Genes for hormone-free plant regeneration

Abstract

The present invention relates to a method for regenerating plant cells, preferably regenerating shoots from plant cells, by altering the expression levels of at least WOX5 and PLT proteins, preferably WOX5 and PLT1. Additionally, the expression levels of additional proteins, such as WIND1, SHR, SCR, RBR, PLT4, and PLT5, can be altered to regenerate shoots from plant cells. Preferably, the expression levels are transiently altered. The present invention further relates to nucleic acid constructs suitable for transient protein expression and the use of said protein combinations for regenerating shoots from plant cells. [Selection diagram] None

Description

Technical Field

[0001] The present invention relates to the field of molecular plant biology, in particular, to the field of plant regeneration. The present invention relates to a method for improving regeneration of plant cells without the need for plant growth hormones.

Background Art

[0002] It remains undetermined whether modern plant regeneration technologies will succeed in manipulating desired traits in important crop species. Current regeneration protocols still largely rely on the application of two key plant hormones, auxin and cytokinin, that control new plant regeneration in a two-step process (Skoog and Miller, 1957). Generally, auxin-rich medium is used to induce regeneration-competent callus, from which shoot organogenesis is induced using cytokinin-rich medium. Manipulation of plant species refractory to this protocol has resulted in the ineffectiveness of using modern biotechnology for their genetic improvement. Therefore, there is a need for methods that increase regeneration efficiency enabling genotype-independent shoot organogenesis.

[0003] Experiments in *Arabidopsis thaliana* have revealed that the ability of callus to produce shoots is achieved through the emergence of root traits that indicate the acquisition of root identity by callus cells (Sugimoto et al., 2010). After the expression of root traits in regeneration-competent callus, the expression of shoot-specific genes occurs, demonstrating the importance of transient root acquisition to generate founder cells for the initiation of shoot regeneration (Rosspopoff et al., 2017). Genetic studies support the importance of root trait acquisition for the regeneration capacity of callus tissue (Sugimoto et al., 2010) (Kareem et al., 2015) (Fan et al., 2012). While these studies reveal the involvement of additional regulatory factors for shoot regeneration, they still rely on hormone induction during the regeneration process. Therefore, this dependence on plant hormone induction still hinders the regeneration efficiency of plant cells.

[0004] Iwase et al. (2015) showed that overexpression of WIND1 can bypass auxin pretreatment, but still requires the presence of the plant hormone cytokinin.

[0005] Shoot regeneration in the absence of phytohormones has been previously demonstrated in this field, but requires plant injury (Iwase et al., 2017).

[0006] Therefore, there remains a need in the art for methods to confer regenerative ability to plant species, particularly refractory plant species, or to enhance their regenerative efficiency, without relying on externally administered plant hormones and without the need to harm the plants. In addition, there is a need for recombinant DNA constructs that, when introduced into plant cells, increase or induce regenerative ability in, for example, refractory plants. [Overview of the initiative]

[0007] In one aspect, the present invention relates to a method for regenerating shoots from plant cells, comprising the following steps: a) at least i) WUSCHEL-associated homeobox 5 (WOX5) protein; and ii) A PLETHORA(PLT) protein selected from the group consisting of PLETHORA(PLT)1, PLT2, PLT3, PLT4, PLT5, and PLT7, preferably PLT1 A step of introducing or increasing the expression of a protein combination into plant cells, wherein the expression of at least one protein of the protein combination is transiently introduced or increased; and b) A step that allows the plant cells to regenerate into shoots.

[0008] In one embodiment, the protein combination is iii) WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) protein It also includes.

[0009] In one embodiment, the protein combination is iv) Short root (SHR) protein; v) SCARECROW (SCR) protein; and vi) At least three PLETHORA (PLT) proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. It also includes.

[0010] In one embodiment, the protein combination comprises at least three selected PLT proteins, including at least one or more of PLT1, PLT4, and PLT5, preferably the at least three selected PLT proteins being PLT1, PLT4, and PLT5.

[0011] In one embodiment, step a) further comprises reducing the expression of an endogenous Retinoblastoma-related (RBR) protein, preferably transiently.

[0012] Preferably, the total protein expression of a protein combination as defined herein is transiently introduced or increased, and optionally, the RBR protein expression is transiently decreased.

[0013] Preferably, the expression of all proteins in a protein combination as defined herein is simultaneously and transiently introduced or increased, and optionally, the expression of RBR proteins is transiently decreased simultaneously with the protein combination.

[0014] In one embodiment, the expression of at least one protein of a protein combination as defined herein, preferably the expression of all proteins in the protein combination, is transiently introduced or increased by transient activation of their expression, and optionally, the expression of RBR proteins is transiently decreased by transient activation of RBR inhibitor expression.

[0015] In a further embodiment, i) The amino acid sequence of the SHR protein has at least 60% sequence identity with SEQ ID NO: 1; ii) The amino acid sequence of the SCR protein has at least 60% sequence identity with SEQ ID NO: 2; iii) The amino acid sequence of the WOX5 protein has at least 60% sequence identity with SEQ ID NO: 3; iv) The amino acid sequences of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins have at least 60% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 30, respectively; v) The amino acid sequence of the RBR protein has at least 60% sequence identity with SEQ ID NO: 17; and vi) The amino acid sequence of the WIND1 protein has at least 60% sequence identity with SEQ ID NO: 28.

[0016] In one embodiment, i) The SHR protein is encoded by a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: 9; ii) The SCR protein is encoded by a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: 10; iii) The WOX5 protein is encoded by a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: 11; iv) The PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins are encoded by nucleotide sequences having at least 60% sequence identity with SEQ ID NOs. 12, 13, 14, 15, 16, and 19, respectively; v) The RBR protein is encoded by a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 18; and vi) The WIND1 protein is encoded by a nucleotide sequence that has at least 60% sequence identity with SEQ ID NO: 29.

[0017] In further embodiments, the plant cells are part of a multicellular tissue, preferably callus tissue, a plant organ, or an explant. Preferably, the plant organ is a root.

[0018] In one embodiment, plant cells can be obtained from plants selected from the group consisting of Arabidopsis, barley, cabbage, canola, cassava, cauliflower, chicory, chrysanthemum, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, oilseed rape, potato, pumpkin, rice, rye, sorghum, soybean, squash, sugarcane, sugar beet, sunflower, sweet pepper, tomato, watermelon, wheat, and zucchini. Optionally, plant cells can be obtained from plants of the Solanaceae family, optionally from the Solanum genus, optionally from the tomato species (Solanum lycopersicum) or the eggplant species (Solanum melongena). Optionally, plant cells can be obtained from the Brassicaceae family, optionally from its species or subspecies, such as radish (Raphanus sativus), cabbage (Brassica oleracea), turnip (Brassica rapa), rapeseed (Brassica napus), horseradish (Armoracia rusticana), or Arabidopsis thaliana.

[0019] In one embodiment, the method comprises step c) of forming a plant or a plant part from a regenerated shoot.

[0020] In a further aspect, the present invention provides a composition comprising at least two nucleic acid molecules, wherein i) the first nucleic acid molecule comprises a nucleotide sequence encoding a WOX5 protein operably linked to an inducible promoter; and ii) the second nucleic acid molecule comprises a nucleotide sequence encoding a PLT protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, preferably PLT1, operably linked to an inducible promoter, relating to the composition.

[0021] In another aspect, the present invention relates to a nucleic acid construct comprising the nucleotide sequences of the first and second nucleic acid molecules as defined herein.

[0022] Preferably, the nucleic acid construct further comprises an additional nucleotide sequence encoding a transactivator that is preferably operably linked to a promoter, wherein the transactivator activates the inducible promoter upon binding an inducer. Preferably, said inducible promoter is part of at least one expression cassette. Preferably, said inducible promoter is operably linked to at least one of the nucleotide sequences encoding SHR, SCR, WOX5, WIND, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins, and an RBR repressor, as defined herein.

[0023] Preferably, the transactivator is i) encoded by a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 21, wherein said transactivator is capable of binding dexamethasone, corticoid, or a derivative thereof; or ii) Encoded by a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 27, the transactivator has the ability to bind to β-estradiol or its derivatives.

[0024] In a further embodiment, the present invention is i) a first nucleic acid molecule comprising a nucleotide sequence encoding a WOX5 protein operably linked to an inductive promoter, and a second nucleic acid molecule comprising a nucleotide sequence encoding a PLT protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, preferably PLT1, operably linked to an inductive promoter; and ii) Nucleic acid constructs as defined herein This relates to plant cells containing at least one of the following.

[0025] In another embodiment, the present invention relates to a shoot, plant, or plant part that can be obtained by a method as defined herein.

[0026] In one embodiment, the present invention relates to the use of a protein combination as defined herein, and optionally, an RBR inhibitor as defined herein, or a nucleic acid composition or construct as defined herein, for regenerating shoots from plant cells.

[0027] [Definition] Various terms relating to the methods, compositions, uses, and other embodiments of the present invention are used throughout this specification and the claims. Unless otherwise indicated, such terms shall be given their common meanings in the art to which the present invention pertains. Other clearly defined terms shall be construed to be consistent with the definitions provided herein.

[0028] It will be apparent to those skilled in the art that any methods and materials similar to or equivalent to those described herein may be used to carry out the present invention.

[0029] The methods for carrying out the conventional techniques used in the methods of the present invention will be apparent to those skilled in the art. The practices of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields are well known to those skilled in the art and are discussed, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodically updated; and the series Methods in Enzymology, Academic Press, San Diego.

[0030] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Therefore, for example, a reference to "a plant cell" includes combinations of two or more plant cells. Thus, the indefinite articles "a" or "an" usually mean "at least one."

[0031] The term "and / or" refers to a situation that may occur, either individually or in combination with at least one of the stated cases, up to all of the stated cases.

[0032] As used herein, the term “about” is used to describe and explain small variations. For example, the term may mean ±(+ or -) 10% or less, e.g., ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less.

[0033] In addition, quantities, ratios, and other numerical values ​​may be presented herein in range form. Such range forms should be understood as being used for convenience and conciseness, and should be flexibly understood to include not only numerical values ​​that are clearly specified as limits of the range, but also all individual numerical values ​​or partial ranges contained within that range, as if each numerical value and partial range were clearly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include not only the clearly listed limits of about 1 and about 200, but also individual ratios, e.g., about 2, about 3, and about 4, as well as partial ranges, e.g., about 10 to about 50, about 20 to about 100, etc.

[0034] The term “comprising” is to be interpreted as inclusive, open-ended, and non-exclusive. Specifically, the term and its declensions mean that a specified feature, step, or component is included. These terms should not be interpreted as excluding the presence of other features, steps, or components.

[0035] The terms “plant hormone,” “plant growth hormone,” “plant growth regulator,” or “phytohormone” should be understood herein as chemical substances that affect the growth and development of plant cells and tissues. Plant growth regulators include chemical substances from the following five groups: auxins, cytokinins, gibberellins, abscisic acid (ABA), and ethylene. In addition to these five main groups, the following two other classes of chemical substances are often considered plant growth regulators: brassinosteroids and polyamines. For inducing shoot regeneration in plant tissues, a combination of one or more cytokinins and one or more auxins is commonly used.

[0036] The terms "protein" or "polypeptide" are used interchangeably to refer to molecules consisting of chains of amino acids, without regard to their specific mode of action, size, three-dimensional structure, or origin. Therefore, a "fragment" or "part" of a protein can still be called a "protein." "Isolated protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or within recombinant bacterial or plant host cells.

[0037] "Plant" refers to the entire plant, or any part of a plant that can be obtained from a plant, such as cells, tissues, or organs (e.g., pollen, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruits, etc.), as well as any derivatives thereof, and offspring derived from such plants by self-pollination or hybridization.

[0038] "(One or more) plant cells" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., whether isolated or found within a tissue, organ, or organism. Plant cells may be part of a multicellular structure, such as a callus, a meristematic plant organ, or an explant.

[0039] "Similar conditions" for culturing plants / plant cells means, among other things, similar temperature, humidity, nutrient, and light conditions, as well as the use of similar watering and day / night rhythms.

[0040] The terms “homology” and “sequence identity” are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences, or between two or more nucleic acid (polynucleotide) sequences, as determined by comparing their sequences. In the art, “identity” also means the degree of sequence relevance between amino acid sequences or nucleic acid sequences, as determined by, in some cases, by matching strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods. Percentage sequence identity / similarity can be determined over the full length of the sequence.

[0041] Sequence identity and sequence similarity can be determined by the alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). The sequences may then be called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined below) when they are optimally aligned using default parameters (e.g., by the program GAP or BESTFIT). GAP aligns two sequences over their entire length (full length) using the Needleman and Wunsch global alignment algorithms while maximizing the number of matches and minimizing the number of gaps. Global alignment is appropriately used to determine sequence identity when two sequences have similar lengths. Generally, default GAP parameters are used, with a gap generation penalty of 50 (nucleotides) / 8 (proteins) and a gap elongation penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Scores for sequence alignment and percentage sequence identity can be determined using computer programs such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open-source software such as the "needle" program (using the global Needleman-Wunsch algorithm) or "water" program (using the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters or default settings as for GAP above (for both "needle" and "water," and for both protein and DNA alignments, the default gap opening penalty is 10.0 and the default gap elongation penalty is 0.5; the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA). If the sequences have substantially different total lengths, local alignment, such as that using the Smith-Waterman algorithm, is preferred.

[0042] Alternatively, percentage similarity or identity can be determined by searching public databases using algorithms such as FASTA and BLAST. Therefore, the nucleic acid and protein sequences of the present invention can further be used as "query sequences" for performing searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol. 215:403~10. A BLAST nucleotide search can be performed using the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search can be performed using the BLASTx program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain a gapped alignment for comparison, gapped BLAST can be used as described by Altschul et al., (1997) Nucleic Acids Res. 25(17):3389~3402. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0043] The term “complementarity” is defined herein as the sequence identity of a sequence with its complete complementary strand. For example, a sequence that is 100% complementary (completely complementary) is understood herein to have 100% sequence identity with its complementary strand, and a sequence that is 80% complementary is understood herein to have 80% sequence identity with its (complete) complementary strand.

[0044] The terms “nucleic acid construct,” “nucleic acid vector,” “vector,” and “expression vector” are interchangeable herein and are defined herein as artificial nucleic acid molecules resulting from the use of recombinant DNA technology. Therefore, while a nucleic acid construct may include (some of) naturally occurring nucleic acid molecules, the terms “nucleic acid construct” and “nucleic acid vector” do not include naturally occurring nucleic acid molecules.

