system) may include one or more polynucleotides and / or one or more polypeptides, including but not limited to a nucleic acid binding polypeptide (e.g., a DNA binding domain), a nuclease, another polypeptide, and / or a polynucleotide. In some embodiments, a CRISPR-Cas editing system comprising a CRISPR-Cas effector protein is provided and / or used.

[0029] In some embodiments, the editing system comprises one or more sequence-specific nucleic acid binding polypeptides (e.g., DNA binding domains), which can be derived from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, the editing system comprises one or more cleavage polypeptides (e.g., nucleases), including but not limited to endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs).

[0030] As used herein, "nucleic acid binding polypeptide" refers to a polypeptide or domain that binds and / or is capable of binding to a nucleic acid (e.g., a target nucleic acid). A DNA binding polypeptide is an exemplary nucleic acid binding polypeptide, and may be a site-specific and / or sequence-specific nucleic acid binding polypeptide. In some embodiments, the nucleic acid binding polypeptide may be a sequence-specific nucleic acid binding polypeptide, such as, but not limited to, sequence-specific binding domains from, for example, polynucleotide-guided endonucleases, CRISPR-Cas effector proteins (e.g., CRISPR-Cas endonucleases), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, the nucleic acid binding polypeptide comprises a cleavage domain (e.g., a nuclease domain), such as, but not limited to, endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases, zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs). In some embodiments, the nucleic acid binding polypeptide is associated with and / or is capable of associating with (e.g., forming a complex with) one or more nucleic acid molecules (e.g., forming a complex with a guide nucleic acid as described herein), which can guide and / or direct the nucleic acid binding polypeptide to a specific target nucleotide sequence (e.g., a genomic locus) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof), thereby causing the nucleic acid binding polypeptide to bind to a nucleotide sequence at a specific target site. In some embodiments, the nucleic acid binding polypeptide is a CRISPR-Cas effector protein as described herein.

[0031] In some embodiments, the editing system comprises a ribonucleoprotein or is a ribonucleoprotein, such as an assembled ribonucleoprotein complex (e.g., a ribonucleoprotein comprising a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase). In some embodiments, the ribonucleoproteins of the editing system can be assembled together (e.g., a preassembled ribonucleoprotein comprising a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase), such as when contacted with a target nucleic acid or when introduced into a cell (e.g., a plant cell). In some embodiments, the ribonucleoproteins of the editing system can be assembled into a complex (e.g., a covalently and / or non-covalently bound complex) while a portion of the ribonucleoprotein contacts the target nucleic acid and / or can be assembled after and / or during introduction into a plant cell. In some embodiments, the editing system can be assembled when introduced into a plant cell (e.g., assembled into a covalently and / or non-covalently bound complex). In some embodiments, the ribonucleoprotein can comprise a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase.

[0032] As used herein, "CRISPR-Cas effector protein" is a protein or polypeptide that cuts, cuts or nicks nucleic acids; binds nucleic acids (e.g., target nucleic acids and / or guide nucleic acids); and / or identifies, recognizes or binds to guide nucleic acids as defined herein. In some embodiments, the CRISPR-Cas effector protein can be an enzyme (e.g., a nuclease, an endonuclease, a nickase, etc.) and / or can act as an enzyme. In some embodiments, the CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease. In some embodiments, the CRISPR-Cas effector protein comprises nuclease activity and / or nickase activity, comprises a nuclease domain whose nuclease activity and / or nickase activity has been reduced or eliminated, comprises single-stranded DNA cleavage activity (ssDNAse activity) or it has ssDNAse activity that has been reduced or eliminated, and / or comprises self-processing RNAse activity or it has self-processing RNAse activity that has been reduced or eliminated. The CRISPR-Cas effector protein can bind to a target nucleic acid. The CRISPR-Cas effector protein can be a type I, type II, type III, type IV, type V, or type VI CRISPR-Cas effector protein. In some embodiments, the CRISPR-Cas effector protein can be from a type I CRISPR-Cas system, a type II CRISPR-Cas system, a type III CRISPR-Cas system, a type IV CRISPR-Cas system, a type V CRISPR-Cas system, or a type VI CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein of the present invention can be from a type II CRISPR-Cas system or a type V CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein can be a type II CRISPR-Cas effector protein, for example, a Cas9 effector protein. In some embodiments, the CRISPR-Cas effector protein can be a type V CRISPR-Cas effector protein, for example, a Cas12 effector protein. In some embodiments, the CRISPR-Cas effector protein may be Cas12a, and optionally may have an amino acid sequence of any one of SEQ ID NOs: 1-17 or 18 and / or a nucleotide sequence of any one of SEQ ID NOs: 19-21. In some embodiments, the CRISPR-Cas effector protein may be an active Cas12a, and optionally may have an amino acid sequence of SEQ ID NOs: 9 or 18. In some embodiments, the CRISPR-Cas effector protein may be an inactive (i.e., dead) Cas12a, and optionally may have an amino acid sequence of SEQ ID NO: 1.In some embodiments, the CRISPR-Cas effector protein can be Cas12b, and optionally can have the amino acid sequence of SEQ ID NO:22.

[0033] Exemplary CRISPR-Cas effector proteins include, but are not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3 , Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein can be a Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c effector protein.

[0034] In some embodiments, the CRISPR-Cas effector protein useful in the present invention may include a mutation in its nuclease active site and / or nuclease domain (e.g., RuvC, HNH, e.g., RuvC site of the Cas12a nuclease domain; e.g., RuvC site and / or HNH site of the Cas9 nuclease domain). CRISPR-Cas effector proteins that have mutations in their nuclease active sites and / or nuclease domains and therefore no longer contain nuclease activity are often referred to as "inactive" or "dead", e.g., dCas9. In some embodiments, a CRISPR-Cas effector protein having a mutation in its nuclease active site and / or nuclease domain may have impaired activity or reduced activity (e.g., nickase activity) compared to the same CRISPR-Cas effector protein without a mutation.

[0035] CRISPR Cas9 effector proteins or Cas9 that can be used in the present invention can be any known or later identified Cas9 nuclease. In some embodiments, the Cas9 of the present invention can be a protein from, for example, Streptococcus (e.g., Streptococcus pyogenes, Streptococcus thermophilus), Lactobacillus, Bifidobacterium, Kandleria, Leuconostoc, Oenococcus, Pediococcus, Weissella, and / or Oulsonella. In some embodiments, the CRISPR-Cas effector protein can be Cas9, and optionally can have a nucleotide sequence of any one of SEQ ID NO:23-33 or 34-37 and / or an amino acid sequence of any one of SEQ ID NO:38-39.

