Method for improving plant regeneration efficiency

Abstract

The invention relates to a method for improving the regeneration capacity of plants. In particular, the methods of the invention relate to increasing the levels and / or activity of peptides involved in local wound signaling and plant regeneration. Plants expressing the peptides and methods of producing the plants are also described.

Description

Technical Field

[0001] The present invention relates to a method for improving the regeneration efficiency of a plant or a part of the plant, and describes tissue regeneration factors and plant tissue culture media that can be used to improve the regeneration efficiency, as well as genetically altered plants expressing tissue regeneration factors and tissue regeneration factor receptors. Background Art

[0002] Organisms are often subject to a variety of biotic and abiotic stresses that induce severe injuries leading to partial or complete organ loss. Upon injury, both plants and animals are able to regenerate new tissues or organs to minimize the effects of daily loss (Birnbaum and Sanchez Alvarado, 2008; Mathew and Prasad, 2021; Pulianmackal et al., 2014; Sena and Birnbaum, 2010; Sugimoto et al., 2011; Sugimoto et al., 2019). Plants experience trauma more frequently than their animal counterparts and have evolved remarkable regenerative abilities to repair damaged tissues and to regenerate new organs or entire plants, although the extent of these abilities varies by species and tissue type (Birnbaum and Sanchez Alvarado, 2008; Ikeuchi et al., 2019; Ikeuchi et al., 2016; Mathew and Prasad, 2021; Sena and Birnbaum, 2010; Sugimoto et al., 2019). As a result, plants display different ways of regenerating when body parts are lost or damaged. For example, when local damage occurs, most plants rebuild the shoot apical meristem or the root apical meristem. More strikingly, new organs or entire plants can grow from small pieces of injured tissue or even from single cells through de novo organogenesis or somatic embryogenesis (Ikeuchi et al., 2019; Ikeuchi et al., 2016; Kareem et al., 2016; Mathew and Prasad, 2021; Mendez-Hernandez et al., 2019). De novo organogenesis is characterized by the reactivation of cell proliferation at the site of injury and the formation of a cell mass called callus that is capable of undergoing a regenerative response (Ikeuchi et al., 2013).

[0003] The regenerative capacity of plant cells in vitro can be enhanced when cuttings and similar explants are cultured on nutrient media supplemented with auxins and cytokinins (George et al., 2008; Skoog and Miller, 1957). Conventionally used protocols for in vitro regeneration involve pre-cultivation of explants on auxin-rich media to generate callus tissue capable of organ regeneration (Valvekens et al., 1988). The intrinsic regenerative capacity of plants provides the fundamental basis for different agricultural and biotechnological procedures used for in vitro propagation and production of many plant species under controlled conditions (Ikeuchi et al., 2019; Ikeuchi et al., 2016; Sugiyama, 2015).

[0004] Given that wounding is necessary for the initiation of regeneration in nature and in vitro (Birnbaum and Sanchez-Alvarado, 2008; Ikeuchi et al., 2013; Iwase et al., 2015; Sugiyama, 2015), it is widely assumed that wounding stimuli may, in fact, provide the primary inductive trigger for this phenomenon. However, what exactly plants perceive as a wound signal and how they initiate regeneration has remained unknown for decades.

[0005] Therefore, there is a need to understand the mechanisms of plant regeneration. In addition, there is a need to be able to modulate the regeneration capacity of plants for agricultural and biotechnology applications, particularly for plants that are difficult to cultivate. In addition, there is a need to modulate the regeneration capacity of plants with important commercial value, rare or endangered species, and plants with advantageous characteristics. Traditionally, plant regeneration has been limited to specific genotypes, and regeneration of transgenic plants is often more difficult. The present invention addresses the need to improve the regeneration efficiency of all plants, including transgenic plants. Summary of the invention

[0006] Here, we report that Plant Elicitor Peptide (PEP) is a local wound signal that enhances plant regeneration capacity. In contrast to systemins, which primarily regulate systemic defense responses, we found that mutations in PEP, PEP precursor (PROPEP), and PEP receptor (PEPR) impaired both local and systemic defense responses, suggesting that PEP is a local wound signal. Remarkably, although deletion of PROPEP or PEPR abolished the regenerative capacity of tomato plants, overexpression of these genes resulted in enhanced regeneration capacity, and exogenous application of PEP significantly increased regeneration and transformation efficiency. These observations suggest that PEP is the long-sought wound signal that promotes plant regeneration. Therefore, PEP was designated as regeneration factor 1 ( RE GENERATION F ACTOR 1 , REF1), the regeneration factor precursor is designated PROPEP, PROSIPEP, PROREF1 PRR or PRP (PROREF1), and the REF1 receptor is designated PEPR, SIPEPR1, RER or PORK1 (REF1 receptor); the terms are used interchangeably herein. Further, in contrast to systemin, which is found only in Solanaceae plants, orthologues of REF1, PRR and RER can be identified in most (if not all) sequenced angiosperm species, indicating that the function of PEP is highly conserved in the plant kingdom. Therefore, the present invention is applicable to all plants. Further evidence for this is provided in the Examples. Our studies have shown that exogenous application of REF1, or overexpression or PRR and / or RER significantly improved the regeneration capacity of all plants tested, including some different varieties and plants that are known to be difficult to regenerate.