[0045] The vector backbone may be, for example, a binary or super-binary vector (see, for example, U.S. Patent No. 5,591,616, U.S. Patent Application Publication No. 2002138879, and International Publication No. 95 / 06722), a co-incorporation vector, or a T-DNA vector, into which a chimeric gene is incorporated, or, if a suitable transcriptional regulatory sequence already exists, a desired nucleic acid sequence (e.g., coding sequence, antisense, or reverse repeat sequence) is incorporated downstream of that transcriptional regulatory sequence. The vector may include, for example, selectable markers, multiple cloning sites, or other genetic elements to facilitate their use in molecular cloning.

[0046] The term "gene" refers to a DNA fragment that contains a region (transcription region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. Genes can be operably ligated with appropriate regulatory regions (e.g., promoters). Genes typically consist of several operably ligated fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing polyadenylation sites.

[0047] "Genetic expression" refers to the process by which appropriate regulatory regions, particularly DNA regions operably linked to promoters, are transcribed into RNA, and if that RNA encodes a bioactive protein or peptide, it is subsequently translated into a bioactive protein or peptide.

[0048] The term "operably ligated" refers to the ligation of polynucleotide elements in a functional relationship. A nucleic acid is "operably ligated" if it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or more precisely, a transcriptional regulatory sequence, is operably ligated to a coding sequence if it influences the transcription of that sequence. "Operatably ligated" can sometimes mean that the ligated DNA sequences are in close proximity.

[0049] A “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acids. A promoter fragment is located upstream (5') of the transcription start site of its gene with respect to the direction of transcription, is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, and (one or more) transcription start sites, and may further include any other DNA sequences, including, but not limited to, transcription factor binding sites, repression and activation protein binding sites, and any other nucleotide sequences known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0050] Optionally, the term “promoter” may also include the 5'UTR region (5' untranslated region) (for example, a promoter may, as used herein, include one or more parts upstream of the translation start codon in a transcription region, because this region may play a role in regulating transcription and / or translation). A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is regulated physiologically (e.g., by the external application of a particular compound) or developmentally. A “tissue-specific” promoter is active only in a particular type of tissue or cell.

[0051] The term “regeneration” is defined herein as the formation of new tissue and / or new organs from a single plant cell, callus, explant, tissue, or organ. Preferably, regeneration is at least one of shoot regeneration, ectopic apical meristem formation, and root regeneration. Regeneration can occur through somatic somatic embryogenesis or organogenesis. In the context of the present invention, regeneration includes at least organogenesis, and preferably, regeneration is through the process of organogenesis. Preferably, regeneration as defined herein relates to at least the formation of new shoots. Regeneration may further include the formation of new plants from a single plant cell or, for example, from callus, explant, tissue, or organ. The plant cell for regeneration may be an undifferentiated plant cell. Thus, the regeneration process can occur directly from the parent tissue or indirectly, for example, via callus formation.

[0052] "Conditions that enable regeneration" are understood herein to mean an environment in which plant cells or tissues can regenerate. Such conditions include, at a minimum, appropriate temperature, nutrients, diurnal rhythm, and irrigation.

[0053] The “altered expression level” of a protein is understood herein as an expression level that deviates from the endogenous expression level of that protein in unmodified wild-type plant cells. Unmodified plant cells and plant cells with altered protein expression preferably have the same genetic background. The altered expression level may be increased or decreased compared to the endogenous expression level. In the context of the present invention, the altered expression level of WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 is preferably an increase or introduction of expression. The altered expression level of RBR is preferably a decrease in expression. The expression level can be measured by any method suitable in the art, such as, but not limited to, qPCR, Northern blotting, or microarrays. [Modes for carrying out the invention]

[0054] Plant cell regeneration requires cell exposure to plant hormones such as cytokinins and / or auxins. We have now discovered that the timely expression of a specific group of proteins renders such plant hormones obsolete. In other words, we have found that the expression of this specific group of proteins induces spontaneous regeneration, particularly spontaneous organogenesis.

[0055] Therefore, in a first aspect, the present invention relates to a method for regenerating plant cells. In one embodiment, the method relates to regenerating meristem from plant cells, preferably the meristem growing to form a shoot. Thus, the present invention relates to a method for regenerating a shoot from plant cells. The shoot can be cut from a basal cell mass and induced to form roots. Alternatively, the shoot can be cut from a basal cell mass and roots can form without any further induction.

[0056] Therefore, the present invention also relates to a method for regenerating shoots from plant cells without the need for the plant cells to be exposed to plant growth hormones to induce or stimulate regeneration. Thus, a method as defined herein is a hormone-independent method for regenerating shoots from plant cells.

[0057] Preferably, shoot regeneration occurs through the process of organogenesis. Therefore, methods for regeneration as defined herein are preferably methods for organogenesis, preferably for shoot organogenesis. Shoot organogenesis may be direct or indirect.

[0058] In this context of plant tissue culture, there are two selective pathways for the novel formation (i.e., regeneration) of new plants from callus or tissue explants: organogenesis and somatic somatic embryogenesis. Organogenesis involves inducing callus or tissue to form organs (shoots or roots). The preferred type of organogenesis in tissue culture is shoot organogenesis. Somatic somatic embryogenesis is the process by which callus or tissue explants typically develop a structure resembling a zygote through the embryogenetic callus phase, which then germinates into a complete plant body (Chieng, LMN et al. (2014) Induction of organogenesis and somatic embryogenesis of Gonystylus bancanus (Miq.) Kurz (Ramin) in Sarawak. SARAWAK FORESTRY Corporation & ITTO, Kuching, Malaysia & ITTO; ISBN 978-967-12855-3-4). These routes differ in several characteristics, as shown herein.

[0059] Explants are small pieces of primary tissue taken from plants that can serve as a source of regeneration through either organogenesis or somatic embryogenesis. Examples of explants include seedling parts such as stem and root segments, leaf sections, inflorescence sections, cotyledons, and hypocotyls, as well as immature and mature seed embryos (Thorpe, TA (1993) In vitro Organogenesis and Somatic Embryogenesis: Physiological and Biochemical Aspects. Roubelakis-Angelakis KA, Van Thanh KT (eds.) Morphogenesis in Plants. NATO ASI Series (Series A: Life Sciences), Vol. 253. Springer, Boston, MA). Depending on the manipulation of plant growth regulators and culture conditions, explants can directly develop shoots (or roots). This is called direct organogenesis. When shoot formation passes through the callus phase, this is called indirect organogenesis. Similarly, in somatic somatic embryogenesis, the embryo can develop directly from the explant (direct somatic somatic embryogenesis) or via the callus phase (indirect somatic somatic embryogenesis, Thorpe, see above; Chieng et al., see above).

[0060] Shoot organ formation: Shoot organ formation is a regenerative pathway in which cells of the callus or explant form a new shoot apical meristem that develops into a shoot with leaf primordia and leaves. Because there is only one apical meristem, this is a unipolar structure, and roots are not formed at this stage. The vascular system of the shoot is often connected to the parent tissue. Root formation can only be induced in a separate root induction step in a different medium after the shoot has fully formed and elongated and separated from the callus or explant (Thorpe, above). In the art, shoot organ formation is induced by plant growth regulators (PGRs), usually cytokinins, either alone or in combination with auxin at different concentrations, and the cytokinins remain as a component of the medium until a new shoot apical meristem and shoot have formed and have elongated sufficiently to separate them from the primary explant or callus.

[0061] Cytokinins include, for example, 6-aminopurine (BAP), zeatin, kinetin, thidiazurone (TDZ), and 6-(γ,γ-dimethylallylamino)purine (2-iP). For shoot organ formation by the combination of cytokinin and auxin, the ratio of cytokinin to auxin must be >1 (Dodds, JH and Roberts, LW (1985) Experiments in plant tissue culture. Cambridge University Press, Cambridge, UK).

[0062] Somatic somatic embryogenesis: In contrast, somatic somatic embryogenesis results in the formation of a bipolar structure resembling a zygote, containing a root-shoot axis with a closed, independent vascular system. In other words, both the root primordium and shoot primordium are formed simultaneously, without vascular connections to the basal tissue (Dodds and Roberts, above). Somatic somatic embryogenesis can be indirectly induced from callus or cell suspension, or they can be directly induced from cells in the explant (Thorpe, above). Somatic embryogenesis progresses through several distinct stages, from a spherical stage (small, equidiameter cell clusters) to a heart-shaped stage (bilateral symmetrical structure) and then to a torpedo-shaped stage (elongation). The transition from spherical to heart-shaped is characterized by the growth of two cotyledons and the initiation of radicle development (Zimmerman, JL (1993) Somatic Embryogenesis: A Model for Early Development in Higher Plants. The Plant Cell 5:1411~1423; Von Arnold et al. (2002) Developmental pathways of somatic embryogenesis. Plant Cell, Tissue and Organ Culture 69:233~249). Finally, the torpedo-shaped somatic embryo can develop into a plant body containing green cotyledons, an elongated hypocotyl, and developed radicles with clearly differentiated root hairs in a process called "germination" (similar to the zygote), "transition," or "maturation" (Von Arnold et al., above) (Zimmerman, above). In the direct or indirect induction of somatic somatic embryogenesis, auxins are used in the early stages to induce embryogenesis in the callus, but embryos are formed only after subculturing cultures in auxin-free or reduced-auxin media. Examples of auxins used for somatic embryo induction include 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picoram, and dicamba.

[0063] [Table 1]

[0064] In one embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the step of introducing or increasing the expression of at least SHORT ROOT (SHR) protein. The terms SHR, SGR7, SHOOT GRAVITROPISM 7, and SHORT ROOT are used interchangeably herein. Preferably, their expression is introduced or increased transiently. The SHR protein is a transcription factor that can be expressed in the central column and translocates to surrounding cells, including the quiescent center.

[0065] The quiescent center is a group of cells that act as organizers, preventing the differentiation of surrounding stem cells. Each stem cell adjacent to the quiescent center divides asymmetrically to renew itself and to produce daughter cells that divide multiple times in the meristem zone before exiting the cell cycle in the transition zone. Subsequently, the cells elongate and acquire a specific differentiation state. Stem cells distal to the quiescent center produce daughter cells that differentiate (Heidstra and Sabatini, 2014). In the quiescent center, SHRs can activate SCARECROW (SCR).

[0066] In addition, or otherwise, the method includes the step of introducing or increasing the expression of at least the SCARECROW (SCR) protein. The terms SCR, SCARECROW, SGR1, and SHOOT GRAVITROPISM 1 are used interchangeably herein. Preferably, the expression is introduced or increased transiently. The SCR protein is known to be required for the identification of quiescent centers and is involved in both stem cell maintenance and the differentiation of their offspring. In quiescent centers, SCR can directly repress the expression of the pro-differentiation cytokinin-responsive transcription factor ARR1.

[0067] In addition, or otherwise, the method includes at least the step of introducing or increasing the expression of WUSCHEL-associated homeobox 5 (WOX5) protein. Preferably, the expression is introduced or increased transiently. WOX5 is a homolog of WUS and marks the quiescent center beyond the proximal end. Loss of WOX5 function is known to result in the differentiation of distal columella stem cells without altering root growth and meristematic size. Nevertheless, mutant analysis has shown that WOX5 can redundantly regulate proximal stem cell maintenance (Sarkar et al. 2007).

[0068] In addition, or otherwise, the method includes the step of introducing or increasing the expression of at least a plethora (PLT) protein. Preferably, the expression is introduced or increased transiently. The PLT protein accumulates in the quiescent center to form a directive gradient and a peak protein level in the stem cell niche that coincides with the maximum auxin level. PLT can regulate the expression of the PIN gene family, which suggests a feedforward loop for maintaining high auxin and PLT levels in the stem cell niche (Heidstra and Sabatini, 2014). The PLT protein may be selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7.

[0069] In addition, the method may include the step of introducing or increasing the expression of at least two, three, four, or five PLT proteins, preferably at least three PLT proteins, and preferably the step of introducing or increasing the expression of three PLT proteins. Preferably, the expression of at least two, three, four, five, or six PLT proteins is introduced or increased transiently. The PLT proteins may be selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the PLT proteins used in the method of the present invention are PLT1, PLT4, and PLT5. Preferably, the expression of these three proteins is introduced or increased transiently. The terms PLT4 and BBM are used interchangeably herein. Similarly, the terms PLT5, AIL5, AINTEGUMENTA-LIKE 5, CHO1, CHOTTO 1, EMBRYOMAKER, and EMK may be used interchangeably herein. In addition, the terms PLT7, AIL7, AINTEGUMENTA-LIKE 7, and PLETHORA 7 may be used interchangeably herein.

[0070] In addition, or alternatively, the method includes the step of reducing the expression of Retinoblastoma Related (RBR) protein in plant cells. The terms RBR, ATRBR1, RB, RB1, RBR1, RETINOBLASTOMA 1, RETINOBLASTOMA-RELATED, RETINOBLASTOMA-RELATED 1, and RETINOBLASTOMA-RELATED PROTEIN 1 are used interchangeably herein. Preferably, the expression of the RBR protein is transiently reduced. As a plant homolog of the RB tumor suppressor protein, the RBR protein plays a crucial role in both the shoot stem cell niche and the root stem cell niche. As in animals, RBR inhibits cell cycle progression by interacting with the E2F transcription factor homolog. Furthermore, a decrease in RBR levels results in an increase in stem cell number, and an increase in RBR levels leads to stem cell differentiation, which demonstrates the significant role of RBR in stem cell maintenance (Heidstra and Sabatini, 2014).

[0071] In addition, or otherwise, the method includes the step of introducing or increasing the expression of at least WOUND INDUCED DEDIFFERENTIATION 1 (WIND1). The terms WIND1, ATWIND1, RAP2.4, RELATED TO AP2 4, and WOUND INDUCED DEDIFFERENTIATION 1 may be used interchangeably herein. Preferably, the expression of the WIND1 protein is introduced or increased transiently. The WIND1 protein is a central regulator of wound-induced cellular reprogramming in plants. It has been previously demonstrated that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ESR1 gene in Arabidopsis thaliana (Iwase et al., 2017).