[0036] In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Streptococcus pyogenes and / or may recognize the PAM sequence motifs NGG, NAG, NGA (Mali et al., Science 2013; 339(6121): 823-826). In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Streptococcus thermophilus and / or may recognize the PAM sequence motifs NGGNG and / or NNAGAAW (W=A or T) (see, e.g., Horvath et al., Science, 2010; 327(5962): 167-170, and Deveau et al., J Bacteriol 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus mutans and / or may recognize the PAM sequence motif NGG and / or NAAR (R=A or G) (see, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Staphylococcus aureus and / or may recognize the PAM sequence motif NNGRR (R=A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Staphylococcus aureus and / or may recognize the PAM sequence motif N GRRT (R=A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Staphylococcus aureus and / or may recognize the PAM sequence motif N GRRV (R=A or G). In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Neisseria meningitidis and / or may recognize a PAM sequence motif N GATT or N GCTT (R = A or G, V = A, G or C) (see, e.g., Hou et al., PNAS 2013, 1-6). In the aforementioned embodiments in this paragraph, N in the PAM sequence motif may be any nucleotide residue, e.g., any one of A, G, C or T. In some embodiments, the CRISPR-Cas effector protein may be Cas13a derived from Leptotrichia shahii and / or may recognize a single 3'A, U or C pre-spacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif, which may be located within the target nucleic acid.

[0037] The V-type CRISPR-Cas effector protein that can be used in embodiments of the present invention can be any V-type CRISPR-Cas nuclease. Exemplary V-type CRISPR-Cas effector proteins include, but are not limited to, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c nuclease. In some embodiments, the V-type CRISPR-Cas effector protein can be Cas12a. In some embodiments, the V-type CRISPR-Cas effector protein can be a nickase, optionally, a Cas12a nickase. In some embodiments, the V-type CRISPR-Cas effector protein can be Cas12b (e.g., SEQ ID NO: 22).

[0038] As used herein, “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA,” “crRNA,” or “crDNA” means a sequence comprising at least one spacer sequence that is complementary to (and hybridizes to) a target DNA (e.g., a pre-spacer sequence) and at least one repeat sequence (e.g., a repeat sequence of a type V Cas12a CRISPR-Cas system, or a fragment or portion thereof; a repeat sequence of a type II Cas9 CRISPR-Cas system, or a fragment thereof; a type V C2c1 CRISPR Cas system repetitive sequence, or its fragment; For example, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, C In some embodiments, the guide nucleic acid comprises DNA. In some embodiments, the guide nucleic acid comprises RNA (e.g., a guide RNA). The design of the gRNA of the present invention can be based on a type I, type II, type III, type IV, type V, or type VI CRISPR-Cas system.

[0039] Any deaminase domain / polypeptide that can be used for base editing can be used in the present invention. As used herein, "cytosine deaminase" and "cytidine deaminase" refer to a polypeptide or its domain that catalyzes or can catalyze the deamination of cytosine, because the polypeptide or domain catalyzes or can catalyze the removal of an amine group from a cytosine base. Therefore, cytosine deaminase can cause cytosine (cystosine) to be converted to thymidine (via a uracil intermediate), thereby causing C to T conversion or G to A conversion in the complementary chain in the genome. Therefore, in some embodiments, the cytosine deaminase encoded by the polynucleotide of the present invention produces C→T conversion in the sense (e.g., "+"; template) chain of the target nucleic acid or produces G→A conversion in the antisense (e.g., "-", complementary) chain of the target nucleic acid. In some embodiments, the cytosine deaminase encoded by the polynucleotide of the present invention produces C to T, G or A conversion in the complementary chain in the genome.

[0040] The cytosine deaminase that can be used in the present invention can be any known or later identified cytosine deaminase from any organism (see, e.g., U.S. Patent No. 10,167,457 and Thuronyi et al., Nat. Biotechnol. 37: 1070–1079 (2019), each of which is incorporated herein by reference for the cytosine deaminase disclosed by them). The cytosine deaminase can catalyze the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Therefore, in some embodiments, the deaminase or deaminase domain that can be used in the present invention can be a cytidine deaminase domain that catalyzes the hydrolysis and deamination of cytosine to uracil. In some embodiments, the cytosine deaminase can be a variant of a naturally occurring cytosine deaminase, including but not limited to primates (e.g., humans, monkeys, chimpanzees, gorillas), dogs, cattle, rats, or mice. Thus, in some embodiments, a cytosine deaminase useful in the present invention can be about 70% to about 100% identical to a wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a naturally occurring cytosine deaminase, and any range or value therein).

[0041] In some embodiments, the cytosine deaminase useful in the present invention may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation-induced deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570) and an evolved version thereof. Evolved deaminases are disclosed in, e.g., U.S. Pat. No. 10,113,163, Gaudelli et al. (Nature 551(7681):464-471(2017)), and Thuronyi et al. (Nature Biotechnology 37:1070-1079(2019)), each of which incorporates their disclosed deaminases and evolved deaminases herein by reference. In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase having an amino acid sequence of SEQ ID NO:40. In some embodiments, the cytosine deaminase may be an APOBEC3A deaminase having an amino acid sequence of SEQ ID NO:41. In some embodiments, the cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 having an amino acid sequence of SEQ ID NO:42. In some embodiments, the cytosine deaminase may be a FERNY deaminase, optionally a FERNY having an amino acid sequence of SEQ ID NO:43. In some embodiments, the cytosine deaminase may be a rAPOBEC1 deaminase, optionally a rAPOBEC1 deaminase having the amino acid sequence of SEQ ID NO: 44. In some embodiments, the cytosine deaminase may be a hAID deaminase, optionally a hAID having the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46.In some embodiments, a cytosine deaminase useful in the present invention can be about 70% to about 100% identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to the amino acid sequence of a naturally occurring cytosine deaminase (e.g., an "evolved deaminase") (see, e.g., SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49). In some embodiments, a cytosine deaminase useful in the present invention can be about 70% to about 99.5% identical (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) to the amino acid sequence of any one of SEQ ID NOs: 40-49 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical). In some embodiments, a polynucleotide encoding a cytosine deaminase can be codon-optimized for expression in plants, and the codon-optimized polypeptide can be about 70% to 99.5% identical to a reference polynucleotide.

[0042] As used herein, "adenine deaminase" and "adenosine deaminase" refer to polypeptides or domains thereof that catalyze or are capable of catalyzing the hydrolytic deamination of adenine or adenosine (e.g., removing an amine group from adenine). In some embodiments, an adenine deaminase can catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, an adenosine deaminase can catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the present invention can produce an A→G conversion in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C conversion in the antisense (e.g., "-", complementary) strand of a target nucleic acid. Adenine deaminases that can be used in the present invention can be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Pat. No. 10,113,163, which incorporates herein by reference the adenine deaminase disclosed therein).

[0043] In some embodiments, the adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, the adenosine deaminase may be about 70% to 100% identical to a wild adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a naturally occurring adenine deaminase, and any ranges or values ​​therein). In some embodiments, the deaminase or deaminase does not exist in nature and may be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or adenine deaminase domain can be about 70% to 99.9% identical to a naturally occurring adenine deaminase polypeptide / domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264 In some embodiments, the adenine deaminase polypeptide / domain may be codon-optimized for expression in plants.