[0007] As used herein, tissue regeneration factor can be used interchangeably with REF1. Tissue regeneration factor precursor or PROREF1 can be used interchangeably herein. Tissue regeneration factor receptor can be used interchangeably with RER herein.

[0008] In one aspect of the present invention, a tissue regeneration factor comprising an amino acid sequence as defined in SEQ ID NO: 2 or a functional variant or homolog thereof is provided, wherein the homolog is preferably selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homolog thereof of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20.

[0009] In one embodiment, the homologue or functional variant comprises a conserved motif. The sequence of the conserved motif can be selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

[0010] In another aspect of the present invention, a plant tissue culture medium comprising the tissue regeneration factor of the present invention is provided. In one embodiment, the tissue culture medium is a callus induction medium, a shoot induction medium or a root induction medium. In a further embodiment, the plant tissue culture medium further comprises an auxin and / or a cytokinin.

[0011] In one embodiment, the plant tissue culture medium comprises a tissue regeneration factor at a concentration between 0.00001 and 100 nM. In further embodiments, the plant tissue culture medium comprises a tissue regeneration factor at a concentration of about 0.M nM, 1 nM, 10 nM, 50 nM, or 100 nM.

[0012] In another aspect of the present invention, a nucleic acid construct is provided, the nucleic acid construct comprising a nucleic acid sequence encoding at least one of a tissue regeneration factor, a tissue regeneration factor precursor, and a tissue regeneration factor receptor;

[0013] wherein the tissue regeneration factor is selected from SEQ ID NO: 2 or a functional variant or homologue thereof, wherein the homologue is preferably selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homologue thereof; and

[0014] wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homologue thereof, wherein the homologue is preferably selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof; and

[0015] The tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is preferably selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

[0016] In one embodiment, the homologue or functional variant of tissue regeneration factor or tissue regeneration factor precursor comprises a conserved motif, wherein the sequence of the conserved motif is selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

[0017] In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding a tissue regeneration factor precursor or a tissue regeneration factor receptor.

[0018] In another embodiment, the nucleic acid construct comprises nucleic acid sequences encoding a tissue regeneration factor precursor and a tissue regeneration factor receptor.

[0019] In a further embodiment, the nucleic acid sequence encoding at least one of tissue regeneration factor, tissue regeneration factor precursor and tissue regeneration factor receptor is operably linked to one or more regulatory sequences. The regulatory sequence can be a constitutive or strong promoter or an inducible promoter, wherein the inducible promoter is preferably a nopaline synthase promoter.

[0020] In another aspect of the present invention, a host cell comprising the nucleic acid construct of the present invention is provided.

[0021] In another aspect of the present invention, a transgenic plant, plant part thereof or plant cell is provided, wherein the plant, plant part thereof or plant cell is characterized by increased expression and / or level of at least one of the following:

[0022] i. tissue regeneration factors; and / or

[0023] ii. tissue regeneration factor precursor; and / or

[0024] iii. Tissue regeneration factor receptor

[0025] wherein the tissue regeneration factor is selected from SEQ ID NO: 2 or a functional variant or homologue thereof, wherein the homologue is preferably selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homologue thereof; and

[0026] wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homologue thereof, wherein the homologue is preferably selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof; and

[0027] The tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is preferably selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

[0028] In one embodiment, the homologue or functional variant of tissue regeneration factor or tissue regeneration factor precursor comprises a conserved motif, wherein the sequence of the conserved motif is selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

[0029] In a further embodiment, the plant, part thereof or plant cell comprises the nucleic acid construct of the present invention. Preferably, the nucleic acid construct is stably incorporated into the plant genome.

[0030] In yet another aspect of the present invention, a transgenic plant, a plant part thereof or a plant cell thereof is provided, wherein the plant is characterized by one or more mutations in the plant genome, wherein the mutation is the insertion of at least one additional copy of a nucleic acid sequence encoding a tissue regeneration factor precursor and / or at least one additional copy of a nucleic acid sequence encoding a tissue regeneration factor receptor, such that the one or more nucleic acid sequences are operably linked to a regulatory sequence, wherein the mutation is preferably introduced using targeted genome editing;

[0031] wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homolog thereof, wherein the homolog is selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homolog thereof; and

[0032] wherein the tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

[0033] In another aspect of the present invention, there is provided use of the tissue regeneration factor of the present invention, the plant tissue culture medium of the present invention or the nucleic acid construct of the present invention for improving the regeneration capacity of a plant or a part thereof.

[0034] In one embodiment, the plant, part thereof or plant cell is a monocot or a dicot.

[0035] In another aspect of the present invention, there is provided a method for increasing the regeneration efficiency of a plant, a plant part thereof or one or more plant cells, the method comprising

[0036] i. applying the tissue regeneration factor of the present invention to the plant or part thereof or one or more plant cells; or

[0037] ii. applying the plant tissue culture medium of the present invention to the plant or part thereof or one or more plant cells; and / or

[0038] iii. introducing and expressing the nucleic acid construct of the invention in said plant, part or one thereof or in a plant cell.