[0072] In one embodiment, the present invention relates to a method for regenerating plant cells, preferably a shoot from plant cells, wherein the method includes increasing the expression of at least one of the protein combinations detailed above herein, for example, the following protein combinations, in plant cells: SHR protein and SCR protein; SHR protein and WOX5 protein; SHR protein and at least one or more PLT proteins; SHR protein and WIND1 protein; SCR protein and WOX5 protein; SCR protein and at least one or more PLT proteins; SCR protein and WIND1 protein; WOX5 protein and at least one or more PLT proteins; WOX5 protein and WIND1 protein; One or more PLT proteins and WIND1 proteins; SHR protein, SCR protein, and WOX5 protein; SHR protein, SCR protein, and at least one or more PLT proteins; SHR protein, SCR protein, and WIND1 protein; SHR protein, WOX5 protein, and at least one or more PLT proteins; SHR protein, WOX5 protein, and WIND1 protein; SCR protein, WOX5 protein, and at least one or more PLT proteins; SCR protein, WOX5 protein, and WIND1 protein; WOX5, at least one or more PLT proteins, and WIND1 protein; SHR protein, SCR protein, WOX5 protein, and at least one or more PLT proteins; SHR protein, SCR protein, WOX5 protein, and WIND1 protein; SHR protein, SCR protein, at least one or more PLT proteins, and WIND1 protein SHR protein, WOX5 protein, at least one or more PLT proteins, and WIND1 protein; SCR protein, WOX5 protein, at least one or more PLT proteins, and WIND1 protein; and SHR protein, SCR protein, WOX5 protein, at least one or more PLT proteins, and WIND1 protein.

[0073] Preferably, the expression of the proteins listed above is transiently introduced or increased in plant cells.

[0074] Preferably, one or more PLT proteins are selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, and preferably, the selected PLT proteins include at least one or more of PLT1, PLT4, and PLT5. Preferably, one or more PLT proteins are PLT1 or at least PLT1. Preferably, the PLT proteins are PLT1, PLT4, and PLT5.

[0075] In addition to each of the combinations listed above, the expression of endogenous RBR proteins may be reduced in plant cells. Preferably, the expression of RBR proteins is transiently reduced.

[0076] In a particularly preferred embodiment, the present invention relates to a method for regenerating plant cells, preferably a method for regenerating shoots from plant cells, the method comprising the step of altering the expression of at least one of the following protein combinations in the plant cells: Steps to increase the expression of WOX5 protein and PLT1 protein. A step to increase the expression of WIND1 protein, WOX5 protein, and PLT1 protein; A step of increasing the expression of SHR protein, SCR protein, WOX5 protein, PLT1 protein, PLT4 protein, and PLT5 protein; and A step of increasing the expression of WIND1 protein, SHR protein, SCR protein, WOX5 protein, PLT1 protein, PLT4 protein, and PLT5 protein, and downregulating RBR protein, preferably transiently downregulating RBR protein.

[0077] Preferably, the introduction or increase of the expression of the proteins listed above is transiently introduced or increased in plant cells.

[0078] In one embodiment, the method includes the step of allowing plant cells to regenerate, preferably into shoots.

[0079] In one embodiment, the proteins in the protein combination originate from different families, for example, two, three, four, five, or six different families. The first family includes RBR, the second family includes WIND1, the third family includes SHR, the fourth family includes SCR, the fifth family includes WOX5, and the sixth family includes PLT proteins, in particular the sixth family includes PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Those skilled in the art will understand that other family members may be equally suitable in the methods of the present invention.

[0080] In one embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of WOX5 protein and at least one PLT protein in plant cells. Preferably, the at least one PLT protein is selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the at least one PLT protein is PLT1. Preferably, the expression of at least one of the WOX5 protein and the at least one PLT protein is transiently introduced or increased. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0081] In one embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of WOX5 protein, at least one PLT protein, and WIND1 into plant cells. Preferably, the at least one PLT protein is selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the at least one PLT protein is PLT1. Preferably, the expression of at least one of the WOX5 protein, the WIND1 protein, and the at least one PLT protein is transiently introduced or increased. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0082] In one embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of SHR protein, SCR protein, WOX5 protein, and at least one PLT protein into plant cells. Preferably, the at least one PLT protein is selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the expression of at least one of the SHR protein, SCR protein, WOX5 protein, and at least one PLT protein is transiently introduced or increased. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0083] In a further embodiment, the present invention relates to a method for regenerating plant cells, and preferably, regenerating shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of SHR protein, SCR protein, WOX5 protein, and at least two PLT proteins into plant cells. Preferably, the at least two PLT proteins are selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the expression of at least one of the SHR protein, the SCR protein, the WOX5 protein, and at least one of the two PLT proteins is transiently introduced or increased. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0084] In another embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of SHR protein, SCR protein, WOX5 protein, and at least three PLT proteins into plant cells. Preferably, the at least three PLT proteins are selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the at least three selected PLT proteins include at least one or more of PLT1, PLT4, and PLT5. Preferably, the at least three selected PLT proteins are PLT1, PLT4, and PLT5. Preferably, the expression of at least one of the SHR protein, the SCR protein, the WOX5 protein, and at least one of the PLT proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least one of the SHR protein, SCR protein, WOX5 protein, PLT1 protein, PLT4 protein, and PLT5 protein is transiently introduced or increased. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0085] In another embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a1) A step of introducing or increasing the expression of SHR protein, SCR protein, WOX5 protein, WIND1 protein, and at least three PLT proteins into plant cells. Preferably, the at least three PLT proteins are selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. Preferably, the at least three selected PLT proteins include at least one or more of PLT1, PLT4, and PLT5. Preferably, the at least three selected PLT proteins are PLT1, PLT4, and PLT5. Preferably, the expression of at least one of the SHR protein, the SCR protein, the WOX5 protein, the WIND1 protein, and at least one of the PLT proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least one of the following proteins is transiently introduced or increased: the SHR protein, the SCR protein, the WOX5 protein, the WIND1 protein, the PLT1 protein, the PLT4 protein, and the PLT5 protein. a2) Optionally, a step of reducing the expression of endogenous RBR protein in plant cells, preferably a step in which the expression of the RBR protein is transiently reduced; and b) A step that enables the plant cells to regenerate, preferably into shoots.

[0086] Protein used in the method of the present invention The protein combination used in the present invention includes at least one of SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, PLT7, RBR, and WIND1. Preferably, the protein combination used in this method includes at least WOX5 and a PLT protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7.

[0087] Particularly preferred protein combinations include at least or at most the following: WOX5 protein and PLT1 protein WIND1 protein, WOX5 protein, and PLT1 protein; SHR protein, SCR protein, WOX5 protein, PLT1, PLT4, and PLT5 protein; and WIND1 protein, SHR protein, SCR protein, WOX5 protein, PLT1, PLT4, and PLT5 proteins, and RBR protein.

[0088] Preferably, the proteins in the protein combination, excluding the RBR, exhibit induction or increase in expression. Preferably, the RBR protein exhibits decrease in expression.

[0089] The amino acid sequence of the SHR protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1. SEQ ID NO: 1 is the Arabidopsis thaliana SHR protein (see Table 5 for an overview of all SEQ ID NOs used herein). In one embodiment, the SHR amino acid sequence is AT4G37650, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT4G37650 or its homologue, or derived therefrom.

[0090] The amino acid sequence of the SCR protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2. SEQ ID NO: 2 is the Arabidopsis thaliana SCR protein. In one embodiment, the SCR amino acid sequence is AT3G54220, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G54220 or its homologue, or derived therefrom.

[0091] The amino acid sequence of the WOX5 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 3. SEQ ID NO: 3 is the Arabidopsis thaliana WOX5 protein. In one embodiment, the WOX5 amino acid sequence is AT3G11260, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G11260 or its homologue, or derived therefrom.

[0092] The amino acid sequence of the PLT1 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4. SEQ ID NO: 4 is the Arabidopsis thaliana PLT1 protein. In one embodiment, the PLT1 amino acid sequence is AT3G20840, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G20840 or its homologue, or derived therefrom.

[0093] The amino acid sequence of the PLT2 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5 is the Arabidopsis thaliana PLT2 protein. In one embodiment, the PLT2 amino acid sequence is AT1G51190, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT1G51190 or its homologue, or derived therefrom.

[0094] The amino acid sequence of the PLT3 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6, which is the Arabidopsis thaliana PLT3 protein. In one embodiment, the PLT3 amino acid sequence is AT5G10510, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G10510 or its homologue, or derived therefrom.

[0095] The amino acid sequence of the PLT4 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, which is the Arabidopsis thaliana PLT4 protein. In one embodiment, the PLT4 amino acid sequence is AT5G17430, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G17430 or its homologue, or derived therefrom.

[0096] The amino acid sequence of the PLT5 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, which is the Arabidopsis thaliana PLT5 protein. In one embodiment, the PLT5 amino acid sequence is AT5G57390, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G57390 or its homologue, or derived therefrom.

[0097] The amino acid sequence of the PLT7 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30. SEQ ID NO: 30 is the Arabidopsis thaliana PLT7 protein. In one embodiment, the PLT7 amino acid sequence is AT5G65510, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G65510 or its homologue, or derived therefrom.

[0098] The amino acid sequence of the WIND1 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28. SEQ ID NO: 28 is the Arabidopsis thaliana WIND1 protein. In one embodiment, the WIND1 amino acid sequence is AT1G78080, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT1G78080 or its homologue, or derived therefrom.

[0099] The amino acid sequence of the RBR protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17 is the Arabidopsis thaliana RBR protein. In one embodiment, the RBR amino acid sequence is AT3G12280, its homologue, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G12280 or its homologue, or derived therefrom.

[0100] Proteins as defined herein include, for example, non-limitingly, T7 tags (e.g., T7 tags having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 53), Myc tags (e.g., sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43) The tags may further include Myc tags, FLAG tags (e.g., FLAG tags having a sequence with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 46), V5 tags (e.g., V5 tags having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 50), or His tags. Preferably, the tags are located at the C-terminus of the protein, before the original stop codon.

[0101] Nucleic acids used in the method of the present invention In one embodiment, the SHR protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. The nucleotide sequence encoding the SHR protein may be, or may be derived from, the gene AT4G37650, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT4G37650 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0102] Examples of homologs include Brachypodium distachyon (Purple false brome) (BRADI1G23060), soybean (Glycine max) (GLYMA01G40180, GLYMA05G22460, GLYMA11G05110, GLYMA11G23690, or GLYMA17G17400), glutinous rice (Oryza sativa) (SHR1 and SHR2), false moss (Physcomitrella patens) (PHYPADRAFT_14911 and PHYPADRAFT_22633), cottonwood (Populus trichocarpa) (black cottonwood) (POPTR_0012S06430G), and tomato (Solanum) Examples include lycopersicum (tomato) (SOLYC02G092370.1), sorghum bicolor (sorghum) (SB01G031720 and SB02G037890), and European grape (Vitis vinifera) (grape) VIT_07S0129G00030.

[0103] In one embodiment, the SCR protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. The nucleotide sequence encoding the SCR protein may be, or may be derived from, the gene AT3G54220, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G54220 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0104] Examples of homologs include Brachypodium distachyon (BRADI4G44090), soybean (Glycine max) (GLYMA09G40620 and GLYMA18G45220), glutinous rice (Oryza sativa) (SCR1 and SCR2), false moss (Physcomitrella patens) (PHYPADRAFT_150910, PHYPADRAFT_22273, PHYPADRAFT_42008 and PHYPADRAFT_65480), cottonwood (Populus trichocarpa) (black cottonwood) (POPTR_0006S11500G and POPTR_0016S15060G), and tomato (Solanum Examples include lycopersicum (tomato) SOLYC10G074680.1, sorghum bicolor (sorghum) SB05G001500, and European grape (Vitis vinifera) VIT_08S0056G00050.

[0105] In one embodiment, the WOX5 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. The nucleotide sequence encoding the WOX5 protein may be, or may be derived from, the gene AT3G11260, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G11260 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0106] Examples of homologs include Arabidopsis thaliana (AT5G05770.1), Brachypodium distachyon (BRADI2G55270), Glycine max (GLYMA02G42200), Oryza sativa (WOX9), cottonwood (Populus trichocarpa) (POPTR_0008S06560G and POPTR_0010S19950G), tomato (Solanum lycopersicum) (SOLYC03G096300.2), and sorghum. Examples include bicolor sorghum (SB03G040210) and European grape (Vitis vinifera) (VIT_13S0019G03460).

[0107] In one embodiment, the PLT1 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. The nucleotide sequence encoding the PLT1 protein may be, or may be derived from, the gene AT3G20840, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G20840 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0108] Examples of homologs include Arabidopsis thaliana (AT1G51190.1), soybean (Glycine max) (GLYMA11G14040 and GLYMA12G06010), cottonwood (Populus trichocarpa) (POPTR_0001S05580G and POPTR_0003S20470G), tomato (Solanum lycopersicum) (SOLYC11G061750.1), and European grape (Vitis vinifera) (VIT_06S0004G01800).

[0109] In one embodiment, the PLT2 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13. The nucleotide sequence encoding the PLT2 protein may be, or may be derived from, the gene AT1G51190, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT1G51190 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0110] Examples of homologs include Arabidopsis thaliana (AT3G20840.1), soybean (Glycine max) (GLYMA11G14040 and GLYMA12G06010), cottonwood (Populus trichocarpa) (POPTR_0001S05580G and POPTR_0003S20470G), tomato (Solanum lycopersicum) (SOLYC11G061750.1), and European grape (Vitis vinifera) (VIT_06S0004G01800).

[0111] In one embodiment, the PLT3 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14. The nucleotide sequence encoding the PLT3 protein may be, or may be derived from, the gene AT5G10510, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G10510 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0112] In one embodiment, the PLT4 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15. The nucleotide sequence encoding the PLT4 protein may be, or may be derived from, the gene AT5G17430, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G17430 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0113] Examples of homologs include Brachypodium distachyon (BRADI3G48697 and BRADI5G14960), soybean (Glycine max) (GLYMA09G38370 and GLYMA10G31440), glutinous rice (Oryza sativa) (OSJNBB0116K07.8), cottonwood (Populus trichocarpa) (POPTR_0008S07610G and POPTR_0010S18840G), tomato (Solanum lycopersicum) (SOLYC11G008560.1), and sorghum bicolor (SB04G025960).

[0114] In one embodiment, the PLT5 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. The nucleotide sequence encoding the PLT5 protein may be, or may be derived from, the gene AT5G57390, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G57390 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0115] Examples of homologs include Arabidopsis thaliana (AT4G37750.1), Brachypodium distachyon (BRADI1G07290), Glycine max (GLYMA0041S50, GLYMA01G40380, GLYMA02G31035, GLYMA05G22970, GLYMA06G05170, GLYMA11G04910, GLYMA14G10130, and GLYMA17G17010), Oryza sativa (OSJNBA0072F13.9), and Physcomitrella. Patens (moss) (PHYPADRAFT_127673, PHYPADRAFT_127688, PHYPADRAFT_136724, and PHYPADRAFT_189336), cottonwood (Populus trichocarpa) (black cottonwood) (POPTR_0002S11550G, POPTR_0005S19220G, POPTR_0007S14690G, and POPTR_0014S01260G), tomato (Solanum lycopersicum) (tomato) (SOLYC02G092050.2, SOLYC03G123430.2, and SOLYC04G077490.2), sorghum (Sorghum bicolor) (sorghum) (SB01G006830), and European grape (Vitis Examples include vinifera (grape) (VIT_07S0151G00440 and VIT_18S0001G08610).