[0044] In some embodiments, the adenine deaminase domain can be a wild-type tRNA-specific adenosine deaminase domain, e.g., a tRNA-specific adenosine deaminase (TadA) and / or a mutant / evolved adenosine deaminase domain, e.g., a mutant / evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, the TadA domain can be from Escherichia coli. In some embodiments, TadA may be modified, e.g., truncated, such that one or more N-terminal and / or C-terminal amino acids are deleted relative to full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C-terminal amino acid residues may be deleted relative to full-length TadA. In some embodiments, the TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, wild-type E. coli TadA comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the mutant / evolved E. coli TadA* comprises the amino acid sequence of any one of SEQ ID NOs: 51-54. In some embodiments, the polynucleotide encoding TadA / TadA* may be codon-optimized for expression in plants. In some embodiments, the adenine deaminase may comprise all or a portion of the amino acid sequence of any one of SEQ ID NOs: 55-60. In some embodiments, the adenine deaminase may comprise the amino acid sequence of any one of SEQ ID NOs: 56-61. The whole or part of the amino acid sequence of any one of NO:50-60.

[0045] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof, refer to placing the components of a desired reaction together under conditions suitable for the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking and / or cleavage). Thus, for example, a target nucleic acid can be contacted with a nucleic acid construct encoding, for example, a nucleic acid binding polypeptide (e.g., a DNA binding domain, such as a sequence-specific DNA binding protein (e.g., a polynucleotide-guided endonuclease, a CRISPR-Cas effector protein (e.g., a CRISPR-Cas endonuclease), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein)), a guide nucleic acid, and optionally a cytosine deaminase and / or an adenine deaminase, under conditions whereby the nucleic acid binding domain (e.g., a CRISPR-Cas effector protein) is expressed, and the nucleic acid binding domain forms a complex with the guide nucleic acid, the complex hybridizes to the target nucleic acid, and optionally the cytosine deaminase and / or adenine deaminase is recruited to the nucleic acid binding domain (and thereby to the target nucleic acid), or the cytosine deaminase and / or adenine deaminase is fused to the nucleic acid binding domain, thereby modifying the target nucleic acid. In some embodiments, the cytosine deaminase and / or adenine deaminase and the nucleic acid binding domain are localized at the target nucleic acid, optionally through covalent and / or non-covalent interactions.

[0046] In some embodiments, a target nucleic acid can be contacted with a nucleic acid construct encoding a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a cytosine deaminase and / or an adenine deaminase under conditions whereby the CRISPR-Cas effector protein is expressed, or a target nucleic acid can be contacted with a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a cytosine deaminase and / or an adenine deaminase. The CRISPR-Cas effector protein can form a complex with the guide nucleic acid, the complex can hybridize with the target nucleic acid, and optionally the cytosine deaminase and / or the adenine deaminase is recruited to the CRISPR-Cas effector protein (and thus to the target nucleic acid), or the cytosine deaminase and / or the adenine deaminase is fused to the CRISPR-Cas effector protein, thereby modifying the target nucleic acid. The cytosine deaminase and / or the adenine deaminase and the CRISPR-Cas effector protein can be optionally localized at the target nucleic acid by covalent and / or non-covalent interactions. In some embodiments, a target nucleic acid can be contacted with a ribonucleoprotein, such as an assembled ribonucleoprotein complex (e.g., a ribonucleoprotein comprising a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase), optionally wherein all or a portion of the ribonucleoprotein is introduced into a cell.

[0047] As used herein, "modification (modifying or modification)" about target nucleic acid includes editing (e.g., mutation), covalent modification, exchange / replacement of nucleic acid / nucleotide base, deletion, cutting and / or nicking target nucleic acid to thereby provide modified nucleic acid and / or change the transcriptional control of target nucleic acid to thereby provide modified nucleic acid. In some embodiments, modification may include insertion and / or deletion of any size and / or single base change (SNP) of any type. In some embodiments, modification includes SNP. In some embodiments, modification includes exchange and / or replacement of one or more (e.g., 1, 2, 3, 4, 5 or more) nucleotides. In some embodiments, insertion or deletion may be a length of about 1 base to about 30,000 bases (e.g., about 1, 10, 100, 500, 1,000 or 2,000 bases to about 5,000, 10,000, 20,000 or 30,000 bases or longer, or any value or range therein).

[0048] "Introducing, introducing, introduced" (and grammatical variations and derivatives thereof) means presenting a nucleotide sequence of interest (e.g., a polynucleotide, a nucleic acid construct, and / or a guide nucleic acid) to a host organism or a cell of the organism (e.g., a host cell; e.g., a plant cell) in a manner that allows the nucleotide sequence, polypeptide, and / or ribonucleoprotein to gain access to the interior of the cell. Introduction can be equivalent to transformation, and any known or later developed means of transforming a plant or part thereof (e.g., a cell thereof) can be used. Thus, for example, a polynucleotide from an Agrobacterium strain such as Agrobacterium tumefaciens or Agrobacterium rhizogenes (which can encode or contain at least a portion of an editing system) can be introduced into a cell of an organism, thereby transforming the cell with the polynucleotide. In some embodiments, a nucleic acid construct of the invention encoding a CRISPR-Cas effector protein, a guide nucleic acid, and a deaminase (e.g., a cytosine deaminase and / or an adenine deaminase) can be introduced into a cell of an organism, thereby transforming the cell with the CRISPR-Cas effector protein, the guide nucleic acid, and the deaminase. In some embodiments, the organism is a eukaryotic organism (eg, a plant or a mammal such as a human).

[0049] As used herein, the term "transformation" refers to the introduction of nucleic acids, polypeptides and / or ribonucleoproteins (e.g., heterologous nucleic acids, polypeptides and / or ribonucleoproteins) into cells. The transformation of cells can be stable or transient. Therefore, in some embodiments, host cells or host organisms can be stably transformed with polynucleotides / nucleic acid molecules of the present invention. In some embodiments, host cells or host organisms can be transiently transformed with nucleic acid constructs, polypeptides and / or ribonucleoproteins of the present invention.

[0050] In the context of polynucleotide polypeptides and / or ribonucleoproteins, "transient transformation" means that the polynucleotide, polypeptide and / or ribonucleoprotein is introduced into the cell (e.g., by transformation and / or transfection methods) without being integrated into the genome of the cell; therefore, the cell is transiently transformed by the polynucleotide. As used herein, a "transiently expressed" nucleic acid refers to a nucleic acid that has been introduced into the cell and the nucleic acid is not integrated into the genome of the cell, so the cell is transiently transformed by the nucleic acid.

[0051] In the context of introducing a polynucleotide into a cell (e.g., by transformation and / or transfection methods), "stably introducing" or "stably introduced" is intended to stably incorporate the introduced polynucleotide into the genome of the cell, and thus, the cell is stably transformed with the polynucleotide. As used herein, a "stably expressed" nucleic acid refers to a nucleic acid that has been introduced into a cell and the nucleic acid integrates into the genome of the cell, and thus the cell is stably transformed with the nucleic acid.

[0052] As used herein, "stable transformation" or "stably transformed" means that a nucleic acid molecule is introduced into a cell (e.g., by transformation and / or transfection methods) and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, more specifically, by multiple successive generations of progeny.

[0053] As used herein, "genome" includes nuclear and plastid genomes, and thus includes integration of the nucleic acid into, for example, the chloroplast or mitochondrial genome. As used herein, stable transformation can also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or plasmid.

