Methods of modifying plant tissue

Abstract

Described herein are methods for preparing a modified plant or plant part using a root-based transformation method. The method can include culturing a transformed plant tissue on a root induction medium comprising a selection agent to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells and a gene that confers resistance to the selection agent, and wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant, wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

Description

METHODS OF MODIFYING PLANT TISSUESTATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0001] A Sequence Listing in XML format, entitled 1499-146WO_ST26.xml, 4,042 bytes in size, generated on June 10, 2025, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.FIELD

[0002] The present invention relates to methods of modifying plant tissue using a rootbased transformation method that includes selection of transgenic and / or edited plant tissue.SUMMARY OF THE INVENTION

[0003] One aspect of the present invention is directed to a method of producing a modified plant or plant part, the method comprising: culturing a transformed plant tissue on a first root induction medium comprising a selection agent to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells and a gene that confers resistance to the selection agent, and wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant, wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

[0004] In another aspect, a method of producing a modified plant or plant part is provided that comprises: wounding a plant tissue to provide a wounded tissue comprising root forming competent cells, wherein the plant tissue comprises a shoot including meristem tissue and the wounded tissue comprises at least a portion of the meristem tissue; contacting the wounded tissue with a polynucleotide of interest and a gene that confers resistance to a selection agent to provide a transformed tissue that comprises the meristem tissue, the polynucleotide of interest, and the gene; culturing the transformed tissue on a first root induction medium comprising the selection agent to produce a composite plant, wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

[0005] A further aspect of the present invention is directed to a method of producing a modified plant or plant part, the method comprising: culturing a transformed plant tissue on a first root induction medium to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells, a rolA gene, a rolB gene, and a rolC gene, and wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant, wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

[0006] Another aspect of the present invention is directed to a method of producing a modified plant or plant part, the method comprising: wounding a plant tissue to provide a wounded tissue comprising root forming competent cells, wherein the plant tissue comprises a shoot including meristem tissue and the wounded tissue comprises at least a portion of the meristem tissue; contacting the wounded tissue with an Agrobacterium that comprises a rolA gene, a rolB gene, and a rolC gene to provide a transformed tissue that comprises the meristem tissue, the rolA gene, the rolB gene, and the rolC gene; culturing the transformed tissue on a first root induction medium to produce a composite plant, wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

[0007] These and other aspects of the present invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 is an exemplary schematic illustrating regeneration of a wild-type shoot; a transgenic, non-edited shoot; a transgenic, edited shoot; and a non-transgenic, edited shoot from a composite plant including a chimeric root according to some embodiments of the invention.

[0009] FIG. 2 is an exemplary schematic illustrating regeneration of transgenic shoots that may be edited from a composite plant including a transgenic root according to some embodiments of the invention.DETAILED DESCRIPTION

[0010] The present invention will now be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown and / or discussed. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0011] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented. In case of a conflict in terminology, the present specification is controlling.

[0012] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0013] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0014] Also 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 the lack of combinations when interpreted in the alternative (“or”).

[0015] The term “about” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant 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 the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0016] As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”

[0017] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0018] The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the 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.

[0019] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

[0020] As used herein, the terms “increase,” “increasing,” “enhance,” “enhancing,” “improve” and “improving” (and grammatical variations thereof) describe an elevation of 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 such as compared to another measurable property or quantity (e.g., a control value).

[0021] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of 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% such as compared to another measurable property or quantity (e.g., a control value). In some embodiments, the reduction can result in no or essentially no (z.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0022] The term “plant part,” as used herein, includes, but is not limited to, reproductive tissues (e.g, petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, and embryos); vegetative tissues (e.g, petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term “plant part” also includes plant cells, including plant cells that are intact in a plant, plant part, plant protoplast, plant tissue, plant organ, plant cell tissue culture, plant callus, plant clump, and / or the like.

[0023] As used herein, “shoot” refers to above ground (or above the media in the case of in-vitro) (i.e., aerial) growth of a plant including a sprout, seedling, and / or stem and optionally one or more appendages such as, a leaf, bud, flowering stem, and / or flower bud. In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week(s) post-germination (i.e., the shoot is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week old plant tissue). In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week(s) post-introduction into culture (i.e., the shoot is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week old plant culture tissue). In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 week(s) post-germination and / or post-introduction into culture. In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 month(s) post-germination and / or postintroduction into culture. In some embodiments, a shoot comprises a meristem cell and / or a root forming competent cell. In some embodiments, a shoot comprises a shoot meristem, a shoot apical meristem, and / or lateral meristem tissue. A shoot includes the mainstem of a plant and may have a branch and optionally a secondary branch. A branch isolated away from therest of the plant is not considered a shoot as defined herein.

[0024] As used herein, the term “tissue culture” encompasses a culture of tissue (e.g., plant tissue), cells, protoplasts, and / or callus.

[0025] As used herein, “plant cell” refers to a cell of a plant and includes a plant cell with a cell wall and a plant cell without a cell wall such as a protoplast. In some embodiments, a plant cell is in the form of an isolated single cell or can be a cultured cell. In some embodiments, a plant cell is a part of a higher-organized unit such as, for example, a plant tissue or a plant organ. A “protoplast” is a plant cell without a cell wall or with only parts of the cell wall. Thus, in some embodiments of the invention, a transgenic cell comprising a nucleic acid molecule and / or nucleotide sequence of the invention is a cell of any plant or plant part including, but not limited to, a root cell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, a fruit cell, a pollen cell, and the like. In some aspects of the invention, the plant part can be a plant germplasm. In some aspects, a plant cell can be non-propagating plant cell that does not regenerate into a plant.

[0026] “Plant tissue” as used herein means a group of plant cells organized into a structural and / or functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, explants, tissue culture and any group of plant cells assembled together to provide a structural and / or functional unit. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue. In some embodiments, a plant tissue comprises or is all or a portion of a shoot, a leaf, and / or a petiole.

[0027] An “explant” as used herein refers to a plant tissue that is used as starting material for a tissue culture. In some embodiments, an explant comprises a shoot, optionally a wounded shoot or at least a portion of a shoot. In some embodiments, an explant of the invention is a fragment of a shoot. In some embodiments, an explant comprises meristem tissue (e.g., apical meristem tissue and / or lateral meristem tissue). In some embodiments, an explant comprises shoot meristem cells. In some embodiments, an explant comprises nodal meristem cells and / or tissue and / or apical shoot meristem cells and / or tissue. In some embodiments, an explant is a wounded plant tissue (e.g., a wounded shoot) that comprises meristem tissue and root forming competent cells. Meristematic tissue can be readily identified as it includes actively dividing cells that are typically small and nearly spherical. In addition, cells in the meristem have a dense cytoplasm and relatively few small vacuoles.

[0028] “Meristem” refers to undifferentiated plant tissue that includes meristem cells, which are cells that are capable of cell division and can become specialized cells. “Meristematic tissue” as used herein refers to plant tissue from the meristem or the meristem that includes meristem cells. Meristem cells and meristematic tissue can differentiate to produce one or more different plant structures such as stem, roots, leaves, germline tissue and / or seeds. A meristem may be primary or secondary. Primary meristems include the above ground meristem, referred to as the “shoot apical meristem,” and the below ground meristem, referred to as the “root apical meristem.” Secondary meristems include intercalary meristems and lateral meristems. In some embodiments, meristematic tissue of the present invention is devoid of below ground meristematic tissue (e.g., consists only of aerial meristematic tissue).

[0029] As used herein, a “root forming competent cell” and grammatical variations thereof is any cell that can form a root and / or is competent to form a root. In some embodiments, a root forming competent cell is a cell that is able to respond directly to an inducing stimulus (e.g., wounding and / or a hormone such as an auxin) with the direct formation of root primordia. In some embodiments, a root forming competent cell is a cell of a plant shoot (e.g., a cell of a leaf, slip, petiole, hypocotyl, and / or stem) that can form a root. In some embodiments, a root forming competent cell is a cell of a plant root (e.g., a root cell or root meristem cell). In some embodiments, a root forming competent cell is not a cell of a plant root (e.g., a root forming competent cell is in a plant tissue other than a root). A root forming competent cell may be preformed (e.g., latent primordia) or may form de novo (e.g., induced primordia). Thus, in some embodiments, a root forming competent cells may have the capacity to form a root with or without stimulation (e.g., wounding, root inducing hormones, and / or specific culture conditions). In some embodiments, a root forming competent cell may be triggered to form a root by wounding, cutting, flooding or excessive water supply, loss of primary root growth, absence of light, and / or root growth induction regulators.

[0030] As used herein, “modified” in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has undergone a physical (e.g., genetic) change compared to the plant, plant part, shoot, root, tissue, or cell, respectively, at a prior time point (e.g., prior to contact with a composition of the present invention and / or prior to a method of the present invention). In some embodiments, “modified” in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has been subjected to genome editing, genetic transformation (e.g., introduction of a transgene), or a combination thereof.Accordingly, in some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic cell and / or an edited cell (i.e., a target nucleic acid of the cells has been modified). In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic, non-edited cell and / or a transgenic, edited cell. In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a non-transgenic, edited cell.

[0031] An “edited cell,” “edited plant,” “edited plant part,” “edited root,” “edited tissue,” “edited plantlet,” and / or the like as used herein refer to a cell, plant, plant part, root, tissue, plantlet, and / or the like, respectively, that comprises a modified nucleic acid in that a target nucleic acid has been modified using an editing system as described herein to provide the modified nucleic acid. Thus, an “edited cell,” “edited plant,” “edited plant part,” “edited root,” “edited tissue,” “edited plantlet,” and / or the like comprise a nucleic acid that has been modified and / or changed compared to its unmodified or native sequence and / or structure (z.e., a modified nucleic acid). The term “non-edited” refers to a condition in which a nucleic acid in the genome of a host cell or tissue or organism of interest has not been modified using an editing system as described herein.

[0032] As used herein, "modifying" or "modification" in reference to a target nucleic acid includes editing (e.g., mutating), covalent modification, exchanging / substituting nucleic acids / nucleotide bases, deleting, cleaving, and / or nicking of a target nucleic acid to thereby provide a modified nucleic acid and / or altering transcriptional control of a target nucleic acid to thereby provide a modified nucleic acid. In some embodiments, a modification may include an insertion and / or deletion of any size and / or a single base change (SNP) of any type. In some embodiments, a modification comprises a SNP. In some embodiments, a modification comprises exchanging and / or substituting one or more (e.g., 1, 2, 3, 4, 5, or more) nucleotides. In some embodiments, an insertion or deletion may be about 1 base to about 30,000 bases or more in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11.500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500,17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000,22.500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500,28,000, 28,500, 29,000, 29,500, 30,000 bases in length or more, or any value or range therein). Thus, in some embodiments, an insertion or deletion may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 to about 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000 bases in length, or any range or value therein; about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 bases to about 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 bases or more in length, or any value or range therein; about 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 bases to about 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bases or more in length, or any value or range therein; or about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 bases to about 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500,4000, 4500, or 5000 bases or more in length, or any value or range therein. In some embodiments, an insertion or deletion may be about 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,8000, 8500, 9000, 9500, or 10,000 bases to about 10,500, 11,000, 11,500, 12,000, 12,500,13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500,24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000,29,500, or 30,000 bases or more in length, or any value or range therein.

[0033] The term “transgene” as used herein refers to at least one nucleic acid sequence that is taken from the genome of one organism or produced synthetically, and which is then introduced into a host cell (e.g., a plant cell) or organism or tissue of interest and which is subsequently integrated into the host’s genome by means of “stable” transformation or transfection approaches. In contrast, the term “transient” transformation or transfection or introduction refers to a way of introducing molecular tools including at least one nucleic acid (DNA, RNA, single-stranded or double-stranded or a mixture thereof) and / or at least one amino acid sequence, optionally comprising suitable chemical or biological agents, to achieve a transfer into at least one compartment of interest of a cell, including, but not restricted to, the cytoplasm, an organelle, including the nucleus, a mitochondrion, a vacuole, a chloroplast, or into a membrane, resulting in transcription and / or translation and / or association and / or activity of the at least one molecule introduced without achieving a stable integration or incorporation into the genome and thus without inheritance of the respective at least one molecule introduced into the genome of a cell. A “transgenic” as used herein refers to a condition in which a transgene is present in the genome of a cell, tissue, or organism of interest. Thus, a “transgenic cell” refers to a cell that includes a transgene. The term “transgene-free” or “non-transgenic” refers to a condition in which a transgene is not present or found in the genome of a host cell or tissue or organism of interest.

[0034] A “mosaic” or “genetic mosaic” plant or plant tissue refers to a plant, plant part (e.g., a shoot, root, or the like), or plant tissue that is composed of cells that are not genetically identical, i.e., the plant or plant tissue has at least two different populations of cells that each have a different genotype. In some embodiments, a mosaic plant or plant tissue may comprise wild-type cells, transgenic cells (e.g., transgenic, edited cells and / or transgenic, non-edited cells), and / or edited cells (e.g., non-transgenic, edited cells and / or transgenic, edited cells). In some embodiments, a mosaic plant or plant tissue may comprise wild-type cells and transgenic cells (e.g., transgenic, edited cells and / or transgenic, non-edited cells). In some embodiments,a mosaic plant or plant tissue may comprise wild-type cells and edited cells (e.g., non- transgenic, edited cells and / or transgenic, edited cells). In some embodiments, a mosaic plant or plant tissue may be chimeric in that a portion of the cells of the plant or plant tissue include a transgene (i.e., are transgenic cells) and a portion of the cells of the plant or tissue are transgene-free (e.g., wild-type cells and / or non-transgenic, edited cells). For example, a chimeric root may comprise wild-type cells and transgenic cells. In some embodiments, a mosaic plant or plant tissue may comprise at least two different populations of edited cells, wherein the first population of edited cells comprises a first modified nucleic acid and the second population of edited cells comprises a second modified nucleic acid, wherein the first and second modified nucleic acids are different (e.g., a different modification was made), optionally wherein the at least two different populations of edited cells are non-transgenic.

[0035] In some embodiments, a plant or plant tissue is non-mosaic. “Non-mosaic” as used herein refers to a plant, plant part (e.g., a shoot, root, or the like), or plant tissue that is composed of cells that are genetically identical. In some embodiments, a plant tissue (e.g., a shoot) or plant comprises non-mosaic, edited cells, wherein the non-mosaic, edited cells each comprise a nucleic acid that has been modified and / or changed in the same manner compared to its unmodified or native sequence and / or structure (i.e., a modified nucleic acid) such that the non- mosaic, edited cells have the same genotype.

[0036] A “composite plant” as used herein refers to a plant that comprises a shoot and a root, wherein a portion (e.g., all or a portion of a root) is modified (e.g., transgenic and / or edited) and another portion (e.g., a shoot) is not modified (e.g., wild-type) such as modified as a result of a composition and / or method of the present invention. In some embodiments, a composite plant includes a modified root (e.g., the root comprises transgenic and / or edited cells) and one or more other portion(s) (e.g., the aerial portions) of the plant are not modified (e.g., the aerial portions of the plant comprise cells that are not transgenic and / or edited optionally as a result of a composition and / or method of the present invention). In some embodiments, a composite plant includes a modified root (e.g., the root comprises transgenic and / or edited cells) and substantially all of the cells (e.g., greater than 90% of the total number of cells) in the aerial parts of the plant are not modified. In some embodiments, a composite plant includes a modified root (e.g., the root comprises transgenic and / or edited cells) and substantially all of the cells (e.g., greater than 90% of the total number of cells) in a shoot of the composite plant are wild-type cells. In some embodiments, a composite plant includes a modified root (e.g., the root comprises transgenic and / or edited cells) and substantially all of the cells (e.g., greater than 90% of the total number of cells) in a shoot of the composite plantare native cells. In some embodiments, a composite plant includes a modified root (e.g., the root comprises transgenic and / or edited cells) and wild-type (non-modified) aerial tissue (e.g., a wild-type shoot).

[0037] A “native” plant or plant part refers to a naturally occurring plant or plant part or a plant or plant part prior to a method of the present invention. In some contexts, a “native plant” plant or plant part is a plant or plant part that is non-transgenic or transgene-free and does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. A “native plant” plant or plant part is a plant or plant part that does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. A “native cell” as used herein refers to a naturally occurring plant cell or to a plant cell prior to a method of the present invention. A native cell does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. In some embodiments, a native plant cell is non-transgenic.

[0038] As used herein, a “wild-type” plant or plant part (e.g., a wild-type shoot, wild-type root, or wild-type cell) refers to a plant or plant part that comprises native cells in an amount greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the plant or plant part are native cells). A “wild-type shoot” as used herein refers to a shoot that comprises native cells in an amount greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the plant or plant part are native cells). In some embodiments, all cells of a wild-type shoot are native cells. Thus, in some embodiments, a wild-type shoot may comprise less than 10% transgenic and / or edited cells. A “wild-type cell” as used herein refers to a cell that does not include a transgene that is introduced by a method of the present invention and has not been modified (e.g., a nucleic acid in the cell is not edited) via a method of the present invention. A wild-type cell is genetically identical to a native cell and may be present in a wild-type shoot and / or a transgenic root.

[0039] A "native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, amino acid sequence, or bacteria refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, amino acid sequence, or bacteria. Thus, for example, a "native nucleic acid" is a nucleic acid that is naturally occurring in or endogenous to a reference organism. A "homologous" nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.

[0040] As used herein, a “suckering plant” refers to a plant that is capable of producing (or produces) a sucker, which is a new growth (e.g., a shoot) from an adventitious bud of a stem or root of the plant. In some embodiments, a suckering plant is capable of producing (orproduces) an adventitious shoot from a root. In some embodiments, a suckering plant is capable of producing (or produces) a shoot from an internodal portion of a plant tissue (e.g., an explant). A plant that produces an adventitious shoot may also be referred to as surculose. Suckering plants include plants that naturally are capable of producing (or produce) a sucker and plants that can be induced to sucker (i.e., produce or grow a sucker) by growing the plant or plant part in-vitro under suitable conditions.

[0041] As used herein, “contact,” “contacting,” “contacted,” and grammatical variations thereof, refer to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking, and / or cleavage).

[0042] The term “transformation” or “transformed” as used herein refers to the introduction of a nucleic acid, polypeptide, and / or ribonucleoprotein (e.g., a heterologous nucleic acid, polypeptide, and / or ribonucleoprotein) into a cell (e.g., into at least one cell of a plant tissue). Transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell, host tissue, or host organism may be stably transformed with a polynucleotide / nucleic acid molecule of the invention. In some embodiments, a host cell, host tissue, or host organism may be transiently transformed with a nucleic acid construct, a polypeptide, and / or a ribonucleoprotein as described herein. A “transformed plant tissue” as used herein refers to a plant tissue that comprises a cell that has been transformed (i.e., the cell comprises a nucleic acid, polypeptide, and / or ribonucleoprotein that has been introduced into the cell). In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is transgenic and not edited. In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is non-transgenic and edited. In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is transgenic and edited. In some embodiments, a transformed plant tissue comprises or is all or a portion of a shoot, a leaf, and / or a petiole that comprises a cell that has been transformed.

[0043] Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into a cell (e.g., by a transformation and / or transfection approach) and does not integrate into the genome of the cell; thus, the cell is transiently transformed with the polynucleotide. A nucleic acid that is “transiently expressed” as used herein refers to a nucleic acid that has been introduced into a cell and the nucleic acid is not integrated into the genome of the cell, thereby the cell is transiently transformed with the nucleic acid.

[0044] By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell (e.g., by a transformation and / or transfection approach) is intended thatthe introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. A nucleic acid that is “stably expressed” as used herein refers to a nucleic acid that has been introduced into a cell and the nucleic acid is integrated into the genome of the cell, thereby the cell is stably transformed with the nucleic acid.

[0045] Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell (e.g., by a transformation and / or transfection approach) and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast or mitochondrial genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome or a plasmid.

[0046] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgenes introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a mammal, plant, etc.). Stable transformation of a cell can be detected by, for example, a northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a host organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, 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.

[0047] “Introducing,” “introduce,” “introduced” (and grammatical variations and derivatives thereof) in the context of a polynucleotide of interest means presenting a polynucleotide of interest (e.g., polynucleotide, a nucleic acid construct, and / or an editing system (e.g., a polynucleotide, polypeptide, and / or ribonucleoprotein)) to a host organism or cell of said organism (e.g., host cell; e.g., a plant cell) in such a manner that the polynucleotide gains access to the interior of a cell. Thus, for example, a polynucleotide from an Agrobacterium strain, such as A. tumefaciens or A. rhizogenes (that may encode or compriseat least a portion of an editing system), may 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 that encodes and / or comprises a CRISPR-Cas effector protein, a guide nucleic acid, reverse transcriptase, and / or a deaminase (e.g., a cytosine deaminase and / or adenine deaminase) may be introduced into a cell of an organism, thereby transforming the cell with the CRISPR-Cas effector protein, guide nucleic acid, reverse transcriptase, and / or deaminase. In some embodiments, a CRISPR-Cas effector protein and a guide nucleic acid may be introduced into a cell of a plant, optionally wherein the CRISPR-Cas effector protein and guide nucleic acid may be comprised in a complex (e.g., a ribonucleoprotein).

[0048] A polynucleotide may be introduced into a plant cell by any method known to those of skill in the art. In some embodiments, transformation methods include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide and / or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, particle bombardment (e.g., microparticle bombardment), calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and / or biological mechanism that results in the introduction of a nucleic acid into the plant cell, including any combination thereof. In some embodiments of the invention, transformation of a cell comprises nuclear transformation. In some embodiments, transformation of a cell comprises plastid transformation (e.g., chloroplast transformation). In some embodiments, a recombinant nucleic acid construct of the invention can be introduced into a cell via conventional breeding techniques. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via Agrobacterium transformation. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via biolistic transformation. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via particle bombardment.