[0116] In one embodiment, the PLT7 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. The nucleotide sequence encoding the PLT7 protein may be, or may be derived from, the gene AT5G65510, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT5G65510 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0117] Examples of homologs include Arabidopsis thaliana (AT5G10510.2), Brachypodium distachyon (BRADI1G31337), Glycine max (GLYMA01G02760, GLYMA08G38190, GLYMA09G33241, and GLYMA18G29400), Oryza sativa (P0677B10.15), cottonwood (Populus trichocarpa) (POPTR_0007S14210G), and tomato (Solanum). Examples include lycopersicum (tomato) (SOLYC05G051380.2 and SOLYC11G010710.1), sorghum bicolor (sorghum) (SB10G026150), and European grape (Vitis vinifera) (grape) (VIT_00S0772G00020 and VIT_00S1291G00010).

[0118] In one embodiment, the WIND1 protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29. The nucleotide sequence encoding the WIND1 protein may be, or may be derived from, the gene AT1G78080, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT1G78080 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0119] Examples of homologs include Arabidopsis thaliana (AT1G22190.1, AT1G36060.1, AT1G64380.1, AT2G20880.1, AT2G22200.1, AT4G13620.1, AT4G28140.1, AT4G39780.1, and AT5G65130.1), Brachypodium distachyon (BRADI1G45470), and soybean (Glycine max) (Soybean) (GLYMA01G43450, GLYMA05G31370, GLYMA06G45010, GLYMA08G14600, GLYMA10G33700, GLYMA11G02050, GLYMA12G12270, GLYMA12G33020, GLYMA13G01930, GLYMA13G37451, GLYMA14G34590, GLYMA18G02170, and GLYMA20G33890), glutinous rice (Oryza sativa) (rice) (OS06G0222400 and P0516A04.31), false bellflower (Physcomitrella) (Patens) (Moss) (PHYPADRAFT_142112 and PHYPADRAFT_151367), Cottonwood (Populus trichocarpa) (Black Cottonwood) (POPTR_0001S10540G, POPTR_0001S32250G, POPTR_0002S09480G, POPTR_0003S13910G, POPTR_0005S07900G, POPTR_0005S16690G, POPTR_0007S05690G, POPTR_0013S13920G, POPTR_0017S08250G, and POPTR_0019S13330G), Tomato (Solanum) Lycopersicum (tomato) (DREB3, SOLYC04G054910.2, SOLYC07G054220.1, SOLYC08G082210.2, SOLYC09G091950.1, SOLYC12G013660.1, and SOLYC12G056980.)1) Examples include sorghum bicolor (SB01G044410, SB02G023230, SB07G020090, SB08G007411, and SB10G007780), and European grape (Vitis vinifera) (VIT_00S0662G00030, VIT_00S0662G00040, VIT_02S0025G01360, VIT_05S0029G00140, VIT_12S0059G00280, VIT_18S0001G05250, and VIT_19S0014G03180).

[0120] Sequences encoding SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, PLT7, or WIND1 proteins are preferably codon-optimized for expression in plant cells, preferably codon-optimized for expression in plant cells of the method of the present invention, and preferably codon-optimized for the species of plant cells used in the method of the present invention. As a non-limiting example, an overexpressed or newly expressed protein, as defined herein, may be an endogenous protein, but the sequence encoding this endogenous protein is an exogenous codon-optimized sequence. Alternatively, a codon-optimized sequence may encode a protein that is exogenous to that plant cell.

[0121] In one embodiment of the present invention, a protein having increased or introduced expression, as defined herein, is a functional protein. SHR, as defined herein, preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 1 in Arabidopsis thaliana. SCR, as defined herein, preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 2 in Arabidopsis thaliana. WOX5, as defined herein, preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 3 in Arabidopsis thaliana. PLT1, as defined herein, preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 4 in Arabidopsis thaliana. PLT2 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 5 in Arabidopsis thaliana. PLT3 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 6 in Arabidopsis thaliana. PLT4 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 7 in Arabidopsis thaliana. PLT5 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 8 in Arabidopsis thaliana.PLT7 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 30 in Arabidopsis thaliana. WIND1 as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 28 in Arabidopsis thaliana.

[0122] In one embodiment, the endogenous RBR protein is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18. The nucleotide sequence encoding the RBR protein may be, or may be derived from, the gene AT3G12280, its homologues, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with AT3G12280 or its homologues. Percentage identity can be determined over the full length of the genome sequence. Alternatively, percentage identity can be determined over the full length of the coding sequence of the gene.

[0123] Examples of homologs include Brachypodium distachyon (BRADI3G41630), Chlamydomonas reinhardtii (MAT3), Glycine max (GLYMA04G36700, GLYMA13G26170, and GLYMA15G36890), Oryza sativa (RBR1), Physcomitrella patens (PHYPADRAFT_88833, RBL1502, and RBR), cottonwood (Populus trichocarpa) (RBL901), and tomato (Solanum) Examples include lycopersicum (tomato) (SOLYC09G091280.2), sorghum bicolor (sorghum) (SB07G025760), and European grape (Vitis vinifera) (grape) (VIT_04S0008G02780).

[0124] In one embodiment of the present invention, the RBR protein having reduced expression is a functional protein. Therefore, the RBR as defined herein preferably performs the same or similar function in plant cells as the protein having the amino acid sequence of SEQ ID NO: 17 in Arabidopsis thaliana.

[0125] In one embodiment, those skilled in the art may use a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17 or SEQ ID NO: 18 to find a sequence encoding a homologous RBR protein, preferably a genomic sequence, in a plant cell, preferably in the genome of the plant cell as defined herein. This sequence can then be used to target and downregulate the expression of endogenous RBR proteins, for example, by using an RNAi mechanism.

[0126] Transiently increased (SHR, SCR, WOX5, PLT1-PL5, PLT7, WIND1) or decreased (RBR) expression In one embodiment, the expression of at least one of the SHR protein, SCR protein, WOX5 protein, and WIND1 protein, as well as the expression of at least one, two, three, four, five, or six PLT proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, is transiently introduced or increased. Optionally, in addition, the expression of endogenous RBR protein is transiently decreased.

[0127] In one embodiment, the expression of at least WOX5 and a PLT protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least WOX5 and PLT1 is transiently introduced or increased. The expression of WOX5 and PLT1 alone may be transiently introduced or increased.

[0128] In one embodiment, the expression of at least WIND1, WOX5, and a PLT protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least WIND1, WOX5, and PLT1 is transiently introduced or increased. The expression of WIND1, WOX5, and PLT1 alone may be transiently introduced or increased.

[0129] In one embodiment, the expression of at least SHR, SCR, WOX5, and one, two, or three PLT proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least SHR, SCR, WOX5, PLT1, PLT4, and PLT5 is transiently introduced or increased. The expression of only SHR, SCR, WOX5, PLT1, PLT4, and PLT5 may be transiently introduced or increased.

[0130] In one embodiment, the expression of at least WIND1, SHR, SCR, WOX5, and one, two, or three PLT proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is transiently introduced or increased. Preferably, the expression of at least WIND1, SHR, SCR, WOX5, PLT1, PLT4, and PLT5 is transiently introduced or increased. The expression of only WIND1, SHR, SCR, WOX5, PLT1, PLT4, and PLT5 may be transiently introduced or increased. In addition, the expression of endogenous RBR proteins is transiently decreased.

[0131] An increase or decrease in the expression of proteins as defined herein induces the regeneration of plant cells, preferably shoot regeneration. Transient introduction or increase in expression of proteins as defined herein, and optionally, transient decrease in expression, may be continuous or simultaneous. For example, transient expression of one or more proteins in a protein combination as defined herein, such as WIND1, may occur before transient expression of one or more subsequent proteins as defined herein. The increase or introduction of these subsequent proteins in the protein combination may occur during or after the increase or introduction of the first one or more proteins.

[0132] Similarly, a transient decrease in the expression of RBR proteins as defined herein may occur before, during, or after a transient introduction or increase in the expression of one or more proteins as defined herein. Likewise, the increase or introduction of the expression of one or more proteins may occur before, during, or after a decrease in the expression of RBR proteins.

[0133] Preferably, there is a simultaneous increase in expression or introduction of at least two, three, four, five, or six proteins selected from the group consisting of WIND1, SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 in plant cells at a given time. Optionally, the expression of RBR proteins is simultaneously or concurrently decreased.

[0134] Preferably, there is a simultaneous increase in expression or introduction of at least two, three, four, five, or six proteins selected from the group consisting of WIND1, SHR, SCR, WOX5, PLT1, PLT4, and PLT5 in plant cells at one time.

[0135] For example, at some point in time, there may be a simultaneous increase or introduction of expression of at least WOX5 and PLT1, or a simultaneous increase or introduction of expression of at least WOX5, PLT1, and WIND1, or a simultaneous increase or introduction of expression of at least SHR, SCR, WOX5, PLT1, PLT4, and PLT5, or a simultaneous increase or introduction of expression of at least WIND1, SHR, SCR, WOX5, PLT1, PLT4, and PLT5. Optionally, the expression of RBR proteins may be decreased simultaneously or during the same period.

[0136] Alternatively, the expression of one or more proteins as defined herein may be transiently introduced or increased and optionally decreased prior to the introduction or increase of the expression of the following one or more proteins. In one embodiment, the expression of WIND1 may be transiently introduced or increased and the expression of RBR may be transiently decreased prior to the introduction or increase of the expression of one or more of the SHR, SCR, WOX5, and one or more PLT proteins as defined herein.

[0137] As a non-limiting example, WIND1 expression may be transiently increased or introduced at least before transient induction or increase of WOX5 and PLT1 expression. WIND1 expression may be transiently increased or introduced at least before transient induction or introduction of WOX5, PLT1, SHR, SCR, PLT4, and PLT5 expression. RBR protein expression may be decreased simultaneously with the increase or introduction of WIND1 expression.

[0138] Introducing or increasing WIND1 expression, and optionally decreasing RBR expression, may occur before the expression of any of the other proteins as defined herein. WIND1 expression levels, and optionally RBR expression levels, may remain altered during the introduction or increase of expression of the other proteins in the protein combination as defined herein. Alternatively, WIND1 expression levels, and optionally RBR expression levels, may have returned to endogenous levels before the increase or introduction of expression of the other proteins in the protein combination as defined herein.

[0139] The period between introducing a change in the expression level of WIND1 and optionally RBR and inducing a change in the expression level of other proteins in the protein composition as defined herein is preferably at least about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 days, or at least about 14 days.

[0140] period The period during which a protein, as defined herein, has increased or altered expression, or optionally decreased expression (as specified herein for a particular protein), in a plant cell is preferably long enough to induce plant cell regeneration. However, maintaining altered expression levels may hinder the further development of the regenerated plant cells into plants. Therefore, in preferred embodiments, the changes in the expression of at least one, two, three, four, five, or six of the aforementioned proteins are transient. The expression levels of a protein, as defined herein, can be returned to endogenous levels, i.e., the protein levels before the increase, alteration, or decrease in expression, preferably before one or more of the proteins induce uncontrolled meristem formation. Returning protein levels to endogenous levels preferably induces tissue differentiation.

[0141] In one embodiment, the period during which a protein combination as defined herein has increased, introduced, or decreased expression in plant cells is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 days, or at least about 50 days. The period may be at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or at least about 10 weeks. After this period, the protein level of at least one protein, preferably all proteins, can return to endogenous levels. Therefore, the said period may be less than about 100 days, less than 95 days, less than 90 days, less than 85 days, less than 80 days, less than 75 days, less than 70 days, less than 65 days, less than 60 days, less than 55 days, less than 50 days, less than 45 days, less than 40 days, less than 35 days, less than 30 days, less than 25 days, less than 20 days, or less than about 15 days. The said period may be less than about 20 weeks, less than 15 weeks, less than 12 weeks, less than 10 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, or less than about 2 weeks. The period during which one or more proteins, as defined herein, have increased or introduced expression may be about 0.5 to 10 weeks, 1 to 8 weeks, 1 to 6 weeks, 1 to 4 weeks, about 1 to 3 weeks, or about 2 weeks.

[0142] Preferably, for the same period defined above herein, at least one, two, three, four, five, or six proteins selected from the group consisting of WIND1, SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, and PLT5 have increased or introduced expression in the plant cells, and preferably for the same period as defined herein, at the same time that each of the proteins in the protein combination as defined herein has increased or introduced expression, the plant cells also have decreased expression of endogenous RBR proteins.

[0143] The duration for introducing, increasing, and optionally decreasing the expression of one or more proteins as defined herein may depend on the type of plant cell and / or regeneration conditions, and appropriate durations can be determined using conventional means known in the art. In non-limiting examples, the expression of such proteins may be introduced or increased and optionally decreased as defined herein, and the morphology of the plant during regeneration can be monitored. Protein levels can be returned to normal or endogenous levels immediately before or after regeneration. For example, the introduction or increase and optionally decrease of protein expression in a protein combination as defined herein can be returned to endogenous levels approximately two weeks, four weeks, six weeks, eight weeks, or ten weeks after regeneration, for example, from the appearance of the first shoot, or approximately two months, four months, six months, eight months, or ten months after regeneration.

[0144] Transiently introduced or increased expression In one embodiment, the expression of at least one of WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 is introduced or increased in plant cells. Such increase may be by increased expression of endogenous proteins (i.e., via mutations in the endogenous genes encoding these proteins that result in increased expression of functional proteins, or via mutations in the regulatory or coding sequences that result in increased protein function), or via transgenic introduction of constructs encoding the protein sequences. The introduced or increased expression is preferably transient. Thus, the expression is introduced or increased for a specific period as defined above herein, and preferably, the expression is introduced or increased only for a specific period. Transient increase or introduction of expression can be achieved using any suitable means known in the art. For example, the expression of one or more previously introduced proteins can be knocked out, for example, using targeted mutagenesis or RNAi.

[0145] Alternatively, transient expression enhancement can be achieved by transient introduction of one or more proteins as defined herein into plant cells. Alternatively, transient expression can be achieved by introducing one or more nucleic acid constructs into plant cells, wherein the nucleic acid construct comprises a coding sequence encoding one or more proteins as defined herein, and the coding sequence is operably linked to a regulatory element, such as a constitutive or tissue-specific promoter. A “constitutive” promoter is understood herein to be a promoter that is active in most tissues under most physiological and developmental conditions. A “tissue-specific” promoter is active only in specific types of tissues or cells.

