Hydrogel carriers for inducing plant morphogenesis

Abstract

The present invention relates to a method for producing plant shoots, said method comprising the step of locally contacting plant tissue containing a sequence encoding at least one regeneration factor under the control of an inducible promoter with an inducible hydrogel. The present invention further relates to inducible hydrogels and the use of said inducible hydrogels to induce regeneration.

Description

[Technical Field]

[0001] The present invention relates to the field of molecular plant biology, in particular to the field of plant regeneration.The present invention relates to a method for improving the regeneration of plant tissues by topical application of inducers of morphogenetic regulators. [Background technology]

[0002] Regeneration, the process of propagating whole plants from a single or group of cells, is often a bottleneck in plant biotechnology workflows. Non-limiting examples of such biotechnology workflows include general propagation of plant material (clonal propagation, haploid, polyploid, and / or heterozygous F1), but also more advanced plant biotechnology workflows such as, but not limited to, targeted plant genome editing, production of transformants (stable or transient), and doubled haploid derivation.

[0003] Inducible constructs containing regeneration factors have been applied relatively effectively in various plant types. These (trans)genes, also referred to as morphogenetic genes, have been shown to induce shoot regeneration and / or somatic embryogenesis in plant tissues and / or explants (see, e.g., WO 2019 / 211296 and WO 2017 / 074547, which are incorporated herein by reference). Non-limiting examples of such genes include genes encoding the WUS / WOX homeobox polypeptide (van der Graaff et al., 2009, Genome Biology 10:248), transcription factors containing two AP2 DNA-binding domains, PLETHORA (PLT) proteins, and related polypeptides such as Ovule Development Protein 2 (ODP2) and Babyboom (BBM), WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) protein, SHORT ROOT (SHR) protein, and SCARECROW (SCR) protein (Galinha et al., 2007 Nature 449:1053-1057; Wildwater et al., 2005, Cell 123, 1337-1349). Summary of the Invention

[0004] In many cases, expression of such (trans)genes in explants, such as leaves, results in the appearance of early somatic embryos, but these embryos often fail to develop into whole plants. Thus, there is a need for improved protocols for inducing regeneration in plant explants that leads to the formation of mature plants.

[0005] Embodiment 1. A method for producing a plant shoot, comprising: a) providing plant tissue containing a sequence encoding at least one regeneration factor under the control of an inducible promoter; b) locally contacting the plant tissue with inducing hydrogel beads containing an inducing agent, the inducing agent optionally being a compound capable of inducing an inducible promoter upon binding to a transactivator contained in the plant tissue; and c) Producing shoots from the plant tissue A method comprising:

[0006] Embodiment 2. The method for producing plant shoots according to embodiment 1, wherein the beads are macrospheres having a diameter of about 0.1 to 10 millimeters (mm), or microspheres having a diameter of about 0.1 to 100 micrometers (μm).

[0007] Embodiment 3. The method of producing a plant shoot according to embodiment 1 or 2, wherein the inducer is a steroid.

[0008] Embodiment 4. The method of producing plant shoots according to embodiment 3, wherein the steroid is dexamethasone or at least one of its derivatives, and the steroid is β-estradiol or a derivative thereof.

[0009] Embodiment 5. The inducer is dexamethasone or a derivative thereof, and the plant tissue of step a) contains a transactivator that is a GVG protein; and / or 10. The method of any one of the preceding embodiments, wherein the inducer is β-estradiol or a derivative thereof, and the plant tissue of step a) comprises a transactivator that is an XVE protein.

[0010] Embodiment 6. The method of any one of the preceding embodiments, wherein the concentration of the inducer in the hydrogel beads is about 1 nM to 100 μM.

[0011] Embodiment 7. The at least one regeneration factor comprises at least one of a PLETHORA (PLT) polypeptide and a WUS / WOX homeobox polypeptide, preferably: i) the PLT polypeptide is selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5 and PLT7, preferably PLT1; ii) The method of any one of the preceding embodiments, wherein the WUS / WOX homeobox polypeptide is selected from the group consisting of WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, or WOX9, preferably WOX5.

[0012] Embodiment 8. The method of embodiment 7, wherein the at least one regeneration factor comprises PLT1 and WOX5, and the PLT1 coding sequence and the WOX5 coding sequence are each operably linked to an inducible promoter, preferably a GVG-inducible promoter.

[0013] Embodiment 9 The method of any one of the preceding embodiments, wherein the at least one regeneration factor comprises WIND1, and wherein the WIND1 coding sequence is operably linked to an inducible promoter, preferably an XVE-inducible promoter.

[0014] Embodiment 10. The method of any one of the preceding embodiments, wherein the hydrogel is alginate or a derivative thereof.

[0015] Embodiment 11. The method of any one of the preceding embodiments, wherein the plant tissue of step a) comprises an expression construct encoding at least one regeneration factor under the control of an inducible promoter, and optionally encoding a transactivator.

[0016] Embodiment 12. The method of any one of the preceding embodiments, wherein the plant tissue of step a) is a hypocotyl or a leaf, wherein the plant tissue is preferably any one of a cotyledon, a first true leaf, and a young (lateral) leaf.

[0017] Embodiment 13 The method of any one of the preceding embodiments, further comprising regenerating a plant from the plant shoot produced.

[0018] Embodiment 14. A combination of hydrogel beads containing an inducer of gene expression according to any one of embodiments 1 to 9 and plant cells.

[0019] Embodiment 15. Use of hydrogel beads according to embodiment 14 for inducing regeneration of plant tissue. [Brief explanation of the drawings]

[0020] [Figure 1] Hydrogel carriers for inducing regeneration in tomato (S. lycopersicum) are shown. (A) Alginate macrospheres. (B) Inducing alginate beads placed on the surface of a young leaf explant. (C) Local morphogenetic response 2.5 weeks after application of inducing beads. The boxed area corresponds to the boxed area shown in (B). (D) Example of a typical morphogenetic response observed in a conventional induction assay. (E) Formation of embryonic structures (indicated by *) after local application of hydrogel beads. (F) Resulting regenerated shoot. [Figure 2-1] Hydrogen carriers for regeneration induction in pepper cv. Maor (C. annuum cv.). (A) Induced alginate beads placed on the leaf surface. (B) Local morphogenetic response 19 days after application of the inducing beads. (C-G) Developmental stages of Maor somatic embryos. (C) Early, (D) Globular, (E) Heart, (F) Early Torpedo, and (G) Late Torpedo stages. (H) Shoot regenerating from embryonic structures while attached to callus. (I) Resulting regenerated shoot. Note the presence of the root system and internodes. (J) Fluorescence recording of the shoot apex of the regenerated shoot depicted in (I). Note the presence of a regular phyllotactic pattern. (K) Regenerated Maor plant with mature transgenic fruit. (L-N) Later developmental stages of the flower during fruit setting. (L) Closed flower bud. (M) Open flower bud. (N) Fertilized flower bud. (O) Mature transgenic fruit and transgenic seed set. (P) Transgenic Maor seed. Green fluorescent signal indicating the presence of the stem cell niche transgene. [Figure 2-2] (As mentioned above.)

[0021] definition Various terms relating to the methods, compositions, uses, and other aspects provided in the specification and claims are intended to have their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed consistent with the definition set forth herein.

[0022] Those skilled in the art will understand how to practice the conventional techniques used in the methods provided herein. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, breeding by crossing and selection, and related fields is well known to those skilled in the art and is discussed, for example, in the following references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and series Methods in Enzymology, Academic Press, San Diego.

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

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

[0025] As used herein, the term "about" is used to describe and account for slight variations. For example, this term can refer to ±(+ or -) 10% or less, e.g., ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. Furthermore, quantities, ratios, and other numerical values ​​may be expressed in range format herein. It is understood that such range formats are used for convenience and clarity and should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly stated. For example, a ratio in the range of about 1 to about 200 should be understood to include not only the explicitly stated limits of about 1 to about 200, but also individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc.

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

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

[0028] "Plant" refers to a whole plant, or a portion of a plant tissue or organ obtained from a plant (e.g., pollen, seeds, roots, stems, leaves, flowers, flower buds, anthers, fruit, etc.), and any derivatives thereof, and progeny obtained from such a plant by self-pollination or crossbreeding or apomixis. Non-limiting examples of plants include crop plants and cultivated plants such as African eggplant, allium, artichoke, asparagus, barley, beet, bell pepper, bitter melon, ground cherry, bottle gourd, cabbage, cannabis, canola, carrot, cassava, cauliflower, celery, chicory, kidney bean, corn salad, cotton, cucumber, eggplant, endive, fennel, gherkin, grape, hops, and tomato. These include mustard, lettuce, corn, melon, rapeseed, okra, parsley, parsnip, pepino, pepper, potato, pumpkin, radish, rice, loofah, rocket, rye, snake gourd, sorghum, spinach, loofah, pumpkin, sugar beet, sugarcane, sunflower, tomatillo, tomato, tomato rootstock, Brassica vegetables, watermelon, wax gourd, wheat, and zucchini.

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

[0030] "Similar conditions" for culturing plants / plant cells means, inter alia, the use of similar temperature, humidity, nutrient and light conditions, as well as similar irrigation and day / night rhythms.

[0031] As used herein, the terms "homology," "sequence identity," and the like are used interchangeably. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleotide (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as the case may be, as determined by the match between stretches of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The percentage of sequence identity / similarity can be determined over the entire length of the sequences.

[0032] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences remain constant throughout the window of alignment of components, e.g., nucleotides or amino acids. The "fractional identity" of an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller, defined portion of the reference sequence. "Percent identity" is the fractional identity multiplied by 100.

[0033] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide sequences or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences can be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined herein) (e.g., when optimally aligned using the programs GAP or BESTFIT with default parameters). The percentage of sequence identity is preferably determined using "BESTFIT" or "GAP" from the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). GAP uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, Journal of Molecular Biology 48:443-453, 1970) to align two sequences over their full length, maximizing the number of matches and minimizing the number of gaps. Global alignment is suitable for use in determining sequence identity when two sequences are similar in length. Generally, the GAP default parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension 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).Sequence alignments and percent sequence identity scores 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 the programs "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP described above, or using default settings (for both "needle" and "water," and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5; the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA). The gap extension penalty is 0.5; the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA). "BESTFIT" uses the local homology algorithm of Smith and Waterman (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981; Smith et al., Nucleic Acids Research 11:2205-2220, 1983) to optimally align the most similar segments between two sequences and insert gaps to maximize the number of matches. When the total length of the sequences differs substantially, local alignments such as the Smith-Waterman algorithm are preferred.

[0034] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Applied Math (1988) 48:1073. Among other computer programs suitable for determining sequence identity are the Basic Local Alignment Search Tool (BLAST) programs, publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. Mol. Biol. 215:403-410 (1990); BLAST program versions 2.0 and later allow gaps (deletions and insertions) to be introduced into the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0035] Alternatively, percent similarity or identity may be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences described herein can also be used as "query sequences" to search 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. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschulet et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0036] As used herein, "nucleic acid" or "polynucleotide" can include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). Any deoxyribonucleotide, ribonucleotide, or nucleic acid component, and any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of the above bases, are contemplated. The polymer or oligomer can be heterogeneous or homogeneous in composition and can be isolated from naturally occurring sources or produced artificially or synthetically. Furthermore, the nucleic acid can be DNA (optionally cDNA) or RNA, or a mixture thereof, and can exist permanently or transiently in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0037] "Isolated nucleic acid" is used to refer to a nucleic acid that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial or plant cell. The nucleic acid and / or protein may be at least one of a recombinant, synthetic, or artificial nucleic acid and / or protein.

[0038] The term "expression construct" (or nucleic acid construct or vector) is used to refer to an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, the terms "nucleic acid construct," "nucleic acid vector," or "expression construct" do not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (parts of) naturally occurring nucleic acid molecules.