[0054] Transient transformation can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of peptides or polypeptides encoded by one or more transgenics introduced into an organism. Stable transformation of cells can be detected by, for example, Southern blot hybridization assays of genomic DNA of cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenics introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays of RNA of cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenics introduced into a host organism. Stable transformation of cells can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, using specific primer sequences that hybridize with target sequences of transgenics, resulting in amplification of transgenic sequences, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0055] Therefore, in some embodiments, nucleotide sequences, polynucleotides, nucleic acid constructs and / or expression cassettes of the present invention can be transiently expressed and / or they can be stably incorporated into the genome of a host organism. Therefore, in some embodiments, nucleic acid constructs of the present invention can be transiently introduced into cells together with guide nucleic acids, and therefore, no DNA is maintained in the cell.

[0056] Nucleic acid construct of the present invention, polypeptide and / or ribonucleoprotein can be introduced into cell by any method known to those skilled in the art.In some embodiments, introduction and / or conversion method include but are not limited to: via bacterial-mediated nucleic acid delivery (for example, via Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide and / or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery conversion, microinjection, microparticle bombardment, calcium phosphate-mediated conversion, cyclodextrin-mediated conversion, electroporation, nanoparticle-mediated conversion, ultrasonic treatment, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism causing nucleic acid to be introduced into cell (for example, plant cell or animal cell), including any combination thereof.In some embodiments of the present invention, the conversion of cell includes nuclear conversion.In some embodiments, the conversion of cell includes plastid conversion (for example, chloroplast conversion).In some embodiments, the recombinant nucleic acid construct of the present invention can be introduced into cell via conventional breeding techniques.

[0057] Procedures for transforming eukaryotic and prokaryotic organisms are well known and routine in the art and are described throughout the literature (see, e.g., Jiang et al., 2013. Nat. Biotechnol. 31: 233-239; Ran et al., Nature Protocols 8: 2281–2308 (2013)). General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, Eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858 (2002)).

[0058] Therefore, nucleotide sequence, polypeptide and / or ribonucleoprotein can be introduced into host organism or its cell in many ways well known in the art.The method of the present invention does not rely on the ad hoc method for introducing one or more nucleotide sequences, polypeptides and / or ribonucleoproteins into organism, but only relies on them to enter the inside of at least one cell of organism.When introducing more than one nucleotide sequence, polypeptide and / or ribonucleoprotein, they can be assembled into a part of a single nucleic acid construct, or assembled into a separate nucleic acid construct, and can be located on the same or different nucleic acid constructs.Therefore, nucleotide sequence, polypeptide and / or ribonucleoprotein can be introduced into interested cell in a single transformation event and / or in a separate transformation event, or alternatively, in the case of relevance, nucleotide sequence can be, for example, incorporated into plant as a part of a breeding scheme.In some embodiments, cell is a eukaryotic cell (for example, plant cell or mammal such as human cell).

[0059] According to an embodiment of the present invention, a method comprising producing organogenic callus is provided. The organogenic callus of the present invention can be produced from a plant of an outcrossing species, such as, but not limited to, sweet cherry (European sweet cherry). The method of the present invention may include: wounding a plant or plant part comprising stem tissue including a node to provide injured tissue (e.g., cutting tissue); and culturing the injured tissue on a culture medium comprising thidiazuron (TDZ) at a concentration of at least about 5 mg / L culture medium, thereby producing organogenic callus. In some embodiments, the organogenic callus is not an organogenic callus of cotyledon origin (i.e., the organogenic callus is not prepared by cotyledons) and / or the step of wounding provides injured tissue without cotyledons. The organogenic callus of the present invention may be the same as the original species relative to the plant or plant part from which the injured tissue is obtained and / or compared to the plant from which the organogenic callus is produced. As used herein, "same as the original" refers to a plant (e.g., a progeny plant) or a precursor thereof that will provide (e.g., regenerate into) a plant having the same unique characteristics (e.g., growth habit, growth type, flowering time, color, etc.) and / or phenotype as the plant (e.g., parent plant) from which the plant or precursor is derived. Thus, "organogenic callus identical to the original" refers to an organogenic callus that can be grown into (e.g., regenerated into) a plant having the same unique characteristics and / or phenotype as the plant from which the organogenic callus was developed.

[0060] The method of wounding a plant or plant part is known in the art. In some embodiments, wounding includes cutting a plant or plant part with a device such as a knife or a surgical scalpel, optionally by hand (e.g., manual operation). In some embodiments, wounding includes blending plants or plant parts in a blender, thereby providing injured tissue. As used herein, "blender" refers to a mechanical device configured to blend, chop, dice and / or slice materials such as food. In some embodiments, the blender is driven by a motor (i.e., an electric blender). Exemplary electric blenders include, but are not limited to, a kitchen blender including a rotating blade (e.g., a metal blade, such as a stainless steel blade) driven by a motor and a food processor including a blade (e.g., a cutting blade, such as a metal (e.g., stainless steel) cutting blade) driven by a motor. The motor can be a 350 watt motor. In some embodiments, the blender used in the method of the present invention is a Cuisinart blender of model CPB-300P1 or a blender equivalent thereto, and the blender can be equipped with a chopping blade. The chopping blade can also be referred to as an S blade. Exemplary chopping blades and / or S blades include, but are not limited to, blades having model number CPB-300CHA or equivalent included in a Cuisinart chopping assembly. The blade may have a planar configuration. In some embodiments, the blade assembly 10 provided in the blender includes a single elongated blade 20 that rotates about its center 20c so that it provides edge "fins" 20f for chopping and / or cutting on either side of its pivot, such as Figure 1 In some embodiments, the blade assembly 30 disposed in the blender includes a blade 40 having only a single edge "fin" 40f (see Figure 2 ); blade 40 rotates around fixed axis 45 and uses only a single "fin" 40f for chopping and / or cutting. The blender of the present invention can be attached to a container operably associated with the blender, such as a Cuisinart cup with model CTC-16. In some embodiments, the blender used in the method of the present invention is an electric blender (e.g., a kitchen blender and / or a food processor) that includes and / or is equipped with a chopping blade and / or an S blade with metal (e.g., stainless steel) fins. One or more parts of the blender used in the method of the present invention may be capable of being sterilized and / or configured for sterilization (e.g., alcohol sterilization and / or steam sterilization). In some embodiments, the blender portion that contacts the plant and / or plant part can be sterilized and / or configured for sterilization. For example, the blender used in the method of the present invention may have a container (e.g., a cup), a blade and / or a blade assembly that can be sterilized and / or configured for sterilization.

[0061] In the method of the present invention, the blender can be operated at a speed of about 1, 5, 10, 15 or 20 Hz to about 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or 380 Hz. In some embodiments, the blender can be set to the lowest speed setting of the blender and / or run with the lowest speed setting of the blender, optionally set to a chopping setting and / or run with a chopping setting. In some embodiments, blending includes blending plant materials (e.g., plants or plant parts) at a speed of about 1, 5, 10, 15 or 20 Hz to about 25, 30, 35, 40, 45 or 50 Hz. Blending can be carried out for a period of time, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds or longer, optionally using a pulse setting. In some embodiments, blending the plant material comprises blending the plant or plant parts at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) time intervals, each time interval comprising a time period during which the blender is operated to blend the plant material and optionally a time period during which the blender is not operated to provide a time period for stopping the blending. In some embodiments, blending the plant material comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) time intervals, wherein each time interval comprises about 1 second to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds during which the blender is operated to blend the plant material and optionally about 1 second to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds during which the blender is not operated. In some embodiments, the blending step and / or the time for providing the injured tissue is performed in about 10 seconds or less, such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second. In some embodiments, blending parameters and / or settings (eg, speed, time, number of pulses, etc.) of the blender may depend on the plant tissue type and / or the purpose of wounding (eg, wounding for propagation, regeneration, or transformation).