[0146] Alternatively, or in addition, transient expression of one or more proteins as defined herein is achieved by transient activation of their gene expression. Thus, the expression of at least one of the WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins, preferably a combination of proteins as defined herein, is transiently introduced or increased by transient activation of their expression.

[0147] Transient activation of expression can be achieved by placing one or more sequences encoding proteins as defined herein under the control of an inductive promoter. An "inductive" promoter is defined herein as a promoter that is physiologically (e.g., by the external application of a particular compound) or developmentally regulated. Preferably, at least one, two, three, four, five, six, or at least seven genes encoding the WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 proteins, respectively, are placed under the control of an inductive promoter. The expression of proteins as defined herein may be controlled by the same type of inductive promoter, or the expression of different proteins may be controlled by different types of inductive promoters.

[0148] An inducible promoter may be placed upstream of one or more endogenous genes encoding proteins as defined herein, for example, upstream of endogenous genes encoding WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7, and optionally operably ligated to that endogenous gene. Preferred examples of inducible promoters are described in the following second aspect of this specification.

[0149] Alternatively, plant cells may be stably transformed with a construct in which the expression of at least one of WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, or PLT5 is controlled by an inducible promoter. Preferably, plant cells are transformed with a construct as described in the second embodiment below. Preferably, plant cells are stably transformed with one or more expression constructs as defined in the second embodiment of this specification.

[0150] As a non-limiting example, a transactivator can bind to an inductive promoter and subsequently induce the transcription of one of the proteins as defined herein. Such a transactivator may first be activated by binding to a specific compound (inducer). Alternatively, the binding of the transactivator to the inductive promoter may be inhibited in the presence of a specific compound (inhibitor). Preferably, the transactivator used in the method of the present invention is activated upon binding to a specific compound (inducer). Preferably, the inducer is an inducer as described in the second aspect below herein.

[0151] Transient decrease in expression In some embodiments, in combination with increased or introduced expression of at least one protein as defined above herein, the expression of the RBR protein is reduced in plant cells, preferably transiently. Reduced RBR protein expression is understood herein to be reduced expression of the endogenous protein. Such reduction may occur through mutations in one or more endogenous genes resulting in reduced expression of the endogenous functional protein, i.e., mutations in a regulatory sequence (e.g., promoter) or coding sequence, or through transgenic introduction of a construct encoding a repressor.

[0152] The expression is preferably reduced for a specific period as defined above herein, and preferably, the expression is reduced only for a specific period as defined above herein. Transient reduction in expression can be achieved using any suitable means known in the Art. For example, transient reduction in expression can be achieved by introducing a small RNA transcript (e.g., a miRNA or siRNA targeting an RBR gene transcript) into cells, or by introducing an RNAi construct into plant cells, the expression of the small RNA (e.g., a miRNA or siRNA) can be controlled by a regulatory element, such as a constitutive or tissue-specific promoter.

[0153] Alternatively, or in addition, transient reduction in RBR expression can be achieved by transient expression of RBR repressors. Non-limiting examples of RBR repressors are non-coding small RNAs, such as miRNAs or siRNAs, that target RBR RNA transcripts. Mature siRNAs or miRNAs may contain at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides. Mature siRNAs or miRNAs may contain at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides that have at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with the consecutive sequence in the endogenous RBR transcript.

[0154] The endogenous RBR transcript is preferably an endogenous RBR mRNA molecule, and preferably contains 3' and 5' untranslated RBR sequences. Therefore, the sequence of the non-coding small RNA may be partially or completely complementary to the sequence contained in the RBR coding sequence, or to the sequence contained in the 3' or 5' untranslated region of the RBR transcript. Preferably, the siRNA or miRNA may be partially or completely complementary to the sequence contained in the RBR coding sequence. For example, at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides of a small RNA molecule have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with a sequence of at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides of an endogenous RBR transcript.

[0155] Those skilled in the art will understand a method for designing a small RNA molecule capable of downregulating endogenous RBR protein expression using conventional RNAi, wherein the RBR protein is an RBR protein as defined above herein.

[0156] In one embodiment, a small RNA molecule for inhibiting RBR expression may contain a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23. The small RNA molecule may contain a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24.

[0157] Transient activation of RBR repressor expression as defined herein can be achieved by placing the sequence encoding the RBR repressor upstream of an inductive promoter and operably ligated to the inductive promoter. The inductive promoter controlling the expression of the RBR repressor may be the same type of inductive promoter controlling the expression of at least one of the following proteins: WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7. Alternatively, the inductive promoter may be a different type of inductive promoter. Transient expression of an RBR repressor results in a transient decrease in the expression of an RBR protein as defined herein.

[0158] A preferred example of an inducible promoter is described in the second aspect of this specification below.

[0159] Use of the structure of the present invention In one embodiment, plant cells can be stably transformed with one or more nucleic acids comprising an expression cassette in which the expression of at least one of WOX5, WIND1, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, preferably at least one of WOX5, WIND1, SHR, SCR, PLT1, PLT4, or PLT5, is controlled by an inductive promoter. Optionally, plant cells can also be stably transformed with a nucleic acid construct comprising an expression cassette in which the expression of an RBR repressor is also controlled by an inductive promoter. Some or all of the nucleic acids may be included in a single construct.

[0160] Preferably, plant cells are transformed with one or more constructs as described in the second embodiment below. Therefore, in one embodiment, the present invention relates to a method for regenerating plant cells, and preferably shoots from plant cells, comprising the following steps: a) The step of introducing, preferably stably, one or more nucleic acids or nucleic acid constructs as defined in the second embodiment of this specification into plant cells; b) A step of maintaining the cells in a culture medium containing an inducing factor; c) Optionally, the step of detecting the expression levels of at least one or more proteins as defined herein, and optionally, selecting plant cells having altered expression levels of at least one or more proteins as defined herein; d) optionally, the step of maintaining the selected plant cells in a culture medium containing the inducing factor for a period of time as defined herein; and e) A step that allows the plant cells to regenerate, preferably into shoots.

[0161] After the plant cells have regenerated, the inducing factors can be removed from the culture medium.

[0162] The constructs can be introduced into plant cells using any conventional means known in the art. Non-limiting examples of transformation methods include Agrobacterium transformation of plant tissue, particulate gun irradiation, and electroporation. A preferred transformation method is Agrobacterium transformation, for example, using the floral dipping method (Clough et al., 1998).

[0163] The preferred concentration of the inductor in step b) is a concentration known in the art to effectively activate a transactivator, as defined herein, which leads to the subsequent activation of the inductive promoter. The preferred concentration is about 0.05 μM to 50 μM, preferably about 0.1 μM to 15 μM, and preferably about 10 μM.

[0164] In one embodiment, the present invention relates to a method for regenerating plant cells as defined herein, wherein the plant cells are not exposed to plant growth hormones before, after, and / or during the regeneration of the plant cells. The plant growth hormones may be at least one of cytokinins or auxins. Preferably, the plant cells are not exposed to concentrations of plant growth hormones that would induce plant cell regeneration, such as unmodified, e.g., wild-type plant growth hormones.

[0165] While plant cells may be exposed to plant growth hormones, it is further intended herein that the presence of such plant hormones is not an essential requirement for the regeneration of plant cells.

[0166] In a preferred embodiment, the present invention relates to a method for hormone-independent shoot regeneration from plant cells, comprising the following steps: a) at least i) WUSCHEL-associated homeobox 5 (WOX5) protein; and ii) A PLETHORA (PLT) protein selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, preferably PLT1 A step of introducing or increasing the expression of a protein combination including into plant cells, wherein the expression of at least one protein of the protein combination is transiently introduced or increased; and b) A step that allows the plant cells to regenerate into shoots.

[0167] "Hormone-independent shoot regeneration" is understood herein as shoot regeneration that does not require exposure of plant cells to plant growth hormones. Preferably, shoot regeneration is shoot organ formation.

[0168] In one embodiment, plant tissue is not damaged to stimulate plant regeneration. Damage is a well-known step in tissue culture techniques, and those skilled in the art know how to damage plant cells and how to induce wound stress. While plant tissue can be damaged, it is intended herein that such damage is not an essential step for regeneration.

[0169] multicellular plant tissue In one embodiment, plant cells are part of a multicellular tissue. The plant multicellular tissue may include differentiated cells. Or, in addition, the multicellular tissue may include undifferentiated cells. In one embodiment, all cells of the multicellular tissue have increased or introduced expression of one or more proteins as defined herein at a particular time or for a period of time. Preferably, 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 about 100% of the cells in a multicellular tissue have, at a particular time or period, increased or novel expression of at least one of the WOX5, WIND1, SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins, and optionally decreased expression of RBR proteins as defined herein.

[0170] In one embodiment, 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 about 100% of the cells in a multicellular tissue have a change in the expression of all proteins of a protein combination as defined herein at a particular time or over a period of time.

[0171] All cells of a multicellular plant tissue can be transformed to have increased or altered expression of one or more proteins as defined herein, preferably transient increased or altered expression. In addition, all cells of a multicellular tissue can be transformed to have decreased expression of RBR proteins as defined herein. Preferably, 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 about 100% of the cells of the multicellular tissue are transformed to have altered expression of all proteins in a protein combination as defined herein.

[0172] Multicellular tissues may be callus tissue, plant organs, or explants. In some embodiments, plant cells having increased or introduced expression of one or more proteins as defined herein may be part of a plant organ. Plant organs may be vegetative or reproductive organs. Vegetative organs may originate from shoots or root systems. Organs may be at least one of roots, stems, and leaves. Reproductive plant organs may be selected from the group consisting of flowers, seeds, fruits, cones, sporangia, sporangiocarps, and gametophoes. Preferably, the plant organ is a root. Roots are understood herein as the part of the plant body that has neither leaves nor nodes. A typical arrangement of cells in a root is root hairs, epidermis, epibrem, cortex, endodermis, endothelium, and vascular tissue. In one embodiment, at least one cell or cell portion of the root hair, epidermis, epibrem, cortex, linden, endothelium, and vascular tissue has novel expression or increased expression of at least one of the WOX5, WIND1, SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins as defined herein, and optionally has decreased expression of endogenous RBR protein. Preferably, at least one cell or cell portion of the root hair, epidermis, epibrem, cortex, linden, endothelium, and vascular tissue has a change in the total protein expression of the protein combination as defined herein for the period defined above herein.

[0173] Alternatively, or in addition, plant cells having altered expression of one or more proteins as defined herein may be part of a seedling.

[0174] Alternatively, or in addition, plant cells having altered expression of one or more proteins as defined herein may be part of the callus. The callus is a group of undifferentiated cells, preferably derived from adult cells. Callus cells may be capable of embryogenesis and the formation of a completely new plant. Plant callus can be considered a growth mass of unorganized plant parenchyma cells. The callus can arise from a single differentiated cell, and the callus cell may be totipotent and capable of regenerating an entire plant body. The plant callus may originate from one or more somatic tissues, for example, tissues available for explant culture. The cells that give rise to the callus and somatic embryos are preferably rapidly dividing and / or partially undifferentiated, such as meristematic tissue. The callus cells used in the methods of the present invention may be fragile or compact. In addition, or alternatively, the callus cells may be root-forming, shoot-forming, or embryo-forming callus (Ikeuchi M, Plant Cell. 2013 Sept; 25(9):3159~3173).

[0175] In one embodiment, a plant cell having altered expression of one or more proteins as defined herein may be part of an explant. An explant can be defined herein as a sample obtained from a part of a plant. The plant sample may be placed in a solid or liquid medium. Explants can be isolated from many different parts of a plant, including shoots, leaves, stems, flowers, roots, portions of a single undifferentiated cell, and from mature cells. The cells preferably contain living cytoplasm and nucleic acids, and can dedifferentiate and resume cell division. Explants may be, for example, the ends of a plant's meristem, such as shoot apex, axillary bud apex, or root tip, or may be obtainable from there. In one embodiment, the explant is selected from the group consisting of hypocotyl explants, stem explants, cotyledon explants, root explants, leaf explants, floral explants, and meristem.

[0176] In further embodiments, plant cells can be obtained from plants selected from the group consisting of Arabidopsis, barley, cabbage, canola, cassava, cauliflower, chicory, chrysanthemum, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, rapeseed, potato, pumpkin, rice, rye, sorghum, soybean, pumpkin, sugarcane, sugar beet, sunflower, paprika, tomato, watermelon, wheat, and zucchini. Optionally, plant cells can be obtained from plants of the Solanaceae family, optionally from the Solanum genus, optionally from the tomato species (Solanum lycopersicum) or the eggplant species (Solanum melongena). Optionally, plant cells can be obtained from the Brassicaceae family, optionally from its species or subspecies, such as radish (Raphanus sativus), cabbage (Brassica oleracea), turnip (Brassica rapa), rapeseed (Brassica napus), horseradish, or Arabidopsis thaliana.

[0177] In one embodiment, the plant cells are refractory plant cells, i.e., plant cells whose regeneration efficiency is not useful or whose regeneration efficiency is poor. Non-limiting examples include pepper, soybeans, cucumbers, and sugar beets.

[0178] In a further embodiment, the plant cells are selected from the group consisting of Arabidopsis, tomato, and paprika.

[0179] Preferably, the plant is not of the genus Nicotiana, or is not available from it. Preferably, the plant is not of the species Nicotiana tabacum, or is not available from it.

[0180] In one embodiment, the method includes the step of forming a plant or plant part from a regenerated shoot. "Forming," "generating," or "regenerating" a plant or plant part preferably includes the step of elongating the formed shoot. Preferably, the elongated shoot is removed from the callus or explant.

[0181] Subsequent root formation can be induced in a separate root induction step, for example, by incubating the shoot in a different medium (Thorpe, above). Alternatively, roots may form spontaneously through any further induction. The regenerated plant can then be grown in soil, for example, to produce seeds.

[0182] The resulting plant, plant part, or plant product may consist of cells transformed to have a change in the total protein expression of a protein combination as defined herein, preferably a transient change.

[0183] Preferably, at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or about 100% of the cells of the formed plant, plant part, or plant product are transformed to have a change in the expression of all proteins of the protein combination as defined herein, preferably a transient change.

[0184] Preferably, the formed plant or plant part does not have introduced or increased expression of at least one of the WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 proteins as defined herein. Preferably, the formed plant or plant part does not have decreased expression of the RBR protein as defined herein. Therefore, at least one of the WIND1, WOX5, SHR, SCR, WOX5, PLT1, PLT2, PLT3, PLT4, PLT5, PLT7, and RBR proteins as defined herein has an endogenous expression level in the formed plant or plant part. Preferably, the WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, PLT7, and RBR proteins as defined herein have an endogenous expression level in the formed plant or plant part. Preferably, the total protein expression level of a protein combination as defined herein has an endogenous expression level in the formed plant or plant part. Nevertheless, the formed plant or plant part may include constructs as defined in the second embodiment herein, which may be present in at least a detectable amount.