[0039] The vector backbone is known in the art and described elsewhere herein, and may be, for example, a binary or superbinary vector (see, e.g., U.S. Pat. No. 5,591,616, U.S. Patent Application No. 2002138879, and WO 95 / 06722), an integrative vector, or a T-DNA vector, into which the chimeric gene is incorporated, or, if appropriate transcriptional regulatory sequences are already present, into which only the desired nucleic acid sequence (e.g., coding sequence, antisense sequence, or inverted repeat sequence) is incorporated downstream of the transcriptional regulatory sequence. Vectors may contain additional genetic elements, such as selectable markers, multiple cloning sites, etc., to facilitate their use in molecular cloning.

[0040] The term "gene" refers to a DNA fragment containing a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell and operably linked to an appropriate regulatory region (e.g., a promoter). A gene usually contains several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing a polyadenylation site.

[0041] "Gene expression" refers to the process by which a DNA region operably linked to appropriate regulatory regions, particularly a promoter, can be transcribed into biologically active RNA, such as RNA that can be translatable into a biologically active protein or peptide, or, for example, regulatory non-coding RNA.

[0042] The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked can mean that the DNA sequences being linked are contiguous.

[0043] "Promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acids. A promoter fragment is preferably located upstream (5') of the transcription initiation site of a gene in the direction of transcription, and is structurally distinguished by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and may further include any other DNA sequences, such as, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0044] A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically (e.g., by the external application of certain compounds or "inducing agents") or developmentally regulated. A "tissue-specific" promoter is active only in particular tissue or cell types.

[0045] Optionally, the term "promoter" may also include the 5' UTR region (5' untranslated region) (e.g., as used herein, a promoter may include one or more portions upstream of the translation start codon of a transcribed region, as this region may have a role in regulating transcription and / or translation).

[0046] "3'UTR" or "3' untranslated sequence" (often called the 3' untranslated region or 3' end) refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, transcription termination sites and (in most, but not all, eukaryotic mRNAs) polyadenylation signals (e.g., AAUAAA or variants thereof). After transcription termination, the mRNA transcript can be cleaved downstream of the polyadenylation signal and a poly(A) tail can be added, which is involved in transport of the mRNA into the cytoplasm (where translation occurs).

[0047] The term "cDNA" refers to complementary DNA. Complementary DNA is produced by reverse transcribing RNA into a complementary DNA sequence. Thus, a cDNA sequence corresponds to an RNA sequence expressed from a gene. Because RNA sequences expressed from the genome may undergo splicing before being translated into proteins in the cytoplasm, i.e., introns are spliced ​​out from the pre-mRNA and exons are joined together, expression of a cDNA is understood to refer to expression of the mRNA encoding the cDNA. Thus, in the case of a protein, a cDNA may only encode a complete open reading frame consisting of joined exons, whereas a genomic DNA sequence may contain exon sequences flanked by intron sequences, and therefore a cDNA sequence may not be identical to its corresponding genomic DNA sequence. Genetic modification of a protein-encoding gene may involve not only modification of the protein-encoding sequence but also mutations in the intron sequences and / or other gene regulatory sequences of the genomic DNA.

[0048] The term "regeneration" is defined herein as the formation of new tissues and / or new organs from a single plant cell, a group of cells, a callus, an explant, a tissue, or an organ. Regeneration can include the formation of new plants from a single plant cell, or from, for example, a callus, an explant, a tissue, or an organ. Plant cells for regeneration can be undifferentiated plant cells. Preferred plant cells are protoplasts. The regeneration process can occur directly from parent tissue or indirectly, for example, via callus formation. The regeneration pathway can be somatic embryogenesis or organogenesis. Somatic embryogenesis is understood herein as the formation of a somatic embryo that can develop into a whole plant. Organogenesis is understood herein as the formation of a new organ from an (undifferentiated) cell. Organogenesis can be at least one of meristem formation, adventitious shoot formation, inflorescence formation, root formation, adventitious shoot elongation, and (subsequent) formation of a complete plant. Preferably, regeneration is at least one of shoot regeneration, (ectopic) apical meristem formation, and root regeneration. Shoot regeneration, as defined herein, is de novo shoot formation. For example, regeneration can be the regeneration of (n) (inflorescence) shoots from (n) (elongated) hypocotyl explants.

[0049] As used herein, "normal growing conditions" refers to the environment in which plants grow, including at least the appropriate temperature (i.e., 0°C to 60°C), nutrition, day-night rhythm, and irrigation.

[0050] The term "conditions allowing regeneration" is understood herein as an environment in which plant cells or tissues are able to regenerate, and preferably includes normal growth conditions.

[0051] "Shoot organogenesis" is a regeneration pathway in which cells, preferably cells from a callus or explant, form a de novo shoot apical meristem, which develops into a shoot with leaf primordia and leaves. Because there is only one apical meristem, it is a monopolar structure, and roots do not form at this stage. The vascular system of the shoot is often connected to the parent tissue. Only after the shoot is fully formed and elongated, e.g., removed from the callus or explant, can root formation be induced in a separate root induction step on a different medium (Thorpe, TA (1993) In vitro Organogenesis and Somatic Embryogenesis: Physiological and Biochemical Aspects. In: Roubelakis-Angelakis KA, Van Thanh KT (eds) Morphogenesis in Plants. NATO ASI Series (Series A: Life Sciences), Vol. 253. Springer, Boston, MA).

[0052] Shoot organogenesis can occur spontaneously, i.e., without any external addition of plant growth regulators (PGRs). Shoot organogenesis can be induced by plant growth regulators, usually cytokinins at different concentrations, alone or in combination with auxins; preferably, cytokinins remain as components of the medium until new shoot apical meristems and shoots have formed and fully elongated, e.g., for removing them from primary explants or calli. Preferably, the concentration of cytokinins exceeds the concentration of auxins to induce shoot formation.

[0053] Somatic embryogenesis results in the formation of a bipolar structure similar to a zygotic embryo, but containing a root-shoot axis with a closed, independent fiber bundle system. In other words, root and shoot primordia form simultaneously, and the fiber bundles are not connected to the underlying tissue (Dodds, JH and Roberts, LW (1985) Experiments in plant tissue culture. Cambridge University Press, Cambridge, UK). Somatic embryogenesis can be induced indirectly, for example, from callus or cell suspensions, or directly from explant cells (Thorpe, supra). Somatic embryo formation proceeds through several distinct stages, from a globular stage (a small, uniformly sized group of cells) to a cardiac stage (a bilaterally symmetrical structure) to a torpedo stage (elongation). The transition from globular to cardiac is marked by the development 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). Ultimately, the torpedo-stage somatic embryo can develop into a plantlet containing green cotyledons, an elongated hypocotyl, and a developed radicle with clearly differentiated root hairs (Zimmerman, supra), in a process called "germination" (similar to a zygotic embryo) or "transition" or "maturation" (Von Arnold et al., supra). In the induction of somatic embryogenesis, auxins are preferably used directly or indirectly at an early stage to induce an embryogenic state in the callus, with embryos forming after subculture onto a medium lacking or containing reduced levels of auxin. Examples of auxins used in the induction of somatic embryos include 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picloram, and dicamba.

[0054] As used herein, the term "endogenous" when used in conjunction with a protein or nucleic acid means that the protein or nucleic acid is still contained within the plant, i.e., is present in its natural environment. In many cases, an endogenous gene is present in its normal genetic background in the plant.

[0055] "Plant hormones," "plant growth hormones," "plant growth regulators," or "phytohormones" are chemicals that affect the growth and / or development of plant cells and tissues. Plant growth regulators include chemicals from five groups: auxins, cytokinins, gibberellins, abscisic acid (ABA), and ethylene. In addition to these five groups, two other classes of chemicals, brassinosteroids and polyamines, are often considered plant growth regulators.

[0056] "Targeted mutagenesis" refers to mutagenesis that can be designed to modify specific nucleotides or nucleic acid sequences, for example, but not limited to, oligonucleotide-directed mutagenesis, mutagenesis using RNA-guided endonucleases (e.g., CRISPR technology), meganucleases, TALENs, or zinc finger technology.

[0057] The term "sequence of interest" includes, but is not limited to, genetic sequences that are preferably present in a cell, such as genes, portions of genes, and non-coding sequences within or adjacent to genes. Sequences of interest can be present in, for example, chromosomes, episomes, organelle genomes such as mitochondrial or chloroplast genomes, or genetic material, but they can also exist independently of the body of genetic material, such as infectious viral genomes, plasmids, episomes, and transposons. A sequence of interest can be present within the coding sequence of a gene or within a transcribed non-coding sequence, such as a leader sequence, trailer sequence, or intron. The sequence of interest can be present in a double-stranded or single-stranded nucleic acid molecule. Preferably, the nucleic acid sequence is present in a double-stranded nucleic acid molecule. A sequence of interest can be any sequence within a nucleic acid, such as a gene, gene complex, locus, pseudogene, regulatory region, highly repetitive region, polymorphic region, or portion thereof. A sequence of interest can also be a region containing a gene or epigenetic mutation that is indicative of a phenotype or disease. Preferably, the sequence of interest is a short or long contiguous stretch of nucleotides (i.e., a polynucleotide) of double-stranded DNA, said double-stranded DNA further comprising a sequence complementary to the sequence of interest in the complementary strand of said double-stranded DNA. The sequence of interest may be a gene of interest, preferably an endogenous gene of interest, or a portion thereof. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present inventors have discovered a versatile, simple, and improved protocol for the regeneration of plant tissue, preferably explants. To this end, the present inventors have developed a method in which plant tissue containing one or more sequences encoding regeneration factors under the control of an inducible promoter is locally contacted with a hydrogel matrix containing an inducing compound (referred to herein as an inducing hydrogel), i.e., a compound capable of activating or inducing (directly or indirectly) an inducible promoter operably linked to one or more sequences encoding the regeneration factors. The present inventors have found that efficient plant cell regeneration can be achieved by locally applying such an inducing hydrogel to explants. For example, local application of the inducing hydrogel to plant leaves containing a construct encoding at least one regeneration factor under the control of an inducible promoter results in the formation of shoots that can be developed into viable seedlings (i.e., whole plants). Preferably, the shoot formation is carried out under auxin-free conditions, and more preferably, under plant hormone-free conditions.

[0059] Without wishing to be bound by theory, the hydrogel can define a dose gradient of the inducing compound in the plant tissue, wherein the dose can be a spatially or temporally decreasing gradient. Thus, an easy and simple method for applying a compound to plant tissue in a non-uniform dose-dependent manner, e.g., both spatially and temporally, is provided. The method defined herein preferably comprises a step of locally contacting the plant tissue with an inducing hydrogel containing an inducing agent to obtain a concentration gradient of the inducing agent. Preferably, the concentration gradient is a spatial and / or temporal gradient.

[0060] Topical application of inducers has been described in the art. For example, sprays, paints, and lanolin pastes, preferably in the presence of a surface tension-reducing agent such as Silwet L-77 (Samalova et al., supra; Borghi, supra), have been used for topical application to plants (Samalova et al., The Plant Journal, 2005; 41, 919-935; Borghi, "Plant Developmental Biology: Methods and Protocols," vol. 655, pages 65-75; and Wyrzykowska et al., Plant Physiology, August 2006, Vol. 141, pp. 1338-1348). However, the use of surface tension-reducing agents such as Silwet L-77 in sprays and / or sticky pastes such as lanolin (which require heating before application) are not preferred for fragile plant materials such as leaf structures. Furthermore, these are laborious procedures and not easy for topical application of inducers.

[0061] Methods for regenerating shoots and / or plants are provided, comprising topically applying an inducible hydrogel to plant tissue. Preferably, the plant tissue comprises at least one regeneration factor under the control of an inducible promoter, and the inducible hydrogel optionally comprises a compound (inducer) capable of inducing or activating the inducible promoter through binding to a transactivator present in the plant tissue. Preferably, at least a portion of the cells of the plant tissue comprises a construct encoding the at least one regeneration factor under the control of an inducible promoter, and optionally a transactivator for inducing the inducible promoter upon binding of an inducer. Optionally, the transactivator is encoded on a construct (optionally the same construct encoding the at least one regeneration factor and an inducible promoter operably linked thereto) contained in the at least a portion of the cells of the plant tissue, which construct may further comprise a promoter constitutively active in plant cells operably linked to a sequence encoding the transactivator. Preferably, the construct is an isolated construct.