[0039] In another aspect of the present invention, there is provided a method for producing a plant, a plant part thereof or one or more plant cells having improved regeneration efficiency, the method comprising

[0040] i. applying the tissue regeneration factor of the present invention to the plant or part thereof or one or more plant cells; or

[0041] ii. applying to the plant or part thereof or one or more plant cells any of the plant tissue culture medium of the present invention; and / or

[0042] iii. introducing and expressing the nucleic acid construct of the invention in said plant or part thereof or in one or a plant cell.

[0043] In one embodiment, the method further comprises producing a plant from the one or more plant cells.

[0044] In another embodiment, 0.01-500 nmol / L of the tissue regeneration factor is exogenously administered to the plant, part thereof or plant cell.

[0045] In another embodiment, 0.1 to 0.5 mL of plant tissue culture medium is applied exogenously to the plant, part thereof or plant cell.

[0046] In another embodiment, the method increases one or more of callus formation, shoot regeneration, and root regeneration.

[0047] In another embodiment, the method increases the transformation efficiency of a plant, part thereof, or plant cell.

[0048] In a further embodiment, the plant part is an explant, wherein preferably the explant is selected from but not limited to a node, an apical meristem, a root, a cotyledon, an embryo, a leaf disk, a leaf blade, a pedicel, a petiole, anther, pollen, a microspore ovary, a hypocotyl.

[0049] In any of the aspects described herein, the plant may be a monocot or a dicot. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The invention is further illustrated in the following non-limiting figures.

[0051] Figure 1 - Schematic diagram of signal transduction in plants after wounding, (a) showing the formation of root meristems, (b) showing de novo root formation, (c) showing shoot regeneration, and (d) showing stem cell formation in moss.

[0052] Figure 2 - Identification of SIPep (REF1) as a Pep homolog in tomato.

[0053] Figure 3 - (a) Schematic showing SIPEP (REF1), a 23 amino acid peptide cleaved from a precursor protein called PROSIPEP (also known as PROREF1 and PRR). (b) Sequence alignment of PROSIPEP (PRR) and a PROSIPEP knockout mutant (prr).

[0054] Figure 4 -PI-II local expression (red) and systemic expression (blue) in wounded wild-type tomato plants, PROSIPEP (PRR) knockout mutants (prr-1 and prr-2) and PROSYSTEMIN knockout mutants (prs-1 and prs-2), and the PROSIPEP PROSYSTEMIN double mutant (prr1prs1).

[0055] Figure 5 - (a) Images of callus formation in tomato varieties Ailsa Craig (AC), where varieties are wild type (WT), PROSIPEP knockout (prr-1 and prr-2), or PROSYSTEMIN knockout (prs-1). (b) Relative callus area in WT, prr-1, prr-2, and prr-2 Aisla Craig tomato varieties.

[0056] Figure 6- (a) Hypocotyl explants of WT (Ailsa Craig, AC) and prr mutants were cultured on callus induction medium (CIM) without SIPEP (REF1) and 1 nM concentration of CIM REF1 for 14 days, and photographs of callus formation in these plants are shown. (b) Distribution of projected callus area of ​​WT and prr mutants grown on callus induction medium without REF1 (control) and in callus induction medium with 1 nM SIPEP (REF1). Projected callus area was quantified by Image J, and individual values ​​(black dots) are shown, with error bars representing standard deviations from three independent experiments.

[0057] Figure 7 -Hypocotyl explants of wild type (WT) Ailsa Craig and prr mutants. (a) shows a photograph of the explants. (b) shows the relative regeneration frequency, (c) shows the number of hypocotyls of the explants, and the data are the mean ± SD of three independent experiments. ***P<0.001, **P<0.01, *P<0.05 (Student's t-test).

[0058] Figure 8 -Hypocotyl explants of wild-type (WT) Ailsa Craig and prr mutants were cultured in root induction medium in the presence or absence of 1 nM SIPEP (REF1) for 21 days. (a) shows the photograph of the explants. (b) shows the relative regeneration frequency, (c) shows the number of hypocotyls of the explants, and the data are the mean ± SD of three independent experiments. ***P<0.001, **P<0.01, *P<0.05 (Student's t-test).

[0059] Fig. 9 -WT (Ailsa Craig, AC) hypocotyl explants were cultured on callus induction medium (CIM) containing the indicated concentrations of REF1, and callus phenotypes were scored after 14 days of incubation. (a) shows a photograph of the explants. (b) shows a box plot representing the distribution of projected callus area. The projected area of ​​callus was quantified by Image J, and individual values ​​(black dots) are shown, with error bars representing the standard deviation of three independent experiments. ***P<0.001, **P<0.01, *P<0.05 (Student's t-test).

[0060] Fig.10- Callus formation in wild type (Ailsa Craig, AC), PROSIPEP knockout (prr-1 and prr-2) and PROSIPEP (PRR) overexpressing tomato plants (PRR-OE-1 and PRR-OE-2). (a) Photographs of callus formation in the plants. (b) Relative callus area in the plants.