[0185] Endogenous protein expression levels are understood herein as unmodified, naturally occurring protein expression levels. Therefore, endogenous expression levels are the expression levels in plant cells that are not modified to have alterations in the expression of one or more proteins as defined herein, such as introduction, increase, or decrease, and are otherwise identical. In a formed plant or plant part, the expression levels of one or more proteins as defined herein may be the same as or similar to their endogenous protein expression levels. Alternatively, a formed plant or plant part may maintain elevated expression of one or more proteins as defined herein.

[0186] nucleic acid construct In a second embodiment, the present invention relates to a nucleic acid molecule comprising at least one expression cassette, wherein the expression cassette comprises a nucleotide sequence encoding at least one of the following as defined herein in the first embodiment: WIND1 protein, WOX5 protein, SHR protein, SCR protein, PLT1 protein, PLT2 protein, PLT3 protein, PLT4 protein, PLT5 protein, PLT7 protein, and an RBR repressor. The terms “nucleic acid” and “nucleic acid molecule” may be used interchangeably herein.

[0187] In one embodiment, the nucleotide sequence encoding at least one of the WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins is a sequence as defined in the first embodiment. In one embodiment, the nucleotide sequence encoding the RBR repressor has a sequence as defined in the first embodiment.

[0188] Preferably, the nucleotide sequence encoding the SHR protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. Preferably, the nucleotide sequence encoding the SCR protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. Preferably, the nucleotide sequence encoding the WOX5 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. Preferably, the nucleotide sequence encoding the PLT1 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. Preferably, the nucleotide sequence encoding the PLT2 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13. Preferably, the nucleotide sequence encoding the PLT3 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14. Preferably, the nucleotide sequence encoding the PLT4 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15. Preferably, the nucleotide sequence encoding the PLT5 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. Preferably, the nucleotide sequence encoding the PLT7 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19.Preferably, the nucleotide sequence encoding the WIND1 protein has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29. Preferably, the nucleotide sequence encoding the RBR repressor has at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23, or preferably, the nucleotide sequence encoding the RBR repressor has at least about 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24.

[0189] In one embodiment, a single promoter can control the expression of two or more proteins as defined herein. In such cases, sequences encoding two or more proteins are preferably separated by, for example, intra-sequence ribosome entry sites (IRESs) or other suitable elements that enable translation initiation in a cap-independent manner.

[0190] In one embodiment, WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, PLT7, and RBR inhibitors are each independently controlled by promoters. Therefore, the expression cassette may include a promoter that controls the expression of an SHR protein as defined herein, or an expression cassette may include a promoter that controls the expression of an SCR protein as defined herein, or an expression cassette may include a promoter that controls the expression of a WOX5 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT1 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT2 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT3 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT4 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT5 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a PLT7 protein as defined herein, or an expression cassette may include a promoter that controls the expression of a WIND1 protein as defined herein, or an expression cassette may include a promoter that controls the expression of an RBR repressor as defined herein.

[0191] The promoter in the expression cassette is preferably a constitutive promoter, a tissue-specific promoter, or an inductive promoter. Preferably, a nucleotide sequence encoding a protein or repressor as defined herein is operably ligated to an inductive promoter, preferably an inductive promoter as defined below herein.

[0192] Inducible promoters are well known in the art. A preferred inducible promoter can be switched on by an inducer and is typically active as long as it is exposed to the inducer (i.e., the inducer). The inducer may be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed on plant cells, such as cold, heat, salt, toxin, or the action of a microbial pathogen or pest.

[0193] Therefore, inducible promoters can be used to regulate the expression of one or more proteins, as defined in the first embodiment.

[0194] The inductive promoter in the expression cassette of the present invention may be a stress-inducible promoter, a photo-inducible promoter, or a chemical-inducible promoter.

[0195] Examples of abiotic stress-inducible promoters include, but are not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., 1993); desiccation-inducible promoters such as the maize rabl7 gene promoter (Pla et al., 1993), the maize rab28 gene promoter (Busk et al., 1997), and the maize Ivr2 gene promoter (Pelleschi et al., 1999); and heat-inducible promoters such as the tomato-derived heat tomato hsp80 promoter (U.S. Patent No. 5,187,267) and the PHS1 heat shock protein gene (Takahashi et al., 1989).

[0196] Examples of light-inducible promoters include three chlorophyll a / b light-harvesting protein promoters (Leutwiler et al., 1986) and the preferedoxin promoter (Vorst et al., 1990).

[0197] Other examples of inducible promoters include the promoter derived from the 27kD subunit (GST-II-27) of glutathione-S-transferase, isoform II. This promoter is induced by a chemical compound known as a "herbicide antidote," which can be applied to plant cells to induce the promoter. See international applications PCT / GB92 / 01187 and PCT / GB90 / 00101, incorporated herein by reference. This promoter functions in both monocots and dicots. Similarly, the alcA / alcR gene activation system of Aspergillus nidulans (for example, including the AlcA element of SEQ ID NO: 32, or any sequence having at least approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, and the AlcR transactivator sequence of SEQ ID NO: 31, or any sequence having at least approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31) can be used for chemically inducible gene expression.

[0198] The AlcR / AlcA gene activation system is ethanol-inducible. Other suitable systems for chemical induction include the alc-switch, GVE / VGE, GVG, pOp6 / LhGR (Craft et al., 2005), and the XVE system (incorporated herein by reference, Moore et al., 2006, in particular Figure 3 and Table 5).

[0199] The LhGR and GVG systems use dexamethasone as an inductor. The XVE system uses 17-β-estradiol as an inductor, and the VGE system uses methoxyphenozide (Intrepid-F2). Those skilled in the art know how to use these inductive systems to clone a suitable promoter element upstream of a protein-encoding nucleotide sequence, as defined in the first embodiment of this specification, in order to achieve inductive expression.

[0200] In one embodiment, the induction system used for transient activation of expression as defined herein is at least one of the LhGR, GVG, or XVE systems, or a combination thereof.

[0201] One or more nucleic acid molecules, as defined herein, may be part of a nucleic acid construct.

[0202] The present invention further relates to nucleic acid constructs comprising two or more expression cassettes, such as two, three, four, five, six, seven, or eight different expression cassettes as defined herein. Alternatively, each expression cassette as defined herein may be present in the nucleic acid construct in more than one-fold, for example, two-fold, three-fold, four-fold, or five-fold. Preferably, each expression cassette comprises, under the control of an inducible promoter, a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein, or an RBR repressor, as defined herein.

[0203] In one embodiment, the nucleic acid construct is at least A first nucleic acid sequence comprising an expression cassette having a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein, or an RBR repressor, as defined herein, under the control of an inducible promoter; and A second nucleic acid sequence comprising a second expression cassette having a sequence encoding a control element and a transactivator operably linked to it. The transactivator may include the following transactivators as defined below in this specification. Preferably, the regulatory element is a strong constitutive promoter such as CaMV, G10-90, CsV, TCTP2, or UBQ10 promoter. Upon binding the inducing factor, the expressed transactivator can bind to the inducible promoter of the first nucleic acid molecule to induce transcription.

[0204] The nucleic acid molecules or constructs of the present invention may include expression cassettes for the expression of each protein combination and / or repressor as defined in the first embodiment of this specification. Optionally, the protein combination and / or repressor of the first embodiment may be in separate constructs, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve different constructs, but preferably the combination may be in a single construct.

[0205] GVG series A nucleic acid molecule and an expression cassette contained within a nucleic acid molecule may contain one or more UAS elements, the elements preferably linked to a minimal promoter such as the -46 35S minimal promoter. The minimal promoter and the elements are preferably located upstream of a sequence encoding a protein or repressor as defined in the first embodiment and operably linked to it. Preferably, a nucleic acid molecule and an expression cassette contained within a nucleic acid molecule may contain at least about four or five UAS elements located upstream of a sequence encoding a protein or repressor as defined in the first embodiment. The UAS sequence preferably has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22. The UAS elements are preferably operably linked to a sequence encoding a protein or repressor as defined in the first embodiment. One or more of these UAS elements may be bound by a transactivator, resulting in transcription of a protein or repressor as defined herein.

[0206] The transactivator that binds one or more UAS elements preferably comprises a GAL4 DNA-binding domain, a VP16 domain, and a glucocorticoid receptor (GR) domain. Such GVG systems are well known in the art and are described, for example, by Moore et al. (2006). The transactivator can induce transcription upon binding of dexamethasone or a derivative thereof. Therefore, in one embodiment of the present invention, the nucleic acid may comprise an expression cassette, the expression cassette comprising a sequence encoding the transactivator. Preferably, the transactivator is a protein comprising a domain that binds to the UAS element, preferably a GAL4 domain. Preferably, the transactivator further comprises a GR domain and a VP16 domain. In a preferred embodiment, the nucleotide sequence encoding the transactivator has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0207] LhGR series In one embodiment, the nucleic acid molecule and the expression cassette contained within the nucleic acid molecule may include one or more LacOp elements, the elements preferably linked to a minimal promoter such as the -46 35S minimal promoter. The minimal promoter and the elements are preferably located upstream of a sequence encoding a protein or repressor as defined in the first embodiment and are operably linked to it. Preferably, the nucleic acid molecule and the expression cassette contained within the nucleic acid molecule may include at least about five or six LacOp elements located upstream of a sequence encoding a protein or repressor as defined in the first embodiment. The LacOp sequence preferably has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25. The LacOp elements are preferably operably linked to a sequence encoding a protein or repressor as defined in the first embodiment. One or more of these LacOp elements may be bound by a transactivator, resulting in the transcription of a protein or repressor as defined herein.

[0208] A transactivator that binds one or more LacOp elements preferably comprises a GAL4 DNA-binding domain, a VP16 domain, and a glucocorticoid receptor (GR) domain. Such LacOp systems are well known in the art and are described, for example, by Moore et al. (2006). The transactivator can induce transcription upon binding of dexamethasone or a derivative thereof. Therefore, in one embodiment of the present invention, the nucleic acid may comprise an expression cassette, the expression cassette comprising a sequence encoding the transactivator. Preferably, the transactivator is a protein comprising a domain that binds to the LacOp element, preferably a GAL4 domain. Preferably, the transactivator further comprises a GR domain and a VP16 domain. In a preferred embodiment, the nucleotide sequence encoding the transactivator has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21. Preferably, the transactivator has an amino acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 58.

[0209] XVE series The nucleic acid molecule and the expression cassette contained within the nucleic acid molecule may contain one or more LexAop elements, which are preferably linked to a minimal promoter such as the -46 35S minimal promoter. The minimal promoter and the elements are preferably located upstream of a sequence encoding a protein or repressor as defined in the first embodiment and are operably linked to it. Preferably, the nucleic acid molecule and the expression cassette contained within the nucleic acid molecule may contain at least about seven or eight LexAop elements located upstream of a sequence encoding a protein or repressor as defined in the first embodiment. The LexAop sequence preferably has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26. The LexAop elements are preferably operably linked to a sequence encoding a protein or repressor as defined in the first embodiment. One or more of these LexAop elements may be bound by a transactivator, resulting in the transcription of a protein or repressor as defined herein.

[0210] A transactivator that binds one or more LexAop elements preferably comprises a LEXA DNA-binding domain, a VP16 domain, and an estrogen receptor (ER) domain. Such XVE systems are well known in the art and are described, for example, by Moore et al. (2006). The transactivator can induce transcription upon binding of β-estradiol or a derivative thereof. Therefore, in one embodiment of the present invention, the nucleic acid may comprise an expression cassette, the expression cassette comprising a sequence encoding the transactivator. Preferably, the transactivator is a protein comprising a domain that binds to the LexAop element, preferably a LEXA domain. Preferably, the transactivator further comprises an ER domain and a VP16 domain. In a preferred embodiment, the sequence encoding the transactivator has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27.

[0211] In one embodiment, the nucleic acid construct comprises at least one or more expression cassettes as defined herein. Preferably, the expression cassette comprises a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein or an RBR repressor as defined herein, wherein the sequence is operably linked to an inducible promoter as defined herein. The construct may comprise more than one such expression cassette. In one embodiment, the construct may comprise the following elements: A nucleic acid sequence comprising an expression cassette encoding the WIND1 protein as defined herein, operably linked to an inducible promoter as defined herein; and A nucleic acid sequence comprising an expression cassette encoding a WOX5 protein as defined herein, operably linked to an inducible promoter as defined herein. In a further embodiment, the structure may include the following elements: A nucleic acid sequence comprising an expression cassette encoding the WIND1 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a WOX5 protein as defined herein, operably linked to an inducible promoter as defined herein; and A nucleic acid sequence comprising an expression cassette encoding a PLT1 protein as defined herein, operably linked to an inducible promoter as defined herein.

[0212] In one embodiment, the structure may include the following elements: A nucleic acid sequence comprising an expression cassette encoding an SHR protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding an SCR protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a WOX5 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a PLT1 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a PLT4 protein as defined herein, operably linked to an inducible promoter as defined herein; and A nucleic acid sequence comprising an expression cassette encoding a PLT5 protein as defined herein, operably linked to an inducible promoter as defined herein. In a further embodiment, the structure may include the following elements: A nucleic acid sequence comprising an expression cassette encoding the WIND1 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding an SHR protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding an SCR protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a WOX5 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a PLT1 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a PLT4 protein as defined herein, operably linked to an inducible promoter as defined herein; A nucleic acid sequence comprising an expression cassette encoding a PLT5 protein as defined herein, operably linked to an inducible promoter as defined herein; and A nucleic acid sequence comprising an expression cassette encoding an RBR repressor as defined herein, operably linked to an inducible promoter as defined herein.

[0213] In one embodiment, the expression of SHR, SCR, PLT, and WOX5 proteins as defined herein is controlled by a GVG system as defined above herein.

[0214] In one embodiment, the expression of the WIND1 protein and RBR repressor as defined herein is controlled by the XVE system as defined above herein.

[0215] In one embodiment, the construct further comprises at least one expression cassette for the expression of a transactivator. Preferably, the construct further comprises two expression cassettes for the expression of two transactivators, wherein the sequences encoding the transactivators preferably have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20 or SEQ ID NO: 27, respectively.

[0216] In further embodiments, the construct may include resistance genes for selecting plants that stably incorporate the construct, the resistance genes being, for example, the Streptomyces hygroscopicus BASTA herbicide resistance marker.