[0062] Optionally, the construct is present in substantially all cells of the plant tissue (preferably stably integrated into its genome), and thus preferably the plant tissue is derived from a stable transgenic line having the construct stably integrated into its genome.

[0063] Preferably, at least a portion of the cells of the plant tissue contain at least one regeneration factor under the control of an inducible promoter, and the induced hydrogel may optionally contain a compound (inducing agent) capable of inducing (activating) the inducible promoter through binding of a transactivator present in the cells of the plant tissue. The induced hydrogel is preferably prepared by dissolving the inducing agent in the hydrogel, and topical application is preferably performed by applying the induced hydrogel to only a portion of the surface of the plant tissue, for example, by applying the induced hydrogel to the plant tissue as beads (spheres or granules). Preferably, the induced hydrogel contacts at most about 45%, 40%, 35%, 30%, 25%, 20%, 10%, or 5% of the surface of the plant tissue in the methods provided herein. Preferably, the induced hydrogel contacts less than 50%, preferably less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 5%, 10%, or 5% of the surface of the plant tissue. Preferably, the plant tissue is a detached leaf. In a preferred embodiment, the compound is a gene expression inducer.

[0064] The inventors have recognized that when an inducer is used to express factors involved in regeneration, the methods provided herein can overcome the problem of induced organ cessation and allow shoot formation and elongation. Therefore, preferably, the methods of the present invention result in increased shoot formation compared to otherwise similar methods that differ primarily in that in step b), at least about 50% of the plant tissue structure (i.e., the portion in contact with the growth medium) is exposed to the inducer hydrogel by attaching the plant tissue structure to a growth medium containing an inducer, thereby exposing at least one entire surface of the plant tissue to the inducer hydrogel. "Otherwise similar" in this context should be understood to mean the use of the same or similar hydrogel composition, the same or similar plant tissue, and otherwise the same or similar conditions (except for the surface area of ​​the plant tissue contacted with the hydrogel).

[0065] In one aspect, a hydrogel, preferably a hydrogel bead, containing a compound is provided. Preferably, the hydrogel is an induced hydrogel. In the present specification, an induced hydrogel is understood to mean a hydrogel containing a compound, in which case the compound is a gene expression inducer. The gene expression inducer may be a compound that disables a repressor of gene transcription or binds to an activator of gene transcription. Preferably, the gene expression inducer binds to the activator of gene transcription, thereby activating the activator of gene transcription.

[0066] The inducer may be an activator of gene expression, preferably a (chemical) inducer of gene expression. Preferably, the (chemical) inducer interacts with, and preferably binds to, a (chemically) responsive transcription factor, also referred to herein as a transactivator. This interaction preferably results in activation of gene transcription by binding of the transcription factor to an element that controls the expression of the gene.

[0067] Those skilled in the art will recognize suitable responsive transcription factors or combinations of inducers and responsive transcription factors (induction systems). Suitable combinations are disclosed, for example, in Omelina, E. S. et al. (Optogenetic and Chemical Induction Systems for Regulation of Transgene Expression in Plants: Use in Basic and Applied Research, Int. J. Mol. Sci. (2022), 23, 1737) and Moore I. et al. (Transactivated and chemically inducible gene expression in plants, The Plant Journal (2006) 45, 651-683), which are incorporated herein by reference.

[0068] Suitable induction systems include, but are not limited to, the Tet-derepression system, the Tet-off system, the GVG / UAS system, the pOp6 / LhGR system, the β-estradiol (E2) induction system, the XVE system, the TGV system, the insecticide induction system, the copper induction system, and the ethanol induction system. These systems, along with appropriate inducers and responsive transcription factors, are shown in Table 1 below.

[0069] [Table 1]

[0070] The inducer may be an antibiotic, preferably tetracycline (Tet) or a derivative thereof. A preferred derivative is doxycycline. Tetracycline and / or its derivatives can be used as inducers in the Tet-derepression system and / or as inhibitors in the Tet-off system. A Tet-dependent promoter can be created by placing a Tet response element (TRE), consisting of repeats of the tetracycline operator (tetO) sequence, upstream of a promoter, preferably a minimal promoter. In the Tet-derepression system, in the absence of tetracycline or a derivative thereof, the tetR repressor binds to the TRE sequence and prevents the promoter from initiating transcription. Upon addition of Tet, the tetR protein binds to the Tet molecule and separates it from the TRE, thereby allowing gene expression.

[0071] In the Tet-off system, the TRE is recognized by the tetR protein fused to the VP16 activation domain. In the absence of Tet, the chimeric tetR-VP16 protein binds to the TRE and activates gene transcription. When Tet is added, the tetR-VP16 protein is no longer able to bind to the TRE, and the gene is not transcribed. Thus, Tet may function as an inhibitor of gene expression, and the hydrogels provided herein may contain an inhibitor of gene expression, or optionally a combination of an inhibitor of gene expression and an activator of gene expression. In the Tet-inducible system, gene expression can be controlled by Tet and its derivatives, such as, but not limited to, doxycycline (Omelina et al., supra).

[0072] Preferably, the inducer is a compound that can passively pass through the cell membrane. Preferably, the inducer is a lipophilic compound. The inducer may be a steroid, preferably dexamethasone, β-estradiol, or a derivative thereof. The steroid is preferably used in combination with a responsive transcription factor (trans-transcription factor), where the transcription factor is a fusion of a DNA-binding domain, an activation domain, and a steroid receptor regulatory domain.

[0073] The inducer dexamethasone or a derivative thereof can interact with a transactivator, where the transactivator comprises a GAL4 DNA-binding domain, a VP16 (activation) domain, and a glucocorticoid receptor (GR) domain (GVG). Such GVG systems are known in the art and are described, for example, in Moore et al., (supra) (2006) and Omelina et al., (supra). For example, upon interaction with a steroid such as dexamethasone or a derivative thereof, the transactivator can bind to one or more UAS elements and initiate transcription, which elements are preferably linked to a (minimal) promoter, such as, but not limited to, a 35S minimal promoter. Optionally, at least about four or five UAS elements are located upstream of the promoter.

[0074] Alternatively or additionally, the inducer dexamethasone or a derivative thereof can interact with a transactivator, wherein the transactivator LhGR contains a domain that binds to a LacOp element, preferably the DNA-binding mutant lacl His17 DNA-binding domain. Preferably, the transactivator further contains a GR domain and a GAL4 activation domain. Such LacOp systems are known in the art and are described, for example, in Moore et al. (supra) and Omelina et al. (supra). Upon interaction of the transactivator with a steroid, such as dexamethasone or a derivative thereof, the transactivator can initiate transcription upon binding to one or more LacOp elements, which are preferably linked to a minimal promoter, such as a 35S minimal promoter. Optionally, at least about five or six LacOp elements are located upstream of the promoter.

[0075] Optionally, the promoter comprising one or more binding sites for binding a transactivator may be a non-specific, tissue-specific, or cell-type specific promoter. Combining the inducible system defined herein with a tissue- or cell-type-specific promoter can result in inducible and tissue- or cell-type-specific expression.

[0076] The inducer may be β-estradiol or a derivative thereof. The inducer, β-estradiol or a derivative thereof, preferably binds to the chimeric ER-C1 protein or XVE protein and induces transcription. The transactivator ER-C1 comprises a fusion of the activation domain of the corn activator C1 with the estrogen receptor (ER). Upon interaction with β-estradiol or a derivative thereof, ER-C1 can bind to an ER element (ERE) that may be located upstream of a 35S minimal promoter. The chimeric XVE protein contains a DNA-binding domain, a VP16 (activation) domain, and an estrogen receptor ligand-binding domain derived from the bacterial LexA protein. Upon interaction with β-estradiol or a derivative thereof, the transinducer can bind to one or more LexA operator sequences (OlexAs) and activate gene expression. Preferably, two or more OlexA sequences are located upstream of the promoter, preferably upstream of a minimal promoter, preferably upstream of a 35S minimal promoter. Optionally, at least 5, 6, 7, or 8 copies of OlexA are located upstream of the (minimal) promoter.

[0077] The inducer may be ethanol, which can interact with the transactivator AlcR. Upon interaction, AlcR binds to the promoter sequence of the AlcA gene, which can be fused to a minimal promoter, for example, as described in Moore et al. (supra) and Omelina et al. (supra).

[0078] The inducer may be an insecticide, preferably at least one of tebufenozide and methoxyfenozide. The insecticide can interact with a transactivator to activate gene expression, preferably the transactivator is based on a chimeric GVEcR protein containing a glucocorticoid receptor activation domain and a DNA binding domain, a VP16 activation domain, and a worm ecdysone receptor ligand binding domain, as described, for example, in Moore et al. (supra) and Omelina et al. (supra), or a variant thereof (e.g., GVGE(Gal4-VP16-GR-EcR), GVE(Gal4-VP16-EcR), or VGE(VP16-Gal4-EcR)).

[0079] The inducer may be a metal, preferably copper. Copper can bind to a transactivator comprising the yeast-copper-regulated transcription factor ACE1 fused to the VP16 activation domain. After interaction with copper, the transactivator can bind to a metal-responsive element (MRE), preferably linked to a minimal promoter, and induce transcription, as described, for example, in Omelina et al. (supra).

[0080] The hydrogels provided herein may contain a combination of two or more inducers, each of which may regulate gene expression through interaction with a respective transactivator. Preferably, the hydrogel contains a combination of steroids, preferably dexamethasone and β-estradiol, or derivatives thereof.

[0081] Combinations of hydrogels as defined herein are also provided, and preferably, the combination of hydrogels is used in the methods provided herein. Combinations of hydrogels may be applied, for example, to different parts of the same plant tissue or sequentially to the same tissue. Those skilled in the art will be familiar with how to select suitable combinations of hydrogels. As a non-limiting example, one hydrogel may contain dexamethasone or a derivative thereof, and the second hydrogel may contain β-estradiol or a derivative thereof. Alternatively, or in addition, the first hydrogel may contain tetracycline or a derivative thereof, and the second hydrogel may contain dexamethasone or a derivative thereof. The dual-regulatory TGV system is based on the combination of a dexamethasone-inducible expression system and a tetracycline-dependent expression system. The chimeric TGV protein contains the tetR DNA-binding domain, the GR ligand-binding domain, and the VP16 activation domain. In the absence of dexamethasone, TGV is inactive due to its binding to Hsp90 proteins. Upon addition of dexamethasone, TGV dissociates from Hsp90 and interacts with the promoter, activating expression. The promoter further contains one or more, preferably, for example, heptad repeats, of tetracycline operator (tetO) sequences. Upon interaction with tetracycline, the TGV protein dissociates again from the promoter, reducing expression. The TGV system is further described, for example, in Omelina et al., supra.

[0082] The hydrogels provided herein may contain one or more additional compounds, preferably one or more biological compounds. Those skilled in the art will readily appreciate that additional compounds and / or combinations of compounds may be included in hydrogels for use in the methods described herein. Such additional compounds may be related to and / or useful in the regeneration and / or shoot production processes of the methods of the present invention. Additionally or alternatively, such additional compounds may serve other purposes, such as, but not limited to, inducing (germline) mutations (e.g., programmed nucleases), gene insertion (e.g., transgenes), and / or selection (e.g., selection markers).