[0062] As used herein, "blending (blending, blending)" and grammatical variants thereof refer to mechanical cutting (e.g., chopping, dicing, etc.) of materials (e.g., plant materials). Blending can cut materials into pieces smaller than the size of the materials before blending. In some embodiments, blending can include coarse cutting and / or fine cutting of materials. In some embodiments, blending is configured to coarsely cut plant materials and is intended to limit the blending of plant materials to a smoothie-like consistency. In some embodiments, blending provides cutting of plant materials (e.g., injured plant tissues), such as measured at the widest part of the plant material, having a size of about 0.2 or 0.3 cm to about 0.4 or 0.5 cm. Plants or plant parts can be blended in a blender. In some embodiments, blending includes blending the entire plant or one or more parts thereof (e.g., the above-ground and / or non-aerial parts of the plant). In some embodiments, blending includes blending stems, leaves, nodes, buds and / or callus. In some embodiments, blending of plant materials is carried out in the presence of water and / or an aqueous composition (e.g., a liquid growth medium). Plant material and water and / or aqueous composition can be added to the container of the blender and blended together. In some embodiments, plant material and bacteria (optionally in water and / or aqueous composition) are blended together. In some embodiments, organogenesis callus of the present invention and bacteria (optionally in water and / or aqueous composition) are blended together. The bacteria can be Agrobacterium strains, such as Agrobacterium tumefaciens and / or Agrobacterium rhizogenes. The bacteria can include a nucleic acid construct encoding all or part of an editing system (e.g., CRISPR-Cas editing system) configured to modify polynucleotides in plant cells. Blending plant materials (e.g., organogenesis callus) can inoculate bacteria into plant materials and / or introduce polynucleotides from bacteria into cells of plant materials, optionally into cells of injured tissues. In some embodiments, the method of the present invention may include introducing polynucleotides into cells of injured plant tissues using bacteria blended with plant tissues. In some embodiments, the method of the present invention may include introducing polynucleotides into cells of organogenesis callus of the present invention using bacteria blended with organogenesis callus. In some embodiments, plant tissue is admixed in water and / or an aqueous composition without bacteria to provide wounded tissue, and the wounded tissue is contacted with bacteria, optionally to introduce a polynucleotide into cells of the wounded tissue.

[0063] The methods and / or blending steps of the present invention can process about 8, 9, 10, 11, 12, 13, 14, 15, or 16 ounces to about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of plant material to provide injured tissue in about 10 seconds or less, such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second. In some embodiments, the methods and / or blending steps of the present invention can process about 50 clumps of plant material (e.g., plant buds, such as about 6-week-old plant buds) to provide injured tissue in about 10 seconds or less, such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second. In some embodiments, the methods and / or blending steps of the present invention can process about 5 grams to about 250 grams of plant material to provide injured tissue in about 10 seconds or less, such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second.

[0064] In some embodiments, about 8, 9, 10, 11, 12, 13, 14, 15, or 16 ounces to about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of plant material are blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition, optionally comprising bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant sprouts, such as about 6 week old plant sprouts) are blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition, optionally comprising bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant sprouts, such as about 6 week old plant sprouts) are blended with about 300 mL of liquid (e.g., water and / or an aqueous composition, optionally comprising bacteria). In some embodiments, about 5 grams to about 250 grams of plant material are blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition, optionally comprising bacteria).

[0065] In some embodiments, the stem tissue used in the method of the present invention comprises meristem. The meristem used in the method of the present invention may comprise a node. Stem tissue and / or meristem may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nodes. A node is a region of stem with a large amount of cell growth. A node may include buds and / or leaves and / or petioles that are emerging and / or growing. In some embodiments, callus (e.g., organogenesis callus) may be generated from meristem, optionally from injured meristem.

[0066] The step of wounding a plant or plant part to provide wounded tissue (e.g., wounded meristem) may include providing a plant tissue mixture comprising a stem tissue (e.g., meristem) comprising a node and / or wounding a plant tissue mixture comprising a stem tissue (e.g., meristem) comprising a node. Therefore, a wounded plant tissue mixture comprising wounded stem tissue and / or wounded meristem may be provided. In some embodiments, before the cultivation step, wounded stem tissue and / or wounded meristem are isolated from wounded plant tissue. In some embodiments, before the cultivation step, wounded stem tissue and / or wounded meristem are not isolated from the mixture of wounded tissue. In some embodiments, the cultivation step includes cultivating a mixture of wounded plant tissue, optionally wherein the mixture of said wounded plant tissue comprises wounded stem tissue and / or wounded meristem.

[0067] The method of the present invention may include a cultivation step using methods known in the art. For example, cultivating injured plant tissue may include exposing plant material (optionally injured plant tissue) to certain conditions (e.g., light, darkness, hormones, nutrients, humidity, etc.) for a period of time (e.g., about 1, 2, 3, 4 or 5 days or weeks to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more days or weeks). In some embodiments, cultivating plant material includes providing temperature, nutrients and / or light conditions sufficient to maintain and / or grow plant material. Plant material can be cultivated on solid and / or gelled medium. In some embodiments, cultivating plant material is carried out before and / or after the wounding step. In some embodiments, the method of the present invention includes cultivating injured plant tissue (e.g., injured stem tissue and / or injured meristem). The plant material and / or wounded plant tissue may be cultured in the presence of and / or on a culture medium that may contain antibiotics and / or growth hormones (e.g., auxins and / or nutrients).

[0068] In some embodiments, the culturing step comprises culturing the injured plant tissue on a medium comprising thidiazuron (TDZ) at a concentration of about 5, 6, 7, 8, 9, or 10 mg / L to about 11, 12, 13, 14, or 15 mg / L of the medium. In some embodiments, TDZ is present in the medium on which the injured plant tissue is cultured at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / L of the medium. As will be readily appreciated by one of ordinary skill in the art, the concentration of a component such as TDZ in a solid and / or gelled medium based on the weight of the component per volume of the medium (e.g., mg / L) refers to the weight amount of the component (e.g., TDZ) in the liquid medium prior to solidification and / or gelling of the medium and optionally prior to autoclaving. Thus, for example, when a component (e.g., TDZ) is present in the culture medium in an amount of 5 mg / L, the component is present in the liquid culture medium in an amount of 5 mg / L of culture medium, and the liquid culture medium can then be autoclaved and / or allowed to solidify and / or gel, thereby providing a culture medium on which to culture plant tissue (e.g., wounded plant tissue).