[0217] composition In a third embodiment, the present invention relates to a composition. The composition may include nucleic acid constructs as defined in the second embodiment described herein. In some embodiments, the composition includes, for example, a stabilizer, a salt, or a diluent.

[0218] Alternatively, the composition may comprise two nucleic acid constructs, the first of which comprises a first expression cassette having a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein, or an RBR repressor, as defined herein, under the control of an inducible promoter. The composition further comprises a second construct, the second of which comprises an expression cassette having a sequence encoding a transactivator operably linked to a control element. Preferably, the control element is a strong constitutive promoter such as CaMV, G10-90, CsV, TCTP2, or UBQ10 promoter. Upon binding the inducible factor, the expressed transactivator can bind to the inducible promoter of the first construct and induce transcription.

[0219] The first construct may include additional expression cassettes, each having a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein, or an RBR repressor, as defined herein, under the control of an inducible promoter.

[0220] Alternatively, the composition may include additional constructs comprising an expression cassette having a sequence encoding a WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, or PLT7 protein, or an RBR repressor, as defined herein, under the control of an inducible promoter.

[0221] Plants and plant parts In a fourth embodiment, the present invention is i) nucleic acid molecules as defined in the second embodiment; and ii) Nucleic acid constructs as defined in the second embodiment This relates to plant cells containing at least one of the following.

[0222] Preferably, plant cells may, upon exposure to an inducer as defined herein, exhibit altered or increased expression of at least one of the following proteins: WIND1, WOX5, SHR, SCR, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7, and optionally, decreased expression of endogenous RBR protein. Preferably, at a point in time or over a period as defined in the first embodiment, plant cells may exhibit altered or increased expression of at least WOX5 and PLT1 upon exposure to an inducer as defined herein.

[0223] Plant cells can preferably be obtained from Arabidopsis, barley, cabbage, canola, cassava, cauliflower, chicory, chrysanthemum, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, rapeseed, potato, pumpkin, rice, rye, sorghum, soybean, pumpkin, sugarcane, sugar beet, sunflower, paprika, tomato, watermelon, wheat, and zucchini. Optionally, plant cells can be obtained from plants of the Solanaceae family, optionally from the Solanum genus, optionally from the tomato species (Solanum lycopersicum) or the eggplant species (Solanum melongena). Optionally, plant cells can be obtained from the Brassicaceae family, optionally from its species or subspecies, radish (Raphanus sativus), cabbage (Brassica oleracea), turnip, rapeseed (Brassica napus), horseradish, or Arabidopsis thaliana.

[0224] In a fifth aspect, the present invention relates to a shoot, plant, or plant part that can be obtained or obtained by the method of the present invention as defined herein. Plant cells obtained from the plant or plant part preferably have an endogenous expression level of at least one of WOX5, SHR, SCR, WIND1, PLT1, PLT2, PLT3, PLT4, PLT5, and RBR. Preferably, plant cells obtained from the plant or plant part have endogenous WOX5, SHR, SCR, WIND1, PLT1, PLT2, PLT3, PLT4, PLT5, and RBR protein levels.

[0225] The plant cells of a shoot, plant, or plant part that can be obtained or obtained by the method of the present invention as defined herein are at least i) Nucleic acid molecules in the second embodiment; and ii) Nucleic acid constructs as defined in the second embodiment, or may include at least some or one of them.

[0226] The plants that can be obtained or obtained by the method of the present invention are preferably selected from the group consisting of Arabidopsis, barley, cabbage, canola, cassava, cauliflower, chicory, chrysanthemum, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, rapeseed, potato, pumpkin, rice, rye, sorghum, soybean, pumpkin, sugarcane, sugar beet, sunflower, paprika, tomato, watermelon, wheat, and zucchini. Optionally, the plants that can be obtained or obtained by the method of the present invention are derived from the Solanaceae family, optionally from the Solanum genus, and optionally from the tomato species (Solanum lycopersicum) or the eggplant species (Solanum melongena). Optionally, the plants that can be obtained or obtained by the method of the present invention are of the Brassicaceae family, and optionally, the species or subspecies may be radish (Raphanus sativus), cabbage (Brassica oleracea), turnip, rapeseed (Brassica napus), horseradish, or Arabidopsis thaliana.

[0227] Alternatively, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or about 99% of plant cells are at least i) nucleic acid molecules as defined in the second embodiment; and ii) Nucleic acid constructs as defined in the second embodiment, or having at least some or one of them. In one embodiment, the plant part is a seed, fruit, or non-reproductive material.

[0228] In a sixth aspect, the present invention relates to products derived from plants or plant parts that can or can be obtained by the methods of the present invention, such as any fruits, leaves, plant organs, plant fats, plant oils, plant starches, and plant protein fractions that have been crushed, milled, or still intact, mixed with other materials, dried, or frozen. These products may be non-reproductive. Preferably, the plant products are at least i) nucleic acid molecules as defined in the second embodiment; and ii) Nucleic acid constructs as defined in the second embodiment, or comprising at least a portion or one thereof. Preferably, these products comprise at least a fraction of the nucleic acid and / or construct, which allows for assessment that the plant product is derived from a plant obtained by a method of a first aspect of the present invention as defined herein.

[0229] In a seventh aspect, the present invention relates to plant cells, plantlets, or plant offspring that can be obtained or obtained by the method of the present invention. The offspring plant cells may, upon exposure to an inducer as defined herein, have increased or induced expression of at least one of the proteins WOX5, SHR, SCR, WIND1, PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 as defined herein, and optionally, decreased expression of the RBR protein as defined herein.

[0230] The offspring may include nucleic acids and / or expression constructs as defined in the second embodiment of the present invention, as defined herein. Preferably, the offspring can generate shoots from plant cells after exposure to one or more inducers as defined herein.

[0231] In an eighth aspect, the present invention relates to a method for producing a plant, wherein the method includes the step of regenerating a shoot from plant cells as defined in the first aspect above, and the shoot developing into a plant.

[0232] Use of the present invention's protein for regeneration In a ninth aspect, the present invention relates to the use of protein combinations and / or inhibitory factor combinations as defined herein for regenerating shoots from plant cells. Preferably, the present invention relates to the use of protein combinations and / or inhibitory factor combinations as defined herein for regenerating shoots from plant cells. WOX5 protein and PLT1 protein; WIND1 protein, WOX5 protein, and PLT1 protein; SHR protein, SCR protein, WOX5 protein, PLT1, PLT4, and PLT5 protein; and WIND1 protein, SHR protein, SCR protein, WOX5 protein, PLT1, PLT4, and PLT5 proteins, and RBR inhibitors The present invention relates to the use of at least one combination of the following. The plant cells are preferably plant cells as defined above herein.

[0233] In a tenth aspect, the present invention relates to the use of nucleic acid molecules or nucleic acid constructs, as defined in the second aspect, for regenerating shoots from plant cells. The plant cells are preferably plant cells as defined above herein. [Examples]

[0234] Example 1 A composition and method for inducing shoot regeneration from plant cells using a set of predefined Arabidopsis (transcription) factors are provided. The composition includes constructs having the following factors: WIND1 (Iwase et al., 2011), artificial microRNA targeting RETINOBLASTMA RELATED (amiRBR) (Cruz-Ramirez et al., 2013), SHORT ROOT (SHR) (Benfey et al., 1993), SCARECROW (SCR) (Di Laurenzio et al., 1996), PLETHORA1 (PLT1) (Aida et al., 2004), BABYBOOM / PLETHORA4 (PLT4) (Boutilier et al., 2002, Galinha et al., 2007), PLETHORA5 (PLT5) (Tsuwamoto et al., 2010, Prasad et al., 2011), and WUSCHEL RELATED HOMEOBOX 5 (WOX5) (Sarkar et al., 2007). The selected factors were divided into two distinct sets and placed under the control of different constitutively expressive chemically inducible transactivation systems to enable time-limited and controlled expression. The constructs were built from modular parts using the golden gate cloning method (Engler et al., 2014). The transactivation system includes GVG / UAS (Aoyama and Chua, 1997), which consists of a chimeric transcription GVG assembled by the fusion of the DNA-binding domain (G) of the yeast transcription factor GAL4, the transactivation domain (V) of the herpesvirus protein VP16, and the receptor domain (G) of the rat glucocorticoid receptor, and a promoter (UAS) containing six copies of the upstream activation sequence of GAL4 fused with the -46 35S minimal promoter; and XVE / lexA (Zuo et al., 2000), which consists of a chimeric transcription activator XVE assembled by the fusion of the DNA-binding domain (X) of the bacterial suppressor LexA, the transactivation domain (V) of the herpesvirus protein VP16, and the regulatory region (E) of the human estrogen receptor, and a promoter (lexA) containing eight copies of the LexA operator sequence fused with the -46 35S minimal promoter.

[0235] Methods for the timed induction of factors are provided, including the application of chemical inducers to induce regulatory transcription separately or simultaneously, as further detailed below in this specification. Methods using these constructs to induce shoot regeneration from plant cells without relying on externally added plant hormones are also provided.

[0236] Materials and methods Construction We constructed a vector containing the following transcription elements: Streptomyces hygroscopicus BASTA herbicide resistance marker (BAR; Thompon et al., 1987) is operably linked to the Agrobacterium tumefaciens nopalin synthase promoter (NOSp; SEQ ID NO 33) and the A. tumefaciens nopalin synthase terminator (NOST; SEQ ID NO 34). The coding sequence for the chimeric transcription factor XVE (Zuo, 2000; SEQ ID NO: 27), operably linked to the Arabidopsis thaliana (A. thaliana) TRANSLATIONALLY-CONTROLLED TUMOR PROTEIN 1 promoter (AtTCTP1) (Czechowski et al., 2005; SEQ ID NO: 35) and the Arabidopsis thaliana (A. thaliana) UBIQUITIN 10 terminator (AtUBQ10Ter; SEQ ID NO: 36); The G10:90 synthetic promoter (Ishige, 1999; SEQ ID NO 37) and the coding sequence for the chimeric transcription factor GVG (Aoyama, 1997; SEQ ID NO 20), operably linked to the pea (Pisum sativum) ribulose-1,5-bisphosphate carboxylase small subunit terminator (rbcST; SEQ ID NO 38) and NOST (SEQ ID NO 34); Artificial microRNA (AmiRBR) of a gene-silencing overcome specific to the Arabidopsis thaliana (A. thaliana) Retinoblastoma Related coding sequence (Cruz-Ramirez, 2013; SEQ ID NO: 18), operably linked to the Escherichia coli (Escherichia coli) LexA (Zuo, 2000) promoter (SEQ ID NO: 39), the cauliflower mosaic virus 35Smini promoter (35Smini) (Odell et al., 1985; SEQ ID NO: 40), and the tomato (Solanum lycopersicum) ATPase (SlATPase) terminator (Engler, 2014; SEQ ID NO: 41); The Arabidopsis thaliana (A. thaliana) WOUND INDUCED DEDIFFERENTIATION1 code sequence (AtWIND1; SEQ ID NO: 29) contains a 4xMyc C-tag (4xMyc; SEQ ID NO: 42) operably linked to the LexA+35Smini promoter (LexAop; SEQ ID NO: 26) and the Arabidopsis thaliana (A. thaliana) UBIQUITIN3 terminator (AtUBQ3; SEQ ID NO: 44); A. thaliana (A. thaliana) short-root coding sequence (AtSHR; SEQ ID NO: 9) containing a 3xFLAG octapeptide C-tag (3xFLAG; SEQ ID NO: 45), operably linked to the Saccharomyces cerevisiae upstream activation sequence promoter (UASp; SEQ ID NO: 47), 35Smini (SEQ ID NO: 40), and the A. tumefaciens purified octopine synthase terminator (AtuOCS; SEQ ID NO: 48); Arabidopsis thaliana (A. thaliana) SCARECROW coding sequence (AtSCR; SEQ ID NO: 10) containing a simian virus 5 C-tag (V5; SEQ ID NO: 49), operably linked to the UAS+35Smini promoter and the A. tumefaciens mannopin synthase terminator (AtuMas; SEQ ID NO: 51); *Arabidopsis thaliana* PLETHORA 1 coding sequence (AtPLT1) comprising bacteriophage T7 gene 10 C-tag (T7; SEQ ID NO: 52) operably linked to UAS+35S mini promoter (UAS; SEQ ID NO: 22) and *A. thaliana* heat shock protein 18.2 terminator (AtHSP; SEQ ID NO: 54); *Arabidopsis thaliana* BABYBOOM coding sequence (AtPLT4; SEQ ID NO: 15) comprising T7 C-tag (SEQ ID NO: 52) operably linked to UAS+35S mini promoter (UAS; SEQ ID NO: 22) and *A. thaliana* UBIQUITIN5 terminator (AtUBQ5; SEQ ID NO: 55); *Arabidopsis thaliana* PLETHORA 5 coding sequence (AtPLT5; SEQ ID NO: 16) comprising 3xFLAG C-tag (3xFLAG; SEQ ID NO: 45) operably linked to UAS+35S mini promoter (UAS; SEQ ID NO: 22) and *A. thaliana* alcohol dehydrogenase terminator (AtADH; SEQ ID NO: 56); and *Arabidopsis thaliana* WUSCHEL RELATED HOMEOBOX 5 coding sequence (AtWOX5; SEQ ID NO: 11) comprising V5 C-tag (SEQ ID NO: 49) operably linked to UAS+35S mini promoter (UAS; SEQ ID NO: 22) and cauliflower mosaic virus 35S terminator (T35S; SEQ ID NO: 57).

[0237] Arabidopsis transformation: For plant transformation, the vector SHOOT REGENERATION was introduced into *A. tumefaciens* strain C58C1.pMP90 by electroporation. Using the transformed *A. tumefaciens*, Col-0 ecotype *A. thaliana* plants were transformed by the floral dip method (Clough, 1998).

[0238] Germination: The seeds were surface-sterilized using chlorine gas by steam sterilization (Lindsey, 2017). After sterilization, the seeds were suspended in a 0.1% agarose solution before cryogenic treatment at 4°C in darkness for 48 hours. Subsequently, the seeds were plated onto germination medium under sterile conditions. The seeds were plated onto a fine nylon mesh (100 μm) to allow contact with the medium without allowing the seedlings to penetrate the mesh. The plates containing the seedlings were sealed with surgical tape (3M, Micropore) to prevent drying. The plates were placed in a growth chamber (22°C, 120-150 μmol / m²). 2 The plates were placed upright for 5 days under these conditions (photoperiod of 16 hours of light / 8 hours of dark).

[0239] Induction: After 5 days of growth, the seedlings were transplanted under sterile conditions by moving the mesh in which they grew from the germination medium to the induction medium. The plate containing the seedlings was sealed with surgical tape (3M, Micropore) to prevent drying. The plate was returned to the growth chamber in an upright position. The plate was left to grow in this state for 14 days. Shoots began to appear in the second week of induction.