[0083] Preferably, the compound can diffuse from and / or through the hydrogel. Preferably, the additional compound is a lipophilic, hydrophilic, or amphiphilic compound. Preferably, the additional compound is lipophilic. Preferably, the additional compound is a compound that enters cells via passive diffusion through the lipid bilayer of the cell membrane. The additional compound can be a bioactive compound or a non-bioactive compound. A bioactive compound refers to a compound that effects a biological or chemical change in an organism or its cells, including, but not limited to, mammals, vascular plants, non-vascular plants (eukaryotic algae, bryophytes), fungi, yeast, and prokaryotes (bacteria, cyanobacteria, etc.).

[0084] Preferably, the further compound is selected from the group consisting of an inducer of gene expression, a plant hormone, a peptide, an enzyme, a guide RNA, a (viral) vector, a bacterium, an oligonucleotide, a nucleic acid, a protein, or any combination thereof.

[0085] The further compound may be BSA (bovine serum albumin), GFP (green fluorescent protein), DNA, antisense RNA or messenger RNA. The further compound may be, for example, an enzyme selected from the group consisting of carbohydrases (such as cellulases, amylases, pectinases and lactases), proteases, lipases, phytases, laccases, polymerases and nucleases.

[0086] Preferred additional compounds are at least one of a gene expression inducer, a plant hormone, a guide RNA, a site-specific endonuclease, a (pre-)mRNA, a (viral) vector and a bacterium, or any combination thereof.

[0087] A preferred compound contained in the hydrogel is a plant hormone. Plant hormones (also known as plant hormones or "plant growth substances") are chemicals that can regulate plant growth and development. Plant hormones can affect at least one of plant shape, seed development, flowering time, flower sex, leaf senescence, and fruit senescence. Alternatively or additionally, plant hormones can affect at least one of tissue upgrowth, tissue downgrowth, leaf formation, stem growth, fruit development, fruit ripening, plant lifespan, and plant death.

[0088] The plant hormone may be selected from the group consisting of auxin, cytokinin, gibberellin, abscisic acid (ABA), ethylene, brassinosteroids, polyamines, salicylic acid, jasmonates, plant peptide hormones, polyamines, nitric oxide, strigolactones, karrikins, and triacontanol. A preferred plant hormone is a cytokinin or an auxin. Optionally, the hydrogel comprises two or more plant hormones, e.g., two, three, four, five, or more plant hormones. Optionally, the hydrogel comprises a combination of a cytokinin and an auxin, or multiple cytokinins and / or multiple auxins.

[0089] The plant hormone contained in the hydrogel can be an auxin. Auxins are a class of plant hormones that can have morphogen-like properties. The auxin can be an endogenously synthesized auxin. The endogenously synthesized auxin can be selected from the group consisting of indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid. The auxin can be a synthetic auxin, such as an auxin analog. The synthetic auxin may be at least one of 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (α-NAA), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (trudon or picloram), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The auxin may be 1-naphthaleneacetic acid (NAA). Additionally or alternatively, the plant hormone included in the hydrogel may be a gibberellin. The gibberellin may be a 19-carbon gibberellin or a 20-carbon gibberellin. The gibberellin may be a dihydroxylated gibberellin. The gibberellin may be at least one of GA1, GA3, GA4, and GA7.

[0090] The plant hormone contained in the hydrogel can be a cytokinin. The cytokinin can be either an adenine-type cytokinin or a phenylurea-type cytokinin. Similarly, the cytokinin can be a naturally occurring plant hormone or a synthetic compound. Adenine-type cytokinins can be plant hormones typically synthesized in at least one of roots, seeds, and fruits. In addition, the cambium and other actively dividing tissues can also synthesize cytokinins. Non-limiting examples of naturally occurring adenine-type cytokinins include zeatin and its metabolic precursor, 2-isopentenyladenine (2iP). Non-limiting examples of synthetic adenine-type cytokinins are kinetin and 6-benzylaminopurine (BAP). Substituted urea compounds, such as thidiazuron and CPPU, are not present in plants but can act as cytokinins in tissue culture. The adenine-type cytokinin can be selected from the group consisting of kinetin, zeatin, trans-zeatin, cis-zeatin, dihydrozeatin, 6-benzylaminopurine, and 2iP, and combinations thereof. The phenylurea-type cytokinin can be diphenylurea or thidiazuron.

[0091] The additional compound contained in the hydrogel may be a vector. Preferred expression vectors are naked DNA, DNA complexes, or viral vectors. Preferred naked DNA is a linear or circular nucleic acid molecule, such as a plasmid. A plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. The DNA complex may be a DNA molecule bound to any carrier suitable for delivering DNA into cells. Preferred carriers are selected from the group consisting of lipoplexes, liposomes, polymersomes, polyplexes, PEG, dendrimers, inorganic nanoparticles, virosomes, and cell-penetrating peptides. The vector is preferably a viral vector. Preferably, the viral vector is suitable for infecting plant cells. Preferably, the viral vector is at least one of Tobacco Rattle Virus (TRV), Bean yellow dwarf virus (BeYDV), Cabbage leaf curl virus (CaLCuV), tobravirus, and Wheat dwarf virus (WDV). Optionally, the viral vector comprises a transgene for expression in plant cells.

[0092] The additional compound may be a bacterium. Preferably, the bacterium is an Agrobacterium, preferably Agrobacterium tumefaciens. Optionally, the bacterium is modified to contain a transgene for expression in plant cells.

[0093] An additional compound contained in the hydrogel may be a guide RNA, preferably a guide RNA that guides a CRISPR protein to a location in the plant genome. The guide RNA may be suitable for directing, for example, a Cas9 protein, a Cpf1 protein, or a Mad7 protein to a specific location in the plant genome. In this embodiment, at least a portion of the cells of the plant tissue may contain a sequence encoding a CRISPR protein, preferably at least one of Cas9 (SpCas9, StCas9, SaCas9, ScCas9, and dead Cas9), CasX, CasY, Cas-Phi, Cas12a (Cpf1), Cas13, Cas14, and MAD7. The guide RNA contained in the hydrogel may be a crRNA (e.g., in the case of Cpf1), a combination of crRNA and tracrRNA, or a single guide RNA (e.g., in the case of Cas9 protein).

[0094] The additional compound may be a gene-modifying molecule, for example, but not limited to, a gene silencing molecule such as an siRNA and / or an miRNA, a gene replacement and / or gene insertion molecule, or a molecule that induces targeted modification in the genome, or a messenger RNA encoding such a molecule. Optionally, the miRNA or siRNA may target an RBR RNA transcript. The mature siRNA or miRNA may comprise at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides. The mature siRNA or miRNA may comprise 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. The endogenous RBR transcript is preferably an endogenous RBR mRNA molecule, and preferably includes 3' and 5' untranslated RBR sequences. Therefore, the sequence of the non-coding small RNA may be partially or completely complementary to a sequence contained in the RBR coding sequence or to a 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 a sequence contained in the RBR coding sequence. For example, at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides of the small RNA molecule have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with the consecutive sequence of at least 20, 21, 22, 23, 24, or at least 25 consecutive nucleotides of the endogenous RBR transcript, respectively. Those skilled in the art will understand how to use conventional RNAi to design small RNA molecules that can down-regulate the expression of endogenous RBR proteins, where the RBR protein is the RBR protein defined herein above. Optionally, a small RNA molecule for inhibiting RBR expression can comprise a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34 (RBR miRNA precursor).The small RNA molecule can comprise a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35 (RBR mature miRNA).

[0095] The additional compound may be a molecule that induces targeted modification (targeted mutagenesis) in the genome, such as a CRISPR ribonucleoprotein (RNP), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and an oligonucleotide (such as a small interfering RNA (siRNA) and a short hairpin RNA (shRNA)), and / or an mRNA encoding a programmable nuclease such as a CRISPR endonuclease and / or a TALEN. The targeted modification is preferably located within the sequence of interest. In some embodiments, the additional compound is a CRISPR protein, or a DNA or RNA molecule encoding a CRISPR protein. The CRISPR protein may be at least one of Cas9 (SpCas9, StCas9, SaCas9, ScCas9, and inactive Cas9), CasX, CasY, Cas-Phi, Cas12a (Cpf1), Cas13, Cas14, and MAD7. The additional compound may be a complex of a CRISPR protein and a guide RNA.

[0096] The additional compound may be a messenger RNA (mRNA) or a pre-mRNA. The protein encoded by the (pre-)mRNA is preferably at least one of an enzyme, a hormone, and a transcription factor. Optionally, the (pre-)mRNA encodes a CRISPR-protein, preferably a CRISPR-protein as defined herein.

[0097] A hydrogel is a three-dimensional hydrophilic network. A hydrogel can be formed by gelation of a polymer solution, preferably in the presence of a compound defined herein. The terms "gelation" and "gelation" can be used interchangeably herein. During the gelation process, crosslinks are formed between polymers contained in the polymer solution. The crosslinks can include covalent bonds, ionic bonds, molecular entanglements, hydrogen bonds, hydrophobic interactions, van der Waals forces, and / or dipole-dipole interactions. Preferably, the crosslinks include covalent bonds and / or ionic bonds. The nature of these crosslinks is primarily determined by the chemical structures of the polymers and, optionally, the compounds defined herein. For example, a solution containing a polymer with ionic pendant groups can form ionic bonds upon gelation. The crosslinks can include one or more linking agents, such as ions.

[0098] Gelation preferably occurs under specific conditions, which may be characterized by one or a combination of one or more parameters, such as temperature, pH, the concentration of specific ions, and the presence of a gelation inducer. A polymer solution may have a lower critical gelation temperature (LCGT), around which gelation occurs when heated. A polymer solution may have an upper critical gelation temperature (UCGT), around which gelation occurs when cooled. In addition to temperature, specific conditions that induce gelation may include the concentration of specific ions. For example, gelation of an aqueous alginate solution occurs in the presence of calcium ions. Without intending to be bound by any theory, it is generally believed that calcium ions act as a linking agent, allowing ionic crosslinks to form between the carboxyl pendant groups of alginate chains.

[0099] Preferably, the hydrogels provided herein are formed from a polymer solution, preferably aqueous, containing an ionic polymer, preferably an anionic polymer, preferably an anionic polymer with pendant carboxylic acid groups, wherein the hydrogel comprises ionic crosslinks. Such ionic crosslinks include ions, preferably calcium, as a linking agent. Thus, preferably, the hydrogels provided herein comprise crosslinked ionic polymers, preferably anionic polymers with pendant carboxylic acid groups.

[0100] In the context of this application, anionic polymers are understood to refer to polymers that are negatively charged under the conditions present in the corresponding polymer solution, although said compounds or groups may nevertheless be neutral or positively charged under different conditions. Corresponding definitions apply to neutral and cationic polymers.

[0101] Preferably, the hydrogels provided herein are formed from a polymer solution, preferably aqueous, comprising a polysaccharide or a derivative thereof. More preferably, the polysaccharide or derivative thereof comprises one or more sugar acids, preferably uronic acids. Preferably, the hydrogels provided herein are formed from a polymer solution comprising alginic acid or a derivative thereof. Thus, preferably, the hydrogels provided herein comprise cross-linked polysaccharides or derivatives thereof, and preferably, the polysaccharides or derivatives thereof comprise uronic acids. Preferably, the hydrogels provided herein comprise alginic acid or a derivative thereof.

[0102] Alginic acid is a family of linear copolymers of (1,4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues or monomers. The M and G monomers are arranged in alginic acid as consecutive G residues, consecutive M residues, or alternating M and G residues. The ratio of the number of M residues to the number of G residues in alginic acid, as well as the length of blocks of residues, blocks of M residues, and blocks of MG residues, depends on the source from which the alginic acid is obtained. Preferred sources of alginic acid are those from species within the class of Phaeophycea (brown algae). Alginic acid can be obtained from at least one species of the genera Macrocystis, Sargassum, and Laminaria. Preferably, the Laminaria is at least one species of Laminaria digitate, Laminaria hyperborea, and Laminaria Durvillaea.

[0103] Preferably, the G / M ratio is ≧1.5. Preferably, the M / G ratio is at least about 0.7 or 0.8, preferably about 0.7 to 1.4 or about 0.8 to 1.6.