[0049] Procedures for transforming both eukaryotic and prokaryotic organisms are well known and routine in the art and are described throughout the literature (See, for example, 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 MolecularBiology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0050] A polynucleotide and / or polypeptide can be introduced into a host organism or its cell (e.g., a plant, plant part, and / or plant cell) in any number of ways that are well known in the art such as, but not limited to, by particle bombardment, transfection, a non-viral chemical method, and / or viral mediated transformation. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into the organism (e.g., a plant), only that they gain access to the interior of at least one cell of the organism. When more than one polynucleotide is to be introduced, it can be assembled as part of a single nucleic acid construct, as separate nucleic acid constructs, can be located on the same or different nucleic acid constructs, and / or as a complex (e.g., a ribonucleoprotein). A polynucleotide and / or polypeptide can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, a polynucleotide and / or polypeptide can be incorporated into a plant as part of a breeding protocol.

[0051] “Root induction medium” refers to a medium that induces and / or promotes the development and / or growth of a root. In some embodiments, a root induction medium comprises a basal medium and is devoid of a growth regulator. In some embodiments, a root induction medium comprises a basal medium at a concentration that is about half to about a quarter of the recommended medium strength (e.g., concentration) (e.g., about 25% or about 50% of the recommended concentration) and optionally a basal salt and / or vitamin. In some embodiments, a root induction medium comprises activated charcoal, a carbohydrate, a gelling agent, a growth regulator, a cytokinin, and / or an auxin. In some embodiments, a root induction medium comprises a high auxin to cytokinin ratio, e.g., an auxin to cytokinin ratio of greater than 1. In some embodiments, a root induction medium comprises a growth regulator that comprises or consists of an auxin (e.g., a synthetic or endogenous auxin) and / or is devoid of a cytokinin. In some embodiments, a root induction medium comprises indole-3 -acetic acid (IAA), indole-3 -butyric acid (IB A), 1 -naphthaleneacetic acid (NAA), 6-isopentenyladenine (IPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), and / or picloram.“Shoot induction medium” refers to a medium that induces and / or promotes the development and / or growth of a shoot. In some embodiments, a shoot induction medium comprises a basal medium. In some embodiments, a shoot induction medium comprises a medium comprising a carbohydrate, a gelling agent, a growth regulator, a cytokinin, an auxin, an amino acid, casein hydrolysate, an ethylene inhibitor (e.g., silver nitrateand / or polyvinylpyrrolidone (PVP)), and / or a pH stabilizer (e.g., 2-(N- morpholino)ethanesulfonic acid (MES). In some embodiments, a shoot induction medium comprises a high cytokinin to auxin ratio, e.g., a cytokinin to auxin ratio of greater than 1. In some embodiments, a shoot induction medium comprises a growth regulator that comprises or consists of a cytokinin (e.g., a synthetic or endogenous cytokinin) and / or is devoid of an auxin. In some embodiments, a shoot induction medium comprises 6-benzylaminopurine (BAP), zeatin, zeatin riboside, 6-(y,y-dimethylallylamino) purine (2iP), kinetin, adenine, adenine hemisulfate, and / or meta-topolin. In some embodiments, a shoot induction medium comprises thidiazuron (TDZ) and / or forchlorfenuron (4-CPPU) optionally in a concentration that may cause a cytokinin-like effect due to their inhibition of cytokinin oxidase that optionally results in accumulation of endogenous cytokinin.

[0052] A “selection agent” refers to a substance (e.g., a chemical compound) whose exposure to and / or contact with favors or disfavors growth of plant cells and / or mortality of a plant cell and / or tissue. In some embodiments, a selection agent is a substance that, upon exposure to and / or contact with cells that are devoid of a gene that confers resistance to the selection agent, terminates, decreases, or suppresses the growth of all or a portion of the cells that are devoid of a gene that confers resistance to the selection agent, whereas growth is not decreased or suppressed for cells that comprise a gene that confers resistance to the selection agent upon exposure and / or contact with the substance.

[0053] According to some embodiments, provided are methods for producing a modified plant or plant part comprising culturing a transformed plant tissue on a root induction medium comprising a selection agent to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells and a gene that confers resistance to the selection agent, and wherein the composite plant comprises a wild-type shoot and a root (e.g., a modified root), wherein the root comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant (e.g., a modified shoot growing from the modified root), wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part. In some embodiments, the modified shoot comprises transgenic and / or edited cells. In some embodiments, the modified shoot comprises non-transgenic, edited cells, optionally wherein the modified shoot is non-mosaic (e.g., the edited cells are genetically identical) or the modified shoot is mosaic and comprises at least two different populations of edited cells.

[0054] Further provided are methods for producing a modified plant or plant part comprising culturing a transformed plant tissue on a root induction medium that optionally comprises a selection agent to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells, a rolA gene, a rolB gene, and a rolC gene, and wherein the composite plant comprises a wild-type shoot and a root (e.g., a modified root), wherein the root comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant (e.g., a modified shoot growing from the modified root), wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part. In some embodiments the root induction medium is devoid of a selection agent. In some embodiments, the root induction medium comprises a selection agent and optionally the transformed plant tissue comprises a gene that confers resistance to the selection agent. In some embodiments, the modified shoot comprises transgenic and / or edited cells. In some embodiments, the modified shoot comprises non-transgenic, edited cells, optionally wherein the modified shoot is non-mosaic (e.g., the edited cells are genetically identical) or the modified shoot is mosaic and comprises at least two different populations of edited cells.

[0055] In some embodiments, a transformed plant tissue can be provided and / or prepared by wounding a plant tissue (e.g, a plant, plant part (e.g., a shoot), or explant) to provide a wounded plant tissue (e.g, a cut tissue), and contacting the wounded plant tissue with a polynucleotide of interest to provide the transformed plant tissue. In some embodiments, the polynucleotide of interest comprises at least one gene that confers resistance to a selection agent and / or comprises and / or encodes all or a portion (e.g., at least one component) of an editing system. In some embodiments, a transformed plant tissue can be provided and / or prepared by wounding a plant tissue (e.g., a plant, plant part (e.g., a shoot), or explant) to provide a wounded plant tissue (e.g., a cut tissue), and contacting the wounded plant tissue with a rolA gene, a rolB gene, and a rolC gene and / or at least one gene that confers resistance to a selection agent and / or a polynucleotide that comprises and / or encodes all or a portion (e.g., at least one component) of an editing system. In some embodiments, a wounded plant tissue is not contacted with a gene that confers resistance to a selection agent and / or a transformed plant tissue is devoid of a gene that confers resistance to a selection agent. In some embodiments, a wounded plant tissue is contacted with a rolA gene, a rolB gene, and a rolC gene and / or a polynucleotide that comprises and / or encodes all or a portion (e.g., at least one component) of an editing system and is not contacted with a gene that confers resistance to a selection agent and / or a transformed plant tissue comprises a rolA gene, a rolB gene, and a rolC gene and / or apolynucleotide that comprises and / or encodes all or a portion (e.g., at least one component) of an editing system and is devoid of a gene that confers resistance to a selection agent. In some embodiments, the wounded plant tissue comprises meristem cells and / or root forming competent cells. In some embodiments, the wounded plant tissue comprises shoot meristem tissue (e.g., primary meristem tissue and / or secondary meristem tissue). In some embodiments, a transformed plant tissue can be prepared and / or provided by wounding a plant tissue comprising shoot meristem tissue and root forming competent cells to provide a wounded plant tissue, and contacting the wounded plant tissue with at least one polynucleotide of interest (e.g., a gene that confers resistance to a selection agent, a rolA gene, a rolB gene, a rolC gene, and / or a polynucleotide that comprises and / or encodes all or a portion (e.g., at least one component) of an editing system). In some embodiments, a transformed plant tissue can be prepared by wounding shoot tissue comprising shoot meristem cells and / or root forming competent cells, and contacting the wounded shoot tissue with at least one polynucleotide of interest (e.g., at least one gene that confers resistance to a selection agent), wherein the transformed plant tissue may comprise the shoot meristem cells and / or root forming competent cells and one or more of the cells of the transformed plant tissue comprise at least one polynucleotide of interest. In some embodiments, a transformed plant tissue can be prepared by wounding plant tissue that is devoid of a root and / or is devoid of a non-aerial plant part. In some embodiments, a wounded plant tissue is devoid of a root and / or is devoid of a non-aerial plant part. In some embodiments, a transformed plant tissue prior to culturing on a growth medium and / or a root induction medium is devoid of a root and / or is devoid of a non-aerial plant part.

[0056] In some embodiments, a method of the present invention includes culturing, propagating, regenerating, and / or transforming a wounded tissue. In some embodiments, prior to wounding a plant or plant part, the method comprises collecting a plant part. For example, in some embodiments, a shoot grown from a node tissue may be collected and then blended in a blender to provide a wounded plant tissue.

[0057] In some embodiments, a wounded plant tissue may be obtained by collecting one or more node(s) from a plant (e.g., a blackberry, raspberry, or cherry plant) optionally a greenhouse grown plant, optionally sterilizing the node(s), wounding (e.g., cutting) the node(s) by cutting (e.g., by hand) or by blending in a blender to provide wounded node tissue optionally having a smaller size than the size of the node(s) prior to wounding, and culturing the wounded node tissue optionally on solid media for about 1 week to about 6 weeks or more. During the culturing step, the wounded node tissue may produce a plant tissue comprising one or more shoot(s) (e.g., about 10 to about 20 shoots per wounded node).

[0058] In some embodiments, a method may comprise collecting an explant and / or wounding a plant tissue in a blender or by cutting (e.g., by hand manipulation) to provide a wounded tissue (e.g., fragmented or cut tissue). Methods of wounding a plant or plant part are known in the art. In some embodiments, the wounding comprises cutting the plant or plant part with a device such as a knife or scalpel, optionally by hand (e.g., human hand manipulation). In some embodiments, the wounding comprises blending the plant or plant part in a blender to thereby provide the wounded tissue.

[0059] A “blender” as used herein refers to a mechanical device that is configured to blend, chop, dice, and / or slice material such as food. In some embodiments, the blender is powered by an electric motor (z.e., an electric blender). Exemplary electric blenders include, but are not limited to, a kitchen blender that includes a rotating blade (e.g., a metal blade such as a stainless steel blade) powered by an electric motor and a food processor that includes a blade (e.g., a cutting blade such as a metal (e.g., stainless steel) cutting blade) that is powered by an electric motor. The electric motor may be a 350-watt motor. In some embodiments, a blender used in a method of the present invention is a Cuisinart blender having model number CPB-300P1 or a blender comparable thereto and the blender may be fitted with a chopping blade. A chopping blade may also be referred to as an S blade. Exemplary chopping blades and / or S blades include, but are not limited to, a blade included in Cuisinart chopping assembly having model number CPB-300CHA or a blade comparable thereto. The blade may have a planar configuration. In some embodiments, the blade assembly provided in a blender comprises a single elongate blade that rotates about its center such that it provides an edged “fin” for chopping and / or cutting on either side of its pivot axis. In some embodiments, a blade assembly provided in a blender comprises a blade with only a single edged “fin”; the blade rotates about a fixed axis and employs only the single “fin” for chopping and / or cutting. A blender of the present invention may be attached to a vessel operably associated with the blender such as Cuisinart cup having model number CTC-16. In some embodiments, the blender used in a method of the present invention is an electric blender (e.g., a kitchen blender and / or food processor) that includes and / or is fitted with a chopping blade and / or an S blade having metal (e.g., stainless steel) fins. One or more part(s) of a blender used in a method of the present invention may be able to be sterilized and / or configured for sterilization (e.g., alcohol sterilization and / or steam sterilization). In some embodiments, the parts of a blender that contact a plant and / or plant part may be sterilized and / or configured for sterilization. For example, a blender used in a method of the present invention may have a vessel (e.g., a cup), blade, and / or blade assembly that can be sterilized and / or that are configured for sterilization.In some embodiments, a step of wounding a plant or plant tissue in a blender is a step in a plant propagation, regeneration, and / or transformation method. Exemplary blenders, blades, systems, and / or methods of wounding a plant or plant tissue may be as described in WO 2023 / 133479, the contents of which are incorporated herein by reference in their entirety.

[0060] In some embodiments, a callus is removed from a plant tissue prior to wounding the plant tissue. In some embodiments, a callus is not removed from a plant tissue prior to wounding the plant tissue and the plant tissue including the callus are blended. In some embodiments, about 10, 15, 20, or 25 clumps to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 clumps of shoots (e.g., blackberry or cherry shoots) may be blended in a blender. In some embodiments, specific plant tissue may be selected and / or isolated such as when wounded tissue is being provided by hand processing. In some embodiments, a step of selection and / or isolation of specific plant tissue is not performed and a plant material is wounded (e.g., blended) such that it includes desired plant material and non-desired plant material to provided wounded plant tissue that includes non-desired plant material and optionally the wounded plant tissue including the non-desired plant material is cultured. For example, in a method that is targeting cut stem tissue, a method providing the cut stem tissue by hand processing would cut off any leaves (e.g., non-desired plant material), but in some embodiments of the present invention an entire stem with the leaves may be blended to provide a wounded tissue.

[0061] In embodiments comprising the use of a blender, the blender may be operated at a speed from 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 may be set to and / or operated at the lowest speed setting for the blender, optionally set to and / or operated at a chop setting. In some embodiments, blending comprises blending plant material (e.g., a plant or plant part) at a speed from about 1, 5, 10, 15, or 20 Hz to about 25, 30, 35, 40, 45, or 50 Hz. The blending may be carried out for a period of time such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds or more, optionally using a pulse setting. In some embodiments, blending the plant material comprises blending the plant or plant part at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) interval(s) with each interval including 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 thereby provide a rest from 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) interval(s) with each interval including 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 to provide the wounded tissue is carried out in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s).

[0062] A method herein and / or wounding step (e.g., a blending step) may 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 tissue (e.g., shoot tissue or explant) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method herein and / or wounding step may process about 35 or 40 to about 45 or 50 clumps of plant tissue (e.g., plant shoots such about 3 or 6 week old plant shoots) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method of herein and / or wounding step may process about 35, 40, 45, or 50 clumps of plant tissue (e.g., plant shoots such about 3 or 6 week old plant shoots) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method herein and / or wounding step may process about 5 grams to about 250 grams of plant tissue to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s).

[0063] 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 is 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 including a bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant shoots such about 3 or 6 week old plant shoots) is 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 including a bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant shoots such about 3 or 6 week old plant shoots) is blended with about 300 mL of liquid (e.g., water and / or an aqueous composition optionally including a bacteria). In some embodiments, about 5 grams to about 250 grams of plant material is 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 including a bacteria).

[0064] “Blending,” “blend,” and grammatical variants thereof as used herein refer to mechanically cutting (e.g., chopping, dicing, etc.) material (e.g., plant material). Blending may cut the material into smaller pieces than the size of the material prior to blending. In some embodiments, blending may include coarsely cutting and / or finely cutting material. In someembodiments, blending is configured to coarsely cut plant material and aims to limit blending the plant material to a smoothie-like consistency. In some embodiments, blending provides cut plant material (e.g., wounded plant tissue) that has a size of about 0.2 or 0.3 cm to about 0.4 or 0.5 cm as measured at the widest part of the plant material. In some embodiments, blending comprises blending a whole plant or one or more part(s) thereof (e.g., an aerial portion). In some embodiments, blending comprises blending a shoot. In some embodiments, the blending of plant material is carried out in the presence of water and / or an aqueous composition (e.g., a liquid growth media). The plant material and water and / or aqueous composition may be added into a vessel of a blender and blended together. In some embodiments, plant material and bacteria, optionally in water and / or an aqueous composition, are blended together.

[0065] In some embodiments, a wounded plant tissue may be contacted with a polynucleotide of interest to provide a transformed plant tissue. The wounded plant tissue and / or transformed plant tissue may comprise meristem cells (e.g., shoot meristem cells) and / or a shoot. In some embodiments, a wounded plant tissue may be contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the transformed plant tissue comprises meristem tissue and the polynucleotide of interest. In some embodiments, a wounded plant tissue may be contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the transformed plant tissue comprises meristem cells and one or more of the meristem cells comprise the polynucleotide of interest. In some embodiments, a wounded plant tissue may be contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the transformed plant tissue comprises shoot meristem cells and the polynucleotide of interest. In some embodiments, a wounded plant tissue is contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the polynucleotide of interest comprises a gene that confers resistance to a selection agent. In some embodiments, a wounded plant tissue is contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the polynucleotide of interest comprises at least one component of an editing system. In some embodiments, a wounded plant tissue is contacted with a polynucleotide of interest to provide a transformed plant tissue, wherein the polynucleotide of interest comprises a rolA gene, a rolB gene, and a rolC gene.

[0066] In some embodiments, a method of the present invention comprises a step of contacting and / or transforming a wounded plant tissue with a polynucleotide of interest (e.g., a gene that confers resistance to a selection agent and / or all or a portion of an editing system). The contacting step may comprise contacting the wounded plant tissue with an Agrobacterium that comprises the polynucleotide of interest and introducing the polynucleotide of interest intoa cell of the wounded plant tissue. Exemplary methods for contacting and / or transforming a plant tissue (e.g., a wounded plant tissue) with an Agrobacterium are known in the art and may be used in a method herein. In some embodiments, a wounded plant tissue is contacted with an Agrobacterium, optionally the wounded plant tissue may be contacted with a composition (e.g., a suspension and / or culture) comprising the Agrobacterium. In some embodiments, a wounded plant tissue is cultured and / or cultivated with the Agrobacterium and / or a composition comprising the Agrobacterium. For example, a plant tissue may be wounded with a scalpel or blender and contacted with and / or provided in an Agrobacterium cell suspension, optionally followed by sonication treatment (e.g., for about 1 minute to about 5 or 10 minutes). In some embodiments, a method of transforming a plant tissue comprises introducing a polynucleotide of interest (e.g, a gene conferring resistance to a selection agent, editing system, a rolA gene, a rolB gene, a rolC gene, and / or other transgene, e.g., a transgene encoding a disease resistance protein or therapeutic protein) from an Agrobacterium into a wounded plant tissue to provide a transformed plant tissue.

[0067] A bacteria used in a method of the present invention (e.g., in a contacting and / or transformation step) may be an Agrobacterium strain such as A. tumefaciens and / or A. rhizogenes. In some embodiments, the Agrobacterium is an A. tumefaciens strain. In some embodiments, the Agrobacterium is an A. rhizogenes strain. An Agrobacterium may comprise a polynucleotide of interest and the polynucleotide of interest may be transfer DNA (T-DNA) from the tumor-inducing (Ti) plasmid and / or hairy root-inducing (Ri) plasmid of the Agrobacterium strain. In some embodiments, the T-DNA from the Ti plasmid and / or the T- DNA from the Ri plasmid are integrated (optionally concurrently integrated) into the genome of a plant cell to provide a transgenic plant cell. In some embodiments, T-DNA from the Ti plasmid of an Agrobacterium strain is introduced into a plant cell and encodes or comprises a polynucleotide of interest (e.g., a polynucleotide encoding or comprising all or a portion of an editing system, a gene that confers resistance to a selection agent, and / or a reporter gene). In some embodiments, a plant tissue (e.g., a wounded plant tissue) and an Agrobacterium strain, optionally in water and / or an aqueous composition, are blended together. In some embodiments, a wounding step and contacting step are carried out simultaneously. A bacteria (e.g., an Agrobacterium strain) used in a method of the present invention may include a nucleic acid construct encoding all or a portion of an editing system (e.g., a CRISPR-Cas editing system) that is configured to modify a polynucleotide (e.g., a target nucleic acid) in a plant cell. In some embodiments, a bacteria (e.g., an Agrobacterium strain) may include a gene that confers resistance to a selection agent. In some embodiments, a bacteria (e.g., anAgrobacterium strain) includes a gene that confers resistance to a selection agent and a polynucleotide that encodes all or a portion of an editing system (e.g., a CRISPR-Cas editing system) that is configured to modify e.g., edit) a polynucleotide in a plant cell. In some embodiments, a bacteria (e.g., an Agrobacterium strain) includes a gene that confers resistance to a selection agent and a polynucleotide of interest encoding a transgene. In some embodiments, blending a plant tissue in the present of an Agrobacterium may inoculate the plant tissue with the Agrobacterium and / or introduce a polynucleotide from the Agrobacterium into a cell of the plant tissue, optionally into a cell of the wounded tissue.

[0068] In some embodiments, the Agrobacterium strain is a wild-type Agrobacterium strain (e.g., a wild-type Agrobacterium rhizogenes strain or a wild-type Agrobacterium tumefaciens strain). In some embodiments, the Agrobacterium strain may be devoid of and / or have knocked out a wild-type bacterial transfer protein (e.g., a wild-type Agrobacterium virulence protein). For example, an Agrobacterium strain may have VirD2 knocked out. In some embodiments, an Agrobacterium strain may be aAgrobacterium strain. A “disarmed Agrobacterium strain” as used herein refers to an Agrobacterium strain whose genome has been modified compared to its native sequence and / or structure so that introduction of a polypeptide and / or polynucleotide from the strain into a plant cell cannot cause and / or provide the hairy root and / or tumor phenotype in the plant cell including (e.g., expressing) the polypeptide and / or polynucleotide. The hairy root phenotype is characterized by short internodes, wrinkled leaves, abundant roots with extensive lateral branching, and / or increased production certain metabolites. In addition, a root exhibiting the hairy root phenotype can be visibly identified by extensive lateral branching in a root, rapid root growth, root growth that appears fuzzy, and / or being thicker than a standard root.