[0061] Fig.11 - Shoot regeneration in wild type (Ailsa Craig, AC), PROSIPEP knockout (prr-1 and prr-2) and PROSIPEP (PRR) overexpressing tomato plants (PRR-OE-1 and PRR-OE-2). (a) Photographs of shoot regeneration in the plants. (b) Relative regeneration frequency in the plants. (c) Number of hypocotyls in the plants.

[0062] Fig.12 -SIPEPR1 (RER) is the tomato ortholog of the Arabidopsis PEP receptor.

[0063] Fig.13 -SIPEP (REF1), but not systemin, triggers autophosphorylation of REF1 receptor (RER). (a) shows the pull-down assay between the HA-Flag-tagged ectodomain of RER (purified from tobacco leaves), biotinylated systemin, biotinylated REF1, and REF1. (b) shows the autophosphorylation assay of tobacco leaves expressing the constructs in (c) that were treated with 100 nM REF1, systemin, or control for 20 min prior to the autophosphorylation assay.

[0064] Fig.14 - (a) REF1- or systemin-induced expression of pFRK1::LUC in Arabidopsis protoplasts. Protoplasts were transfected with pFRK1::LUC and the indicated constructs and induced with 100 nM REF1 or systemin for 3 h. Data are mean ± SD, n = 3 replicates. The experiment was repeated three times with similar results. ***P < 0.001 (Student's t-test); ns, not significant. (b) REF1- or systemin-induced expression of pFRK1::LUC in Arabidopsis protoplasts. Protoplasts were transfected with pFRK1::LUC and the indicated constructs and induced with the indicated concentrations of REF1 or systemin for 3 h. Data are mean ± SD, n = 3 replicates. The experiment was repeated three times with similar results. ***P < 0.001 (Student's t-test); ns, not significant.

[0065] Fig.15- (a) Sequence alignment of SIPEPR1 (RER) and RER knockout mutants (rer-1 and rer-3). (b) Western blot of RER overexpressing plants (RER-OE-3 and RER-OE-5) compared to wild-type (WT) plants.

[0066] Fig.16 - IPEPR1(RER) knockout mutants are defective in SIPEP(REF1)-induced callus formation. (a) Photographs of wild type (WT) and RER knockout mutants (rer-1 and rer-3) grown in the presence and absence of REF1. (b) Relative callus areas of wild type (WT) and RER knockout mutants (rer-1 and rer-3) grown in the presence and absence of REF1.

[0067] Fig.17 - IPEPR1(RER) knockout mutants are defective in SIPEP(REF1)-induced shoot regeneration. (a) Photographs of shoot regeneration in wild type (WT) and RER knockout mutants (rer-1 and rer-3) grown in the presence and absence of REF1. (b) Relative regeneration frequency in wild type (WT) and RER knockout mutants (rer-1 and rer-3) grown in the presence and absence of REF1. (c) Number of hypocotyls in wild type (WT) and RER knockout mutants (rer-1 and rer-3) grown in the presence and absence of REF1.

[0068] Fig.18 - Plants overexpressing SIPEPR1 (RER) have improved callus formation ability. (a) Photographs of callus formation in wild type (WT), RER knockout mutants (rer-1 and rer-3), and RER overexpressing plants (RER-OE-3 and RER-OE-5). (b) Relative callus area in wild type (WT), RER knockout mutants (rer-1 and rer-3), and RER overexpressing plants (RER-OE-3 and RER-OE-5).

[0069] Fig.19-SIPEPR1 (RER)-overexpressing plants have enhanced shoot regeneration capacity. (a) Photographs of shoot regeneration in wild type (WT), RER knockout mutants (rer-1 and rer-3), and RER overexpressing plants (RER-OE-3 and RER-OE-5). (a) Relative regeneration frequency in wild type (WT), RER knockout mutants (rer-1 and rer-3), and RER overexpressing plants (RER-OE-3 and RER-OE-5). (c) Number of hypocotyls in wild type (WT), RER knockout mutants (rer-1 and rer-3), and RER overexpressing plants (RER-OE-3 and RER-OE-5).

[0070] Fig. 20 - SIPEP (REF1) promotes shoot regeneration in S. habrochaites (LA1777). (a) Photographs of LA1777 grown in the presence and absence of 1 nM REF1. (b) Relative regeneration frequency of LA1777 grown in the presence and absence of 1 nM REF1. (c) Number of hypocotyls of LA1777 grown in the presence and absence of 1 nM REF1.

[0071] Fig.21 - SIPEP (REF1) promotes shoot regeneration in S. peruvianum (PI126944). (a) Photographs of PI126944 grown in the presence and absence of 1 nM REF1. (b) Relative regeneration frequency of PI126944 grown in the presence and absence of 1 nM REF1. (c) Number of hypocotyls of PI126944 grown in the presence and absence of 1 nM REF1.

[0072] Fig. 22 -Constructs for plant transformation and genome editing to induce expression of PROSIPEP (PRR) and SIPEP / REF1 receptor (RER). (a) Plant transformation overexpression vector (PRR+RER-OE). (b) CRISPR-Cas9 vector design (PRR+RER).