[0104] The viscosity of alginic acid when dissolved in water may be high, medium, or low. Preferably, the viscosity (mPa / s) is about 4 to 12 or about 20 to 200. Preferred alginic acid derivatives are amphiphilic alginic acid and alginic acid covalently bonded to an oligopeptide. Amphiphilic alginic acid can be derived from alginic acid by covalently bonding a hydrophobic group, such as an alkyl, to the carboxylic acid pendant group.

[0105] Preferably, a compound, preferably a compound as defined herein, preferably an inducer as defined herein, is added to the polymer solution before gelation so that the compound becomes entrapped within the hydrogel upon gelation. Preferably, said inducer is homogeneously entrapped within said hydrogel. The compound is preferably diffusible from the formed hydrogel.

[0106] Further provided are hydrogel beads, which contain a compound, preferably a compound defined herein, preferably an inducer defined herein. The beads may contain a uniform distribution of the compound. The hydrogel beads may be applied to plant tissue. After applying the hydrogel beads to the plant tissue, the compound may at least partially diffuse out of the hydrogel, forming a gradient in the plant tissue. Exemplary approaches are also outlined in the Examples section below. Optionally, a single hydrogel bead is provided. Alternatively, a combination of two or more hydrogel beads is provided. The two or more beads may contain the same compound, preferably a compound defined herein, or different compounds, preferably a combination of different compounds defined herein. Alternatively, or in addition, the concentration of the optional different compounds may vary between beads or combinations of beads. Optionally, a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 1000, 5000, 10,000, or more hydrogel beads is provided.

[0107] The size of the hydrogel beads can vary, for example, between about 0.1 nm and 100 mm. Preferably, the beads are macrospheres, microspheres, or nanospheres. Macrospheres preferably have a diameter of about 0.1 mm to 100 millimeters (mm), preferably 0.1 mm to 10 mm; microspheres preferably have a diameter of about 0.1 μm to 100 micrometers (μm), preferably 0.1 μm to 10 μm; and nanospheres preferably have a diameter of about 0.1 nm to 100 nanometers (nm), preferably 0.1 nm to 10 nm.

[0108] The hydrogel beads are preferably macrospheres, and the diameter of the macrospheres is preferably about 0.1 mm to 100 mm, 0.5 mm to 70 mm, 1 mm to 50 mm, 2 mm to 10 mm, 3 mm to 8 mm, or about 4 mm to 6 mm. The diameter of the macrospheres is preferably about 0.1 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 70 mm, or about 100 mm. The hydrogel beads are preferably microspheres, and the diameter of the microspheres is preferably about 0.1 μm to 100 μm, 0.5 μm to 70 μm, 1 μm to 50 μm, 2 μm to 10 μm, 3 μm to 8 μm, or about 4 μm to 6 μm. Preferably, the diameter of the microsphere is about 0.1 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 70 μm, or about 100 μm. The hydrogel beads are preferably nanospheres, and the diameter of the nanospheres is preferably about 0.1 nm to 100 nm, 0.5 nm to 70 nm, 1 nm to 50 nm, 2 nm to 10 nm, 3 nm to 8 nm, or about 4 nm to 6 nm. Preferably, the diameter of the microspheres is about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 70 nm, or about 100 nm.

[0109] The concentration of the compound described herein, preferably the inducer defined herein, in the hydrogel is a concentration that produces an effect on one or more cells of the plant tissue. Thus, the concentration of the compound is an effective concentration. Preferably, the concentration of the inducer is such that, when bound to a transactivator contained in the plant tissue, it induces the induction of an inducible promoter.

[0110] The concentration of the compound, preferably the inducer, in the hydrogel, preferably in the hydrogel beads can be about 0.1 pM to 100 mM, preferably about 0.1 mM to 100 mM, 0.1 μM to 100 μM, 0.1 nM to 100 nM, or about 0.1 pM to 100 pM.

[0111] The concentration of the compound, preferably the inducer, in the hydrogel can be about 0.1 mM to 100 mM, 0.5 mM to 70 mM, 1 mM to 50 mM, 2 mM to 10 mM, 3 mM to 8 mM, or about 4 mM to 6 mM. Preferably, the concentration of the compound can be about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 70 mM, or about 100 mM. The concentration of the compound in the hydrogel can be about 0.1 μM to 100 μM, 0.5 μM to 70 μM, 1 μM to 50 μM, 2 μM to 10 μM, 3 μM to 8 μM, or about 4 μM to 6 μM. Preferably, the concentration of the compound can be about 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 70 μM, or about 100 μM. The concentration of the compound in the hydrogel can be about 0.1 nM to 100 nM, 0.5 nM to 70 nM, 1 nM to 50 nM, 2 nM to 10 nM, 3 nM to 8 nM, or about 4 nM to 6 nM. Preferably, the concentration of the compound can be about 0.1 nM, 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 70 nM, or about 100 nM. The concentration of the compound in the hydrogel can be about 0.1 pM to 100 pM, 0.5 pM to 70 pM, 1 pM to 50 pM, 2 pM to 10 pM, 3 pM to 8 pM, or about 4 pM to 6 pM. Preferably, the concentration of the compound can be about 0.1 pM, 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 70 pM, or about 100 pM.

[0112] A hydrogel is a three-dimensional hydrophilic network. As understood herein, fats are not hydrogels. Lanolin or wool wax are not hydrogels as defined herein.

[0113] In one aspect, the following: a) providing a plant tissue; b) locally contacting the plant tissue with an inducing hydrogel containing a compound, wherein the compound is an inducer of gene expression; and c) Producing a plant structure, preferably a plant shoot. A method for producing a plant structure is provided, comprising:

[0114] In step a) of the methods provided herein, plant tissue is provided. The plant tissue may be of any plant species or genus. Preferably, the plant or plant cell is regenerative, preferably capable of at least one of shoot organogenesis and somatic embryogenesis.

[0115] The plant tissue may be tissue from a monocotyledonous or dicotyledonous plant. The plant tissue may be from a crop or cereal plant. Non-limiting examples of cereal plants for use in the methods provided herein are cassava, maize, sorghum, soybean, wheat, oats, or rice. Crop plants are plant species cultivated and bred by humans. Crop plants may be grown for food and feed purposes (e.g., field crops) or ornamental purposes (e.g., cut flower production, lawn grass, etc.). Crop plants, as defined herein, also include plants from which non-food products are harvested, such as fuel oil, plastic polymers, pharmaceuticals, and cork.

[0116] The plant tissue may be or be derived from a plant belonging to the Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae, Asteraceae (Compositae), Rosaceae, or Poaceae families. Optionally, the plant tissue is selected from the group consisting of maize / corn (Zea spp.), wheat (Triticum spp.), barley (e.g., Hordeum vulgare), oats (e.g., Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp., e.g., G. max), cotton (Gossypium spp., e.g., G. hirsutum, G. barbadense), Brassica spp. spp.) (e.g., rapeseed (B. napus), mustard (B. juncea), kale (B. oleracea), Brassica rapa, etc.), sunflower (Helianthus annuus), safflower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species, e.g., the indica or japonica cultivars), forage grasses, pearl millet (Pennisetumspp.), e.g. pearl millet (P. glaucum), tree species (pine, poplar, fir, plantain, etc.), tea, coffee, oil palm, coconut, vegetable species, e.g. pea, zucchini, beans (e.g. Phaseolus spp.), pepper, cucumber, artichoke, asparagus, eggplant, broccoli, garlic, leek, lettuce, onion, radish, turnip, tomato, potato, Brussels sprouts, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fleshy fruiting plants (grape, or derived from a plant selected from the group consisting of: peach, plum, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus fruit, kiwi, fig, lemon, lime, nectarine, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g., rose, petunia, chrysanthemum, lily, Gerbera species), herbs (mint, parsley, basil, thyme, etc.), woody plants (e.g., Populus, Salix, Quercus, Eucalyptus species), fiber species such as flax (Linum usitatissimum) and hemp (Cannabis sativa, etc.). Plant tissue may also be derived from trees or producing plants, fruit or vegetables (e.g., citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; Brassica plants; Lactuca plants; Spinacia plants; Capsicum plants; Solanum plants, preferably tomato (SolanumThe plant may be derived from a sorghum (Solanum lycopersicum) plant. Preferably, the plant is a sorghum (Solanum) plant. Preferred plants for use in the methods provided herein are tomato (Solanum lycopersicum) or pepper (Capsicum annuum) plants.

[0117] In another preferred embodiment, the plant tissue is derived from a plant selected from the following: asparagus, barley, blackberry, blueberry, broccoli, cabbage, canola, carrot, cassava, cauliflower, chicory, cocoa, coffee, cotton, cucumber, eggplant, grape, chili pepper, lettuce, corn, melon, rapeseed, pepper, potato, pumpkin, raspberry, rice, rye, sorghum, spinach, squash, strawberry, sugarcane, sugar beet, sunflower, bell pepper, tobacco, tomato, watermelon, wheat, and zucchini.

[0118] Optionally, the plant tissue is derived from a refractory plant, which is understood herein as a plant that is unable to regenerate or regenerates poorly. Optionally, a refractory plant or cells thereof are unable to regenerate or exhibit poor regeneration efficiency under conditions known in the art to be optimal for regeneration, such as, but not limited to, conditions that allow regeneration in the presence of an exogenously supplied growth regulator. Those skilled in the art will recognize refractory plants. While both refractory and regenerative cultivars, varieties, and / or accessions may exist within a species, pepper, soybean, and sugar beet are generally non-limiting examples of refractory plants, and their cells are non-limiting examples of refractory plant cells. Representative, non-limiting examples of recalcitrant plants known in the art are pepper (Capsicum annuum), sugar beet (Beta vulgaris, more specifically Swiss chard (Beta vulgaris subsp. vulgaris)), soybean (Gycine max), sunflower (Helianthus annuus), cotton (Gossipium hirsutum), hemp or cannabis (Cannabis sativa), strawberry (Fragaria x ananassa), hops (Humulus lupulus), melon (Cucumis melo), and cucumber (Cucumis sativus). However, the method of the present invention may be applied to any plant or plant cell that would benefit, in some circumstances, from improved regeneration efficiency. Non-limiting examples of workflows in which regeneration is a bottleneck include general propagation of (clonal propagation) plant material, especially for haploid plant material or genetically complex (e.g., highly polyploid and heterozygous) F1 populations, but also include advanced plant biotechnology workflows such as, but not limited to, targeted plant genome editing, production of (stable or transient) transformants, and doubled haploid derivation. Thus, the methods of the present invention can be part of such plant biotechnology workflows.

[0119] At least some of the cells of the plant tissue comprise an expression construct as defined herein, wherein the expression construct comprises a sequence encoding a regeneration factor operably linked to an inducible promoter, and the inducible promoter is activated by an inducer present in the inducible hydrogel. Optionally, the inducer can induce or activate the promoter after binding of the inducer to a transactivator present in the at least some of the cells of the plant tissue. The transactivator can be present in the genome of these cells or on the expression construct. Optionally, the transactivator is present on the expression construct as a construct comprising a sequence encoding a regeneration factor operably linked to an inducible promoter. Thus, in certain embodiments, the plant tissue of step a) comprises an expression construct comprising at least one sequence encoding a regeneration factor operably linked to an inducible promoter and a sequence encoding a transactivator operably linked to a promoter, which may be a promoter that is constitutively active in the plant cells comprising the construct.

[0120] Plant tissue comprising an expression construct defined herein can be provided by introducing the expression construct into a plant cell, for example, by Agrobacterium or viral transfection. Optionally, the plant tissue of step a) of the method provided herein develops from said transfected plant cell. Optionally, the plant tissue of step a) is formed by (inducing) the formation of a callus from said transfected plant cell. Preferably, the callus is formed by placing the transfected plant cell on CIM (callus inducing medium) and / or placing the formed callus on SIM (shoot inducing medium). The components of CIM and SIM are well known in the art and may vary for each plant species used in the method provided herein.