[0069] In some embodiments, a disarmed Agrobacterium (e.g., A. rhizogenes) strain is provided by modifying (e.g., deleting) the T-DNA that contains one or more rol gene(s) in a manner such that the hairy root phenotype is not provided when the strain infects a plant. In some embodiments, a disarmed Agrobacterium (e.g., A. rhizogenes) strain is provided by modifying (e.g., deleting) at least a portion (or all) of the T-DNA that contains the rol genes so that the strain lacks the T-DNA and / or a lacks a functional rol gene. In some embodiments, a disarmed Agrobacterium (e.g., A. rhizogenes) strain is provided by knocking out (e.g., deleting) the TL-DNA and TR-DNA and / or by deleting an aux gene. In some embodiments, a disarmed Agrobacterium (e.g., A. rhizogenes) strain is devoid of T-DNA in its hairy root-inducing (Ri) plasmid and the Ri plasmid comprises trans factors (e.g., vir genes). In some embodiments, both T-DNAs in the Ri plasmid pRIA4 have been removed in a disarmed Agrobacterium (e.g.,A. rhizogenes) strain. In some embodiments, a disarmed Agrobacterium (e.g., A. rhizogenes strain is devoid of a rol gene or lacks a functional rol gene (e.g., comprises a nonfunctional rol gene). In some embodiments, an Agrobacterium used in a method of the present invention is a disarmed strain and / or a strain that lacks a functional rol gene (e.g., lacks a functional rolA gene, rolB gene, and / or rolC gene), and a plant tissue that is contacted with the Agrobacterium that is a disarmed strain and / or a strain that lacks a functional rol gene (e.g., a transformed plant tissue) may be devoid of the rolA gene, rolB gene, and rolC gene or lack a functional rolA gene, rolB gene, and / or rolC gene.

[0070] In some embodiments, a rol gene (e.g., a functional rol gene) may be present in an Agrobacterium strain used in a method of the present invention and optionally the rol gene may aid in root formation for a plant cell transformed (e.g., inoculated) with the Agrobacterium strain and / or stimulate plant growth. In some embodiments, an Agrobacterium strain comprises nucleic acids encoding one or more of rolA gene (e.g., GENBANK Accession No. CAA3 1091.1), rolB gene (e.g., GENBANK Accession No. CAA45540.1) and / or rolC gene (e.g., GENBANK Accession No. CAA45541.1). In some embodiments, an Agrobacterium strain comprises a rolA gene, optionally wherein the rolA gene encodes an amino acid sequence comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1; a rolB gene, optionally wherein the rolB gene encodes an amino acid sequence comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and / or a rolC gene, optionally wherein the rolC gene encodes an amino acid sequence comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3. In some embodiments, an A. tumefaciens strain used in a method of the present invention comprises a nucleic acid encoding a rolA gene, rolB gene and / or rolC gene. In some embodiments, an A. rhizogenes strain used in a method of the present invention comprises a nucleic acid encoding a rolA gene, rolB gene and / or rolC gene. In some embodiments, an Agrobacterium strain (e.g., A. tumefaciens or Agrobacterium rhizogenes strain) used in a method of the present invention comprises a nucleic acid encoding a rolA gene, rolB gene and / or rolC gene as extrachromosomal DNA, e.g., in a vector. In some embodiments, an A. tumefaciens strain used in a method of the present invention comprises a nucleic acid encoding rolA gene, rolB gene and rolC gene (rolABC genes), wherein the nucleic acid is provided extrachromasomally (e.g., in a transformation vector). Further exemplary Agrobacterium strains include, but are not limited to, those described in International Application No. WO 2022 / 109448, the contents of which are incorporated herein by referencein their entirety in regard to exemplary Agrobacterium strains. In some embodiments, an Agrobacterium strain (e.g., an A. tumefaciens or Agrobacterium rhizogenes strain) used in a method of the present invention comprises a rolA gene, rolB gene and rolC gene (i.e., the rolABC genes). In some embodiments, an Agrobacterium strain (e.g., an A. tumefaciens strain) is engineered to include a rolA gene, rolB gene and / or rolC gene. In some embodiments, an Agrobacterium strain (e.g., an A. tumefaciens or Agrobacterium rhizogenes strain) used in a method of the present invention comprises the rolABC genes and the rolABC genes are provided extrachromasomally (e.g., in a transformation vector).

[0071] In some embodiments, an Agrobacterium strain (e.g., an A. tumefaciens or Agrobacterium rhizogenes strain) that comprises the rolABC genes is used in a method of the present invention to introduce the rolABC genes into a cell of a wounded plant tissue and provide the hairy root and / or tumor phenotype in the plant cell including (e.g., expressing) the rolABC genes. A method of the present invention may comprise introducing the rolABC genes from an Agrobacterium strain into a cell of a plant tissue (e.g., a wounded plant tissue) to provide a transformed plant tissue that comprises and / or expresses the rolABC genes and culturing the transformed plant tissue on a root induction medium (e.g., a first root induction medium) to provide a composite plant that exhibits and / or has the hairy root phenotype, which is characterized by short internodes, wrinkled leaves, abundant roots with extensive lateral branching, rapid root growth, root growth that appears fuzzy, roots that are thicker than a standard root, and / or increased production certain metabolites. In some embodiments, the hairy root phenotype is used to select a composite plant (e.g., a composite plant that comprises a wild-type shoot and a root that comprises transgenic cells), optionally wherein presence of a root exhibiting the hairy root phenotype is used to select a composite plant (e.g., a composite plant that comprises a wild-type shoot and a root that comprises transgenic cells and exhibits the hairy root phenotype).

[0072] In some embodiments, a method herein comprises introducing a polynucleotide of interest and / or a gene that confers resistance to a selection agent into a cell of a wounded plant tissue, e.g., a wounded plant tissue comprising a shoot meristem and / or root forming competent cells, using an Agrobacterium strain that is contacted with (optionally blended with) the plant tissue thereby providing a transformed tissue. In some embodiments, a plant tissue is blended in water and / or an aqueous composition that is devoid of an Agrobacterium strain to provide a wounded plant tissue and the wounded plant tissue is contacted with (e.g., cultured with and / or in the presence of an Agrobacterium strain, optionally to introduce a polynucleotide of interestand / or a gene that confers resistance to a selection agent into a cell of the wounded plant tissue and provides transformed tissue.

[0073] In some embodiments, at least one cell of a wounded plant tissue is stably transformed with a polynucleotide from an Agrobacterium strain, optionally wherein the polynucleotide comprises a gene that confers resistance to a selection agent and / or a polynucleotide encoding or comprising a portion or all of an editing system. In some embodiments, a polynucleotide (e.g., a polynucleotide from an Agrobacterium strain and / or that encodes or comprises a portion or all of an editing system) is stably expressed in the plant cell and transient expression of the polynucleotide in the plant cell allows and / or provides for a target nucleic acid to be modified in the plant cell and / or another plant cell and thereby provides a modified nucleic acid.

[0074] In some embodiments, a plant cell is transiently transformed with a polynucleotide from an Agrobacterium strain, optionally wherein the polynucleotide comprises a gene that confers resistance to a selection agent and / or a portion or all of an editing system. In some embodiments, a polynucleotide (e.g., a polynucleotide from an Agrobacterium strain and / or that encodes or comprises a portion or all of an editing system) is transiently expressed in the plant cell and transient expression of the polynucleotide in the plant cell allows and / or provides for a target nucleic acid to be modified in the plant cell and / or another plant cell and thereby provides a modified nucleic acid.

[0075] A method of the present invention may comprise one or more culturing steps (e.g.,1, 2, 3, 4, 5, or more), which may be carried out using methods known in the art. In some embodiments, culturing a plant tissue (e.g., a wounded plant tissue, transformed plant tissue, composite plant, explant, and / or modified shoot) may include exposing the plant tissue to certain conditions (e.g., light, dark, nutrients, humidity, etc.) for a period of time (e.g., about 1,2, 3, 4, or 5 day(s) to about 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days). In some embodiments, culturing a plant tissue comprises providing temperature and / or light conditions sufficient to maintain and / or grow the plant tissue. In some embodiments, culturing a plant tissue is carried out before and / or after a wounding step. In some embodiments, a method herein may comprise culturing a wounded plant tissue. In some embodiments, a method herein may comprise culturing a transformed plant tissue. In some embodiments, a method herein may comprise culturing a composite plant. In some embodiments, a method herein may comprise culturing a modified shoot. In some embodiments, a method herein may comprise culturing a plant tissue in vitro, e.g, via tissue culture. A plant material may be cultured in the presence of and / or on media that may include an antibiotic and / or a growth hormone (e.g., a plant growth hormone,and / or a nutrient), optionally wherein the plant material comprises (e.g., is in contact with an Agrobacterium strain).

[0076] A method and / or culturing step herein may include culturing a transformed plant tissue on a root induction medium that optionally comprises a selection agent to produce a composite plant (e.g., a plant comprising a wild-type shoot and a root that comprises transgenic cells). In some embodiments, following contacting a wounded plant tissue with a polynucleotide of interest to provide a transformed plant tissue, the first culturing step in the method is culturing the transformed plant tissue on a root induction medium. In some embodiments, the transformed plant tissue comprises an Agrobacterium strain such that the transformed plant tissue and Agrobacterium strain are co-cultured on the root induction medium. In some embodiments, culturing the transformed plant tissue on the root induction medium comprises contacting at least a portion of the transformed plant tissue with the root induction medium such as by laying, distributing, and / or the like the transformed plant tissue onto the root induction medium and / or staking, pressing, and / or the like the transformed plant tissue into the root induction medium. In some embodiments, prior to and / or during culturing of the transformed plant tissue on the root induction medium, the transformed plant tissue is at least partially in the root induction medium, optionally wherein a root develops and / or grows from the transformed plant tissue into the root induction medium. In some embodiments, a root induction medium may comprise a selection agent, a basal medium, an antioxidant, a micronutrient, a chemoattractant (e.g., a chemoattractant for an Agrobacterium species), a cytokine, a plant growth regulator, an antibiotic, and / or a phenol. In some embodiments, a root induction medium is devoid of a selection agent. In some embodiments, a root induction medium comprises a basal medium, e.g., a medium capable of supporting the development and / or growth of plant tissue optionally containing an inorganic salt, vitamin, sugar (e.g., glucose), buffer system, and / or amino acid (e.g., an essential amino acid), and the basal medium may optionally comprise a selection agent. In some embodiments, a root induction medium comprises a basal medium and optionally a selection agent and the root induction medium is devoid of a plant growth regulator (e.g., auxin and / or an auxin precursor) and / or is devoid of copper (II) sulphate (e.g., cupric sulfate, pentahydrate). In some embodiments, a root induction medium comprises a basal medium, a selection agent, and an auxin or auxin precursor. In some embodiments, a root induction medium is devoid of a selection agent and comprises a basal medium and an auxin or auxin precursor. In some embodiments, a root induction medium comprises a basal medium and the root induction medium is devoid of a selection agent and is devoid of an auxin or auxin precursor. Exemplary auxins or auxinprecursors include, but are not limited to natural auxins and / or natural auxin precursors, such as those obtainable from seaweed and / or algae, synthetic auxins and / or synthetic auxin precursors, and / or auxin conjugates. Examples of auxins include, but are not limited to, indole- 3 -acetic acid (IAA), 4-chloro-indole-3 -acetic acid (4-C1-IAA), phenylacetic acid (PAA), indole-3 -butyric acid (IB A), indole-3 -acetyl- 1-O-P-D-glucose (lAAglc), 1 -naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (tordon), 2,4,5-trichlorophenoxyacetic acid (2,4, 5-T), 2,3,6-trichlorobenzoic acid, 4-chloro-2 methyl phenoxy acetic acid (MCPA) and / orN,N-dimethylethylthiocarbamate. Auxin conjugates may also be used including, e.g., IAA- Inositol, lAA-Inositol-arabinose, IAP1, an lAA-peptide, an IAA glycoprotein, an lAA-glucan, lAA-aspartate, lAA-glucose, IAA-1-O-glucose, lAA-myo-Inositol, IAA-4-O-glucose, IAA-6- O-glucose, lAA-Inositol-galactose, an IAA amide conjugate, and / or an lAA-amino acid conjugate. In some embodiments, a root induction medium comprises a basal medium that comprises a selection agent and indole-3 -butryic acid (IBA). In some embodiments, a root induction medium comprises an auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration of less than about 0.5 mg / L, e.g., less than about 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1 or 0.05 mg / L. In some embodiments, an auxin or auxin precursor is present in a root induction medium at a concentration of about 0.01, 0.05, or 0.1 mg / L to about 0.2 or 0.3 mg / L. In some embodiments, the auxin or auxin precursor is present in a root induction medium at a concentration of aboutO.1 mg / L. In some embodiments, a root induction medium comprises an auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / L. In some embodiments, a root induction medium comprises an auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration about 1, 2, 3, 4, or 5 mg / L to about 6, 7, 8, 9, or 10 mg / L. In some embodiments, a root induction medium comprises an auxin or auxin precursor in a root induction medium and / or a dip treatment (e.g., an IBA dip treatment) at a concentration about 500, 600, 700, 800, 900, 1000, 1100, or 1200 mg / L.

[0077] In some embodiments, the presence of a selection agent in a medium (e.g., a root induction medium) is used in a method of the present invention to select a transformed and / or transgenic cell and / or tissue. In some embodiments, culturing a transformed plant tissue on a root induction medium comprising a selection agent allows for and / or provides a root to grow and / or develop that comprises one or more cells that include a gene that confers resistance to the selection agent, whereas growth of a wild-type root from the transformed plant tissuecultured on the root induction medium comprising the selection agent is stunted or a wild-type root does not develop. A selection agent may be an antibiotic and / or herbicide. In some embodiments, the selection agent is glyphosate, bialaphos, phosphinothricin, Basta, glufosinate, 2,4-D, imazamox, kanamycin or a related aminoglycosides, hygromycin, ampicillin, or dicamba. In some embodiments, a wounded plant tissue and / or transformed plant tissue includes a gene conferring resistance to a selection agent (e.g., a resistance gene). A resistance gene that may be used in a method of the present invention includes, but is not limited to, the neomycin phosphotransferase II (NPT II / Neo) gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra (1982) Gene 19:259-268; Bevan et al. (1983) Nature 304: 184-187); the bar gene, which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl. Acids Res. 18: 1062, Spencer et al. (1990) Theor. AppL Genet. 79:625-631); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann (1984) Mol. Cell Biol. 4:2929-2931); the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642); and the acetolactate synthase (ALS) gene, which confers resistance to imazamox. In some embodiments, the gene that confers resistance is NPT II / Neo and the selection agent is kanamycin. In some embodiments, the gene that confers resistance is acetolactate synthase (ALS) and the selection agent is imazamox. In some embodiments, the selection agent is not spectinomycin.

[0078] In some embodiments, a selection agent is present in a root induction medium at a concentration that is less than (e.g., at least 50% less than) a concentration of the same selection agent in a shoot induction medium. In some embodiments, a selection agent is present in a root induction medium at a concentration that preferences growth of a transgenic root (e.g., fully transgenic root and / or chimeric root) over a wild-type root. A “transgenic root” as used herein refers to a root that includes a transgenic cell. In some embodiments, a transgenic root is fully transgenic (i.e., all root cells include a transgene). In some embodiments, a transgenic root is chimeric (i.e., a portion of the root cells include a transgene and another portion of the root cells are devoid of the transgene). In some embodiments, the selection agent in a method and / or culturing step of the present invention is kanamycin. When used as a selection agent, kanamycin may be present in a medium (e.g., a root induction medium) in an amount of about 10 mg / L to about 75 mg / L, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 mg / L. In some embodiments, kanamycin is present in a root induction medium in an amount of about 10 mg / L to about 75 mg / L, optionally wherein the kanamycin is present in theroot induction medium in an amount of about 25 mg / L to about 50 mg / L. In some embodiments, the selection agent in the methods and culturing steps herein is imazamox. When used as a selection agent, imazamox may be present in a medium (e.g., a root induction medium) in an amount of about 0.1 pM to about 0.75 pM, e.g., about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16,0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33,0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50,0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67,0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 pM. In some embodiments, imazamox is present in a root induction medium at a concentration of about 0.1 pM to about 0.75 pM, optionally wherein the imazamox is present in the root induction medium at a concentration of about 0.25 pM to about 0.5 pM.

[0079] Culturing a transformed plant tissue on a root induction medium optionally comprising a selection agent in accordance with a method of the present invention provides a composite plant that includes a wild-type shoot (e.g., a shoot that is substantially transgene- free and non-edited in accordance with the method used to provide the transformed plant tissue) and a root that comprises one or more transgenic cells, e.g., the root is a modified root and comprises a polynucleotide of interest (e.g., a gene that confers resistance to a selection agent and / or the rolABC genes) that was contacted to (e.g., introduced into) a wounded plant tissue to provide the transformed plant tissue. In some embodiments, the composite plant includes a wild-type shoot (e.g., a shoot that is transgene-free and non-edited in accordance with the method used to provide the transformed plant tissue) and a mosaic root, e.g., a root comprising (i) wild-type cells and transgenic, non-edited cells, (ii) wild-type cells and transgenic, edited cells, (iii) transgenic, non-edited cells and transgene-free edited cells, (iv) transgenic, nonedited cells and transgenic edited cells, (v) transgene-free, edited cells and transgenic edited cells, (vi) wild-type cells, transgenic non-edited cells, and transgene-free edited cells, (vii) wild-type cells, transgenic non-edited cells, and transgenic edited cells, (viii) wild-type cells, transgene-free edited cells and transgenic edited cells, (ix) transgenic non-edited cells, transgene-free edited cells and transgenic edited cells, or (x) wild-type cells, transgenic nonedited cells, transgene-free edited cells and transgenic edited cells. In some embodiments, a composite plant has a wild-type shoot (e.g., a shoot that is substantially transgene-free and nonedited in accordance with the method used to provide the transformed plant tissue) and a chimeric root, e.g., a root comprising transgenic cells (e.g., transgenic non-edited and / or transgenic edited) and transgene-free cells (e.g., wild-type cells and / or transgene-free edited cells).

[0080] One or more root(s) (e.g., 1, 2, 3, 4, 5, or more) may grow from a transformed plant tissue that is cultured on a root induction medium (e.g., during culturing and / or provided at the end of culturing on the root induction medium) in accordance with an embodiment of the present invention. In some embodiments, culturing a transformed plant tissue of the present invention on a root induction medium provides and / or comprises growing a root (e.g., a transgenic root) from the transformed plant tissue, wherein the root is at least 2 cm in length (e.g., has a length of about 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 cm or more). In some embodiments, culturing a transformed plant tissue of the present invention on a root induction medium provides and / or comprises growing a root system (e.g., a full root system), wherein two or more (e.g., 2, 3, 4, 5, 6, or more) roots are 2 cm or more in length and optionally includes one or more branching root(s). In some embodiments, culturing a transformed plant tissue of the present invention on a root induction medium provides and / or comprises growing a wild-type root from the transformed plant tissue that has stunted growth (e.g., a growth less than 2 cm) and / or no branching.

[0081] A method or culturing step of the present invention may further include culturing a composite plant on shoot induction medium to produce at least one modified shoot on the composite plant, e.g., at least one modified shoot growing from a root comprising one or more transgenic cells. In some embodiments, a composite plant is removed (e.g., extracted) from a root induction medium and at least a portion of the composite plant is contacted with a shoot induction medium. In some embodiments, culturing the composite plant on a shoot induction medium comprises contacting at least a portion of the composite plant with the shoot induction medium such as by laying, placing, staking, and / or the like the composite plant tissue onto and / or in the shoot induction medium. In some embodiments, a shoot (e.g., an adventitious shoot) may originate and / or form from one or more cell(s) of a transgenic root, optionally from a cell within the pericycle layer (e.g., the single or two-celled pericycle layer) of the transgenic root. A shoot (e.g., an adventitious shoot) originating and / or formed from a transgenic root may comprise cells that are genetically the same as the cells from which the adventitious shoot was formed, e.g., at least a portion of the cells of the adventitious shoot may be modified in the same manner. Accordingly, in some embodiments, the methods herein provide for and / or produce a modified shoot, wherein cells of the modified shoot are different than (e.g., genetically different than) cells of the wild-type shoot. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention may comprise a polynucleotide of interest. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of thecells of a modified shoot of the present invention may comprise a gene that confers resistance to a selection agent. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention are transgene- free and comprise a modified nucleic acid. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention comprise a modified nucleic acid and a gene the confers resistance to a selection agent. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention comprise a transgene that is not a gene that confers resistance to a selection agent, e.g., a transgene encoding a therapeutic protein. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot comprise a gene the confers resistance to a selection agent and a second transgene. In some embodiments, culturing a composite plant on a shoot induction medium produces at least two modified shoots, wherein two or more of the at least two modified shoots may be the same or may be different (e.g., comprise different edits).

[0082] A shoot induction medium of use in a method and / or culturing step of the present invention may comprise a basal medium that includes one or more (e.g., 1, 2, 3, 4, 5, or more) growth regulator(s). In some embodiments, a shoot induction medium includes a basal medium and a selection agent. In some embodiments, a shoot induction medium is devoid of a selection agent. The term “plant growth regulator,” as used herein, encompasses naturally occurring or synthetic (not naturally occurring) compounds that regulate and / or aid in plant growth and / or development. Exemplary plants growth regulators, include, but are not limited to, auxins or auxin precursors and cytokinins or cytokinin-like compounds. Exemplary cytokinins include, but are not limited to, thidiazuron, BAP (6-benzylaminopurine), kinetin, 4-CPPU (forchlorfenuron), 2iP (6-(y,y-dimethylallylamino) purine), zeatin, zeatin-riboside, dihydrozeatin, adenine, 6-isopentenyladenine (IPA), TIBA (2,3,5-triiodobenzoic acid) and / or PhytoAx™ plant growth regulating preparation commercially available from PhytoTech Labs. In some embodiments, a shoot induction medium comprises 4-CPPU. In some embodiments, a shoot induction medium comprises about 0.5 mg / L to about 10 mg / L 4-CPPU (e.g., about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg / L 4-CPPU), optionally about 1 mg / L to about 5 mg / L 4-CPPU or about 2 mg / L to about 4 mg / L 4-CPPU. In some embodiments, a shoot induction medium comprises BAP. In some embodiments, a shoot induction medium comprises about 0.05 mg / L to about 5 mg / L BAP (e.g., about 0.05, 0.10, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / L BAP), optionally about 0.05mg / L to about 1 mg / L BAP or about 0.25 mg / L to about 0.75 mg / L BAP. In some embodiments, a shoot induction medium comprises 4-CPPU and BAP. In some embodiments, a shoot induction medium comprises about 0.5 mg / L to about 10 mg / L 4-CPPU (e.g., about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg / L 4-CPPU) and about 0.05 mg / L to about 5 mg / L BAP (e.g., about 0.05, 0.10, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / L BAP), optionally about 2 mg / L to about 4 mg / L 4-CPPU and 0.25 mg / L to about 0.75 mg / L BAP.