[0073] Fig.23- The PRR+RER-OE construct improves the regeneration capacity of recalcitrant tomatoes. (a) Photographs of PI26944 tomatoes (WT, PRR-transformed, RER-transformed, and PRR+RER-transformed) grown for 19 days in the presence or absence of 1 nM REF1. (b) Relative regeneration frequency of PI26944 tomatoes (WT, PRR-transformed, RER-transformed, and PRR+RER-transformed) grown for 19 days in the presence or absence of 1 nM REF1.

[0074] Fig.24 - The pepper ortholog of SIPEP (CaREF1) promotes root hair formation. Photographs of pepper roots grown with 0 nM, 10 nM, and 100 nM CaREF1.

[0075] Fig.25 - Pepper ortholog of SIPEP (CaREF1) promotes callus formation. Cotyledon explants were cultured on callus induction medium (CIM) containing the indicated concentrations of CaREF1. (a) Photographs of cotyledon explants grown at 0 nM, 1 nM, and 10 nM CaREF1. (b) Relative callus area of ​​cotyledon explants grown at 0 nM, 1 nM, and 10 nM CaREF1. Box plots represent the distribution of projected callus area. The projected callus area was quantified by Image J, and individual values ​​(black dots) are shown, with error bars representing the standard deviation of three independent experiments. **P<0.01, *P<0.05 (Student's t-test).

[0076] Fig.26 -The soybean ortholog of SIPEP (GmREF1) promotes shoot regeneration. (a) shows photographs of soybean explants grown in the presence (10 nM, 20 nM, 50 nM and 100 nM) and absence (mock) of GmREF1. (b) shows the relative regeneration frequency of soybean shoots grown for 28 days in the presence (10 nM, 20 nM, 50 nM and 100 nM) and absence of GmREF1. The relative regeneration capacity is represented by the relative frequency of explants with regenerated shoots. Data are mean ± SD of three independent experiments. Bars represent mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05 (Student's t-test).

[0077] Fig. 27- The rice ortholog of SIPEP (OsREF1) promotes callus formation in japonica rice. Mature seeds of ZH11 were cultured on CIM containing the indicated concentrations of OsREF1. (a) Photographs of ZH11 grown in the presence of 1 nM and 10 nM of OsREF1 or in the absence of OsREF1 (mock). (b) Callus phenotypes were scored after 20 days of incubation in CIM. Callus formation ability was represented by the percentage of mature seed explants with regenerated embryonic callus. Data are mean ± SD of three independent experiments.

[0078] Fig.28 - The rice ortholog of SIPEP (OsREF1) promotes shoot regeneration in japonica rice. ZH11 was cultured on SIM containing the indicated concentrations of OsREF1. (a) Photographs of ZH11 grown in the presence of 0 nM and 10 nM OsREF1. (b) Shoot regeneration ability of ZH11 grown in the presence of 0 nM or 10 nM OsREF1.

[0079] Fig.29 - The rice ortholog of SIPEP (OsREF1) promotes the formation of shoot regeneration in japonica rice. Nipponbare ZH11 was grown on SIM containing the indicated concentrations of OsREF1. (a) Photographs of Nipponbare grown in the presence of 0 nM and 10 nM OsREF1. (b) Shoot regeneration ability of Nipponbare grown in the presence of 0 nM or 10 nM OsREF1.

[0080] Fig.30 - The rice ortholog of SIPEP (OsREF1) promotes shoot regeneration in indica rice. 9311 rice was grown on SIM containing the indicated concentrations of OsREF1. (a) Photographs of 9311 rice grown in the presence of 0 nM and 10 nM of OsREF1 (b) Shoot regeneration ability of 9311 rice grown in the presence of 0 nM or 10 nM of OsREF1.

[0081] Fig.31 - The rice ortholog of SIPEP (OsREF1) promotes the growth of wild allotetraploid rice. L-04 rice was grown in the presence of 10 nM OsREF1 or in the absence of OsREF1 (mock). (a) Photographs of L-04 rice grown in the presence of 1 nM, 10 nM OsREF1 or in the absence of OsREF1 (mock). (b) (a) Taproot length of L-04 rice grown in the presence of 1 nM, 10 nM OsREF1 or in the absence of OsREF1 (mock).

[0082] Fig.32 - The rice ortholog of SIPEP (OsREF1) promotes rice callus formation in wild allotetraploids. L-04 rice was grown in the presence of 10 nM or 1 nM OsREF1 or in the absence of OsREF1 (mock). (a) Photographs of L-04 rice grown in the presence of 10 nM, 1 nM OsREF1 or in the absence of OsREF1 (mock). (b) Callus formation ability of L-04 rice grown in the presence of 10 nM, 1 nM OsREF1 or in the absence of OsREF1 (mock).

[0083] Fig.33 - The wheat ortholog of SIPEP / REF1 (TaREF1) promotes root and shoot growth in wheat. (a) Photographs of wheat grown in the presence of 100 nM TaREF1 or in the absence of TaREF1 (mock). (b) Taproot length of L-04 rice grown in the presence of 100 nM TaREF1 or in the absence of TaREF1 (mock). (c) Shoot length of wheat grown in the presence of 100 nM TaREF1 or in the absence of TaREF1.