[0121] The plant tissue in step a) of the methods provided herein can be differentiated or undifferentiated tissue, including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, embryos, and callus tissue. The plant tissue may be an (immature) embryo, hypocotyl, cotyledon, leaf, or (detached) shoot.

[0122] Preferably, at least some of the cells of the plant tissue in step a) of the methods provided herein comprise an inducible promoter operably linked to at least one sequence encoding a regeneration factor, also referred to herein as a morphogenetic polypeptide. Thus, the terms "regeneration factor," "morphogenetic polypeptide," and "morphogenic polypeptide" may be used interchangeably herein. The inducible promoter may comprise one or more elements for binding of a (activating) transactivator. The elements that bind the transactivator are preferably as defined herein. As non-limiting examples, dexamethasone-binding GVG preferably binds to one or more UAS elements having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1; dexamethasone-binding LhGR preferably binds to one or more LacOp elements having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2; and β-estradiol-binding XVE preferably binds to one or more LexAop elements having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3. The elements are preferably linked to a minimal promoter such as, but not limited to, the Cauliflower Mosaic Virus (CaMV) 35S promoter. Optionally, the elements are linked to a tissue-specific promoter.

[0123] Upon binding to the inducer, the transactivator binds to and activates the inducible promoter, resulting in the expression of one or more morphogenetic polypeptides. As used herein, the term "morphogenic polypeptide" or "morphogenetic polypeptide" refers to a polypeptide that, when ectopically expressed, stimulates the formation of somatically derived structures capable of producing plants. More precisely, ectopic expression of a morphogenetic polypeptide stimulates the de novo formation of organogenic structures, such as somatic embryos or shoot meristems, which can produce plants. This stimulated de novo formation occurs either in the cell in which the morphogenetic polypeptide is expressed or in adjacent cells. A morphogenetic polypeptide can be a transcription factor that regulates the expression of other genes or a polypeptide that affects hormone levels in plant tissues, either of which can stimulate morphogenetic changes.

[0124] Preferably, the morphogenetic polypeptide is at least one of a WUS / WOX homeobox polypeptide, a PLT (PLETHORA) protein, a polypeptide comprising two AP-2 DNA-binding domains, and WIND1. Preferably, the morphogenetic polypeptide having inducible expression is at least one of a WUS / WOX homeobox polypeptide and a PLT protein. Preferably, the morphogenetic polypeptide having inducible expression is at least one of a WOX5 homeobox polypeptide and a PLT1 protein. Preferably, the morphogenetic polypeptide having inducible expression is a combination of a WOX5 homeobox polypeptide and a PLT1 protein. Preferably, the morphogenetic polypeptide having inducible expression is a combination of a WOX5 homeobox polypeptide, a PLT1 protein, and a WIND1 protein.

[0125] The WUS / WOX homeobox polypeptide is preferably selected from the group consisting of WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX5A, or WOX9 polypeptides (see, e.g., U.S. Pat. Nos. 7,348,468 and 7,256,322 and U.S. Patent Application Publication Nos. 2017 / 0121722 and 2007 / 0271628, which are incorporated by reference in their entireties, and van der Graaff et al., 2009, Genome Biology 10:248). The functional WUS / WOX homeobox polypeptide used in the methods provided herein can be obtained from or derived from any plant. Functional WUS / WOX polypeptides comprising a homeobox DNA-binding domain, a WUS box, and an EAR repressor domain useful in the methods of the disclosure are set forth in Table 1 of WO2020214986, particularly SEQ ID NOs: 246-310 of WO2020214986, which sequences are incorporated herein by reference.

[0126] A preferred WUS / WOX homeobox polypeptide is WOX5. 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 to SEQ ID NO: 4. SEQ ID NO: 4 is the Arabidopsis thaliana WOX5 protein. In one embodiment, the WOX5 amino acid sequence is or is derived from AT3G11260, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT3G11260 or a homolog thereof. 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: 5. The nucleotide sequence encoding the WOX5 protein may be or be derived from the gene AT3G11260, a homolog thereof, 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 a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0127] The PLT protein is preferably selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5 and PLT7. Preferably, the PLT protein is at least one of PLT1, PLT4 and PLT5. Preferably, the PLT protein is PLT1.

[0128] The amino acid sequence of the PLT1 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. SEQ ID NO: 6 is the Arabidopsis thaliana PLT1 protein. In one embodiment, the PLT1 amino acid sequence is or is derived from AT3G20840, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT3G20840 or a homolog thereof. 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: 7. The nucleotide sequence encoding the PLT1 protein may be or be derived from the gene AT3G20840, a homolog thereof, 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 a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0129] The amino acid sequence of the PLT2 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. SEQ ID NO: 8 is the Arabidopsis thaliana PLT2 protein. In one embodiment, the PLT2 amino acid sequence is or is derived from AT1G51190, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT1G51190 or a homolog thereof. 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: 9. The nucleotide sequence encoding the PLT2 protein may be or be derived from the gene AT1G51190, its homologs, 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 homologs. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0130] The amino acid sequence of the PLT3 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. SEQ ID NO: 10 is the Arabidopsis thaliana PLT3 protein. In one embodiment, the PLT3 amino acid sequence is or is derived from AT5G10510, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT5G10510 or a homolog thereof. 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: 11. The nucleotide sequence encoding the PLT3 protein may be or be derived from the gene AT5G10510, its homologs, 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 homologs. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0131] The amino acid sequence of the PLT4 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. SEQ ID NO: 12 is the Arabidopsis thaliana PLT4 protein. In one embodiment, the PLT4 amino acid sequence is or is derived from AT5G17430, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT5G17430 or a homolog thereof. 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: 13. The nucleotide sequence encoding the PLT4 protein may be or be derived from the gene AT5G17430, a homolog thereof, 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 a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0132] The amino acid sequence of the PLT5 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. SEQ ID NO: 14 is the Arabidopsis thaliana PLT5 protein. In one embodiment, the PLT5 amino acid sequence is or is derived from AT5G57390, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT5G57390 or a homolog thereof. 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 to SEQ ID NO: 15. The nucleotide sequence encoding the PLT5 protein may be or be derived from the gene AT5G57390, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT5G57390 or a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0133] The amino acid sequence of the PLT7 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16. SEQ ID NO: 16 is the Arabidopsis thaliana PLT7 protein. In one embodiment, the PLT7 amino acid sequence is or is derived from AT5G65510, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT5G65510 or a homolog thereof. 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: 17. The nucleotide sequence encoding the PLT7 protein may be or be derived from the gene AT5G65510, a homolog thereof, 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 a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0134] The polypeptide comprising two AP-2 DNA-binding domains is preferably a polypeptide selected from the group consisting of ODP2, BBM2, BMN2, or BMN3 polypeptides. The amino acid sequence of the ODP2 protein may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 18. In one embodiment, the OPD2 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 to SEQ ID NO: 19. The amino acid sequence of the BBM2 protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20. In one embodiment, the BBM2 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 to SEQ ID NO: 21.

[0135] The amino acid sequence of the WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) protein can have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22. SEQ ID NO: 22 is the Arabidopsis thaliana WIND1 protein. In one embodiment, the WIND1 amino acid sequence is or is derived from AT1G78080, a homolog thereof, or a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to AT1G78080 or a homolog thereof. 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: 23. The nucleotide sequence encoding the WIND1 protein may be or be derived from the gene AT1G78080, a homolog thereof, 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 a homolog thereof. The identity percentage can be determined over the entire length of the genomic sequence. Alternatively, the identity percentage can be determined over the entire length of the coding sequence of the gene.

[0136] Optionally, the morphogenetic polypeptide is LEC1 (preferably any one of SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 20, or 22 of U.S. Pat. No. 6,825,397, herein incorporated by reference, or a homolog thereof having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity thereto), SHORT a ROOT protein (preferably an SHR having SEQ ID NO: 30 or having a sequence encoded by SEQ ID NO: 31, or a homolog thereof having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity thereto), or a SCARECROW protein (preferably an SCR having SEQ ID NO: 32 or having a sequence encoded by SEQ ID NO: 33, or a homolog thereof having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity thereto).

[0137] Preferably, the inducible promoter operably linked to the (first) sequence encoding a morphogenetic polypeptide is further operably linked to a second or another morphogenetic polypeptide. Additionally or alternatively, at least some of the cells of the plant tissue in step a) of the methods provided herein comprise a (first) inducible promoter operably linked to the (first) sequence encoding a morphogenetic polypeptide and a further inducible promoter linked to a second or another morphogenetic polypeptide. The first and further inducible promoters may be (substantially) the same or different inducible promoters. Transcription from the first and further inducible promoters may be activated by binding to the same transactivator. Optionally, the first and second morphogenetic polypeptides are controlled by the same inducible promoter or by promoters having (substantially) the same sequence. These elements (a first inducible promoter operably linked to the first and any further morphogenetic polypeptides, and / or a second inducible promoter operably linked to any further morphogenetic polypeptide) may all be present on the same construct. Additionally or alternatively, the first and further morphogenetic polypeptides may be contained in separate constructs, each with an inducible promoter.

[0138] At least some of the cells containing a sequence(s) encoding a regeneration factor (i.e., a morphogenetic polypeptide) may further contain one or more sequences encoding a transactivator that, upon binding with an inducer, promotes or activates transcription of one or more regeneration factors. When multiple sequences encoding transactivators are present in a plant cell, these sequences may be different and preferably encode different transactivators that are activated by different inducers. Each transactivator-encoding sequence is preferably operably linked to a constitutively active promoter. Non-limiting examples of constitutive promoters include the CaMV 35S, G10-90, CsV, TCTP2, and UBQ10 promoters. Upon binding with an inducer, the expressed transactivator can bind to the inducible promoter operably linked to at least one sequence encoding a regeneration factor and initiate transcription.

[0139] The transactivator may be any suitable molecule that activates transcription upon binding with an inducer. Preferably, the one or more transactivators are transactivators described herein, preferably selected from the group consisting of GVG, XVE, ER-C1, GVEcR, LhGR, TGV, AlcR, tetR-VP16, and the tetR repressor protein. Preferred transactivators are at least one of GVG, XVE, or LhGR, preferably GVG.

[0140] The one or more transactivators may be GVG, preferably encoded by a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 24. The one or more transactivators may be XVE, preferably encoded by a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 25. The one or more transactivators may be LhGR, preferably encoded by a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 26. The one or more transactivators may be AlcR, preferably encoded by a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 26. Upon binding an inducer, the transactivator can activate an inducible promoter linked to at least one sequence encoding a regeneration factor. As non-limiting examples, when the transactivator is GVG, the transactivator can activate expression induced by an inducible promoter when bound with dexamethasone or a derivative thereof; when the transactivator is LhGR, the transactivator can activate expression induced by an inducible promoter when bound with dexamethasone or a derivative thereof; when the transactivator is XVE, the transactivator can activate expression induced by an inducible promoter when bound with β-estradiol or a derivative thereof. Those skilled in the art will readily recognize that other transactivator-derivative-inducible promoter combinations are similarly suitable.

[0141] In a preferred embodiment, at least some of the plant cells of the plant tissue in step a) of the method provided herein comprise an expression construct comprising an inducible promoter operably linked to a sequence encoding at least one of a PLETHORA (PLT) polypeptide and a WUS / WOX homeobox polypeptide. Preferably, the construct comprises a first inducible promoter operably linked to a sequence encoding a PLETHORA (PLT) polypeptide and an additional inducible promoter linked to a WUS / WOX homeobox polypeptide. The first and additional inducible promoters can be identical or have (substantially) identical or different inducible promoter sequences. Transcription from the first and additional inducible promoters can be activated upon binding with the same transactivator. Optionally, the PLT polypeptide and the WUS / WOX polypeptide are controlled by the same inducible promoter. Thus, at least some of the plant cells of the plant tissue of step a) may comprise a first inducible promoter operably linked to a sequence encoding a PLETHORA (PLT) polypeptide and a second inducible promoter having (substantially) the same sequence as the first inducible promoter linked to a sequence encoding a WUS / WOX homeobox polypeptide. Preferably, the PLT protein is PLT1 and the WUS / WOX homeobox polypeptide is WOX5.