[0083] According to some embodiments, culturing of a composite plant on a shoot induction medium provides a modified shoot and the modified shoot may comprise transgenic, edited cells; transgenic, non-edited cells; or non-transgenic, edited cells. In some embodiments, all or a portion of a modified shoot of the present invention may be cultured on and / or in a growth medium to produce a modified plant or plant part (e.g., a plant or plant, wherein all or substantially all of the cells thereof are transgenic and non-edited; transgene-free and edited; or transgenic and edited). In some embodiments, all or a portion of a modified shoot may be excised from a composite plant or modified root prior to being cultured on and / or in a growth medium. In some embodiments, all or a portion of the modified shoot and a portion of the modified root associated therewith may be excised from the root that comprises transgenic cells prior to being cultured on the growth medium. In some embodiments, the growth medium is a shoot development medium. A shoot development media may comprise a lower cytokinin to auxin ratio than a shoot induction medium (e.g., a lower cytokinin to auxin ratio than a shoot induction medium on which a composite plant was cultured in accordance with this invention). In some embodiments, a shoot development media is devoid of a growth regulator. In some embodiments, a modified shoot may be cultured on a shoot development media, which may result in a larger plant (e.g., compared to a modified shoot grown on root induction medium rather than a shoot induction medium) and / or minimize proliferation as early-stage shoots. In some embodiments, the growth medium is a root induction medium (e.g., a root induction medium that is the same or substantially the same as a prior used root induction medium such as the root induction medium used to grow a transgenic root in accordance with the invention). In some embodiments, the growth medium may or may not include a selection agent. In some embodiments, the step of culturing a modified shoot on a growth medium comprises culturing the modified shoot on a shoot development medium followed by culturing on a root induction medium. In some embodiments, culturing the modified shoot on a growth medium comprisesculturing the modified shoot in tissue culture. In some embodiments, culturing a modified shoot on a growth medium comprises culturing the modified shoot in a greenhouse.

[0084] Referring to FIG. 1, which is an exemplary schematic illustrating regeneration of a wild-type shoot 31a; a transgenic, non-edited shoot 31b; a transgenic, edited shoot 31c; and a non-transgenic, edited shoot 31d from a composite plant 20 including a chimeric root 21 following culturing on a root induction medium 12 including a selection agent and culturing on a shoot induction medium 14 without a selection agent according to some embodiments of the invention. Initially, plant tissue 10 (e.g., a shoot) comprising root forming competent cells 16 is wounded to provide a wounded tissue 11 and the wounded tissue 11 is contacted with a polynucleotide of interest 15 (optionally via contact with an Agrobacterium strain that comprises the polynucleotide of interest 15) to provide a transformed plant tissue that is cultured on and / or in the root induction medium 12 to provide the composite plant 20. The composite plant 20 includes a chimeric root 21 that includes a wild-type cell 21a and a transgenic cell 21b, wherein the transgenic cell 21b optionally comprises a modified nucleic acid (e.g., a nucleic acid that is modified (e.g., edited) by and / or resulting from introduction of the polynucleotide of interest 15). The composite plant 20 also includes a wild-type root 23 that includes wild-type cells 21a, wherein the wild-type cells 21a do not include the polynucleotide of interest 15 and do not include a nucleic acid that is modified (e.g., edited) by and / or resulting from introduction of the polynucleotide of interest 15. Then, the composite plant 20 is cultured on shoot induction medium 14 that is devoid of a selection agent and the culturing on the shoot induction medium 14 results in the growth of a wild-type shoot 31a; a transgenic, non-edited shoot 31b; a transgenic, edited shoot 31c; and a non-transgenic, edited shoot 31d.

[0085] FIG. 2 is an exemplary schematic illustrating regeneration of transgenic shoots that may be edited from a composite plant 40 including a transgenic root 42 according to some embodiments of the invention. Initially, plant tissue 10 (e.g., a shoot) comprising root forming competent cells 16 is wounded to provide a wounded tissue 11 and the wounded tissue 11 is contacted with a polynucleotide of interest 15 (optionally via contact with an Agrobacterium strain that comprises the polynucleotide of interest 15) to provide a transformed plant tissue that is cultured on and / or in the root induction medium 12 to provide the composite plant 40. The composite plant 40 in Fig. 2 includes a transgenic root 42 that includes a transgenic, nonedited cell 42a (i.e., cell 42a includes the polynucleotide of interest 15 but does not include a nucleic acid that is modified (e.g., edited) by and / or resulting from introduction of the polynucleotide of interest 15), a transgenic, edited cell 42b, and a transgenic, edited cell 42c,wherein the transgenic cell 42b and transgenic cell 42c each comprises a modified nucleic acid (e.g., a nucleic acid that is modified (e.g., edited) by and / or resulting from introduction of the polynucleotide of interest 15), but the modified nucleic acid is different in transgenic cell 42b and transgenic cell 42c (i.e., different modifications resulted from introduction of the polynucleotide of interest 15 and / or were made by the polynucleotide of interest 15). Then, the composite plant 40 is cultured on shoot induction medium 44 that optionally comprises a selection agent and the culturing on the shoot induction medium 44 results in the growth of a transgenic, non-edited shoot 52a; a transgenic, edited shoot 52b; and a transgenic, edited shoot 52c, wherein each of the shoots 52a-52c are genetically different from each other.

[0086] According to some embodiments of the present invention, a transgenic root that develops and / or is grown from a transformed plant tissue cultured on a root induction medium according to embodiments of the present invention may be cultured on a shoot induction medium to provide and / or grow a shoot (e.g., a shoot that develops and / or grows from the transgenic root) that includes only transgenic cells (i.e., a fully transgenic shoot), and the fully transgenic shoot may include edited, transgenic cells (e.g., a nucleic acid in the transgenic cell is modified). In some embodiments, a chimeric root (e.g., comprises wild-type cells and transgenic cells) that develops and / or is grown from a transformed plant tissue cultured on a root induction medium according to embodiments of the present invention may be cultured on a shoot induction medium to provide and / or grow a shoot (e.g., a shoot that develops and / or grows from the transgenic root) that includes only non-transgenic cells that are edited (e.g., a nucleic acid in the non-transgenic cell is modified), a shoot that includes only non-transgenic cells that are not edited, a shoot that includes only transgenic cells that are not edited, or a shoot that includes only cells that are transgenic and edited. The modification (e.g., edit) in an edited, transgenic cell or non-transgenic cell may be made by and / or may result from the introduction of a polynucleotide of interest into a wounded plant tissue that provides a transformed plant tissue, which is cultured on the root induction medium according to embodiments of the present invention.

[0087] A modified plant or plant part produced by a method of the present invention may include a gene conferring resistance to a selection agent, one or more other transgenes (i.e., a transgene other than the resistance gene), and / or one or more modified nucleic acid(s) (e.g., one or more edited gene(s)). A transgene that may be included in a modified plant or plant part in accordance with a method of the present invention includes, but is not limited to, those encoding an agronomic trait, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and / or the like. A transgene may be involved in metabolism of oil, starch,carbohydrates, nutrients, etc. Thus, transgenes or traits of interest include, but are not limited to, environmental- or stress-related traits, disease-related traits, and traits affecting agronomic performance. Transgenes may also include genes responsible for the synthesis of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids, hormones, polymers, flavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, glycolipids, marker proteins, etc. In some embodiments, transgenes may include those encoding enzymes responsible for oil production; enzymes that synthesize, or proteins which are themselves, natural medicaments such as pharmaceuticals or veterinary products. Such proteins may include, but are not limited to, retinoblastoma protein, p53, angiostatin, leptin, serum albumin, hemoglobin, or collagen.

[0088] In some embodiments, a modified plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified plant or plant part, and the modified plant or plant part is devoid of a transgene introduced into a plant cell in accordance with a method of the present invention to provide the modified plant or plant part. In some embodiments, a modified plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified plant or plant part, and the modified plant or plant part is transgene-free. In some embodiments, a modified plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified plant or plant part, and optionally the modified plant or plant part is transgenic.

[0089] In some embodiments, a method of the present invention comprises wounding a plant tissue (e.g., a plant tissue that comprises meristem cells and / or root forming competent cells, and optionally a shoot including meristem tissue) to provide a wounded tissue (e.g., a wounded tissue comprising meristem cells and / or root forming competent cells); contacting the wounded tissue with a polynucleotide of interest and a gene that confers resistance to a selection agent to provide a transformed tissue (e.g., that comprises the meristem cells and / or root forming competent cells) that includes the polynucleotide of interest and the gene that confers resistance to the selection agent; culturing the transformed tissue on a first root induction medium comprising the selection agent to produce a composite plant, wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells (e.g., a transgenic root); culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium,thereby producing a modified plant or plant part. In some embodiments, the modified shoot grows from a portion of the transgenic root.

[0090] In some embodiments, a method of the present invention comprises wounding a plant tissue (e.g., a plant tissue that comprises meristem cells and / or root forming competent cells, and optionally a shoot including meristem tissue) to provide a wounded tissue (e.g., a wounded tissue comprising meristem cells and / or root forming competent cells); contacting the wounded tissue with an Agrobacterium that comprises a rolA gene, a rolB gene, and a rolC gene to provide a transformed tissue (e.g., that comprises the meristem cells and / or root forming competent cells) that includes the rolA gene, the rolB gene, the rolC gene; culturing the transformed tissue on a first root induction medium to produce a composite plant, wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells (e.g., a transgenic root); culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part. In some embodiments, the modified shoot grows from a portion of the transgenic root.

[0091] Modification of a target nucleic acid may be accomplished using an editing system. A “gene editing system” or “editing system” as used herein refers to any site-specific (e.g., sequence-specific) nucleic acid editing system now known or later developed, which system can introduce a modification (e.g., a mutation) in a nucleic acid in target specific manner. For example, an editing system (e.g., a site- and / or sequence-specific editing system) can 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 prime editing system, each of which may comprise one or more polypeptide(s) and / or one or more polynucleotide(s) that when present and / or expressed together (e.g., as a system) in a composition and / or cell can modify (e.g., mutate) a target nucleic acid in a sequence specific manner. In some embodiments, an editing system (e.g., a site- and / or sequence-specific editing system) can comprise one or more polynucleotide(s) and / or one or more polypeptide(s), 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 is provided and / or is used that comprises a CRISPR-Cas effector protein.

[0092] In some embodiments, an editing system comprises one or more sequence-specific nucleic acid binding polypeptide(s) (e.g., a DNA binding domain) that can be from, for example, a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein. In some embodiments, an editing system comprises one or more cleavage polypeptide(s) (e.g., nucleases) including, but not limited to, an endonuclease (e.g., Fokl), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein), a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN).

[0093] A “nucleic acid binding polypeptide” as used herein refers to a polypeptide or domain that binds and / or is capable of binding a nucleic acid (e.g., a target nucleic acid). A DNA binding domain is an exemplary nucleic acid binding polypeptide and may be a site- and / or sequence-specific nucleic acid binding domain. In some embodiments, a nucleic acid binding polypeptide may be a sequence-specific nucleic acid binding polypeptide such as, but not limited to, a sequence-specific binding domain from, for example, 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. In some embodiments, a nucleic acid binding polypeptide comprises a cleavage domain (e.g., a nuclease domain) such as, but not limited to, an endonuclease (e.g., Fokl), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease, a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN). In some embodiments, the nucleic acid binding polypeptide associates with and / or is capable of associating with (e.g., forms a complex with) with one or more nucleic acid molecule(s) (e.g., forms a complex with a guide nucleic acid as described herein), which may direct and / or guide the nucleic acid binding polypeptide to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecule(s) (or a portion or region thereof), thereby causing the nucleic acid binding polypeptide to bind to the nucleotide sequence at the specific target site. In some embodiments, the nucleic acid binding polypeptide is a CRISPR-Cas effector protein as described herein.

[0094] In some embodiments, an editing system comprises or is a ribonucleoprotein such as an assembled ribonucleoprotein complex (e.g., a ribonucleoprotein that comprises a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase or a deaminase). In some embodiments, a ribonucleoprotein of an editing system may be assembled together (e.g., a pre-assembled ribonucleoprotein including a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase or a deaminase) such as when contacted to a target nucleic acid or when introduced into a cell (e.g., a mammalian cell or a plant cell). In some embodiments, a ribonucleoprotein of an editing system may assemble intoa complex e.g., a covalently and / or non-covalently bound complex) while a portion of the ribonucleoprotein is contacting a target nucleic acid and / or may assemble after and / or during introduction into a plant cell. In some embodiments, an editing system may be assembled (e.g., into a covalently and / or non-covalently bound complex) when introduced into a plant cell. In some embodiments, a ribonucleoprotein may comprise a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase. In some embodiments, a ribonucleoprotein may comprise a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase.

[0095] In some embodiments, an editing system comprises a reverse transcriptase, an extended guide nucleic acid, and a CRISPR-Cas effector protein, e.g., a Type II CRISPR-Cas effector protein or Type V CRISPR-Cas effector protein. In some embodiments, the Type V CRISPR-Cas effector protein or Type II CRISPR-Cas effector protein, the reverse transcriptase, and the extended guide nucleic acid may form a complex or may be comprised in a complex that is capable of interacting with a target nucleic acid.

[0096] In some embodiments, an editing system is used in prime editing. “Prime editing” and grammatical variants thereof as used herein refer to a nucleic acid editing technology that uses a Cas9 nickase fused to a reverse transcriptase and modifies a target nucleic acid without a double strand break or a donor DNA template. In Prime editing, the Cas9 nickase cuts the non-complementary strand of DNA upstream of the PAM site, thereby providing a 3’ flap that is extended with the extension including a modification. Further details on Prime editing can be found in Anzalone et al. (2019) Nature 576: 149-157 and / or U.S. Patent Application Publication No. 2021 / 0147862, the contents of each of which are incorporated herein by reference in their entirety.

[0097] In some embodiments, an editing system incorporates the Redraw editing system. Further details on the Redraw editing system can be found in U.S. Patent Application Publication No. 2021 / 0130835 and / or in U.S. Patent Application Publication No. 2022 / 0145334, the contents of each of which are incorporated herein by reference in their entirety.

[0098] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.

[0099] A “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA” “crRNA” or “crDNA” as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target nucleic acid (e.g., a target DNA and / or a protospacer), and at least one repeat sequence e.g., a repeat of a Type V Cast 2a CRISPR- Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2cl CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Casl2a (also referred to as Cpfl), Cas 12b, Cas 12c, Cas 12d, Casl2e, Casl2f, Casl2i, Cas 13 a, Cas 13b, Cas 13c, Cas 13d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, 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, or a fragment thereof), wherein the repeat sequence may be linked to the 5’ end and / or the 3’ end of the spacer sequence. In some embodiments, the guide nucleic acid comprises DNA. In some embodiments, the guide nucleic acid comprises RNA (e.g., is a guide RNA). The design of a gRNA of this invention may be based on a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system.

[0100] In some embodiments, a Casl2a gRNA may comprise, from 5’ to 3’, a repeat sequence (full length or portion thereof (“handle”); e.g., pseudoknot-like structure) and a spacer sequence.

[0101] In some embodiments, a guide nucleic acid may comprise more than one repeat sequence-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeat-spacer sequences) e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat- spacer, and the like). The guide nucleic acids of this invention are synthetic, human-made and not found in nature. A gRNA can be quite long and may be used as an aptamer (like in the MS2 recruitment strategy) or other RNA structures hanging off the spacer.

[0102] A “repeat sequence” as used herein, refers to, for example, any repeat sequence of a wild-type CRISPR Cas locus (e.g., a Cas9 locus, a Casl2a locus, a C2cl locus, etc.) or a repeat sequence of a synthetic crRNA that is functional with the CRISPR-Cas effector protein encoded by the nucleic acid constructs of the invention. A repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR-Cas locus (e.g.,Type I, Type II, Type III, Type IV, Type V or Type VI) or it can be a synthetic repeat designed to function in a Type I, II, III, IV, V or VI CRISPR-Cas system. A repeat sequence may comprise a hairpin structure and / or a stem loop structure. In some embodiments, a repeat sequence may form a pseudoknot-like structure at its 5’ end (i.e., “handle”). Thus, in some embodiments, a repeat sequence can be identical to or substantially identical to a repeat sequence from wild-type Type I CRISPR-Cas loci, Type II, CRISPR-Cas loci, Type III, CRISPR-Cas loci, Type IV CRISPR-Cas loci, Type V CRISPR-Cas loci and / or Type VI CRISPR-Cas loci. A repeat sequence from a wild-type CRISPR-Cas locus may be determined through established algorithms, such as using the CRISPRfmder offered through CRISPRdb (see, Grissa et al. (2007) Nucl. Acids Res. 35(Web Server issue):W52-7). In some embodiments, a repeat sequence or portion thereof is linked at its 3’ end to the 5’ end of a spacer sequence, thereby forming a repeat-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0103] In some embodiments, a repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides depending on the particular repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein; e.g., about). In some embodiments, a repeat sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides.

[0104] A repeat sequence linked to the 5’ end of a spacer sequence can comprise a portion of a repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous nucleotides of a wild-type repeat sequence). In some embodiments, a portion of a repeat sequence linked to the 5’ end of a spacer sequence can be about five to about ten consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5’ end) of a wild-type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure at its 5’ end (e.g., “handle”).

[0105] A “spacer sequence” as used herein is a nucleotide sequence that is complementary to a target nucleic acid (e.g., target DNA) (e.g., protospacer). The spacer sequence can be fullycomplementary or substantially complementary (e.g., at least about 70% complementary (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 more)) to a target nucleic acid. Thus, in some embodiments, the spacer sequence can have one, two, three, four, or five mismatches as compared to the target nucleic acid, which mismatches can be contiguous or noncontiguous. In some embodiments, the spacer sequence can have 70% complementarity to a target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to a target nucleic acid. In still other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity, and the like, to the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. A spacer sequence may have a length from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in some embodiments, a spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleic acid (e.g., protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides in length. In some embodiments, the spacer is about 21, 22, or 23 nucleotides in length.

[0106] In some embodiments, the 5’ region of a spacer sequence of a guide nucleic acid may be fully complementary to a target nucleic acid, while the 3’ region of the spacer may be substantially complementary to the target nucleic acid (such as for a spacer in a Type V CRISPR-Cas system), or the 3’ region of a spacer sequence of a guide nucleic acid may be fully complementary to a target nucleic acid, while the 5’ region of the spacer may be substantially complementary to the target nucleic acid (such as for a spacer in a Type II CRISPR-Cas system), and therefore, the overall complementarity of the spacer sequence to the target nucleic acid may be less than 100%. Thus, for example, in a guide nucleic acid for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleic acid. In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, nucleotides, and any range therein) of the 5’ end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 50%complementary (e.g., 50%, 55%, 60%, 65%, 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 more)) to the target nucleic acid.

[0107] As a further example, in a guide nucleic acid for a Type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleic acid. In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range therein) of the 3’ end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 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 more or any range or value therein)) to the target nucleic acid. A recruiting guide RNA further comprises one or more recruiting motifs as described herein, which may be linked to the 5' end of the guide or the 3' end or it may be inserted into the recruiting guide nucleic acid (e.g., within the hairpin loop).

[0108] A “recruiting motif’ as used herein refers to one half of a binding pair that may be used to recruit a compound to which the recruiting motif is bound to another compound that includes the other half of the binding pair (i.e., a “corresponding motif’). The recruiting motif and corresponding motif may bind covalently and / or noncovalently. In some embodiments, a recruiting motif is an RNA recruiting motif (e.g., an RNA recruiting motif that is capable of binding and / or configured to bind to an affinity polypeptide), an affinity polypeptide (e.g., an affinity polypeptide that is capable of binding and / or configured to bind an RNA recruiting motif and / or a peptide tag), or a peptide tag (e.g., a peptide tag that is capable of binding and / or configured to bind an affinity polypeptide). For example, when a recruiting motif is an RNA recruiting motif, the corresponding motif for the RNA recruiting motif may be an affinity polypeptide that binds the RNA recruiting motif. A further example is that when a recruiting motif is a peptide tag, the corresponding motif for the peptide tag may be an affinity polypeptide that binds the peptide tag. Thus, a compound comprising a recruiting motif (e.g., an affinity polypeptide) may be recruited to another compound (e.g., a guide nucleic acid) comprising a corresponding motif for the recruiting motif (e.g., an RNA recruiting motif). Exemplary peptide tags (e.g., epitope) include, but are not limited to, a GCN4 peptide tag (e.g.,Sun-Tag), a c-Myc affinity tag, an HA affinity tag, a His affinity tag, an S affinity tag, a methionine-His affinity tag, an RGD-His affinity tag, a FLAG® octapeptide, a strep tag or strep tag II, a V5 tag, and / or a VSV-G epitope.

[0109] In some embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides in length, about 5 to about 6 nucleotides in length, or about 6 nucleotides in length.