[0240] Culture medium used: Germination medium 5g Sucrose (Duchefa, product number S0809.5000) 1.1g MS + Vitamin (Duchefa, Product No. M0222.0050) 4g Plant-grade agar (Duchefa, product numbers P1001.1000) 0.5g / L MES 5.8mg / L Induction medium 5g Sucrose (Duchefa, product number S0809.5000) 1.1g MS + Vitamin (Duchefa, Product No. M0222.0050) 4g Plant-grade agar (Duchefa, product numbers P1001.1000) 0.5g / L MES 5.8mg / L 10 mM dexamethasone (Sigma Aldrich product number 101152255) dissolved in 400 μL of DMSO (Sigma Aldrich product number 100897077)

[0241] result Plants transformed with SHOOT REGENERATION vectors (XVE trans-activated AmiRBR and AtWIND1; GVG trans-activated AtSHR, AtSCR, AtPLT1, AtPLT4, AtPLT5, and AtWOX5) were grown on a medium containing 10 μM 17β-estradiol (EST: activation of XVE) and / or 10 μM dexamethasone (DEX: activation of GVG).

[0242] Primary root growth arrest was observed in 11 transformant lines containing the SHOOT REGENERATION vector induced by both EST and DEX. Callus formation in the primary roots was observed in transformants of all independent lines. Green callus formation was observed in 55% of the transgenic lines, and 67% of these lines regenerated shoots from the observed green callus without the application of plant hormones (Table 3). This represents 36% of all transformants tested (Table 2).

[0243] In all transformants containing the SHOOT REGENERATION vector induced by DEX alone, primary root growth arrest was observed. Callus formation in the primary roots was observed in 82% of the transgenic transformants. Green callus formation was observed in 44% of the transformants of these transgenic lines, and all (100%) of these transformants regenerated shoots from the observed green callus without the application of plant hormones (Table 3). This represents 36% of all transformants tested (Table 2).

[0244] Plants transformed with the SHOOT REGENERATION-2 vector (XVE trans-activated AtWIND1; GVG trans-activated AtPLT1 and AtWOX5) were grown on a medium containing 10 μM 17β-estradiol (EST: activation of XVE) and / or 10 μM dexamethasone (DEX: activation of GVG).

[0245] Primary root growth arrest was observed in all 25 transgenic lines containing the SHOOT REGENERATION-2 vector induced by both EST and DEX. Callus formation in the primary roots was observed in 20% of the transgenic lines. Green callus formation was observed in 60% of the transgenic lines, and 33% of the transgenic lines regenerated shoots from the observed green callus without the application of plant hormones (Table 3).

[0246] Primary root growth arrest was observed in all transformants containing the SHOOT REGENERATION-2 vector induced by DEX alone. Callus formation in the primary roots was observed in 28% of the transformants of the transgenic lines. Green callus formation was observed in 57% of the transformants of these transgenic lines, and all (100%) of the transformants in these lines regenerated shoots from the observed green callus without the application of plant hormones (Table 3). This represents 16% of all transformants tested (Table 2).

[0247] Neither the dexamethasone nor the estradiol-induced Arabidopsis roots showed any root growth arrest, callus formation, or shoot regeneration. Similarly, the roots of non-transformed Arabidopsis plants induced with 10 μM dexamethasone or estradiol showed no root growth arrest, callus formation, or shoot regeneration (Tables 2 and 3).

[0248] The emerged shoots were cut out from the plant roots with regenerated shoots, transferred to soil, and observed for their formation of complete plants including roots. It was found that they completed their life cycle and were able to produce seeds.

[0249] [Table 2]

[0250] [Table 3]

[0251] Excision of shoots reliably regenerated whole fertile plants without further induction. The excised shoots were cultured on 1 / 2 GM until roots formed spontaneously. Thereafter, the regenerated individuals can grow in soil, where they will produce seeds. This finding was consistent across both construct types and across several different insertion events.

[0252] Example 2 Induction of Shoot Regeneration in Tomato Transformation Construct for Tomato The transformation construct SHOOT REGENERATION from Example 1 above was adapted for tomato transformation by replacing the BASTA herbicide resistance marker with the kanamycin resistance marker nptII (Bevan et al., 1983; SEQ ID NO 59 and 64) under the control of the Agrobacterium tumefaciens noparin synthase promoter (NOSp; SEQ ID NO 33) and the A. tumefaciens octopine synthase terminator (OCST; SEQ ID NO 60). This construct allowed for easy selection of stably transformed tomato tissue on a medium containing 50 mg / l kanamycin for further use (kanamycin selection was not used in this example). Furthermore, the promoters and terminators that drive the expression of transcription factors XVE and GVG in the original construct were replaced with the cauliflower mosaic virus 35S promoter (Odell et al., 1985; SEQ ID NO 61) and the CaMV terminator (T35S; SEQ ID NO 62). Finally, a fluorescent reporter gene consisting of endoplasmic reticulum (ER)-targeted green fluorescent protein genes (erGFP; SEQ ID NOs. 63 and 65), under the control of the same CaMV 35S promoter and CaMV terminator, was placed between the first and second transcription elements. The resulting plasmid construct was named pKG11051, cloned in E. coli, and checked by restriction enzyme digestion. The Miniprep plasmid DNA was electroporated into Agrobacterium tumefaciens GV3101 strain for plant transformation.

[0253] Similarly, the transformation construct SHOOT REGENERATION-2 from Example 1 above was adapted by replacing the BASTA marker with nptII, replacing the promoters for XVE and GVG with the CaMV 35S promoter, and adding the erGFP fluorescent reporter gene. The resulting plasmid construct was named pKG11052. The Miniprep plasmid DNA was electroporated into Agrobacterium tumefaciens GV3101 strain for plant transformation.

[0254] Tomato transformation Following the modified method of Koornneef et al. (1986, 1987), both constructs pKG11051 and pKG11052 were introduced into tomatoes by Agrobacterium-mediated gene transfer. Approximately 50 tomato seeds were sterilized and germinated on 1 / 2 MS10 medium for 11 days. Cotyledon explants were excised from the seedlings and pre-cultured for 24 hours in MS20 medium supplemented with 40 μg / l acetosyringone. They were grown overnight in TY medium containing 20 mg / l streptomycin and 50 mg / l spectinomycin, and then OD 600 Explants were immersed in a 0.138 dilution suspension of Agrobacterium tumefaciens GV3101 containing pKG11051 or pKG11052. The explants were blot-dried and co-cultured for 2 days on plates of MS20 medium containing 40 μg / l acetosyringone. The experimental treatment consisted of pre-induction of a set transcription factor by adding 10 μM β-estradiol during co-culture in the absence of any hormones. The control treatment consisted of adding 2 mg / l NAA and 1 mg / l BAP to the co-culture medium, as per standard techniques for tomato transformation.

[0255] After co-culture, the explants were transferred to MS20CV medium, which consisted of MS20 medium containing 200 mg / l cefotaxime and 200 mg / l vancomycin to suppress further Agrobacterium growth. In addition, 10 μM dexamethasone (experimental treatment) or 1 mg / l zeatin (plant growth regulator, standard technique in tomato transformation) was added to this medium to induce transcription factor genes under the control of GVG. In this method, the effect of transactivated stem cell genes was compared with the effect of adding plant growth regulators. The explants were cultured in a growth chamber at 25°C and 3000 lux (16 / 8 hour photoperiod). The explants were subcultured in fresh medium every two weeks.

[0256] Record of shoot playback efficiency The experiment was conducted twice in an independent manner. Shoot and callus formation in the explants were recorded for 28 days after the start of the experiment (Table 4). The data demonstrate that induction with estradiol and dexamethasone produces hormone-independent shoot formation with slightly higher efficiency than conventional shoot induction with plant growth regulators (i.e., first with NAA+BAP for 2 days, then with zeatin). This effect is also evident in tomato explants transformed with either construct pKG11051 or pKG11052. Callus formed at the wound end of the explants in all induction treatments.

[0257] Control explants without any induction showed no callus or shoot formation. Non-transformed tomato explants showed normal shoot induction in media containing common plant growth regulators (i.e., first with NAA+BAP for 2 days, then with zeatin), but induction with estradiol and dexamethasone in these explants resulted in no shoot formation at all. This experiment demonstrates that the induction and regulation of transcription factor genes present in gene constructs can lead to novel shoot formation without the presence of any growth regulators. The inducing factors estradiol and dexamethasone alone are not sufficient to induce shoot formation in non-transformed tissues.

[0258] [Table 4]

[0259] [Table 5] TIFF0007917282000006.tif187149

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Claims

1. a) at least i) WUSCHEL-associated homeobox 5 (WOX5) protein; and ii) PLETHORA1 (PLT1) protein A step of introducing or increasing the expression of a protein combination into plant cells, wherein the expression of at least one protein of the protein combination is transiently introduced or increased; and b) A plant hormone-independent method for organogenesis of shoots from plant cells, comprising the step of enabling the plant cells to regenerate into shoots.

2. The method according to claim 1, wherein the PLT1 has at least 90% sequence identity with sequence number 4.

3. The aforementioned protein combination iii) WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) protein The method according to claim 1 or 2, further comprising:

4. The aforementioned protein combination iv) Short Root (SHR) protein; v) SCARECROW (SCR) protein; and vi) At least three PLETHORA (PLT) proteins selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7. The method according to any one of claims 1 to 3, further comprising:

5. The method according to any one of claims 1 to 4, wherein the protein combination comprises at least three selected PLT proteins, including PLT1, at least one or more of PLT4, and PLT5.

6. The method according to claim 5, wherein the at least three selected PLT proteins are PLT1, PLT4, and PLT5.

7. The method according to any one of claims 1 to 6, further comprising step a) reducing the expression of endogenous Retinoblastoma Related (RBR) protein.

8. The method according to claim 7, wherein the expression of the RBR protein is transiently reduced.

9. The method according to any one of claims 1 to 8, wherein the expression of all proteins of a protein combination defined in any one of claims 1 to 6 is transiently introduced or increased.

10. The method according to claim 9, wherein the expression of all proteins of the protein combination defined in any one of claims 1 to 6 is simultaneously and transiently introduced or increased.

11. The method according to claim 8, wherein the expression of RBR proteins is transiently introduced or increased simultaneously and transiently decreased at the same time as the expression of all proteins of the protein combination defined in any one of claims 1 to 6 is transiently introduced or increased.

12. The method according to any one of claims 1 to 11, wherein the expression of at least one protein of a protein combination defined in any one of claims 1 to 6 is transiently introduced or increased by transient activation of their expression.

13. The method according to claim 12, wherein the expression of all proteins in the protein combination is transiently introduced or increased by transient activation of their expression.

14. The method according to claim 8, wherein the expression of RBR protein is transiently reduced by transient activation of the expression of an RBR inhibitor.

15. i) The amino acid sequence of the SHR protein has at least 90% sequence identity with SEQ ID NO: 1; ii) The amino acid sequence of the SCR protein has at least 90% sequence identity with SEQ ID NO: 2; iii) The amino acid sequence of the WOX5 protein has at least 90% sequence identity with SEQ ID NO: 3; and iv) The amino acid sequences of the PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins have at least 90% sequence identity with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 30, respectively. and / or a) The SHR protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 9; b) The SCR protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 10; c) The WOX5 protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 11; and d) The method according to claim 4, wherein the PLT1, PLT2, PLT3, PLT4, PLT5, and PLT7 proteins are encoded by nucleotide sequences having at least 90% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 19, respectively.

16. vi) The amino acid sequence of the WIND1 protein has at least 90% sequence identity with SEQ ID NO: 28, and / or f) The method according to claim 3, wherein the WIND1 protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

29.

17. v) The amino acid sequence of the RBR protein has at least 90% sequence identity with SEQ ID NO: 17, and / or e) The method according to claim 7, wherein the RBR protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

18.

18. The method according to any one of claims 1 to 17, wherein the plant cells are part of a multicellular tissue.

19. The method according to claim 18, wherein the multicellular tissue is callus tissue, a plant organ, or an explant.

20. The method according to claim 19, wherein the plant organ is a root.

21. The method according to any one of claims 1 to 20, wherein the plant cells are obtained from a plant selected from the group consisting of Arabidopsis, barley, cabbage, canola, cassava, cauliflower, chicory, chrysanthemum, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, rapeseed, potato, pumpkin, rice, rye, sorghum, soybean, pumpkin, sugarcane, sugar beet, sunflower, paprika, tomato, watermelon, wheat, and zucchini.

22. The method according to any one of claims 1 to 21, comprising step c) forming a plant or plant part from a regenerated shoot.

23. The method according to any one of claims 1 to 22, wherein one or more inducible promoters control the transient expression of the protein combination.

24. The method according to claim 23, wherein the inducible promoter is at least one of a dexamethasone-inducible promoter and a β-estradiol-inducible promoter.

25. A composition comprising at least two nucleic acid molecules, i) The first nucleic acid molecule comprises a nucleotide sequence encoding the WOX5 protein, operably linked to an inducible promoter; and ii) A second nucleic acid molecule comprising a nucleotide sequence encoding the PLT1 protein, operably linked to an inducible promoter, composition.

26. A nucleic acid construct comprising the nucleotide sequences of the first and second nucleic acid molecules defined in claim 25.

27. The nucleic acid construct according to claim 26, comprising a further nucleotide sequence encoding a transactivator that activates the inducible promoter upon binding of the inducible factor.

28. The nucleic acid construct according to claim 27, wherein the transactivator is operably linked to a promoter.

29. The transactivator is i) Encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 21, the transactivator having the ability to bind to dexamethasone or corticoids; or ii) The nucleic acid construct according to claim 27 or 28, which is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 27, and the transactivator has the ability to bind to β-estradiol.

30. i) the first and second nucleic acid molecules as defined in claim 25; and ii) The nucleic acid construct according to any one of claims 26 to 29 A plant cell containing at least one of the following.

31. A shoot, plant, or plant part obtainable by the method of any one of claims 1 to 24, comprising a plant cell having first and second recombinant nucleic acid molecules, The first recombinant nucleic acid molecule comprises a nucleotide sequence encoding WOX5, The second recombinant nucleic acid molecule comprises a nucleotide sequence encoding PLT1, A shoot, plant, or plant part in which the first and second recombinant nucleic acid molecules are operably linked to an inducible promoter.

32. Use of a protein combination as defined in any one of claims 1 to 6, a composition as defined in claim 25, or a nucleic acid construct as defined in any one of claims 26 to 29 for regenerating shoots from plant cells.

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