[0142] Preferably, the sequence encoding the PLT protein and the sequence encoding the WUS / WOX homeobox polypeptide are each linked to a GVG-inducible promoter. Preferably, the sequence encoding the PLT1 protein and the sequence encoding the WOX5 homeobox polypeptide are each linked to a GVG-inducible promoter. Alternatively, the sequence encoding the PLT protein and the sequence encoding the WUS / WOX homeobox polypeptide are each linked to an XVE-inducible promoter. Preferably, the sequence encoding the PLT1 protein and the sequence encoding the WOX5 homeobox polypeptide are linked to an XVE-inducible promoter. Preferably, at least some of the plant cells of the plant tissue in step a) may comprise a first inducible promoter operably linked to a sequence encoding a PLETHORA (PLT) polypeptide, a second inducible promoter having (substantially) the same sequence as the first inducible promoter linked to a sequence encoding a WUS / WOX homeobox polypeptide, and a sequence encoding a transactivator capable of activating the first inducible promoter upon binding to an inducer. When the first inducible promoter is an XVE-inducible promoter, the transactivator may be XVE, which is induced by β-estradiol.When the first inducible promoter is a GVG-inducible promoter, the transactivator may be GVG, which is induced by dexamethasone.

[0143] In a preferred embodiment, at least some of the cells of the plant tissue in step a) further comprise a second inducible promoter operably linked to the sequence encoding WIND1. The second inducible promoter comprises one or more elements for binding a (activated) second transactivator. The element for binding the second transactivator is preferably as defined herein. The element is preferably linked to a minimal promoter (such as a minimal 35S promoter) or a CaMV 35S promoter. Optionally, the element is linked to a tissue-specific promoter. Preferably, the sequence encoding the WIND1 protein is linked to an XVE-inducible promoter. Alternatively, preferably, the sequence encoding the WIND1 protein is linked to a GVG-inducible promoter. Preferably, at least some of the plant cells of the plant tissue in step a) may contain a GVG-inducible promoter operably linked to a sequence encoding a PLETHORA (PLT) polypeptide, a GVG-inducible promoter operably linked to a sequence encoding a WUS / WOX homeobox polypeptide, a sequence encoding a WIND1 protein, a sequence encoding XVE, and an XVE-inducible promoter operably linked to a sequence encoding GVG. Preferably, the sequences encoding XVE and GVG are each operably linked to a promoter constitutively active in plant cells, preferably the CaMV 35S, G10-90, CsV, TCTP2, or UBQ10 promoter. Preferably, the transactivator is operably linked to the CaMV 35S promoter (SEQ ID NO: 28) and the CaMV terminator (SEQ ID NO: 29).

[0144] Preferred expression constructs present in at least some of the cells of the plant tissue in step a) of the methods provided herein are described in WO 2019 / 211296 (incorporated herein in its entirety). Preferably, the expression construct is the shoot regeneration vector (XVE-transactivated AmiRBR and AtWIND1; GVG-transactivated AtSHR, AtSCR, AtPLT1, AtPLT4, AtPLT5, and AtWOX5) or shoot regeneration vector-2 (XVE-transactivated AtWIND1; GVG-transactivated AtPLT1 and AtWOX5) described in Examples 1 and 2 of WO 2019 / 211296. Preferably, the expression construct is stably inserted into the plant cell.

[0145] Optionally, at least some of the plant cells of the plant tissue of step a) may optionally comprise, in addition to at least one sequence encoding a regeneration factor operably linked to an inducible promoter and / or an expression construct as defined herein, a sequence encoding one or more components of a programmable nuclease for genome editing. Preferably, said one or more components are a CRISPR endonuclease and / or a TALEN as defined herein.

[0146] In step b) of the method provided herein, the plant cells / tissues are exposed to a hydrogel. Preferably, the hydrogel is a hydrogel as defined herein above, i.e., a hydrogel comprising one or more compounds as defined herein, i.e., one or more inducers as defined herein.

[0147] Step b) may include topically applying the hydrogel, for example, by applying hydrogel beads to the plant tissue. The beads may have a substantially spherical shape, and thus may be, but are not limited to, spheres. The beads may also have a non-spherical shape (disks, cubes, granules, etc.).

[0148] The hydrogel beads are preferably hydrogel beads as defined herein. In this embodiment, the compound may have a uniform distribution or concentration throughout the hydrogel beads. A gradient of one or more compounds is formed as the compounds diffuse from the hydrogel into the plant tissue. Preferably, the hydrogel beads contain a limited concentration of one or more compounds, so that the concentration of the compound diffusing from the hydrogel is limited and preferably decreases over time. The concentration of the compound diffusing from the hydrogel may be limited due to the concentration of the compound present in the hydrogel and / or because the hydrogel beads dehydrate and / or evaporate over time, thereby reducing or eliminating the diffusion of the compound or compounds from the hydrogel.

[0149] The hydrogel, preferably the hydrogel beads, may contain one or more inducers as defined herein. Preferred compounds are gene expression inducers. Preferably, the gene expression inducer is selected from the group consisting of steroids, antibiotics, copper, ethanol, acetaldehyde, and insecticides. Preferably, the inducer is at least one of a steroid, preferably dexamethasone or a derivative thereof, or β-estradiol or a derivative thereof. Preferably, the hydrogel contains one or more inducers for expressing one or more regeneration factors contained in at least a portion of the cells of the plant tissue in step a) of the method provided herein.

[0150] The methods provided herein may further comprise step c) of producing a regenerated plant structure, preferably a plant shoot. The shoot is preferably derived from a somatic embryonic structure. Optionally, (somatic) embryogenic tissue is formed that does not itself form an embryo but becomes the site of secondary shoot organogenesis. Step c) is therefore preferably carried out under conditions suitable for the formation of a regenerated plant structure, preferably under shoot-forming conditions. These conditions are herein understood to be the minimum requirements for at least plant cells to regenerate, which generally include at least the normal growth conditions of said plant or plant cells. In a preferred embodiment, the regeneration conditions require exposure of the plant tissue to a hydrogel as defined herein, wherein the hydrogel contains one or more inducers, resulting in the expression in the plant tissue of at least one of a PLT polypeptide and a WUS / WOX homeobox polypeptide, preferably both a PLT polypeptide and a WUS / WOX homeobox polypeptide as defined herein, more preferably a PLT polypeptide, a WUS / WOX homeobox polypeptide, and a WIND1 polypeptide as defined herein.

[0151] Optionally, step c) includes callus formation prior to the formation of plant structures, preferably shoot formation. Thus, step c) may comprise the following substeps: c1) allowing plant tissue to form callus; and c2) growing plant structures, preferably shoots and / or plant embryos, from the callus; optionally, the culture conditions for c1 and c2) are different. More specifically, step c1) can be performed under conditions suitable for the plant tissue to form callus; and step c2) can be performed under conditions suitable for the plant callus to form plant structures, preferably shoots. Those skilled in the art will recognize the conditions suitable for callus and / or regeneration. In a preferred embodiment, steps c1) and c2) require exposure of the plant tissue to a hydrogel as defined herein, wherein the hydrogel contains one or more inducers, resulting in the expression of at least one PLT polypeptide and a WUS / WOX homeobox polypeptide in the plant tissue.

[0152] Optionally, the plant structure, preferably a shoot, is produced without the need for exposure to plant growth hormones, i.e., "hormone-independent shoot regeneration." Preferably, the regenerated plant structure, preferably a plant embryo and / or plant shoot, is formed by locally contacting the plant tissue with a hydrogel, preferably by placing hydrogel beads comprising one or more inducers as defined herein on the plant tissue, thereby creating a gradient of the inducer(s) in the plant tissue.

[0153] Plant structures, preferably plant shoots, can be formed by contacting plant tissue containing an expression construct defined herein with a gradient of one or more inducers, wherein the gradient is formed by overlaying hydrogel beads on the plant tissue. The inventors have discovered that overlaying hydrogel beads containing one or more inducers on plant tissue from a plant containing a construct defined herein results in the formation of embryonic structures at a fixed distance from the hydrogel beads, which can then effectively develop into complete plant structures via shoot formation, from which complete plants can be grown. In the latter situation, many morphogenetic structures form on the plant tissue, but all degenerate and fail to develop into complete plant structures.

[0154] The method may further comprise producing a plant from the produced plant structure, preferably from the produced plant shoot. Preferably, the plant comprises at least one inflorescence and / or is capable of vegetative propagation. Optionally, the method may further comprise producing seeds and / or progeny plants of the produced plant by vegetative, sexual or apomictic propagation.

[0155] In yet another aspect, a plant obtained by the method provided herein is provided. Thus, the plant may be, for example, a transgenic plant and / or a mutant plant comprising at least one regeneration factor and / or transactivator as defined herein (preferably stably inserted into its genome), a transgene, and optionally a mutation in a sequence of interest. The plant may also be an artificial plant. The plant may be obtained using targeted genome editing, such as the CRISPR technology or TALEN, described herein. Optionally, the plant may comprise a sequence encoding one or more components of a CRISRP complex, i.e., a CRISPR endonuclease, and / or one or more RNAs for guiding the CRISPR endonuclease (a crRNA, a tracrRNA, and / or a single guide RNA comprising or consisting of a crRNA linked to a tracrRNA). Optionally, the sequence encoding one or more components of the CRISPR complex is removed after introducing a mutation into the sequence of interest, for example, by breeding and selection. Optionally, the plants obtainable by the methods provided herein are plants that differ from naturally occurring plants only by having a mutation in the sequence of interest. Preferably, the transgene or mutation in the sequence of interest is located in a germline or germline progenitor cell and / or tissue and / or plant part for clonal propagation. Preferably, the plants provided herein are not, or are not necessarily, obtained by an essentially biological process. The plants provided herein preferably differ from naturally occurring plants at least in that they contain at least one regeneration factor and / or transactivator as defined herein, a transgene, and optionally a mutation in the sequence of interest. The transgene or mutation in the sequence of interest is preferably located at least in a germline or germline progenitor cell and / or tissue and / or plant part for clonal propagation of the plant. The transgene or mutation in the sequence of interest is preferably located at least in the L2-shoot meristem layer. The transgene or mutation in the sequence of interest is preferably present in at least one of the pollen and eggs of the plant.Optionally, the transgene or construct containing at least one regeneration factor and / or transactivator regeneration construct is outcrossed in subsequent generations.

[0156] Additionally provided are progeny or seeds from plants produced by the methods provided herein. The progeny may be produced by sexual or asexual (vegetative) reproduction. The progeny preferably contain at least one regeneration factor and / or transactivator, transgene, or sequence of interest mutation as defined herein.

[0157] Also provided are plant parts or plant products derived from plants obtained from the methods provided herein. Optionally, the plant parts or plant products are characterized by comprising at least one regeneration factor and / or transactivator as defined herein. Optionally, the plant parts or plant products are characterized by comprising a transgene or mutation in a sequence of interest. Such genetic material may be genomic DNA or a fragment of genomic DNA. Such genetic material may be mitochondrial DNA or a fragment of mitochondrial DNA. Such genetic material may be chloroplast DNA or a fragment of chloroplast DNA. The plant parts may be propagated or non-propagated material.