[0110] A “target nucleic acid”, “target DNA,” “target nucleotide sequence,” “target region,” and “target region in the genome” are used interchangeably herein and refer to a region of an organism’s (e.g., a plant’s) genome that comprises a sequence that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (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%, or more)) to a spacer sequence in a guide nucleic acid as defined herein. A target nucleic acid is targeted by an editing system (or a component thereof) as described herein. A target region useful for a CRISPR-Cas system may be located immediately 3’ (e.g., Type V CRISPR-Cas system) or immediately 5’ (e.g., Type II CRISPR-Cas system) to a PAM sequence in the genome of the organism (e.g., a plant genome). A target region may be selected from any region of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, and the like) located immediately adjacent to a PAM sequence. A target nucleic acid may be in a gene for which it is desired to alter the expression and / or activity of the product encoded thereby (e.g., protein and / or RNA).

[0111] A “protospacer sequence” or “protospacer” as used herein refer to a sequence that is fully or substantially complementary to (and can hybridize to) a spacer sequence of a guide nucleic acid. In some embodiments, the protospacer is all or a portion of a target nucleic acid as defined herein that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat-spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs).

[0112] In the case of Type V CRISPR-Cas (e.g., Casl2a) systems and Type II CRISPR- Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For Type IV CRISPR-Cas systems, the PAM is located at the 5’ end on the non-target strand and at the 3’ end of the target strand (see below, as an example).Spacer3'-AAANNNNNNNNNNNNNNNNNNN-5’ Target strand5'-TTTNNNNNNNNNNNNNNNNNNN-3’ Non-target strand

[0113] In the case of Type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately 3’ of the target region. The PAM for Type I CRISPR-Cas systems is located 5’ of the target strand. There is no known PAM for Type III CRISPR-Cas systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Makarova et al. (2015) Nature Rev. Microbiol. 13:722-736). Guide structures and PAMs are described by R. Barrangou ((2015) Genome Biol. 16:247).

[0114] Canonical Casl2a PAMs are T rich. In some embodiments, a canonical Casl2a PAM sequence may be 5’-TTN, 5’-TTTN, or 5’-TTTV. In some embodiments, canonical Cas9 (e.g., S. pyogenes) PAMs may be 5’-NGG-3’. In some embodiments, non-canonical PAMs may be used but may be less efficient.

[0115] Additional PAM sequences may be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through the transformation of target plasmid DNA (Esvelt et al. (2013) Nat. Methods 10: 1116-1121; Jiang et al. (2013) Nat. Biotechnol. 31 :233-239). In some aspects, a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner & Barrangou (2014) AppL Environ. Microbiol. 80:994-1001; Mojica et al. (2009) Microbiology 155:733-740).

[0116] In some embodiments, a guide nucleic acid further comprises a reverse transcriptase template and may be referred to as an extended guide nucleic acid. An “extended guide nucleic acid” as used herein is a guide nucleic acid as described herein that further comprises a reverse transcriptase template (RTT) and / or a primer binding site (PBS). In some embodiments, an extended guide nucleic acid is an engineered prime editing guide RNA (pegRNA). An extended guide nucleic acid may be a targeted allele guide RNA (tagRNA) or a stabilized targeted allele guide RNA (stagRNA). A “tagRNA” as used herein refers to an extended guide nucleic acid that comprises a PBS and a RTT and has target strand complementarity. A “stagRNA” as used herein refers to a tagRNA that comprises a stabilization motif. A stabilization motif may be present at the 3’ and / or 5’ end of a tagRNA.In some embodiments, a stabilization motif is present at the 3’ end of a tagRNA. Exemplary stabilization motifs include, but are not limited to, recruiting motifs, RNA hairpins, pseudoknot sequences, and / or PP7 motifs (e.g., a PP7 RNA hairpin sequence). In some embodiments, a stagRNA is a tagRNA that comprises a PP7 RNA hairpin sequence. In some embodiments, a CRISPR-Cas effector protein (e.g., a Type II or Type V CRISPR-Cas effector protein), a reverse transcriptase, and an extended guide nucleic acid can form a complex or are comprised in a complex.

[0117] In some embodiments, an extended guide nucleic acid comprises an extended portion that includes a primer binding site and a reverse transcriptase template, wherein the reverse transcriptase template comprises the modification (e.g., edit) to be incorporated into a target nucleic acid. In some embodiments, an extended guide nucleic acid comprises, at its 3' end, a primer binding site and a modification (e.g., an edit) to be incorporated into the target nucleic acid (e.g., a reverse transcriptase template). In some embodiments, an extended guide nucleic acid comprises: (1) a sequence that interacts (e.g., recruits and / or binds) with a CRISPR-Cas effector protein (e.g., a CRISPR-Cas nuclease), (2) a spacer having substantial complementary to a first site on a target nucleic acid (e.g., a CRISPR RNA (crRNA) (a first crRNA) and / or tracrRNA+crRNA (sgRNA)), and (3) a nucleic acid encoded repair template (e.g., an RNA encoded repair template) comprising a primer binding site and an RNA template (e.g., that encodes the modification to be incorporated into the target nucleic acid). In some embodiments, an extended guide nucleic acid (e.g., an extended guide RNA) may comprise, 5'-3', a spacer sequence, a repeat sequence, and an extended portion, the extended portion comprising, 5' to 3', a reverse transcriptase template and a primer binding site. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', a spacer sequence, a repeat sequence and an extended portion, the extended portion comprising, 5' to 3', a primer binding site and a reverse transcriptase template. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', an extended portion, a spacer sequence, and a repeat sequence, wherein the extended portion comprises, 5' to 3', a reverse transcriptase template and a primer binding site. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', an extended portion, a spacer sequence, and a repeat sequence, wherein the extended portion comprises, 5' to 3', a primer binding site and a reverse transcriptase template.

[0118] According to some embodiments, an extended guide nucleic acid (e.g., a pegRNA) may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiments, an extended guide nucleic acid comprises a primer binding site (PBS) optionally having a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 nucleotides and a reverse transcriptase template (RT template) sequence optionally having a sequence of 65 nucleotides or more. In some embodiments, a PBS of an extended guide nucleic acid has a sequence of less than 15 nucleotides and has a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides (e.g., a sequence of 5 or 6 nucleotides in length). The RT template sequence may be after the PBS sequence in the 5' to 3' direction. In some embodiments, the RT template sequence of the extended guide nucleic acid has a length of greater than 65 nucleotides and may comprise about 50 or more nucleotides of heterology relative to the target site (e.g., target nucleic acid), followed by about 15 or more nucleotides of homology relative to the target site. In some embodiments, the RT template sequence of the extended guide nucleic acid is after the PBS sequence and the RT template sequence has a length of greater than 65 nucleotides with the sequence including more than 50 nucleotides of heterology relative to the target site, followed by more than 15 nucleotides of homology relative to the target site. Accordingly, in some embodiments, when the extended guide nucleic acid is reverse transcribed, the resulting newly transcribed sequence may hybridize and / or is configured to hybridize with the unnicked strand of the target site, which may thereby create a heteroduplex DNA with a large insertion into the newly synthesized strand. Upon repair of this mismatched DNA, the resultant repaired DNA may contain a large insertion (e.g., greater than 50 nucleotides) of DNA sequence. In some embodiments, the method may provide a large deletion (e.g., greater than 50 nucleotides) of DNA sequence. In some embodiments, the PBS and the 15 or more nucleotides of homology to the target site may comprise homology arms, which may serve to insert the heterology into the target site optionally using homology directed repair. The inserted DNA may correspond to any functional sequence of DNA such as, but not limited to: a functional transgene; a fragment of DNA that is inserted into a gene in a way that, when the gene is transcribed, would produce a hairpin RNA that is sufficient to silence homologous genes through RNAi; and / or one or more functional site-specific recombination sites, e.g., lox, frt, which could then be used in subsequent Cre or Flp mediated site-specific recombination processes. In some embodiments, an extended guide nucleic acid may be too large to produce using a PolIII promoter in vivo. In some embodiments, an extended guide nucleic acid may be operatively associated with and / or produced using a PolII promoter. In some embodiments, a DNA binding polypeptide (e.g., a DNA binding domain) and / or DNA endonuclease may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiments, a DNA binding domain and / or DNA endonuclease is a CRISPR Cas polypeptide such as a Cas9 nickase, a nicking variant of another CRISPR Cas polypeptide, or Cas 12a.

[0119] In some embodiments, two extended guide nucleic acids (e.g., pegRNAs) may be used (e.g., an editing system may comprise two extended guide nucleic acids). One or both of the two extended guide nucleic acids may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. The two extended guide nucleic acids may comprise a primer binding site (PBS) optionally having a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides and a reverse transcriptase template (RT template) sequence optionally having a sequence of 50 nucleotides or more. The RT template sequences of the two extended guide nucleic acids may be complementary to each other and as such the polynucleotides that are respectively reverse transcribed from each the RT templates will be complementary to each other and will be able to hybridize with each other. This may allow for the intermediates that are produced by this system and / or method to join together two sections of DNA that are otherwise separated by more than 50 nucleotides, e.g., within a chromosome, or that are positioned on two separate pieces of DNA, e.g., on two different chromosomes. After repair of the intermediates, the resultant products may produce, depending on the design of the RT template, large deletions, large inversions, or inter-chromosomal recombinations. Since all of these products are produced by homology directed repair, the products may be predictably precise and / or reproducible. In some embodiments, a DNA binding polypeptide (e.g., a DNA binding domain) and / or DNA endonuclease may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiment, a DNA binding polypeptide and / or DNA endonuclease is a CRISPR Cas polypeptide such as a Cas9 nickase, a similar nicking variant of another CRISPR Cas polypeptide, or Casl2a. In some embodiments, a DNA binding polypeptide and / or DNA endonuclease is a Cas9 nuclease, a similar nuclease from another CRISPR Cas polypeptide, or Casl2a. Using a nuclease (rather than a nickase) may facilitate the intra- or interchromosomal recombination processes through single-strand annealing of the more than 50 nucleotide 3’ overhangs that would be produced at each of the two target sites corresponding to the two pegRNA target nucleic acids. In some embodiments, an editing system comprises one extended guide nucleic acid and a guide nucleic acid that is devoid of a reverse transcriptase template and / or primer binding site.

[0120] An extended guide nucleic acid may comprise a CRISPR nucleic acid (e.g., CRISPR RNA, CRISPR DNA, crRNA, crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid; and (b) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template), wherein the RT template encodes a modification to be incorporated into the target nucleic acid. The CRISPR nucleic acid may be a Type II or TypeV CRISPR nucleic acid and / or the tracr nucleic acid may be any tracr corresponding to the appropriate Type II or Type V CRISPR nucleic acid. In some embodiments, an extended guide nucleic acid comprises: (i) a Type V CRISPR nucleic acid or a Type II CRISPR nucleic acid (e.g., a Type II or Type V CRISPR RNA, Type II or Type V CRISPR DNA, Type II or TypeV crRNA, or Type II or Type V crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid (e.g., a Type II or Type V tracrRNA, Type II or Type V tracrDNA); and (ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template), wherein the Type V CRISPR nucleic acid or Type II CRISPR nucleic acid comprises a spacer that binds to a first strand (e.g., the target strand) of a target nucleic acid (e.g., the spacer is complementary to a portion of consecutive nucleotides in the first strand of the target nucleic acid) and the primer binding site binds to the first strand (e.g., target strand). In some embodiments, the extended portion can be fused to either the 5' end or 3' end of the CRISPR nucleic acid (e.g., from 5' to 3': repeat-spacer-extended portion or extended portion-repeat-spacer) and / or to the 5' or 3' end of the tracr nucleic acid. In some embodiments, the extended portion of an extended guide nucleic acid comprises, 5' to 3', an RT template (RTT) and a primer binding site (PBS) (e.g., 5’-crRNA-spacer-RTT(edit encoded)-PBS-3’) or comprises 5' to 3' a PBS and RTT, depending on the location of the extended portion relative to the CRISPR nucleic acid of the extended guide nucleic acid (e.g., 5’-crRNA-spacer-PBS-RTT(edit encoded)-3’). For example, in some embodiments, an extended portion of the extended guide nucleic acid may comprise, 5' to 3', an RT template and a primer binding site (when the extended guide is linked to the 3' end of the CRISPR nucleic acid). In some embodiments, an extended portion of the extended guide may comprise, 5' to 3', a primer binding site and an RT template (when the extended guide is linked to the 5' end of the CRISPR nucleic acid).

[0121] In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and a primer binding site of an extended guide nucleic acid binds to the second strand (e.g., the non-target, top strand) of the target nucleic acid. In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and a primer binding site of an extended guide nucleic acid binds to the first strand (e.g., binds to the target strand, optionally the same strand to which a CRISPR-Cas effector protein is recruited, bottom strand) of the target nucleic acid. In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and the primer binding site of an extended guide nucleic acid binds to the second strand (e.g., the non-target strand, optionally the opposite strand from that to which the CRISPR-Cas effector protein is recruited) of the target nucleic acid. In some embodiments, a reverse transcriptase (RT) may add to thetarget strand of a target nucleic acid (e.g., the strand to which the spacer of the CRISPR nucleic acid of the extended guide nucleic acid is complementary and to which the CRISPR-Cas effector protein is recruited). In some embodiments, the reverse transcriptase (RT) adds to the non-target strand of a target nucleic acid (e.g., the strand that is complementary to the strand to which the spacer of the CRISPR nucleic acid is complementary and to which the CRISPR- Cas effector protein is recruited). Example methods and editing systems are described in International Patent Publication No. WO 2021 / 092130, International Patent Publication No. WO 2022 / 098993, and U.S. Patent Application Publication Nos. 2021 / 0147862, 2021 / 0130835, 2021 / 0147862, and 2022 / 0145334, each of which are incorporated herein by reference in their entirety.

[0122] The RT template of an extended guide nucleic acid may encode one or more modification(s) (e.g., edit(s)) to be incorporated into a target nucleic acid. The one or more modification(s) may be located in any position within an RT template (e.g., where the position location may be relative to the position of a protospacer adjacent motif (PAM) of the target nucleic acid). In some embodiments, an RT template has a modification at one or more positions from -1 to 23 (e.g., -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) relative to the position of a protospacer adjacent motif (PAM) (e.g., TTTG) in a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. In some embodiments, an RT template may comprise a modification located at nucleotide position 4 to nucleotide position 17 (e.g., position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a PAM of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 10 to nucleotide position 17 (e.g., position 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a PAM of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 12 to nucleotide position 15 (e.g., position 12, 13, 14, or 15) of the RT template relative to the position of a PAM of a target nucleic acid.

[0123] In some embodiments, an extended portion of an extended guide nucleic acid may comprise, 5' to 3', an RT template and a primer binding site (e.g., when the extended portion is linked to the 3' end of a CRISPR nucleic acid). In some embodiments, an extended portion of an extended guide nucleic acid may comprise, 5' to 3', a primer binding site and an RT template (RTT) (e.g., when the extended portion is linked to the 5' end of the CRISPR nucleic acid). In some embodiments, an RT template may have a length of about 1 nucleotide to about 100nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more nucleotides, and any range or value therein), e.g., about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 1 nucleotide to about 25 nucleotides, about 1 nucleotide to about 30 nucleotides, about 1 nucleotide to about 35, 36, 37, 38, 39 or 40 nucleotides, about 1 nucleotide to about 50 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 35, 36, 37, 38, 39 or 40 nucleotides, about 5 nucleotides to about 50 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotide to about 20 nucleotides, about 8 nucleotide to about 25 nucleotides, about 8 nucleotide to about 30 nucleotides, about 8 nucleotide to about 35, 36, 37, 38, 39 or 40 nucleotides, about 8 nucleotide to about 50 nucleotides in length, about 8 nucleotides to about 100 nucleotides, about 10 nucleotide to about 15 nucleotides, about 10 nucleotide to about 20 nucleotides, about 10 nucleotide to about 25 nucleotides, about 10 nucleotide to about 30 nucleotides, about 10 nucleotide to about 36 nucleotides, about 10 nucleotide to about 40 nucleotides, about 10 nucleotide to about 50 nucleotides, about 10 nucleotides to about 100 nucleotides in length and any range or value therein. In some embodiments, the length of an RT template may be at least 8 nucleotides, optionally about 8 nucleotides to about 100 nucleotides. In some embodiments, the length of an RT template is 36, 37, 38, 39 or 40 nucleotides or less (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length, or any value or range therein (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length to about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length). In some embodiments, the length of an RT template may be at least 30 nucleotides, optionally about 30, 31, 32, 33, 34, 35, 36, 37,38, 39, or 40 nucleotides in length to about to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 nucleotides in length, or any range or value therein. In some embodiments, the length of an RT template may be about 36, 40, 44, 47, 50, 52, 55, 63, 72 or 74 nucleotides. Within the length of the RTT one or more modification(s) may be present. The one or more modification(s) may be located anywhere within the RTT, wherein the position of themodification may be described relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. In some embodiments, an RT template may comprise a modification located at nucleotide position 4 to nucleotide position 17 (e.g., position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 10 to nucleotide position 17 (e.g., position 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 12 to nucleotide position 15 (e.g., position 12, 13, 14, or 15) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid.

[0124] As used herein, a “primer binding site” (PBS) of an extended portion of an extended guide nucleic acid (e.g., a tagRNA) refers to a sequence of consecutive nucleotides that can bind to a region or “primer” on a target nucleic acid, e.g., is complementary to the target nucleic acid primer. As an example, a CRISPR Cas effector protein (e.g., a Type II or Type V, e.g., Cas 9 or Casl2a) may nick / cut the DNA and the 3' end of the cut DNA acts as a primer for the PBS portion of the extended guide nucleic acid. The PBS may be complementary to the 3' end of a strand of the target nucleic acid and may bind and / or may be configured to bind to either the target strand or non-target strand. A primer binding site can be fully complementary to the primer or it may be substantially complementary (e.g., at least 70% complementary (e.g., 70% or about 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%, 99.6%, 99.7%, 99.8%, 99.9% or more)) to the primer of a target nucleic acid. In some embodiments, the length of a primer binding site of an extended portion may be about 1 nucleotide to about 100 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 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 or more nucleotides, or any value or range therein), or about 4 nucleotide to about 85 nucleotides, about 10 nucleotide to about 80 nucleotides, about 20 nucleotide to about 80 nucleotides, about 25 nucleotides to about 80 nucleotides about 30 nucleotide to about 80 nucleotides, about 40 nucleotide to about 80 nucleotides, about 45nucleotide to about 80 nucleotides, about 45 nucleotide to about 75 nucleotides, or about 45 nucleotide to about 60 nucleotides, or any range or value therein. In some embodiments, the length of a PBS may be at least 30 nucleotides, optionally about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range or value therein. In some embodiments, the length of a PBS may be about 8, 16, 24, 32, 40, 48, 56, 64, 72, or 80 nucleotides.

[0125] In some embodiments, an RTT may have a length of about 35 nucleotides to about 75 nucleotides and a PBS may have a length of about 30 nucleotides to about 80 nucleotides, optionally wherein the PBS may comprise a length of about 8, 16, 24, 32, 40, 48, 56, 64, 72, or 80 nucleotides and the RTT may comprise a length of about 36, 40, 44, 47, 50, 52, 55, 63, 72 or 74 nucleotides, or any combination thereof of the RTT length and / or PBS length.

[0126] In some embodiments, an extended portion of an extended guide nucleic acid may be fused to either the 5' end or 3' end of a Type II or a Type V CRISPR nucleic acid (e.g., 5' to 3': repeat-spacer-extended portion, or extended portion-repeat-spacer) and / or to the 5' or 3' end of the tracr nucleic acid. In some embodiments, when an extended portion is located 5' of the crRNA, a Type V CRISPR-Cas effector protein is modified to reduce (or eliminate) selfprocessing RNAse activity.

[0127] In some embodiments, the extended portion of an extended guide nucleic acid may be linked to the Type II or Type V CRISPR nucleic acid and / or the Type II or Type V tracrRNA via a linker. In some embodiments, a linker have a length of about 1 to about 100 nucleotides or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more nucleotides in length, and any range therein (e.g., about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 9 to about 40, about 9 to about 50, about 9 to about 60, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 40 to about 100, about 50 to about 100, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more nucleotides in length (e.g., about 105, 110, 115, 120, 130, 140 150 or more nucleotides in length).

[0128] A guide nucleic acid and / or an extended guide nucleic acid may comprise one or more recruiting motifs as described herein, which may be linked to the 5' end and / or the 3' end of the guide nucleic acid and / or it may be inserted into the guide nucleic acid (e.g., within a hairpin loop of the guide nucleic acid). In some embodiments, an extended guide nucleic acid may be linked to an RNA recruiting motif. An extended guide nucleic acid and / or guide nucleic acid may be linked to one or to two or more RNA recruiting motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), optionally wherein the two or more RNA recruiting motifs may be the same RNA recruiting motif or different RNA recruiting motifs. In some embodiments, an RNA recruiting motif may be located on the 3' end of the extended portion of an extended guide nucleic acid (e.g., 5'-3', repeat-spacer-extended portion (RT template-primer binding site)-RNA recruiting motif). In some embodiments, an RNA recruiting motif may be embedded in the extended portion of an extended guide nucleic acid.

[0129] In some embodiments, an editing system comprises an extended guide nucleic acid that is linked to an RNA recruiting motif and a reverse transcriptase that is a reverse transcriptase fusion protein, wherein the reverse transcriptase fusion protein comprises a reverse transcriptase polypeptide fused to an affinity polypeptide that binds to the RNA recruiting motif, wherein the extended guide nucleic acid binds to a target nucleic acid and the RNA recruiting motif binds to the affinity polypeptide, thereby recruiting the reverse transcriptase fusion protein to the extended guide nucleic acid and contacting the target nucleic acid with the reverse transcriptase. In some embodiments, two or more reverse transcriptase fusion proteins may be recruited to an extended guide nucleic acid, thereby contacting the target nucleic acid with two or more reverse transcriptase fusion proteins.