[0084] Fig.34 - The peach ortholog of SIPEP (PpREF1) promotes callus formation. (a) Photographs of peach cv. GF677 grown on CIM in the presence of 100 nM PpREF1 or in the absence of PpREF1 (mock). (b) Callus formation capacity of peach cv. PpREF1 after 24 days of growth on CIM in the presence of 100 nM PpREF1 or in the absence of PpREF1 (mock). Callus formation capacity is represented by the percentage of leaf explants with regenerated embryogenic callus. Data are mean ± SD of three independent experiments.

[0085] Fig.35 — Example vector constructions for transformation and genome editing to increase expression of PROSIPEP / PROREF1 (PRR) genes. (a) Example vector construction for plant transformation to induce overexpression of PRR genes (pCeGFP:PRR). (b) Example vector construction for genome editing using CRISPR to induce expression of PRR genes (pTX041:PRR).

[0086] Fig.36- Example vector constructions for transformation and genome editing to increase expression of the SIPEPR1 (RER) gene. (a) Example vector construction for plant transformation to induce overexpression of the RER gene (pCeGFP:RER). (b) Example vector construction for genome editing using CRISPR to induce expression of the RER gene (pTX041:RER).

[0087] Fig.37 — Example vector constructions for transformation and genome editing to increase expression of SIPEPR1 (RER) genes and PROSIPEP / PROREF1 (PRR) genes. (a) Example vector constructions for plant transformation to induce overexpression of PRR genes and RER genes (pCeGFP:PRR+RER). (b) Example vector constructions for genome editing using CRISPR to induce expression of PRR genes and RER genes (pTX041:PRR+RER).

[0088] Fig.38 - The PRR+RER-OE construct greatly improves the regeneration capacity of recalcitrant tomatoes. (a) Photographs of control LA1777 tomatoes, LA1777 tomatoes expressing PRR, LA1777 tomatoes expressing RER, and LA1777 tomatoes expressing PRR and RER exposed to 1 nM REF1 for 25 days or in the absence of REF1 (mock). (b) Relative regeneration frequencies of control LA1777 tomatoes, LA1777 tomatoes expressing PRR, LA1777 tomatoes expressing RER, and LA1777 tomatoes expressing PRR and RER exposed to 1 nM REF1 for 25 days or in the absence of REF1 (mock).

[0089] Fig.39 -Tomato "PRR+RER-OE" construct greatly improves transformation efficiency of recalcitrant soybean. (a) Photographs of control recalcitrant soybean and PRR+RER overexpressing (OE) recalcitrant soybean grown in the presence of 50 nM gmREF1, 100 nM GmREF1, or vehicle only control (mock). (b) Relative regeneration frequency of control recalcitrant soybean and PRR+RER overexpressing (OE) recalcitrant soybean in the presence of 50 nM GmREF1, 100 nM GmREF1, or the absence of REF1 (mock).

[0090] Fig.40- HaPEP1 promotes shoot regeneration in sunflower. (A) Shoot regeneration capacity is represented by the relative frequency of explants with regenerated shoots. (B) Shoot regeneration capacity is represented by the number of explants with different numbers of regenerated shoots. Bars represent mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05 (Student's t-test). (C) Photographs of sunflower plants grown in the presence of 1 nM HaREF1, 10 nM HaREF1 or in the absence of HaREF1 (mock).

[0091] Fig.41 - GaREF1 promotes callus formation. (a) Photographs of cotton grown in the presence of 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM GaREF1 or in the absence of GaREF1 (mock), (b) Cotton hypocotyl explants were cultured on callus induction medium (CIM) containing the indicated concentrations of GaREF1, and callus phenotypes were scored after 14 days of incubation. Box plots represent the distribution of projected callus area. The projected callus area was quantified by Image J, and individual values ​​(black dots) are shown, with error bars representing the standard deviation of three independent experiments. ***P<0.001, **P<0.01, *P<0.05 (Student's t-test).

[0092] Fig.42 - OsREF1 promotes shoot regeneration in wild allotetraploid rice. (a) Images of L-04 rice grown in the presence of 10 nM OsREF1 or in the absence of OsREF1 (mock). (b) Regeneration frequency of L-04 rice grown in the presence of 10 nM OsREF1 or in the absence of OsREF1 (mock).

[0093] Fig.43 - TaREF1 promotes shoot regeneration in wheat. (a) Photographs of Chinese Spring wheat grown in the presence of 0 nM, 0.01 nM, or 0.1 nM TaREF1. (b) Immature embryos of Chinese Spring were cultured on callus induction medium (CIM) containing the indicated concentrations of TaREF1, and callus phenotypes were scored after 14 days of incubation on CIM. Callus formation ability was expressed as the percentage of immature embryo explants with regenerated embryonic callus. Data are mean ± SD of three independent experiments.

[0094] Fig.44- TaREF1 promotes shoot regeneration in wheat. (a) Photographs of Kn199 wheat grown in the presence of 0.01 nM, 1 nM TaREF1, or in the absence of TaREF1 (mock). (b) Photographs of Kn199 wheat grown in the presence of 0.01 nM, 1 nM TaREF1, or in the absence of TaREF1.