[0158] Also provided is a combination of plant cells and a hydrogel. The plant cells are preferably contained in plant tissue, preferably plant tissue as defined herein. Alternatively or additionally, the hydrogel is a hydrogel as defined herein. Preferably, the hydrogel is a hydrogel bead comprising one or more inducers as defined herein. The hydrogel, preferably the hydrogel beads, is preferably for non-medical use. Preferably, the hydrogel, preferably the hydrogel beads, is not for use in veterinary medicine, preferably not for use in mammalian medicine, preferably not for use in human medicine. The hydrogel beads may be unsuitable for animal, mammalian and / or human medicine. Optionally, the hydrogel beads are for use inside or outside a plant. Preferably, the hydrogel or hydrogel beads further comprise one or more plant-specific compounds, such as, but not limited to, a plant hormone, a programmable nuclease (or a nucleic acid encoding the same) designed to target a genomic plant sequence, a vector designed to express a protein in a plant cell (e.g., encoding a plant protein and / or a plant-specific promoter sequence), an mRNA encoding a plant protein, or an siRNA or miRNA for targeting a plant transcript. Preferably, the hydrogel or hydrogel beads are topically applied to (or in contact with the exterior of) a plant part, plant structure, plant tissue, plant organ, plant organ system, or entire plant. Preferably, the hydrogel or hydrogel beads are topically applied to (or in contact with the exterior of) a plant leaf, plant shoot, or plant callus. Also provided is the use of a hydrogel, preferably hydrogel beads, as defined herein to induce regeneration of plant tissue.

[0159] All patents and publications cited herein are hereby incorporated by reference in their entirety. [Example]

[0160] Example 1. Hydrogel Carrier for Guided Regeneration in Tomato (S. lycopersicum) Plant material: Stable transgenic single-insertion tomato lines were generated by transformation of tomato cells (Moneymaker, The Netherlands) with construct pKG11052 as previously described (Example 2 of WO 2019 / 211296). Construct pKG11052 contains the following promoter-transgene expression cassette: CaMV35S-XVE CaMV35S-GVG XVE inducible promoter -WIND1 GVG-inducible promoter-PLT1 GVG-inducible promoter - WOX5 CaMV35S-erGFP Includes:

[0161] Seeds were sterilized (rinsing in 70% ethanol for 5 min, washing in 1% bleach for 15 min, followed by four washes with sterile tap water) and germinated on moistened sterile Whatman filter paper (100 mm; Tisch Products, product code: 10311810). Germinated seeds were transferred to a sterile container containing 100 ml of solid Murashige & Skoog medium containing vitamins, 20 g / l sucrose, 0.8% microagarose, and 0.5 g / l MES (pH 5.8).

[0162] Cotyledons and young leaves were excised from 1-week-old seedlings and placed on solid Murashige-Skoog medium containing vitamins, 20 g / l sucrose, 0.8% microagarose, and 0.5 g / l MES (pH 5.8).

[0163] Preparation of Induced Hydrogel Beads: Hydrogel beads were prepared by pipetting 10 μl of 1.6% alginate solution into an excess (≥20 ml) of 1% CaCl2 solution. The beads were collected and equilibrated for 1 hour in liquid Murashige and Skoog medium containing vitamins, 20 g / L sucrose, and 0.5 g / L MES (pH 5.8). The beads were then equilibrated in an inducer solution containing liquid Murashige and Skoog medium containing vitamins, supplemented with 20 g / L sucrose, 0.5 g / L MES (pH 5.8), 10 μM dexamethasone (50 mM stock solution in DMSO, diluted 5,000-fold for the final solution), and 1 μM estradiol (10 mM stock solution in DMSO, diluted 10,000-fold for the final solution).

[0164] result Inducing hydrogel supports were prepared as described previously (Figure 1A) and placed on the tissue surface of cotyledon and young leaf tissue segments using fine tweezers (Figure 1B). Localized morphogenetic events with visible organs were observed within 2.5 weeks after application of the inducing hydrogel beads (Figure 1C). In contrast, with conventional methods (supplementation of the growth medium with an inducer), repeated clusters of morphogenetic events were observed across the entire tissue surface. The formed morphogenetic structures subsequently underwent developmental arrest (Figure 1D). In contrast, the morphogenetic events induced by hydrogel bead-mediated localized induction resulted in the development of embryonic structures and viable seedlings (Figure 1E). Subsequently, regenerated shoots developed from the induced embryogenic structures (Figure 1F).

[0165] Example 2. Hydrogel carrier for guided regeneration in pepper cv. (C. Annuum cv.) Maor Plant Material: Stable transgenic Maor peppers (Capsicum annuum cv. Maor; Israel) were generated by transformation of Maor pepper cells with construct pKG11052 (see Example 1, supra). Seeds were sterilized (washed in 70% ethanol for 5 minutes, 1% bleach for 15 minutes, and then rinsed four times with sterile tap water) and then germinated on solid Murashige-Skoog medium containing vitamins and supplemented with 20 g / L sucrose, 0.8% microagarose, and 0.5 g / L MES (pH 5.8). Cotyledons were detached from 10-day-old seedlings and cut twice to obtain triplicate explants. Explants were pre-cultured for 2 days in co-cultivation medium (CCM) (Heidmann et al., 2011 Plant Cell Rep. 30(6):1107-15) supplemented with 40 mg / L acetosyringone, 1.6% (w / v) glucose, 0.7% microagarose, 2 mg / L zeatin riboside, and 0.1 mg / L indole-3-acetic acid. Explants were immersed for 30 minutes in liquid CCM containing a suspension of Agrobacterium tumefaciens GV3101 (OD600 = 0.3) harboring pKG11052 and 40 mg / L acetosyringone. The explants were wiped dry and then co-cultured for 2 days under dim light in co-culture medium supplemented with 40 mg / L acetosyringone, 1.6% (w / v) glucose, 0.7% microagarose, 2 mg / L zeatin riboside, and 0.1 mg / L indole-3-acetic acid. The light was attenuated by placing cellulose chromatography paper (GE Whatman, Grade: 3 mm CHR, Cat. no.: 3030-931) on top of the plate. After co-culture, the explants were cultured in co-culture medium supplemented with 1 mg / L thidiazuron, 100 mg / L kanamycin sulfate, and 500 mg / L cefotaxime at 23°C under full light conditions (3000 lux, 16 / 8-h photoperiod). Explants were subcultured monthly in fresh medium. GFP-expressing microcalli were isolated from the tissue and subcultured until they showed the first signs of greening and leaf-like structure formation (3-4 months after transformation).Calli bearing initial leaf-like structures were further subcultured on elongation medium (Heidmann et al., supra) supplemented with 1.6% (w / v) glucose, 1 mg / L inositol, 20 mg / L adenine sulfate, 200 mg / L casein hydrolysate, 1 mg / L gibberellic acid 3, 4 mg / L benzylaminopurine, and 30 μM silver thiosulfate. Once calli elongated, leaf-like structures were detached from the calli and cultured on solid Murashige and Skoog medium containing vitamins, 20 g / L sucrose, 0.8% microagarose, and 0.5 g / L MES (pH 5.8). Freshly detached leaf-like structures were used in induction assays.

[0166] Preparation of Induced Hydrogel Beads: Hydrogel beads were prepared by pipetting 10 μl of 1.6% alginate solution into an excess (≥20 ml) of 1% CaCl2 solution. The beads were collected and equilibrated for 1 hour in vitamin-containing liquid Murashige and Skoog medium containing 20 g / L sucrose, 0.5 g / L MES (pH 5.8), and 10 μM dexamethasone and 1 μM estradiol. The beads were then equilibrated in an inducer solution containing vitamin-containing liquid Murashige and Skoog medium containing 20 g / L sucrose, 0.5 g / L MES (pH 5.8), 10 μM dexamethasone, and 1 μM estradiol to produce the induced beads.

[0167] result Pepper (Capsicum annuum) is known to be a recalcitrant plant and has not demonstrated regeneration using conventional tissue culture regeneration methods. Inducing hydrogels were prepared and applied to the surface of freshly detached leaf-like tissue segments using fine tweezers (Figure 2A). Within 19 days after application of the inducing hydrogel beads, visible somatic embryos were present, and localized morphogenetic events were induced (Figure 2B). The morphology of the somatic embryos was clear, and all subsequent stages of embryogenesis were present within the clusters (Figure 2C–G). Embryos germinated while remaining attached to the callus (Figure 2H). Regenerated plants also developed from isolated mature embryos (detached from the callus) cultured on solid Murashige-Skoog medium containing vitamins, 20 g / L sucrose, 2 mg / L GA3, 0.8% microagarose, and 0.5 g / L MES (pH 5.8) (Figure 2I, J). The plantlets further developed into regenerated plants bearing mature transgenic fruit (Figure 2K). Figures 2L-2N show developmental stages after the flower during fruit set. More specifically, Figure 2L shows a closed flower bud, Figure 2M shows an open flower bud, and Figure 2N shows a fertilized flower bud. Figure 2O shows a mature transgenic fruit with transgenic seed set, and Figure 2P shows clear GFP expression in the majority of the seeds.

[0168] Therefore, exposure of plant cells to the inducing hydrogel beads results in plant regeneration. Furthermore, the transgene (GFP) remains in the germplasm of the regenerated plants, allowing the transgene to be carried over to progeny plants.

Claims

1. 1. A method for producing a plant shoot, comprising: a) providing plant tissue containing a sequence encoding at least one regeneration factor under the control of an inducible promoter; b) locally contacting the plant tissue with inducing hydrogel beads comprising an inducing agent, optionally a compound capable of inducing the inducible promoter upon binding to a transactivator contained in the plant tissue; and c) Producing said shoot from said plant tissue The method comprising:

2. 2. The method for producing plant shoots according to claim 1, wherein the hydrogel is hydrogel beads, and the beads are macrospheres having a diameter of about 0.1 to 10 millimeters (mm) or microspheres having a diameter of about 0.1 to 100 micrometers (μm).

3. The method for producing plant shoots according to claim 1 or 2, wherein the inducer is a steroid.

4. 4. The method for producing plant shoots according to claim 3, wherein the steroid is at least one of dexamethasone or a derivative thereof, and the steroid is β-estradiol or a derivative thereof.

5. the inducing agent is dexamethasone or a derivative thereof and the plant tissue of step a) contains a transactivator which is a GVG protein; and / or 5. The method of claim 1, wherein the inducing agent is β-estradiol or a derivative thereof, and the plant tissue of step a) comprises a transactivator that is an XVE protein.

6. The method of any one of claims 1 to 5, wherein the concentration of the inducer in the hydrogel beads is about 1 nM to 100 µM.

7. The at least one regeneration factor comprises at least one of a PLETHORA (PLT) polypeptide and a WUS / WOX homeobox polypeptide, preferably: i) the PLT polypeptide is selected from the group consisting of PLT1, PLT2, PLT3, PLT4, PLT5 and PLT7, preferably PLT1; ii) The method of any one of claims 1 to 6, wherein the WUS / WOX homeobox polypeptide is selected from the group consisting of WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, or WOX9, preferably WOX5.

8. 8. The method of claim 7, wherein the at least one regeneration factor comprises PLT1 and WOX5, and the PLT1 coding sequence and WOX5 coding sequence are each operably linked to an inducible promoter, preferably a GVG-inducible promoter.

9. 9. The method according to any one of claims 1 to 8, wherein the at least one regeneration factor comprises WIND1, and the WIND1 coding sequence is operably linked to an inducible promoter, preferably an XVE-inducible promoter.

10. The method according to any one of claims 1 to 9, wherein the hydrogel is alginic acid or a derivative thereof.

11. 11. The method according to any one of claims 1 to 10, wherein the plant tissue of step a) comprises an expression construct encoding the at least one regeneration factor under the control of an inducible promoter and optionally encoding a transactivator.

12. 12. The method according to any one of claims 1 to 11, wherein the plant tissue in step a) is a hypocotyl or a leaf, wherein the plant tissue is preferably any one of a cotyledon, a first true leaf, and a young (lateral) leaf.

13. 13. The method of any one of claims 1 to 12, further comprising the step of regenerating a plant from the plant shoot produced.

14. A combination of hydrogel beads containing the inducer of gene expression according to any one of claims 1 to 13 and plant cells.

15. 15. Use of the hydrogel beads of claim 14 for inducing regeneration of plant tissue.

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