[0069] The culture medium used in the culture step and / or method of the present invention can also include sugar (for example, sucrose, glucose, etc.). In some embodiments, the sugar is glucose. In some embodiments, the sugar is sucrose. Sugar (for example, glucose) can be present in the culture medium with the amount of about 10,11,12,13,14,14,16,17,18 or 19g / L to about 20,21,22,23,24,25,26,27,28,29 or 30g / L culture medium. In some embodiments, sugar (for example, glucose) is present in the culture medium with the amount of about 10,11,12,13,14,14,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30g / L culture medium. Sugar (e.g., glucose) can be present in the culture medium in an amount of about 10, 11, 12, 13, 14, 14, 16, 17, 18, or 19 g / L to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L culture medium. In some embodiments, the culture medium comprises sugar in an amount less than 25 or 30 g / L culture medium.

[0070] In some embodiments, the medium comprising TDZ may comprise a sugar (e.g., glucose), a basal medium such as, but not limited to, Murashige and Skoogs (MS) medium and / or a woody plant medium (WPM) basal medium (e.g., from Phytotech), and agar (e.g., Phytagar TMIn some embodiments, the medium comprising TDZ has a pH of about 5 or 5.5 to about 6 or 6.5 (optionally a pH of about 5.8) and can include WPM basal medium in an amount of about 1, 1.5, or 2 g / L to about 2.5, 3, 3.5, or 4 g / L medium; glucose in an amount of about 10, 11, 12, 13, 14, 14, 16, 17, 18, or 19 g / L to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L medium; a sugar (e.g., Phytagar) in an amount of about 4, 5, or 6 g / L to about 7, 8, 9, or 10 g / L medium. TM ); and TDZ in an amount from about 5, 6, 7, 8, 9 or 10 mg / L to about 11, 12, 13, 14 or 15 mg / L culture medium.

[0071] In some embodiments, the culture medium used in the method of the present invention (for example, a culture medium comprising TDZ) includes antibiotics such as but not limited to cefotaxime.Antibiotics (for example, cefotaxime, see, for example, Liang et al., Plants 2019, 8, 66) can be present in a culture medium in an amount effective to reduce, slow down or prevent the growth of endophytes (for example, bacterial endophytes) compared to the growth of endophytes in the absence of antibiotics.In some embodiments, antibiotics can be used to control and / or reduce endophyte contamination in culture medium and / or plant materials (for example, injured plant tissue).In some embodiments, antibiotics (for example, cefotaxime) can be present in a culture medium in an amount of about 100, 150 or 200 mg / L to about 250, 300, 350 or 400 mg / L culture medium.The method of the present invention may include exposing injured tissue to antibiotics (for example, cefotaxime) and / or culturing injured tissue on a culture medium comprising antibiotics (for example, cefotaxime).

[0072] An exemplary culture medium of the invention can include TDZ in an amount of at least about 5 mg / L culture medium (e.g., about 5, 6, 7, 8, 9, or 10 mg / L to about 11, 12, 13, 14, or 15 mg / L culture medium); a sugar (e.g., sucrose) in an amount of about 10, 11, 12, 13, 14, 14, 16, 17, 18, or 19 g / L to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L culture medium; and optionally an antibiotic (e.g., cefotaxime) in an amount of about 100, 150, or 200 mg / L to about 250, 300, 350, or 400 mg / L culture medium.

[0073] In some embodiments, the culturing step (e.g., culturing injured stem tissue) includes culturing plant tissue (optionally injured tissue) at a temperature of about 20°C, 21°C, 22°C, 23°C or 24°C to about 25°C, 26°C, 27°C, 28°C, 29°C or 30°C. In some embodiments, the culturing step is carried out at room temperature or at about 25°C. The culturing step can be carried out in the dark or under illumination conditions. In some embodiments, the culturing step is carried out in the dark. In some embodiments, the culturing step is carried out under illumination conditions, such as about 30 μM, 35 μM or 40 μM to about 45 μM or 50 μM light (e.g., about 40 μM light) for a photoperiod of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 hours. The culture step can be carried out for about 1,2,3,4,5,6,7 or 8 days or weeks to about 9,10,11,12,13,14,15,16,17 or 18 days or weeks.In some embodiments, the culture step is carried out for about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18 days or about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18 weeks.In some embodiments, the culture step can provide and / or produce organogenesis callus, optionally wherein said organogenesis callus develops into after cultivating injured stem tissue.In some embodiments, organogenesis callus develops into somatic embryo.

[0074] The method of the present invention may include providing and / or forming a plurality of organogenic calli from one or more (e.g., 1, 2, 3, 4, 5, 10, 50, 100 or more) injured tissues. In some embodiments, the injured tissue may produce one or more (e.g., 1, 2, 3, 4, 5, 10, 50, 100 or more) organogenic calli and / or the method of the present invention may include providing one or more (e.g., 1, 2, 3, 4, 5, 10, 50, 100 or more) organogenic calli. The method of the present invention has a success rate of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% in forming at least one organogenic callus from injured tissue. In some embodiments, organogenic callus is formed on at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the injured tissue used in the methods of the present invention. For example, when there are ten injured tissues, the formation of at least one organogenic callus on eight injured tissues means that organogenic callus is formed on about 80% of the total amount of injured tissue.

[0075] In some embodiments, method of the present invention comprises producing organogenesis callus and using the organogenesis callus in the method for breeding, regeneration and / or conversion.In some embodiments, plant and / or plant part can grow and / or regenerate from organogenesis callus of the present invention.In some embodiments, method of the present invention comprises from the organogenesis callus propagation plant prepared according to the method of the present invention, and the method is included in the presence of light and cultivates the organogenesis callus on culture medium (for example, regeneration culture medium).Can cultivate organogenesis callus on bud regeneration culture medium and / or on root regeneration culture medium.In some embodiments, cultivate organogenesis callus on bud regeneration culture medium to form the tissue comprising bud and then cultivate the tissue comprising bud on root regeneration culture medium.

[0076] The regeneration medium can be a regeneration medium known in the art. In some embodiments, the regeneration medium is a bud regeneration medium containing plant hormones that can promote bud development, such as but not limited to cytokinins (e.g., kinetin and / or 6-benzylaminopurine) and / or auxin-type plant hormones (e.g., naphthaleneacetic acid). In some embodiments, the regeneration medium is a root regeneration medium containing plant hormones, such as but not limited to auxin-type plant hormones that can promote root development (e.g., indole-3-butyric acid).

[0077] The propagation medium can be a propagation medium known in the art. An exemplary propagation medium can have a pH of about 5 or 5.5 to about 6 or 6.5 (optionally a pH of about 5.8) and can include a WPM basal medium in an amount of about 1, 1.5 or 2 g / L to about 2.5, 3, 3.5, 4, 4.5 or 5 g / L of the medium; a nitrate (e.g., potassium nitrate and / or ammonium nitrate) in an amount of about 0.1, 0.2, 0.3 or 0.4 g / L to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5 or 2 g / L of the medium; a sugar (e.g., glucose) in an amount of about 10 or 15 g / L to about 20 or 25 g / L of the medium; an agar (e.g., Phytagar) in an amount of about 4, 5 or 6 g / L to about 7, 8, 9 or 10 g / L of the medium. TM ); a cytokinin (e.g., 6-benzylaminopurine) in an amount of about 0.05, 0.1, 0.15, or 0.2 mg / L to about 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mg / L culture medium; and a plant hormone (e.g., gibberellic acid) in an amount of about 0.05, 0.1, 0.15, or 0.2 mg / L to about 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mg / L culture medium.