[0095] Fig.45 -Leaf explants were cultured on callus induction medium (CIM) containing the indicated concentrations of MsREF1, and callus phenotypes were scored after 14 days of incubation. Callus formation ability was represented by the percentage of leaf explants with regenerated embryonic callus. Data are mean ± SD of three independent experiments. Bars represent mean ± SD. ***P<0.001, **P<0.01, *P<0.05 (Student's t-test).

[0096] Fig.46 -Leaf explants were cultured on medium containing the indicated concentrations of CsREF1. Shoot regeneration was scored after 21 days of incubation. Shoot regeneration capacity was represented by the number of shoots regenerated per explant. Data are mean ± SD of three independent experiments. Bars represent mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05 (Student's t-test).

[0097] Fig.47 - GmREF1 improves transformation efficiency in soybean.

[0098] Fig.48 -The tomato "PRP+RER-OE" construct improves the shoot regeneration ability of recalcitrant rice.

[0099] Fig.49- REF1-RER1 regulates regeneration through the SERK co-receptor. (a) RER1 complexes with SERK1, SERK3a, and SERK3b in a REF1-dependent manner. Tobacco leaves transfected with SERKs-myc and RER1-HA-Flag were treated with 100 nM REF1 and then IP was performed with anti-myc agarose. RER1 and SERK were detected by immunoblotting with anti-Flag antibody or anti-myc antibody, respectively. (b and c) REF1-induced callus formation in the indicated genotypes. Hypocotyl explants were cultured on CIM with or without 1 nM REF1 for 14 days and then scored for callus phenotype. Representative callus images (b) and quantification of callus projection area (c) are shown. Bars, 1 mm. Data are mean ± SD. N = 30 hypocotyl explants. (d and e) REF1-induced shoot organogenesis in the indicated genotypes. Hypocotyl explants were cultured on CIM for 14 days and then transferred to SIM with or without 1 nM REF1 for 21 days before scoring for shoot organogenesis phenotype. Representative images of hypocotyl explants (d) and the number of regenerated shoots per explant (e) are shown. Bars, 0.5 cm. Data are mean ± SD. N = 50 hypocotyl explants. In (c), the projected area of ​​callus was quantified by Image J. Box plots represent the distribution of projected callus area. The callus area of ​​each genotype was normalized to the wild type. Individual values ​​(black dots) are shown and error bars represent standard deviations from three independent experiments. In (c) and (e), data points from three biological replicates were analyzed by one-sided Kruskal-Wallis test with Holm correction, followed by post hoc Dunn's test. Data points with different letters indicate significant differences p < 0.05.

[0100] Fig.50-REF1 regulates regeneration by promoting the expression of SIWIND1. (a) Induction of SlWIND1 promoter activity at the wound site of hypocotyl explants. Hypocotyl explants of 7-day-old pSlWIND1::GUS plants were cultured on MS medium with or without 1 nM REF1. Representative images of hypocotyls at the indicated times after wounding are shown. Bars, 1 mm. (b) Wound-induced SlWIND1 expression was detected by RT-qPCR. Hypocotyls of 7-day-old seedlings of the above genotypes were excised, and gene expression analysis was performed after hypocotyl explants were cultured on MS medium for 9 h. Hypocotyls of unwounded seedlings were harvested as controls. Data are mean ± SD, n = 3 biological replicates. (c and d) REF1-induced callus formation in wild type and slwind1 mutants. Hypocotyl explants were cultured on CIM with or without 1 nM REF1 for 14 days and then scored for callus phenotype. Representative callus images (c) and quantification of callus projection area (d) are shown. Bars, 1 mm. Data are mean ± SD. N = 30 hypocotyl explants. (e and f) REF1-induced shoot organogenesis in wild type and slwind1 mutants. Hypocotyl explants were cultured on CIM for 14 days and then transferred to SIM with or without 1 nM REF1 for 21 days before scoring for shoot organogenesis phenotype. Representative images of hypocotyl explants (e) and the number of regenerated shoots per explant (f) are shown. Bars, 0.5 cm. Data are mean ± SD. N = 50 hypocotyl explants. (g and h) Callus formation in wild type and SlWIND1-OE plants. Hypocotyl explants were cultured on CIM for 14 days before scoring for callus phenotype. Representative callus images (g) and quantification of callus projection area (h) are shown. Bars, 1 mm. Data are mean ± SD. N = 30 hypocotyl explants. (i and j) Shoot organogenesis in WT and SlWIND1-OE plants. Hypocotyl explants were cultured on CIM for 14 days and then switched to SIM for 21 days before scoring for shoot organogenesis phenotype. Representative images of hypocotyl explants (i) and the number of regenerated shoots per explant (j) are shown. Bars, 0.5 cm. Data are mean ± SD. n = 50 hypocotyl explants. In (d) and (h), the projected area of ​​callus was quantified by Image J. Box plots represent the distribution of projected callus area. The callus area of ​​each genotype was normalized to the wild type. Individual values ​​(black dots) are shown and error bars represent standard deviations from three independent experiments. In (b), (d), and (f), data points from three biological replicates were analyzed by one-sided Kruskal-Wallis test with Holm correction, followed by post hoc Dunn's test.In (h) and (j), statistical significance was determined by Student's t-test (***p<0.001, **p<0.01, *p<0.05).