[0130] As used herein, a “CRISPR-Cas effector protein” is a protein or polypeptide that cleaves, cuts, or nicks a nucleic acid; binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid); and / or that identifies, recognizes, or binds a guide nucleic acid as defined herein. In some embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, endonuclease, nickase, etc.) and / or may function as an enzyme. In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease. In some embodiments, a 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 (ss DNAse activity) or which has ss DNAse activity that has been reduced or eliminated, and / or comprises self-processing RNAse activity or which has self-processing RNAse activity that has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid. A CRISPR- Cas effector protein may be a Type I, II, III, IV, V, or VI CRISPR-Cas effector protein. In some embodiments, a CRISPR-Cas effector protein may 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, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein of the invention may be from a Type II CRISPR- Cas system or a Type V CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein may be a Type II CRISPR-Cas effector protein, for example, a Cas9 effector protein. In some embodiments, a CRISPR-Cas effector protein may be Type V CRISPR-Cas effector protein, for example, a Cast 2 effector protein.

[0131] Exemplary CRISPR-Cas effector proteins include, but are not limited to, a Cas9, C2cl, C2c3, Cast 2a (also referred to as Cpfl), Cast 2b, Cast 2c, Cast 2d, Casl2e, Cast 3 a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, 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 may be a Cas9, Casl2a (Cpfl), Casl2b, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2g, Casl2h, Casl2i, C2c4, C2c5, C2c8, C2c9, C2cl0, Casl4a, Casl4b, and / or Casl4c effector protein.

[0132] In some embodiments, a CRISPR-Cas effector protein useful with the invention may comprise a mutation in its nuclease active site and / or nuclease domain (e.g., a RuvC, HNH, e.g., a RuvC site of a Casl2a nuclease domain; e.g., a RuvC site and / or HNH site of a Cas9 nuclease domain). A CRISPR-Cas effector protein having a mutation in its nuclease active site and / or nuclease domain, and therefore, no longer comprising nuclease activity, is commonly 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) as compared to the same CRISPR-Cas effector protein without the mutation.

[0133] A CRISPR Cas9 effector protein or Cas9 useful with this invention may be any known or later identified Cas9 nuclease. In some embodiments, a Cas9 of the present invention may be a protein from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus'), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., O enococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp.

[0134] In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus pyogenes and / or may recognize the PAM sequence motif NGG, NAG, NGA (Mali et al. (2013) Science 339(6121 ): 823 -826). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus thermophilus and / or may recognize the PAM sequence motif NGGNG and / or NNAGAAW (W = A or T) (See, e.g., Horvath et al. (2010) Science 327(5962): 167-170; Deveau et al. (2008) J. Bacteriol. 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. (2008) J. Bacteriol. 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus 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 S. aureus and / or may recognize the PAM sequence motif NGRRT (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from S. aureus and / or may recognize the PAM sequence motif NGRRV (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 that is derived from Neisseria meningitidis and / or may recognize the PAM sequence motif NGATT or NGCTT (R = A or G, V = A, G or C) (See, e.g., Hou et al. (2013) PNAS 110(35): 15644-15649). In the aforementioned embodiments in this paragraph, N in the PAM sequence motif can be any nucleotide residue, e.g., any of A, G, C or T. In some embodiments, the CRISPR-Cas effector protein may be a Cast 3a derived from Leptotrichia shahii and / or may recognize a protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif of a single 3’ A, U, or C, which may be located within the target nucleic acid.

[0135] A Type V CRISPR-Cas effector protein useful with embodiments of the invention may be any Type V CRISPR-Cas nuclease. Exemplary Type V CRISPR-Cas effector proteins include, but are not limited, to Casl2a (Cpfl), Casl2b, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2g, Casl2h, Casl2i, C2cl, C2c4, C2c5, C2c8, C2c9, C2cl0, Casl4a, Casl4b, and / or Casl4c nuclease. In some embodiments, a Type V CRISPR-Cas effector protein may be a Cast 2a. In some embodiments, a Type V CRISPR-Cas effector protein may be a nickase, optionally, a Cast 2a nickase.

[0136] In some embodiments, the CRISPR-Cas effector protein may be a Type V Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease. Cast 2a differs in several respects from the more well-known Type II CRISPR Cas9 nuclease. For example, Cas9 recognizes a G-rich protospacer-adjacent motif (PAM) that is 3' to its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, targetnucleic acid, target DNA) (3'-NGG), while Casl2a recognizes a T-rich PAM that is located 5' to the target nucleic acid (5'-TTN, 5'-TTTN). In fact, the orientations in which Cas9 and Casl2a bind their guide RNAs are very nearly reversed in relation to their N and C termini. Furthermore, Cast 2a enzymes use a single guide RNA (gRNA, CRISPR array, crRNA) rather than the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA) found in natural Cas9 systems, and Casl2a processes its own gRNAs. Additionally, Casl2a nuclease activity produces staggered DNA double stranded breaks instead of blunt ends produced by Cas9 nuclease activity, and Cast 2a relies on a single RuvC domain to cleave both DNA strands, whereas Cas9 utilizes an HNH domain and a RuvC domain for cleavage.

[0137] A CRISPR Casl2a effector protein useful with this invention may be any known or later identified Casl2a (previously known as Cpfl) (see, e.g., U.S. Patent No. 9,790,490, which is incorporated by reference for its disclosures of Cpfl (Casl2a) sequences). The term “Cast 2a” refers to an RNA-guided protein that can have nuclease activity, the protein comprising a guide nucleic acid binding domain and an active, inactive, or partially active DNA cleavage domain, thereby the RNA-guided nuclease activity of the Casl2a may be active, inactive or partially active, respectively. In some embodiments, a Casl2a useful with the invention may comprise a mutation in the nuclease active site (e.g., a RuvC site of the Casl2a domain). A Casl2a having a mutation in its nuclease domain and / or nuclease active site, and therefore, no longer comprising nuclease activity, is commonly referred to as deadCasl2a (e.g., dCasl2a). In some embodiments, a Casl2a having a mutation in its nuclease domain and / or nuclease active site may have impaired activity, e.g., may have reduced nickase activity.

[0138] In some embodiments, a CRISPR-Cas effector protein may be optimized for expression in an organism, for example, in an animal (e.g., a mammal such as a human), a plant, a fungus, an archaeon, or a bacterium. In some embodiments, a CRISPR-Cas effector protein (e.g., Casl2a polypeptide / domain or a Cas9 polypeptide / domain) may be optimized for expression in a plant.

[0139] In some embodiments, a CRISPR-Cas effector protein comprising a Cas9 domain (or a nucleic acid construct encoding the same) may be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, and may be in or may form a complex.

[0140] Likewise, a CRISPR-Cas effector protein may comprise a Casl2a domain (or other selected CRISPR-Cas nuclease, e.g., C2cl, C2c3, Casl2b, Casl2c, Casl2d, Casl2f, Casl2i, Casl2e, Casl3a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, 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), which may be used in combination with a Casl2a guide nucleic acid (or the guide nucleic acid for the other selected CRISPR-Cas nuclease) to modify a target nucleic acid, thereby editing the target nucleic acid.

[0141] A method of the present invention may comprise producing a composite plant, which includes a portion (e.g., one or more root(s)) that is modified and another portion that is wild-type (e.g., non-transgenic). In some embodiments, a composite plant includes a transgenic root and one or more other portion(s) of the plant are non-transgenic. In some embodiments, a composite plant includes a transgenic root and optionally non-transgenic aerial tissue and / or non-transgenic roots. In some embodiments, a composite plant is produced from a wounded plant tissue comprising root forming competent cells and optionally meristem tissue following introduction of a polynucleotide from and / or upon infection by an Agrobacterium strain (e.g., an Agrobacterium tumefaciens strain).

[0142] A method of the present invention may comprise producing a modified plant or plant part from a modified root and / or modified shoot produced according to embodiments of the present invention. In some embodiments, the modified plant or plant part comprises a gene that confers resistance to a selection agent and / or polynucleotide of interest (e.g., a polynucleotide encoding or comprising a portion or all of an editing system). In some embodiments, the modified plant or plant part may be a transgenic plant or plant part. In some embodiments, the modified plant or plant part plant may be non-transgenic and / or transgene free. In some embodiments, the modified plant or plant part may be produced from a transgene- free root that comprises cells including a modified nucleic acid (e.g., edited gene), wherein the modified nucleic acid is provided and / or produced in accordance with a method of the present invention (e.g., as a result of introducing a polynucleotide of interest into a cell of wounded plant tissue as descried herein). In some embodiments, a modified plant or plant part is fully modified in that all cells of the plant or plant part include a modified nucleic acid. A transgene- free, edited plant may be produced from a modified root comprising cells including the modified nucleic acid (e.g., an edited gene). In some embodiments, a modified plant is produced from a modified root and / or modified shoot, and the modified plant may comprise cells including a modified nucleic acid.

[0143] A method herein may comprise screening for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. In some embodiments, a root is screened for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. In some embodiments, a progeny plant isscreened for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. Screening may comprise phenotyping. In some embodiments, a method herein may comprise performing molecular screening such as molecular screening on a root, shoot, and / or progeny plant. The molecular screening may be performed before and / or after phenotyping. Methods of screening are known to those of skill in the art and include, but are not limited to, evaluating gene expression levels such as by using quantitative PCR (qPCT) or sequencing and / or by physical and / or visual evaluation of the phenotype.

[0144] In some embodiments, a method and / or culturing step of the present invention may be used to produce a modified (e.g., edited) plant, plant part, and / or cell thereof. In some embodiments, the plant or plant part is from a species of plant that produces suckers. In some embodiments, a plant may produce a sucker naturally and / or by induction (e.g., by excess water, prolonged or repeated lack of water, saline conditions, and / or exposure to a growth regulator such as naphthylphthalamic acid). Plants used in a method of the present invention and / or that may be modified using a method of the present invention include, but are not limited to, species of plants from the genus Actinidia, Allium, Amaranthus, Amelanchier, Anacardium, Ananas, Arabidopsis, Arachis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica, Capsicum, Carica, Castanea, Quercus, Fagus, Chenopodium, Chusquea, Citrus, Colocasia, Cornus, Corylus, Cucumis, Cydonia, Dioscorea, Diospyros, Fargesia, Ficus, Forsythia, Fragaria, Gymnocladus, Heavea, Helianthus, Hippophae, Hordeum, Illicium, Ipomoea, Juglans, Laurus, Lens, Malus, Manihot, Morus, Musa, Oryza, Panicum, Papaver, Passiflora, Phyllostachys, Pisum, Plantago, Poa, Populus, Prunus, Pseudosasa, Pyrus, Rheum, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Sorghum, Spinacia, Syringa, Triticum, Vaccinium, Vigna, Vitis, Zingiber, or Zizyphus. In some embodiments, a plant or plant part used in a method of the present invention is in the genus Actinidia, Allium, Amelanchier, Anacardium, Arabidopsis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica, Capsicum, Castanea, Quercus, Fagus, Citrus, Cornus, Corylus, Cucumis, Cydonia, Diospyros, Ficus, Forsythia, Gymnocladus, Heavea, Hippophae, Illicium, Ipomoea, Juglans, Laurus, Malus, Morus, Oryza, Papaver, Passiflora, Populus, Prunus, Pyrus, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Spinacia, Syringa, Vaccinium, or Zizyphus. In some embodiments, a plant or plant part used in a method of the present invention is in the genus Carica, Fragaria, Pisum, Sorghum, Vaccinium, or Vitis. In some embodiments, a plant or plant part used in a method of the present invention is in the genus Ananas, Arachis, Chusquea, Colocasia, Cynodon, Dioscorea, Fargesia, Helianthus, Hordeum, Manihot, Musa, Panicum, Phyllostachys, Plantago, Poa, Pseudosasa, Rheum, Triticum, Vigna, or Zingiber.

[0145] In some embodiments, the modified plant or plant part is an Adzuki bean, almond, amaranth, apple, apricot, Arabidopsis, artic bramble, asparagus, bambara groundnut, bamboo, banana, barberry, barley, bay leaf, beech, bermudagrass, birch, blackberry, blueberry, bluegrass (e.g., Kentucky bluegrass), brinjal, broccoli, cashew, cassava, cherry, chestnut, Chinese broccoli, chokeberry, citrus, cranberry, cucumber, currant, dogwood, eggplant, elderberry, fig, forsythia, garlic, ginger, gooseberry, grape, hazelnut, horseradish, Japanese persimmon, Jerusalem artichoke, jujube, Kentucky coffeetree, kiwifruit, kohlrabi, lentil, lilac, millet (e.g., pearl millet, foxtail millet), mulberry, nectarine, oak, onion, papaya, passion fruit, pawpaw, pea, peach, peanut, pear, pepper, pineapple, plantain, plum, poplar, poppy, quince, quinoa, raspberry (e.g., red raspberry, black raspberry), rhubarb, rose, rubber tree, sea buckthorn, serviceberry, sorghum, spinach, star anise, strawberry, sumac, sweet potato, switchgrass, rice, taro, tomato, walnut, wheat or yam. In some embodiments, the modified plant or plant part is an almond, apple, apricot, Arabidopsis, asparagus, barberry, bay leaf, beech, birch, blackberry, blueberry, brinjal, broccoli, cashew, cherry, chestnut, Chinese broccoli, chokeberry, citrus, cucumber, currant, dogwood, eggplant, elderberry, fig, forsythia, garlic, gooseberry, hazelnut, horseradish, Japanese persimmon, jujube, Kentucky coffeetree, kiwifruit, kohlrabi, lilac, mulberry, nectarine, oak, onion, passion fruit, pawpaw, peach, pear, pepper, plum, poplar, poppy, quince, raspberry (e.g., red raspberry, black raspberry), rose, rubber tree, sea buckthorn, serviceberry, spinach, star anise, sumac, sweet potato, rice, tomato, or walnut. In some embodiments, the modified plant or plant part is a blackberry, raspberry (e.g., red raspberry or black raspberry), artic bramble, or cherry. In some embodiments, the modified plant or plant part from a plant in the Rubus family. Exemplary plants in the Rubus family include, but are not limited to, Rubus allegheniensis, Rubus occidentalis, Rubus odoratus, and Rubus fruticosus. In some embodiments, the modified plant or plant part is from a plant in the Prunus family.

[0146] The present invention further comprises a kit or kits to carry out the methods of this invention. A kit of this invention can comprise reagents, buffers, and apparatus for mixing, measuring, sorting, labeling, etc., as well as instructions and the like as would be appropriate for modifying a target nucleic acid.

[0147] In some embodiments, the invention provides a kit for comprising an Agrobacterium strain and / or one or more nucleic acid constructs of the invention, and / or expression cassettes and / or vectors and / or cells comprising the same as described herein, with optional instructions for the use thereof. In some embodiments, a kit may further comprise a CRISPR-Cas guide nucleic acid (corresponding to the CRISPR-Cas effector protein encoded by the polynucleotideof the invention) and / or expression cassettes and / or vectors and or cells comprising the same. In some embodiments, a guide nucleic acid may be provided on the same expression cassette and / or vector as one or more nucleic acid constructs of the invention. In some embodiments, the guide nucleic acid may be provided on a separate expression cassette or vector from that comprising the one or more nucleic acid constructs of the invention.

[0148] Accordingly, in some embodiments, kits are provided comprising an Agrobacterium strain and / or a nucleic acid construct comprising (a) a polynucleotide(s) as provided herein and (b) a promoter that drives expression of the polynucleotide(s) of (a). In some embodiments, the kit may further comprise a nucleic acid construct encoding a guide nucleic acid, wherein the construct comprises a cloning site for cloning of a nucleic acid sequence identical or complementary to a target nucleic acid sequence into backbone of the guide nucleic acid.

[0149] The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.EXAMPLESExample 1: Generation and selection of transgenic roots from blackberry

[0150] For blackberry, transformation explants contained a shoot meristem or a meristem cell. Specifically, tissues used for transformation (i.e., explants or transformation explants) were prepared from micropropagated shoot clusters that contained multiple shoot meristems. Multiple tissue ages were used and were suitable for this method; e.g., testing with shoot tissues 3 -weeks and 6-weeks post-introduction into culture both yielded composite plants that included a transgenic root. For transformation, plant tissues were fragmented, manually or using a blender (Table 1), to provide wounded plant tissue (explants). Wounded plant tissue that included individual nodal and / or apical shoot meristems was used for transformation to provide transformed roots that would develop into full root systems. By comparison, while plant tissue lacking a nodal or apical shoot meristem (e.g., leaf tissue) could be inoculated and would occasionally develop transformed roots, these transformed roots did not grow well and did not develop into full root systems. Generally, wounded plant tissue was inoculated with a disarmed Agrobacterium tumefaciens strain, e.g., during blender fragmentation, in a 0.8 OD solution of Agrobacterium tumefaciens followed by 15 minutes of sonication, 1 minute of vacuum at 25 inHg, and centrifugation at 677 g for 5 minutes. Inoculated plant tissue was then poured througha strainer, blotted on sterile filter paper, and then cultured for 5 to 6 days to facilitate transformation of the plant tissue. The inoculation and culture medium both contained lipoic acid and acetosyringone. After culturing, the transformed plant tissue was transferred to a root induction medium suitable for plant tissue growth and rooting. Transformed plant tissue was either staked into the root induction medium or was gently distributed across the surface without being pressed into the medium. Over a period of 4-8 weeks, the transformed plant tissue grew and recovered from the transformation process and began to form roots. Using low concentrations of kanamycin in the root induction medium, shoot growth was minimally inhibited. Non-transgenic roots would initially form from developing wild-type shoots; however, the non-transgenic roots were largely inhibited as they grew into the root induction medium and remained short (less than 1-2 cm). Transgenic roots formed from the transformed plant tissue almost always exhibited a sector (i.e., a region) of one or more transgenic cell(s) and a sector of one or more non-transgenic cell(s) (e.g., thereby providing a chimeric root) as confirmed by visual marker expression. In some cases, multiple separate sectors of presumably different transformation events could be observed within one chimeric root, alongside one or more non-expressing sector(s) (i.e., a region including one or more cell(s) that do not include a transgene such that the region includes one or more non-transgenic cell()).In other cases, fully transgenic roots were formed. As the transgenic roots grew into the root induction medium, they displayed a clear resistance phenotype as compared to wild-type roots (e.g., non- transgenic roots) growing from a wild-type shoot. Generally, lateral roots that formed from a transgenic sector of a chimeric root would grow and elongate preferentially into the root induction medium, while lateral roots forming from a non-transgenic sector of the chimeric root remained heavily stunted and would not develop further. This process was demonstrated using kanamycin selection at 25 mg / L or 50 mg / L in the root induction medium (Table 1 and Table 2), as well as with imazamox selection in the root induction medium at 0.25 pM or 0.5 pM using a transgenic acetolactate synthase (ALS) resistance vector. After 8 weeks of culture on and / or in the root induction medium, composite plants containing a wild-type shoot and a transgenic root system that included transgenic roots having a length of at least 4 cm and minimal wild-type roots were obtained. These composite plants were advanced to the greenhouse or used for in vitro shoot induction from roots.

[0151] Table 1. Composite plants were successfully generated using different kanamycin doses in PP0216 basal medium with 0.1 mg / L indole-3 -butyric acid (IBA) and other additives and / or antibiotics typically used in plant transformation, using either hand cut or blended(blend) plant tissues of either 3 week (3 w) or 6 week (6 w) blackberry (BK-13) explants (625 explants / treatment) post-introduction into culture, with 0% contamination. Kan25 = 25 mg / L kanamycin; Kan50 = 50 mg / L kanamycin

[0152] Table 2. Composite plants were successfully generated using 3 week explants with kanamycin doses of 25 mg / L (Kan25) or 50 mg / L (Kan50) in blackberry (Genotype BK-13) on PP0216 basal medium with 0.1 mg / L indole-3 -butyric acid (IB A) and other additives and / or antibiotics typically used in plant transformation, with 0% contamination.

[0153] Transgenic roots generated from transformed tissue were able to be induced and grown on a variety of media suitable for root induction (Table 3). Higher levels of auxin were evaluated for increased transgenic root induction, which tended to decrease as auxin concentration increased (Table 4). The standard concentration of indole-3 -butyric acid (IBA) used in the medium was 0.1 mg / L; however, auxin was not required for transgenic root induction since transgenic roots could be induced on root induction medium containing no growth regulators. Kanamycin dose in the root induction medium was selected based on explant size and age to prevent wild-type root overgrowth, and to minimize selective effects on the wild-type shoot. Spectinomycin selection did not perform as well in blackberry compared to kanamycin (Table 5). While not wishing to be bound by any particular theory, it is believed that spectinomycin had a heavy impact on blackberry shoot meristem development, limiting endogenous factors that promote root induction and full composite plant recovery. Some transgenic blackberry roots were induced using spectinomycin, but the root system development was limited and not ideal for subsequent shoot induction from the roots. For larger explants, 50 mg / L kanamycin was used, while smaller explants used 25 mg / L kanamycin, which maximized composite plant formation with transgenic root systems. If the selection level of kanamycin was too low, wild-type roots tended to grow resulting in a lower rate of transgenic root formation. The ideal root induction medium for generating composite plants contained enough kanamycin to effectively allow for preferential growth of transgenic roots (e.g., fully transgenic roots and / or chimeric roots) over wild-type roots, without negatively impactinggrowth and development of the wild-type shoot that supports the transgenic root system development.

[0154] Table 3. Multiple basal media were suitable for composite plant formation in blackberry (BK-13) using 3 week explants, 0.1 mg / L indole-3 -butyric acid (IB A), and 25 mg / L kanamycin with other additives and / or antibiotics typically used in plant transformation, with0% contamination.

[0155] Table 4 Increases in indole-3 -butyric acid (IB A) concentration appeared to lead to reduced composite plant recovery using 3 week blackberry (BK-13) explants (1250 explants / treatment) and PP0216 basal medium with 25 mg / L kanamycin and other additives and antibiotics typically used in plant transformation, with 0% contamination.