[0078] In some embodiments, the method of the present invention includes transforming organogenesis callus and / or conversion callus (e.g., organogenesis callus) and / or plant tissue prepared according to the method of the present invention. The method may include contacting bacterial cells (e.g., Agrobacterium) with organogenesis callus, callus and / or plant tissue, thereby transforming the organogenesis callus, callus and / or plant tissue with the bacterial cells to provide transformed organogenesis callus, transformed callus and / or transformed plant tissue. Exemplary bacterial cells include, but are not limited to, cells of Agrobacterium strains such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0079] The method of the present invention may include modifying the target nucleic acid to provide a modified nucleic acid. The method may include contacting an organogenic callus prepared according to the method of the present invention with a bacterial cell (e.g., Agrobacterium), wherein the organogenic callus comprises a target nucleic acid and wherein the bacterial cell comprises and / or encodes an editing system, and the editing system modifies the target nucleic acid, thereby providing a modified organogenic callus comprising a modified nucleic acid. In some embodiments, the method includes contacting a callus (e.g., an organogenic callus) and / or a plant tissue prepared from an organogenic callus prepared according to the method of the present invention with a bacterial cell (e.g., Agrobacterium), wherein the callus and / or plant tissue comprises a target nucleic acid and wherein the bacterial cell comprises and / or encodes an editing system, and the editing system modifies the target nucleic acid, thereby providing a modified callus comprising a modified nucleic acid and / or a modified plant tissue.

[0080] In some embodiments, the method of the present invention comprises propagating a plant from an organogenic callus prepared according to the method of the present invention, the method comprising culturing the organogenic callus on a culture medium (e.g., a regeneration medium) in the presence of light. The organogenic callus can be a transformed organogenic callus as described herein or a modified organogenic callus as described herein. In some embodiments, culturing the organogenic callus (optionally a transformed organogenic callus or a modified organogenic callus) comprises culturing the organogenic callus on a shoot regeneration medium to form a tissue comprising a bud and then culturing the tissue comprising a bud on a root regeneration medium.

[0081] Non-limiting examples of plants and / or plant parts and / or materials thereof that can be used in the present invention include turf grasses (e.g., bluegrass, bentgrass, ryegrass, fescue), feather reed grass, tuft grass, miscanthus, reeds, switchgrass, vegetables (including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., escarole, leaf lettuce, romaine lettuce), malanga), melons (e.g., cantaloupe, watermelon, Crenshaw melon, honeydew melon, cantaloupe), cole crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, collard greens, Chinese cabbage, pakchoy), spiny thistle, carrots, napa, okra, onions, celery, parsley, chickpeas, parsnips , chicory, peppers, potatoes, cucurbits (e.g., gourds, cucumbers, summer squash, squash, pumpkins, honeydew melons, watermelons, cantaloupes), radishes, bulb onions, rutabagas, eggplants, salsify, kohlrabi, shallots, endive, garlic, spinach, shallots, squash, leafy greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruit crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, figs, nuts (e.g., chestnuts, pecans, pistachios, hazelnuts, pistachios, peanuts, walnuts, macadamia nuts, almonds, etc.), citrus (e.g., clementines, kumquats, , oranges, grapefruits, tangerines, mandarins, lemons, limes, etc.), blueberries, black raspberries, boysenberries, cranberries, currants, gooseberries, loganberries, raspberries, strawberries, blackberries, grapes (wine and table), avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees, field crops such as clover, alfalfa, timothy, evening primrose, meadow foam, corn (non-sweet, sweet, popcorn), hops, jojoba, buckwheat, safflower, quinoa, wheat, rice, barley, rye, millet, sorghum, oats, triticale, sorghum, tobacco, kapok, legumes (legumes (e.g., green beans and dried Beans), lentils, peas, soybeans), oil plants (rapeseed, canola, mustard, olives, sunflowers, coconuts, castor beans, cocoa beans, peanuts, oil palm), duckweed, Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor) or plants such as coffee, sugar cane, tea and natural rubber plants; and / or bedding plants such as flowering plants, cacti, succulents and / or ornamental plants (e.g., roses, tulips, violets), and trees such as forest trees (broadleaf trees and evergreen trees, such as conifers; for example, elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, eucalyptus, willow), and shrubs and other nursery stock. In some embodiments, the plant is a tree or a plant part is from a tree, optionally a stone fruit tree (e.g., cherry, peach, plum, apricot, nectarine or almond). In some embodiments, the plant is a tree or shrub of the genus Prunus or a plant part is from a tree or shrub of the genus Prunus, optionally a tree of the genus Prunus.In some embodiments, the plant is Prunus elaeagnus (sweet cherry) or a plant part is from Prunus elaeagnus (sweet cherry).

[0082] In some embodiments, method of the present invention provides a kind of organogenesis callus identical with original seed, optionally wherein said organogenesis callus identical with original seed comprises modified nucleic acid.When preparing the organogenesis callus identical with original seed, can use the plant material that does not comprise cotyledon and / or the organogenesis callus identical with original seed not prepared by cotyledon.In some embodiments, the plant material that is used to prepare the organogenesis callus identical with original seed is the mixture of plant tissue type, and the mixture of described plant tissue type can be generated by collecting the young stem tissue containing node and / or processing (for example, cutting, blending etc.) stem tissue to produce fragment.Meristem can be included in the mixture of plant tissue type, and in some embodiments, do not make efforts to separate meristem from remaining plant material.

[0083] The method of the present invention may include producing organogenic callus and generating callus (e.g., organogenic callus) from the organogenic callus. In some embodiments, callus can be cultivated to regenerate plants, optionally wherein the plant is identical to the original species compared to the plant from which the organogenic callus is produced. In some embodiments, the plant comprises a modified nucleic acid compared to the plant from which the organogenic callus is produced. The method of the present invention can overcome the problem of low organogenesis efficiency and / or variant organogenic callus. As used herein, "variant" organogenic callus refers to an organogenic callus that can be used to produce (e.g., regenerate) a plant that is not identical to the original species relative to the plant from which the organogenic callus is produced. Variant plants are plants that no longer exhibit the unique characteristics and / or phenotype of the plant from which they are derived. For example, variant plants do not exhibit the unique characteristics and / or phenotype of the parent plant of the variant plant. In some embodiments, the organogenic callus can be derived from stem tissue (optionally, meristem) and can produce organogenic callus identical to the parent species, optionally for outcrossing species such as sweet cherry.

[0084] In some embodiments, the methods of the present invention can produce organogenic callus that: can be formed on at least about 90% of the wounded plant material (e.g., wounded explants) used in the method, can be propagated to produce a stock culture for subsequent use, is regenerable because it is derived from tissue (e.g., explants) taken from a donor plant and is identical to the original stock, and / or is less ergonomically stressful to humans to produce and / or maintain than other systems (e.g., embryogenic systems).

[0085] The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims of the present invention, but are intended to be examples of certain embodiments. Any changes to the exemplary methods that occur to the skilled person are intended to fall within the scope of the present invention.