[0156] Table 5. Spectinomycin selection was not well-suitable for healthy composite plant formation using 3 week blackberry (BK-13) explants (1250 explants / treatment) and 0.1 mg / L indole-3 -butyric acid (IB A) with other additives and antibiotics typically used in plant transformation, with 0% contamination.

[0157] In some cases, Agrobacterium tumefaciens including rolABC genes were used to enhance the rate of composite plant formation with or without use of a selection agent. Root induction medium with and without IBA was used following inoculation with the Agrobacterium tumefaciens including the rolA gene, rolB gene, and rolC gene (i.e., the rolABC genes), and it was determined that IBA was not required (Table 6). Transgenic roots with a transgenic sector and a non-transgenic sector (i.e., chimeric roots) were still observed when using Agrobacterium tumefaciens including rolABC genes, but were less common than when using Agrobacterium tumefaciens without rolABC genes. Instead, fully transgenic roots were more common when using Agrobacterium tumefaciens including rolABC genes. Without wishing to be bound to any particular theory, it is believed that this was because of the excessive branching that tends to occur with rolABC roots. This excessive branching habit and “thin root” phenotype was also observed when Agrobacterium rhizogenes was used in this method. Notably, inclusion of rolABC in the transformation vector could facilitate composite plant recovery with minimal system optimization, as demonstrated in black raspberry (Table 7).

[0158] Table 6. Indole-3 -butyric acid (IBA) is not necessary for composite plant formation; composite plant formation can be achieved without the use of rolABC genes with kanamycin selection; and the use of Agrobacterium tumefaciens with rolABC genes enhances composite plant formation in Blackberry (BK-13) with or without IBA and / or kanamycin using a PP0216 basal medium with other additives and / or antibiotics typically used in plant transformation and 3 week explants (625 explants / treatment). (rolabc) indicates the use of Agrobacterium tumefaciens with the rolABC genes. No reference to rolabc indicates use of an Agrobacterium tumefaciens strain devoid of the rolABC genes.

[0159] Table 7. The use of Agrobacterium tumefaciens with rolABC genes can induce transgenic roots and composite plant formation in black raspberry (Genotype BR-14) explants (400 explants / treatment) using 0.1 m / g indole-3 -butyric acid (IB A) and 25 mg / L kanamycin in a PP0216 basal medium with other additives and / or antibiotics typically used in plant transformation, whereas a roM C-free system was not effective.Example 2: Generation and selection of transgenic roots from cherry

[0160] Sweetheart or Lapins cherry micropropagated shoots containing an apical and lateral meristems were used as starting material for a root-based transformation method, similar to the blackberry and black raspberry transformation method in Example 1. One to two cm shoot apical meristems from in vitro propagated plant tissue were excised using a scalpel and forceps. Leaves of 3 cm or greater were excised, to facilitate easier staking of the shoot posttransformation. Immediately after shoot tip excision, shoot tips were placed into a blackberry inoculation medium containing Agrobacterium rhizogenes disarmed strain DS 101.1 at an OD of 0.8, containing acetosyringone and lipoic acid. Up to 30 shoot tips were placed into 50 ml inoculum in 100 x 25 mm plates, and were allowed to rest in the solution for 30 min. After inoculation, shoot tip explants were removed from the inoculum using forceps and were placed onto sterile paper towels for approximately 5 sec before being transferred into 100 x 25 mm - culture plates containing a single filter paper and 1.5 ml of inoculum medium without Agrobacterium. Culturing was performed at 23 °C for 5 days, after which the transformed shoot tip explants were staked into root induction medium containing 25 mg / L kanamycin selection. About 0.25-0.5 cm of stem was pushed below the surface of the root induction medium, but shoot tips were kept above the root induction medium surface. Approximately 2 weeks after inoculation, transgenic roots (e.g., fully transgenic roots and / or chimeric roots) could be observed growing into the root induction medium. Transgenic roots were observed on wildtype shoot tip explants at a frequency of up to one out of every ten shoot tips (~ 10%). Additional transgenic roots were observed to emerge for up to 8 weeks after inoculation. Table 8 provides the results for composite plants formed from cherry (Genotype SH) explants. Compared to Example 1, the main deviations between this method and that used for blackberry (Example 1) were: (1) hand cutting of explants rather than blending (some explants were hand cut in Example 1 and others blended), (2) shorter co-culture duration of 5 days, and (3) the media used for each of the steps in the process. Root forming competent cells were able to be transformed and transgenic / chimeric roots induced from a broad range of cherry tissues. By using a blender to fragment initial shoot clusters, many transgenic / chimeric roots could be generated originating from excised or intact stems, shoots, leaves or petioles (Table 9). Both the hand-cut method and blender method were suitable for generating transgenic / chimeric roots in genotypes Sweetheart and Lapins.

[0161] A disarmed, non-ro / ABC strain of Agrobacterium tumefasciens AB62 was also successfully used in this method, and performed similarly or better than the disarmed A. rhizogenes strain DS 101.1.

[0162] Table 8. Composite plants formed from cherry (Genotype SH) explants (75 explants / treatment) using disarmed Agrobacterium rhizogenes strain and medium PP0432, with 0% contamination.

[0163] Table 9. Transgenic / chimeric roots (Genotype SH) induced from shoot explants using a hand-cut method, and from many explant types using blender-processing of shoot clusters using disarmed Agrobacterium tumefaciens strain and medium PP0432, with 0% contamination.

[0164] Example 3: Generation and selection of transgenic roots from broccoli

[0165] Seeds of B. oleracea ‘De Cicco’ were surface sterilized and introduced into sterile culture on PP0040 under 16L / 8D conditions at 25°C approximately one week prior to transformation. Using a scalpel and forceps, germinated shoots were excised from the root system by cutting through the hypocotyl above the root radicle such that no pre-existing root system remained. The remaining explant contained hypocotyl tissue, cotyledons, and a shoot apical meristem along with several newly emerging leaves. Transformation explants were then immersed in 50 ml of a 0.8 OD A. tumefaciens resuspension in liquid co-culture medium. Coculture medium contained lipoic acid and acetosyringone. Explants were immersed in inoculum for up to 30 m, and were then placed onto sterile paper towels prior to transfer into 100 X 25 mm plates containing sterile Whatman filter paper wetted with 2 ml co-culture medium for 5 d. Half of the 500 explants were inoculated as described above, while the other half was subjected to 30 second sonication in a 50 ml tube prior to blotting on sterile paper towels. After transformation, each inoculation treatment was further divided evenly between two root induction media, one containing no growth regulators and the other containing 0.1mg / 1 NAA. Both media were comprised of PP0040 with 200 mg / 1 timentin and 10 mg / 1 kanamycin as post-autoclave additives. Transformation explants were staked approximately 0.5 cm into the rooting medium, so that the cut surfaces were below the medium and the shoot apical meristems were above the surface of the medium. Transgenic root data collection was performed after 35 days in root induction medium (Table 10).

[0166] Table 10. Composite plants formed from B. oleracea ‘De Cicco’ (125 explants / treatment) in all inoculation and media treatments using A. tumefaciens and medium PP0040, with 0% contamination. Composite Plant Rate = 100* (Composite Plants / Number of Explants)

[0167] Example 4: Blackberry transgenic shoot regeneration from transgenic roots

[0168] Wild-type roots were used to test different media treatments to induce shoots from excised roots, with limited success (Tables 11-14). Surprisingly, in contrast to what was observed when only excised roots were used, wild-type intact plants that included an intact wild-type shoot and wild-type root were responsive to multiple shoot induction media, forming new shoots from the intact root system of the wild-type plant (Table 15). Media containing forchlorfenuron (4-CPPU) induced new shoots from roots at a higher frequency than other cytokinins or cytokinin-like compounds, while generating less uncontrolled callus growth. Two mg / L 4-CPPU demonstrated maximum new shoot induction from roots of an intact plant. New shoots induced on all media tested were excised from the root systems after between 5 and 6 weeks, moved to a shoot development medium, and were subsequently moved to a rootinduction medium for full plant recovery. Further media development revealed that 2 mg / L 4- CPPU combined with 0.5 mg / L BAP (6-benzyl amino purine) allowed for faster plant recovery and better shoot development. As such, transfer of the new excised shoots to a shoot development medium was not required and the excised shoot could be directly transferred to a root induction medium (Table 16).

[0169] Table 11. Wild-type roots excised from BK-13 failed to support shoot induction under various media conditions.TDZ, Thidiazuron.

[0170] Table 12. Wild-type roots excised from BK-13 failed to support shoot induction under various media conditions.aAuxin Transport Inhibitors.bAnti-auxin.TDZ, Thidiazuron; BAP, 6-Benzyl amino purine; IBA, Indole-3 -butyric acid; TIBA, 2,3,5- Triiodobenzoic acid; PCIB, a(p-Chlorophenoxy)isobutyric acid.

[0171] Table 13. Wild-type roots excised from BK-13 failed to support shoot induction inPP0040 basal medium.TDZ, Thidiazuron; NAA, 1 -Naphthaleneacetic acid; IBA, Indole-3 -butyric acid.

[0172] Table 14. Wild-type roots excised from BK-13 failed to support shoot induction from 24 different source media.BAP, 6-Benzyl amino purine; IB A, Indole-3 -butyric acid; TDZ,

[0173] Table 15. Shoot induction from blackberry BK-13 roots was observed using intact wild-type plants containing both a shoot and root as starting material, and a PP0040 base medium with antibiotics typically used in plant transformation after 6 weeks of shoot induction.a10 Initial composite plants / treatment;b20 Initial composite plants / treatment.BAP, 6-Benzyl amino purine; IB A, Indole-3 -butyric acid; TDZ, Thidiazuron; 2ip, 6-(y,y- dimethylallylamino)purine; 4-CPPU, forchlorfenuron; NPA, N-l -Naphthylphthalamic acid.

[0174] Table 16. Shoot induction from BK-13 roots was observed when wild-type intact plants containing a shoot and root were used as starting material in combination with a PP0040 base medium including antibiotics after 6 weeks of shoot induction.a20 Initial composite plants / treatment;b15 Initial composite plants / treatment.cControl growth regulator composed of 1 mg / L BAP, 0.5 mg / L TDZ, and 0.1 mg / L IB A.BAP, 6-Benzyl amino purine; IB A, Indole-3 -butyric acid; TDZ, Thidiazuron; 4-CPPU, forchlorfenuron.

[0175] Blackberry composite plants containing a wild-type shoot and transgenic roots (e.g., a fully transgenic root and / or chimeric root)were generated after 8 weeks of culture on root induction media. Composite plants were extracted from the root induction medium and placed onto shoot induction medium containing 2 mg / L 4-CPPU with or without 50 mg / L kanamycin (Table 17). After five weeks, transgenic shoots (e.g., expressing a reporter gene) induced from a transgenic cell of a transgenic root could be excised and either (i) moved to shoot development medium containing 50 mg / L kanamycin prior to being placed onto root induction medium that also contained 50 mg / L kanamycin, or (ii) moved directly to root induction medium without the intermediate step of being moved to shoot development medium. All shoots induced on shoot induction medium containing 50 mg / L kanamycin expressed zsGreen, indicating that this is a suitable selection strength to ensure transgenic plant recovery during the shoot induction process from the initial transgenic root systems. This method of regeneration and media used were also suitable for transgenic shoot induction from transgenic black raspberry roots that were grown following transformation using rolABC genes. As an alternative, medium containing 2 mg / L 4-CPPU and 0.5 mg / L BAP with 50 mg / L kanamycin can be used to shorten the transgenic plant recovery process by eliminating the need for a shoot development step after excision of a transgenic shoot from a composite plant for full transgenic plant production.

[0176] Table 17. Regeneration of transgenic, zsGreen expressing shoots induced from composite plants on four different shoot induction media, with PP0040 as the basal medium and including antibiotics typically used in plant transformation. 20 Composite plants were tested per medium treatment.

[0177] Not wishing to be bound by theory, it is believed that the process of shoot induction from roots originates from a few cells within the single- or two-celled pericycle layer. Thus, a transgenic root that is fully transgenic or that is chimeric (e.g., includes transgenic cells and non-transgenic cells) can be used to grow a shoot that includes only transgenic cells (i.e., a fully transgenic shoot), and the fully transgenic shoot may include edited, transgenic cells (e.g., a gene in the transgenic cell is edited). In addition, a chimeric root may be used to grow a shoot that includes only non-transgenic cells that are edited (e.g., one or more genes in the non- transgenic cells are edited), a shoot that includes only non-transgenic cells that are not edited, a shoot that includes only transgenic cells that are not edited, or a shoot that includes only cells that are transgenic and edited. Chimeric roots could be identified visually for the transgene (e.g., by zsGreen expression) and next generation sequence (NGS) data demonstrated that the chimeric roots tended to be heavily mosaic in regard to edited alleles. From one data set, 9 composite plants provided a total of 31 transgenic shoots (which were induced from a transgenic root) of which 22 were edited (22 edited shoots out of 31 total induced transgenic shoots). In some cases, two or more shoots induced from one composite plant contained identical editing profiles and, in other cases, two or more shoots from one composite plant contained different editing profiles. Up to 6 uniquely edited profiles were recovered from the shoots derived from a single composite plant.

[0178] Example 5: Cherry transgenic shoot regeneration from transgenic roots

[0179] Composite plants of cherry plant varieties Sweetheart or Lapins containing a wildtype shoot and a transgenic root were moved intact from root induction medium to a shoot induction medium. In cherry, the shoot induction medium contained 5 mg / L 4-CPPU and 25mg / L kanamycin, and shoot induction could be observed as early as 4 weeks. The shoot induction from roots step was carried out for 6 weeks, after which time induced shoots and shoot primordia were excised from the original transgenic root and placed into various shoot development media. After shoot development, shoots were transferred to a root induction medium. Rooted transgenic plants expressing zsGreen in all tissues were recovered using this method in both genotypes. Edited shoots have been recovered in Sweetheart using this method.

[0180] Example 6: Broccoli transgenic shoot regeneration from transgenic roots.

[0181] Excised roots and intact composite plants were generated using non-transformed seeds of B. oleracea ‘De Cicco’ and were utilized for wild-type shoot induction from roots experiments two weeks after the start of germination. Intact composite plants were removed gently from the PP0040 germination medium and were placed onto the surface of shoot induction medium, and excised roots were removed from the germinated seedlings and were placed onto shoot induction medium shown in Table 18, which is comprised of PP0040 with 200 mg / 1 Timentin. Wild-type shoot induction from roots was assessed 35 days after the transfer to shoot induction medium (Table 18).

[0182] Table 18. Shoot induction from wild-type roots in B. oleracea ‘De Cicco’ using PP0040 basal medium with 200 mg / 1 Timentin.

[0183] Transgenic / chimeric root systems generated in the previously mentioned B. oleracea example were utilized to assess transgenic shoot induction from roots (Table 19). Excised roots or intact composite plants were placed onto shoot induction media, and transgenic shoot induction was assessed after 35 days through visualization of zsGreen.

[0184] Table 19. Transgenic shoot induction from roots in B. oleracea using either excised transgenic / chimeric roots, or intact composite plants containing transgenic / chimeric roots. Shoot induction media contained both 10 mg / 1 kanamycin and 200 mg / 1 Timentin.aGrowth regulators composed of 3 mg / 1 BAP, 0.2 mg / 1 NAA, 0.01 mg / 1 GA3, 5 mg / 1 AgNO3Example 7: Blackberry non-transgenic, edited shoot regeneration from chimeric roots

[0185] For generation of non-transgenic, edited shoots, composite plants containing a wild-type shoot and a chimeric transgenic root system were generated after 8 weeks of culture on root induction media containing kanamycin. Composite plants were extracted from the root induction medium and placed onto shoot induction medium containing 2 mg / L 4-CPPU and BAP without kanamycin. After 5 weeks, shoots without reporter gene expression that were induced from a portion of the root of a composite plant were excised, moved to a propagation medium followed by root induction medium without selection. Because the process of shoot induction from roots is believed to originate from a few cells and from only the pericycle layer, non-mosaic, gene edited, transgene-free plants were generated from one or more non- transgenic cells in a chimeric root (Table 20). Not wishing to be bound by theory, it is believed that edited, non-transgenic plants were induced from transiently edited cells or cell lines contained within the transgene-free sectors of the chimeric root, as the root induction process appears to be of multicellular origin. Resistance provided by the transgenic sector of the chimeric roots could have allowed transiently edited cells in the non-transgenic sectors of the chimeric roots to grow. Transgene absence in these shoots was confirmed by PCR-based assays. Target capture performed on the gene edited and transgene-free plants confirmed the non-transgenic status compared to transgenic and wild-type control samples. Transgene-free edited plants have been generated using this method that contain between one and 12 edited alleles, across three different target genes.

[0186] Using this partial selection system, 103 shoots were induced from 69 root systems. Twenty-seven shoots were transgene-positive, and 74 shoots were transgene-negative (i.e., transgene free). Of the 74 transgene-negative shoots, three unique TFL edit profiles were detected across five total plants. One allele combination (observed in one plant) was confirmed to be non-transgenic and edited via target capture.

[0187] Table 20. Editing data for one non-transgenic and edited shoot (Sample RTS29534 edited with construct pWISE8700) that was negative for the presence of the nuclease which indicates the plants are not transgenic, generated on shoot induction medium without selection.Example 8: Cherry non-transgenic, edited shoot regeneration from chimeric roots.

[0188] A similar approach for recovery of edited and non-transgenic plants in blackberry as described in Example 7 is applicable to cherry. Following a similar approach described in Example 7, cherry composite plants containing a wild-type shoot and a transgenic root were generated using an editing system that included a proprietary CRISPR-Cas effector protein and a guide nucleic acid having complementary to a portion of an endogenous gene, and all subsequent steps were performed without selection (shoot induction, shoot development, rooting). Composite plants that are (1) edited and transgenic, (2) unedited and transgenic, or (3) unedited and non-transgenic have been recovered from a transgenic root of a composite plant comprising a wild-type shoot and the transgenic root. Composite plants that are edited and non-transgenic are expected to be recovered from a transgenic root of a composite plant comprising a wild-type shoot and the transgenic root. Due to the presence of chimeric roots, and the similarity in visual chimerism between blackberry and cherry transformed roots, it is expected that wild-type shoots; transgenic, non-edited shoots; transgenic, edited shoots; and non-transgenic, edited shoots can be produced from cherry roots as in blackberry.

[0189] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a modified plant or plant part, the method comprising: culturing a transformed plant tissue on a first root induction medium comprising a selection agent to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells and a gene that confers resistance to the selection agent, and wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant, wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

2. The method of claim 1, wherein the transformed plant tissue is from a species of plant that produces suckers (e.g., a suckering plant species).

3. The method of claim 1 or 2, wherein the transformed plant tissue is from a plant or plant part in the genus Actinidia, Allium, Amaranthus, Amelanchier, Anacardium, Ananas, Arabidopsis, Arachis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica, Capsicum, Carica, Castanea, Quercus, Fagus, Chenopodium, Chusquea, Citrus, Colocasia, Cornus, Corylus, Cucumis, Cydonia, Dioscorea, Diospyros, Fargesia, Ficus, Forsythia, Fragaria, Gymnocladus, Heavea, Helianthus, Hippophae, Hordeum, Illicium, Ipomoea, Juglans, Laurus, Lens, Malus, Manihot, Morus, Musa, Oryza, Panicum, Papaver, Passiflora, Phyllostachys, Pisum, Plantago, Poa, Populus, Prunus, Pseudosasa, Pyrus, Rheum, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Sorghum, Spinacia, Syringa, Triticum, Vaccinium, Vigna, Vitis, Zingiber, or Zizyphus.

4. The method of any one of the preceding claims, wherein the transformed plant tissue comprises meristem cells (e.g., shoot meristem cells or root meristem cells), optionally wherein the transformed plant tissue comprises a shoot comprising meristem cells.

5. The method of any one of the preceding claims, wherein the first root induction medium further comprises a basal medium, an antioxidant, a micronutrient, a chemoattractant (e.g., a chemoattractant for an Agrobacterium species), a cytokine, a plant growth regulator, an antibiotic, and / or a phenol.

6. The method of any one of the preceding claims, wherein the first root induction medium comprises an auxin or auxin precursor, optionally wherein the first root induction medium comprises indole-3 -butryic acid (IBA).

7. The method of claim 6, wherein the auxin or auxin precursor is present in the first root induction medium at a concentration of less than about 0.5 mg / L.

8. The method of any one of claims 1-5, wherein the first root induction medium is devoid of a plant growth regulator (e.g., an auxin and / or an auxin precursor) and / or is devoid of copper(II) sulphate (e.g., cupric sulfate, pentahydrate).

9. The method of any one of the preceding claims, wherein the selection agent is present in the first root induction medium at a concentration that is less than (e.g., at least 50% less than) a concentration of the same selection agent in a shoot induction medium, optionally wherein the selection agent is present in the first root induction medium at a concentration that preferences growth of transgenic roots over wild type roots.

10. The method of any one of the preceding claims, wherein the selection agent is kanamycin and the kanamycin is present in the first root induction medium in an amount of about 10 mg / L to about 75 mg / L, optionally wherein the kanamycin is present in the first root induction medium in an amount of about 25 mg / L to about 50 mg / L.

11. The method of any one of claims 1-9, wherein the selection agent is imazamox and the imazamox is present in the first root induction medium at a concentration of about 0.1 pM to about 0.75 pM, optionally wherein the imazamox is present in the first root induction medium at a concentration of about 0.25 pM to about 0.5 pM.

12. The method of any one of the preceding claims, wherein, during culturing of the transformed plant tissue on the first root induction medium, the transformed plant tissue is atleast partially in the first root induction medium, optionally wherein the root grows into the first root induction medium.

13. The method of any one of the preceding claims, further comprising contacting a wounded plant tissue with a polynucleotide of interest to provide the transformed plant tissue, optionally wherein the polynucleotide of interest comprises the gene that confers resistance to the selection agent.

14. The method of claim 13, wherein the polynucleotide of interest comprises at least one component of an editing system.

15. The method of any one of claims 13 or 14, wherein contacting the wounded plant tissue with the polynucleotide of interest comprises contacting the wounded plant tissue with an Agrobacterium that comprises the polynucleotide of interest and introducing the polynucleotide of interest into a cell of the wounded plant tissue.

16. The method of claim 15, wherein the Agrobacterium is an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.

17. The method of claim 15 or 16, wherein the Agrobacterium is a disarmed strain and / or a strain that lacks a functional rol gene (e.g., lacks a functional rolA gene, rolB gene, and / or rolC gene).

18. The method of claim 15 or 16, wherein the Agrobacterium is an Agrobacterium strain (e.g., an Agrobacterium rhizogenes strain) that comprises a rolA gene, rolB gene, and / or rolC gene, optionally wherein the Agrobacterium strain comprises the rolA gene, the rolB gene, and the rolC gene and / or optionally wherein the rolA gene, rolB gene, and / or rolC geneis extrachromosomal (e.g., in a transformation vector).

19. The method of any one of claims 13-18, wherein, following contacting the wounded plant tissue with the polynucleotide of interest, the transformed plant tissue is first cultured on the first root induction medium.

20. The method of any one of the preceding claims, wherein the root further comprises edited cells, optionally wherein the transgenic cells are edited cells.

21. The method of any one of the preceding claims, wherein the shoot induction medium comprises for chlorfenuron (4-CPPU), optionally wherein the shoot induction medium comprises 4-CPPU in an amount of about 0.5 mg / L to about 10 mg / L.

22. The method of any one of the preceding claims, wherein the shoot induction medium comprises 6-benzylaminopurine (BAP), optionally wherein the shoot induction medium comprises BAP in an amount of about 0.05 mg / L to about 5 mg / L.

23. The method of any one of the preceding claims, wherein the modified shoot comprises transgenic, edited cells; transgenic, non-edited cells; or non-transgenic, edited cells.

24. The method of any one of the preceding claims, wherein, prior to culturing the modified shoot on the growth medium, the modified shoot is excised from the composite plant to provide an excised modified shoot and the excised modified shoot is cultured on the growth medium.

25. The method of any one of the preceding claims, wherein the growth medium is a second root induction medium.

26. The method of any one of the preceding claims, wherein culturing the composite plant on the shoot induction medium produces at least two modified shoots, optionally wherein the at least two modified shoots are different (e.g., comprise different edits).

27. A method of producing a modified plant or plant part, the method comprising: wounding a plant tissue to provide a wounded tissue comprising root forming competent cells, wherein the plant tissue comprises a shoot including meristem tissue and the wounded tissue comprises at least a portion of the meristem tissue; contacting the wounded tissue with a polynucleotide of interest and a gene that confers resistance to a selection agent to provide a transformed tissue that comprises the meristem tissue, the polynucleotide of interest, and the gene;culturing the transformed tissue on a first root induction medium comprising the selection agent to produce a composite plant, wherein the composite plant comprises a wildtype shoot and a root that comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

28. The method of claim 27, wherein the plant tissue is or is from a species of plant that produces suckers (e.g., a suckering plant species).

29. The method of claim 27 or 28, wherein the plant tissue is in the genus Actinidia, Allium, Amaranthus, Amelanchier, Anacardium, Ananas, Arabidopsis, Arachis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica, Capsicum, Carica, Castanea, Quercus, Fagus, Chenopodium, Chusquea, Citrus, Colocasia, Cornus, Corylus, Cucumis, Cydonia, Dioscorea, Diospyros, Fargesia, Ficus, Forsythia, Fragaria, Gymnocladus, Heavea, Helianthus, Hippophae, Hordeum, Illicium, Ipomoea, Juglans, Laurus, Lens, Malus, Manihot, Morus, Musa, Oryza, Panicum, Papaver, Passiflora, Phyllostachys, Pisum, Plantago, Poa, Populus, Prunus, Pseudosasa, Pyrus, Rheum, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Sorghum, Spinacia, Syringa, Triticum, Vaccinium, Vigna, Vitis, Zingiber, or Zizyphus.

30. The method of any one of claims 27-29, wherein the transformed tissue comprises meristem shoot meristem cells, optionally wherein the transformed tissue comprises a shoot comprising meristem cells.

31. The method of any one of claims 27-30, wherein the first root induction medium further comprises a basal medium, an antioxidant, a micronutrient, a chemoattractant (e.g., a chemoattractant for an Agrobacterium species), a cytokine, a plant growth regulator, an antibiotic, and / or a phenol.

32. The method of any one of claims 27-31, wherein the first root induction medium comprises an auxin or auxin precursor, optionally wherein the first root induction medium comprises indole-3 -butryic acid (IBA).

33. The method of claim 32, wherein the auxin or auxin precursor is present in the first root induction medium at a concentration of less than about 0.5 mg / L.

34. The method of any one of claims 27-31, wherein the first root induction medium is devoid of a plant growth regulator (e.g., an auxin and / or an auxin precursor) and / or is devoid of copper(II) sulphate (e.g., cupric sulfate, pentahydrate).

35. The method of any one of claims 27-34, wherein the selection agent is present in the first root induction medium at a concentration that is less than (e.g., at least 50% less than) a concentration of the same selection agent in a shoot induction medium, optionally wherein the selection agent is present in the first root induction medium at a concentration that preferences growth of transgenic roots over wild type roots.

36. The method of any one of claims 27-35, wherein the selection agent is kanamycin and the kanamycin is present in the first root induction medium in an amount of about 10 mg / L to about 75 mg / L, optionally wherein the kanamycin is present in the first root induction medium in an amount of about 25 mg / L to about 50 mg / L.

37. The method of any one of claims 27-35, wherein the selection agent is imazamox and the imazamox is present in the first root induction medium at a concentration of about 0.1 pM to about 0.75 pM, optionally wherein the imazamox is present in the first root induction medium at a concentration of about 0.25 pM to about 0.5 pM.

38. The method of any one of claims 27-37, wherein, during culturing of the transformed tissue on the first root induction medium, the transformed tissue is at least partially in the first root induction medium, optionally wherein the root grows into the first root induction medium.

39. The method of any one of claims 27-38, wherein the polynucleotide of interest comprises at least one component of an editing system.

40. The method of any one of claims 27-39, wherein contacting the wounded tissue with the polynucleotide of interest and the gene that confers resistance to the selection agentcomprises contacting the wounded plant tissue with an Agrobacterium that comprises the polynucleotide of interest and the gene that confers resistance to the selection agent and introducing the polynucleotide of interest and the gene that confers resistance to the selection agent into a cell of the wounded tissue.

41. The method of claim 40, wherein the Agrobacterium is an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.

42. The method of claim 40 or 41, wherein the Agrobacterium is a disarmed strain and / or a strain that lacks a functional rol gene (e.g., lacks a functional rolA gene, rolB gene, and / or rolC gene).

43. The method of claim 40 or 41, wherein the Agrobacterium is an Agrobacterium strain (e.g., an Agrobacterium rhizogenes strain) that comprises a rolA gene, rolB gene, and / or rolC gene, optionally wherein the Agrobacterium strain comprises the rolA gene, the rolB gene, and the rolC gene and / or optionally wherein the rolA gene, rolB gene, and / or rolC gene is extrachromosomal (e.g., in a transformation vector).

44. The method of any one of claims 27-43, wherein, following contacting the wounded tissue with the polynucleotide of interest and the gene that confers resistance to the selection agent, the transformed tissue is first cultured on the first root induction medium.

45. The method of any one of claims 27-44, wherein the root further comprises edited cells, optionally wherein the transgenic cells are edited cells.

46. The method of any one of claims 27-45, wherein the shoot induction medium comprises for chlorfenuron (4-CPPU), optionally wherein the shoot induction medium comprises 4-CPPU in an amount of about 0.5 mg / L to about 10 mg / L.

47. The method of any one of claims 27-46, wherein the shoot induction medium comprises 6-benzylaminopurine (BAP), optionally wherein the shoot induction medium comprises BAP in an amount of about 0.05 mg / L to about 5 mg / L.

48. The method of any one of claims 27-47, wherein the modified shoot comprises transgenic, edited cells; transgenic, non-edited cells; or non-transgenic, edited cells.

49. The method of any one of claims 27-48, wherein, prior to culturing the modified shoot on the growth medium, the modified shoot is excised from the composite plant to provide an excised modified shoot and the excised modified shoot is cultured on the growth medium.

50. The method of any one of claims 27-49, wherein the growth medium is a second root induction medium.

51. The method of any one of claims 27-50, wherein culturing the composite plant on the shoot induction medium produces at least two modified shoots, optionally wherein the at least two modified shoots are different (e.g., comprise different edits).

52. The method of any one of claims 27-51, wherein the wounding and contacting steps are carried out simultaneously.

53. A method of producing a modified plant or plant part, the method comprising: culturing a transformed plant tissue on a first root induction medium to produce a composite plant, wherein the transformed plant tissue comprises root forming competent cells, a rolA gene, a rolB gene, and a rolC gene, and wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on a shoot induction medium to produce a modified shoot on the composite plant, wherein the modified shoot is different than the wild-type shoot; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

54. The method of claim 53, wherein the transformed plant tissue is from a species of plant that produces suckers (e.g., a suckering plant species).

55. The method of claim 53 or 54, wherein the transformed plant tissue is from a plant or plant part in the genus Actinidia, Allium, Amaranthus, Amelanchier, Anacardium, Ananas, Arabidopsis, Arachis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica,Capsicum, Carica, Castanea, Quercus, Fagus, Chenopodium, Chusquea, Citrus, Colocasia, Cornus, Corylus, Cucumis, Cydonia, Dioscorea, Diospyros, Fargesia, Ficus, Forsythia, Fragaria, Gymnocladus, Heavea, Helianthus, Hippophae, Hordeum, Illicium, Ipomoea, Juglans, Laurus, Lens, Malus, Manihot, Morus, Musa, Oryza, Panicum, Papaver, Passiflora, Phyllostachys, Pisum, Plantago, Poa, Populus, Prunus, Pseudosasa, Pyrus, Rheum, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Sorghum, Spinacia, Syringa, Triticum, Vaccinium, Vigna, Vitis, Zingiber, or Zizyphus.

56. The method of any one of claims 53-55, wherein the transformed plant tissue comprises meristem cells (e.g., shoot meristem cells or root meristem cells), optionally wherein the transformed plant tissue comprises a shoot comprising meristem cells and / or a gene that confers resistance to a selection agent.

57. The method of any one of claims 53-56, wherein the first root induction medium comprises a basal medium, an antioxidant, a micronutrient, a chemoattractant (e.g., a chemoattractant for an Agrobacterium species), a cytokine, a plant growth regulator, an antibiotic, and / or a phenol.

58. The method of any one of claims 53-57, wherein the first root induction medium comprises an auxin or auxin precursor, optionally wherein the first root induction medium comprises indole-3 -butryic acid (IBA).

59. The method of claim 58, wherein the auxin or auxin precursor is present in the first root induction medium at a concentration of less than about 0.5 mg / L.

60. The method of any one of claims 53-57, wherein the first root induction medium is devoid of a plant growth regulator (e.g., an auxin and / or an auxin precursor) and / or is devoid of copper(II) sulphate (e.g., cupric sulfate, pentahydrate).

61. The method of any one of claims 53-60, wherein the composite plant exhibits and / or has the hairy root phenotype, optionally wherein the root that comprises transgenic cells is a hairy root (e.g., a root that exhibits the hairy root phenotype such as has extensive lateral branching).

62. The method of any one of claims 53-61, wherein the first root induction medium is devoid of a selection agent.

63. The method of any one of claims 53-61, wherein the first root induction medium comprises a selection agent at a concentration that is less than (e.g., at least 50% less than) a concentration of the same selection agent in a shoot induction medium, optionally wherein the selection agent is present in the first root induction medium at a concentration that preferences growth of transgenic roots over wild type roots.

64. The method of claim 63, wherein the selection agent is kanamycin and the kanamycin is present in the first root induction medium in an amount of about 10 mg / L to about 75 mg / L, optionally wherein the kanamycin is present in the first root induction medium in an amount of about 25 mg / L to about 50 mg / L, or wherein the selection agent is imazamox and the imazamox is present in the first root induction medium at a concentration of about 0.1 pM to about 0.75 pM, optionally wherein the imazamox is present in the first root induction medium at a concentration of about 0.25 pM to about 0.5 pM.

65. The method of any one of claims 53-64, wherein, during culturing of the transformed plant tissue on the first root induction medium, the transformed plant tissue is at least partially in the first root induction medium, optionally wherein the root grows into the first root induction medium.

66. The method of any one of claims 53-65, further comprising contacting a wounded plant tissue with an Agrobacterium that comprises the rolA gene, the rolB gene, and the rolC gene to provide the transformed plant tissue, optionally wherein the transformed plant tissue and / or the composite plant express the rolA gene, the rolB gene, and the rolC gene such that the transformed plant tissue and / or the composite plant exhibits the hairy root phenotype.

67. The method of claim 66, wherein contacting the wounded plant tissue with the Agrobacterium comprises introducing the rolA gene, the rolB gene, and the rolC gene into a cell of the wounded plant tissue to provide the transformed plant tissue.

68. The method of any one of claims 66 or 67, wherein the Agrobacterium further comprises a polynucleotide of interest and / or a gene that confers resistance to a selection agent, optionally wherein the polynucleotide of interest is at least one component of an editing system, and wherein contacting the wounded plant tissue with Axe Agrobacterium comprises introducing the polynucleotide of interest and / or gene that confers resistance to the selection agent into a cell of the wounded plant tissue to provide the transformed plant tissue.

69. The method of any one of claims 66-68, wherein Axe Agrobacterium is an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.

70. The method of any one of claims 66-69, wherein Axe Agrobacterium is an Agrobacterium strain that comprises the rolA gene, the rolB gene, and the rolC gene extrachromosomally (e.g., in a transformation vector).

71. The method of any one of claims 66-70, wherein, following contacting the wounded plant tissue with the Agrobacterium, the transformed plant tissue is first cultured on the first root induction medium.

72. The method of any one of claims 53-71, wherein the root further comprises edited cells, optionally wherein the transgenic cells are edited cells.

73. The method of claims 53-72, wherein the shoot induction medium comprises forchlorfenuron (4-CPPU), optionally wherein the shoot induction medium comprises 4- CPPU in an amount of about 0.5 mg / L to about 10 mg / L.

74. The method of any one of claims 53-73, wherein the shoot induction medium comprises 6-benzylaminopurine (BAP), optionally wherein the shoot induction medium comprises BAP in an amount of about 0.05 mg / L to about 5 mg / L.

75. The method of any one of claims 53-74, wherein the modified shoot comprises transgenic, edited cells; transgenic, non-edited cells; or non-transgenic, edited cells.

76. The method of any one of claims 53-75, wherein, prior to culturing the modified shoot on the growth medium, the modified shoot is excised from the composite plant to provide an excised modified shoot and the excised modified shoot is cultured on the growth medium.

77. The method of any one of claims 53-76, wherein the growth medium is a second root induction medium.

78. The method of any one of claims 53-77, wherein culturing the composite plant on the shoot induction medium produces at least two modified shoots, optionally wherein the at least two modified shoots are different (e.g., comprise different edits).

79. A method of producing a modified plant or plant part, the method comprising: wounding a plant tissue to provide a wounded tissue comprising root forming competent cells, wherein the plant tissue comprises a shoot including meristem tissue and the wounded tissue comprises at least a portion of the meristem tissue; contacting the wounded tissue with an Agrobacterium that comprises a rolA gene, a rolB gene, and a rolC gene to provide a transformed tissue that comprises the meristem tissue, the rolA gene, the rolB gene, and the rolC gene; culturing the transformed tissue on a first root induction medium to produce a composite plant, wherein the composite plant comprises a wild-type shoot and a root that comprises transgenic cells; culturing the composite plant on shoot induction medium to produce a modified shoot on the composite plant; and culturing the modified shoot on a growth medium, thereby producing a modified plant or plant part.

80. The method of claim 79, wherein the plant tissue is or is from a species of plant that produces suckers (e.g., a suckering plant species).

81. The method of claim 79 or 80, wherein the plant tissue is in the genus Actinidia, Allium, Amaranthus, Amelanchier, Anacardium, Ananas, Arabidopsis, Arachis, Armoracia, Aronia, Asimina, Asparagus, Berberis, Betula, Brassica, Capsicum, Carica, Castanea, Quercus, Fagus, Chenopodium, Chusquea, Citrus, Colocasia, Cornus, Corylus, Cucumis, Cydonia, Dioscorea, Diospyros, Fargesia, Ficus, Forsythia, Fragaria, Gymnocladus,Heavea, Helianthus, Hippophae, Hordeum, Illicium, Ipomoea, Juglans, Laurus, Lens, Mains, Manihot, Morns, Musa, Oryza, Panicum, Papaver, Passiflora, Phyllostachys, Pisum, Plantago, Poa, Populus, Prunus, Pseudosasa, Pyrus, Rheum, Rhus, Ribes, Rosa, Rubus, Sambucus, Solanum, Sorghum, Spinacia, Syringa, Triticum, Vaccinium, Vigna, Vitis, Zingiber, or Zizyphus.

82. The method of any one of claims 79-81, wherein the transformed tissue comprises meristem shoot meristem cells, optionally wherein the transformed tissue comprises a shoot comprising meristem cells and / or a gene that confers resistance to a selection agent.

83. The method of any one of claims 79-82, wherein the first root induction medium comprises a basal medium, an antioxidant, a micronutrient, a chemoattractant (e.g., a chemoattractant for an Agrobacterium species), a cytokine, a plant growth regulator, an antibiotic, and / or a phenol.

84. The method of any one of claims 79-83, wherein the first root induction medium comprises an auxin or auxin precursor, optionally wherein the first root induction medium comprises indole-3 -butryic acid (IBA).

85. The method of claim 84, wherein the auxin or auxin precursor is present in the first root induction medium at a concentration of less than about 0.5 mg / L.

86. The method of any one of claims 79-83, wherein the first root induction medium is devoid of a plant growth regulator (e.g., an auxin and / or an auxin precursor) and / or is devoid of copper(II) sulphate (e.g., cupric sulfate, pentahydrate).

87. The method of any one of claims 79-86, wherein the composite plant exhibits and / or has the hairy root phenotype, optionally wherein the root that comprises transgenic cells is a hairy root (e.g., a root that exhibits the hairy root phenotype such as has extensive lateral branching).

88. The method of any one of claims 79-87, wherein the first root induction medium is devoid of a selection agent.

89. The method of any one of claims 79-87, wherein the first root induction medium comprises a selection agent at a concentration that is less than (e.g., at least 50% less than) a concentration of the same selection agent in a shoot induction medium, optionally wherein the selection agent is present in the first root induction medium at a concentration that preferences growth of transgenic roots over wild type roots.

90. The method of claim 88, wherein the selection agent is kanamycin and the kanamycin is present in the first root induction medium in an amount of about 10 mg / L to about 75 mg / L, optionally wherein the kanamycin is present in the first root induction medium in an amount of about 25 mg / L to about 50 mg / L, or wherein the selection agent is imazamox and the imazamox is present in the first root induction medium at a concentration of about 0.1 pM to about 0.75 pM, optionally wherein the imazamox is present in the first root induction medium at a concentration of about 0.25 pM to about 0.5 pM.

91. The method of any one of claims 79-90, wherein, during culturing of the transformed tissue on the first root induction medium, the transformed tissue is at least partially in the first root induction medium, optionally wherein the root grows into the first root induction medium.

92. The method of any one of claims 79-91, wherein contacting the wounded tissue with the Agrobacterium comprises introducing the rolA gene, the rolB gene, and the rolC gene into a cell of the wounded tissue to provide the transformed tissue.

93. The method of any one of claims 79-92, wherein the Agrobacterium further comprises a polynucleotide of interest and / or a gene that confers resistance to a selection agent, optionally wherein the polynucleotide of interest comprises at least one component of an editing system, and contacting the wounded tissue with the Agrobacterium comprises introducing the polynucleotide of interest and / or the gene that confers resistance to the selection agent into a cell of the wounded tissue to provide the transformed tissue.

94. The method of any one of claims 79-93, wherein the Agrobacterium is an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.

95. The method of any one of claims 79-94, wherein the Agrobacterium is an Agrobacterium strain (e.g., an Agrobacterium rhizogenes strain) that comprises the rolA gene, the rolB gene, and the rolC gene extrachromosomally (e.g., in a transformation vector).

96. The method of any one of claims 79-95, wherein, following contacting the wounded tissue with the. Agrobacterium, the transformed tissue is first cultured on the first root induction medium.

97. The method of any one of claims 79-96, wherein the root further comprises edited cells, optionally wherein the transgenic cells are edited cells.

98. The method of any one of claims 79-97, wherein the shoot induction medium comprises for chlorfenuron (4-CPPU), optionally wherein the shoot induction medium comprises 4-CPPU in an amount of about 0.5 mg / L to about 10 mg / L.

99. The method of any one of claims 79-98, wherein the shoot induction medium comprises 6-benzylaminopurine (BAP), optionally wherein the shoot induction medium comprises BAP in an amount of about 0.05 mg / L to about 5 mg / L.

100. The method of any one of claims 79-99, wherein the modified shoot comprises transgenic, edited cells; transgenic, non-edited cells; or non-transgenic, edited cells.

101. The method of any one of claims 79-100, wherein, prior to culturing the modified shoot on the growth medium, the modified shoot is excised from the composite plant to provide an excised modified shoot and the excised modified shoot is cultured on the growth medium.

102. The method of any one of claims 79-101, wherein the growth medium is a second root induction medium.

103. The method of any one of claims 79-102, wherein culturing the composite plant on the shoot induction medium produces at least two modified shoots, optionally wherein the at least two modified shoots are different (e.g., comprise different edits).

104. The method of any one of claims 79-103, wherein the wounding and contacting steps are carried out simultaneously.

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