[0086] Example

[0087] Embodiment 1:

[0088] Cherry variety Sweetheart was planted in a greenhouse and stem tissue was collected. The stem tissue was grown in vitro on a propagation medium (Table 1) at 25°C, 40 μM light (photoperiod of 16 hours) until lateral buds began to appear. Stem tissue with lateral buds was grown on the propagation medium for no more than 10 days and collected before the 10th day.

[0089] Table 1: Propagation medium.

[0090]

[0091]

[0092] Leaves and petioles were removed from the lateral buds, and cross-sections of the stems (including nodes) of the lateral buds were cut. The cross-sections of the stems were about 0.5 mm wide. In order to induce the formation of callus, the cut material was then placed on Garin medium, medium containing 2 mg TDZ, medium containing 5 mg TDZ, or medium containing 10 mg TDZ as described in Garin, E. et al. (1997). Plant cell, tissue and organ culture, 48 (2), 83-91 and cultured in the dark at 24 ° C for 8 weeks. The medium containing TDZ is provided in Table 2. The plant material was moved to fresh medium approximately every three weeks. If the growth of endophytes was noted during the experiment, the medium was supplemented with 200 mg / L of cefotaxime. About 149 nodes were treated and cultured on a medium containing 5 mg TDZ. About 149 nodes were treated and cultured on a medium containing 10 mg TDZ. About 324 nodes were treated and cultured on Garin medium. The lateral buds were cultured on the above medium for a total of 8 weeks and then observed for callus formation. The callus formation results for each medium are provided in Table 3. The medium containing 10 mg / L TDZ resulted in much more organized callus and also developed larger callus clumps.

[0093] Table 2: Culture medium containing TDZ

[0094] WPM basal medium (Phytytech L449) 2.41mg glucose 16g Make up to 1 liter 1000 Adjust pH to: 5.8 Phytagar 7g Autoclave 17 minutes, 121C Additives after autoclaving TDZ 2, 5, or 10 mg

[0095] Table 3: Callus formation results for the media tested.

[0096]

[0097]

[0098] Example 2: Regeneration of shoots and roots

[0099] First, the organogenic callus of embodiment 1 grown on 10mg TDZ medium was cultivated on the bud regeneration medium containing kinetin, 6-benzylaminopurine and naphthaleneacetic acid to start the process of plant generation from callus. Also use the Garin medium as described in embodiment 1 to regenerate bud as medium.After about 7 weeks, the bud of regeneration was subcultured on the propagation medium containing WPM basic medium with potassium nitrate, ammonium nitrate, glucose, gibberellic acid A3 and 6-benzylaminopurine for about 16 weeks to increase the amount of bud tissue.Notice that the bud of at least 1cm in length is more robust and has a greater chance of survival on the propagation medium than the smaller bud.

[0100] After approximately 16 weeks, the shoots were transferred to a rooting medium containing ethylenediamine-N,N'-bis((2-hydroxyphenyl)acetic acid) and indole-3-butyric acid potassium salt, where the shoots continued to grow and form roots, thereby providing plantlets. It was noted that shoots that were at least 1 inch in length showed a greater chance of surviving and developing roots on the rooting medium. Approximately 76% of the shoots transplanted into the rooting medium formed roots.

[0101] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. A method for producing organogenic callus, the method comprising: wounding a plant or plant part comprising stem tissue including a node to provide wounded tissue (e.g., cutting tissue); as well as The wounded tissue is cultured on a medium comprising thidiazuron (TDZ) at a concentration of at least about 5 mg / L of medium, thereby generating the organogenic callus.

2. The method of claim 1, wherein the stem tissue comprises meristem, optionally wherein the wounded tissue comprises wounded meristem.

3. according to the method for claim 1 or 2, wherein said stem tissue (for example, meristem) is present in a mixture with other plant tissues, wounding comprises wounding said plant tissue present in said mixture, and said cultivating comprises cultivating said wounded tissue from said mixture, optionally wherein said mixture comprises wounded meristem and different wounded plant tissue.

4. The method according to any preceding claim, wherein the culture medium comprises TDZ in an amount of about 5 mg / L to about 15 mg / L of the culture medium.

5. The method of any preceding claim, wherein the culture medium further comprises a sugar (e.g., sucrose), optionally wherein the sugar is present in the culture medium in an amount of about 10 g / L to about 30 g / L of the culture medium.

6. The method of any preceding claim, wherein culturing the wounded tissue comprises culturing the wounded tissue in the dark at a temperature of about 20°C to about 30°C (e.g., optionally at about 25°C), optionally wherein the organogenic callus develops from the wounded tissue.

7. The method of any preceding claim, wherein culturing the injured tissue comprises culturing the injured tissue on the culture medium for about 4 weeks to about 15 weeks.

8. A method according to any preceding claim, wherein the plant or plant part is a plant of or derived from an outcrossing species.

9. A method according to any preceding claim, wherein the organogenic callus is not cotyledon-derived organogenic callus (ie, the organogenic callus is not prepared from cotyledons) and / or the wounded tissue is free of cotyledons.

10. A method according to any preceding claim, wherein the organogenic callus is identical to the stock relative to the plant or plant part from which the wounded stem tissue was obtained.

11. The method of any preceding claim, further comprising forming a plurality of organogenic calli from a plurality of injured tissues.

12. The method of claim 11, wherein organogenic callus is formed on at least about 80%, 85%, 90% or 95% of the plurality of injured tissues.

13. The method according to any preceding claim, further comprising exposing the injured tissue to an antibiotic (eg, cefotaxime), optionally wherein the antibiotic (eg, cefotaxime) is present in the culture medium.

14. A method for propagating plants from organogenic callus, the method comprising: The organogenic callus according to any one of claims 1-13 is cultured on a culture medium (eg, a regeneration medium) in the presence of light.

15. The method of claim 14, wherein culturing the organogenic callus comprises culturing the organogenic callus on a shoot regeneration medium to form shoot-containing tissue and then culturing the shoot-containing tissue on a root regeneration medium.

16. A method for transforming organogenic callus, the method comprising: The organogenic callus according to any one of claims 1-13 is contacted with a bacterial cell (eg, Agrobacterium), thereby transforming the organogenic callus with the bacterial cell to provide a transformed organogenic callus.

17. A method for modifying a target nucleic acid, the method comprising: Contacting the organogenic callus according to any one of claims 1 to 13 with a bacterial cell (e.g., Agrobacterium), wherein the organogenic callus comprises the target nucleic acid and wherein the bacterial cell comprises and / or encodes an editing system, and the editing system modifies the target nucleic acid, thereby providing a modified organogenic callus.

18. A method for propagating plants from organogenic callus, the method comprising: The transformed organogenic callus according to claim 16 or the modified organogenic callus according to claim 17 is cultured on a culture medium (eg, a regeneration medium) in the presence of light.

19. The method of claim 18, wherein culturing the transformed organogenic callus or the modified organogenic callus comprises culturing the transformed organogenic callus or the modified organogenic callus on a shoot regeneration medium to form shoot-containing tissue and then culturing the shoot-containing tissue on a root regeneration medium.

20. The method according to any preceding claim, wherein the plant is or the plant part is from Cherry.

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