[0101] Fig.51- Positive feedback regulation between REF1 and SlWIND1 in response to wounding. (a) Induction of PRP promoter activity at the wound site of hypocotyl explants. Hypocotyl explants of 7-day-old pPRP::GUS plants were cultured on MS medium. Representative images of hypocotyls at the indicated times after wounding are shown. Bars, 1 mm. (b) Wound-induced expression of PRP in the indicated genotypes. Hypocotyls of 7-day-old seedlings were excised and cultured on MS medium without phytohormones for 48 h, and then RNA was extracted and subjected to gene expression analysis. Data are mean ± SD, n = 3 replicates. (c) REF1-induced expression of PRP in the indicated genotypes. 18-day-old seedlings were treated with or without 100 nM REF1. The expression of SlWIND1 was measured 24 h after REF1 treatment. Data are mean ± SD, n = 3 replicates. (d) Schematic diagram of the PRP promoter showing primers and probes used for ChIP-qPCR and EMSA. Blue boxes represent VWRE, red lines represent primers used for ChIP-qPCR, and green triangles represent probes. (e) EMSA shows SlWIND1 binding to the VWRE motif of the PRP promoter. Biotinylated probes were incubated with SlWIND1-His protein, and free and bound DNA (arrows) were separated on an acrylamide gel. Unlabeled and 50-fold mutated probes with 10-, 20-, and 50-fold excess were used for competition as described above. Mu, mutant probe in which the core sequence (AAATTT) of VWRE was replaced by 5'-GGGGGG-3'. (f) ChIP-qPCR shows wound-induced enrichment of SlWIND1 on chromatin of PRP. Chromatin from injured or uninjured SlWIND1-GFP seedlings was immunoprecipitated using anti-GFP antibody, and the immunoprecipitated DNA was quantified by qPCR. Enrichment of the target gene promoter is expressed as a percentage of input DNA. Primers used for PCR amplicons are shown in (d). Data are mean ± SD, n = 3 replicates. (g) Proposed model for the immunomodulatory plant cytokine REF1 to promote wound-triggered regeneration. Following cell damage, REF1 binds and activates the RER1-SERKs receptor complex, thereby initiating both MYC2-dependent defense responses and SlWIND1-dependent regenerative responses. In addition to regulating the reprogramming of regenerative cells, activated SlWIND1 also upregulates the expression of REF1 precursor genes by binding to their promoters, thereby amplifying the primary wound signal for tissue regeneration. In (bc) and (f), data points from three biological replicates were analyzed by one-sided Kruskal-Wallis test with Holm correction, followed by post hoc Dunn's test. Data points with different letters indicate significant differences at p < 0.05.

[0102] Fig.52 - ZmREF1 promotes shoot regeneration.

[0103] Fig.53 - TaREF1 promotes shoot regeneration in Chinese spring wheat.

[0104] Fig.54 - TaREF1 promotes callus formation in JM22 wheat.

[0105] Fig.55 - TaREF1 promotes shoot regeneration in JM22 wheat. DETAILED DESCRIPTION

[0106] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect defined in this way can be combined with any other aspect or aspects, unless there is a clear contrary description. In particular, any feature specified as preferred or advantageous can be combined with any other feature or features specified as preferred or advantageous.

[0107] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of plant biology, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are fully explained in the literature.

[0108] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are meant to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. These terms also include genes. The terms "gene" or "gene sequence" are widely used to refer to DNA nucleic acids associated with biological functions. Therefore, as in a genomic sequence, a gene may include introns and exons, or may include only coding sequences, as in cDNA, and / or may include cDNA combined with regulatory sequences.

[0109] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length linked together by peptide bonds.

[0110] For the purposes of the present invention, a "transgenic plant" is a plant that has been genetically modified compared to a naturally occurring wild-type (WT) plant.

[0111] In vivo SIPEP (REF1) is a 23 amino acid peptide cleaved from its precursor PROSIPEP, also known as PROREF1, PRP, and PRR (these terms are used interchangeably herein). Figure 6 , 8 and 18-44, showed that exogenous application of REF1 increased the regeneration capacity or efficiency (also referred to herein as regeneration frequency) of a plant, its parts or at least one plant cell. Regeneration frequency can be considered as the number of plant regenerating parts, for example, the total number of shoots per inoculated explant. Consistent with these findings, we also Fig.10 , 11 , 18 and 19 show that increasing the expression level of PRR and / or RER in a plant will also increase the regeneration capacity or efficiency of the plant, its parts or at least one plant cell. In certain embodiments, we have shown that the expression of a PRR / RER chimera is particularly effective in increasing the regeneration capacity of a plant, its parts or at least one plant cell.

[0112] Therefore, in a first aspect of the present invention, there is provided a tissue regeneration factor comprising or consisting of an amino acid sequence as defined in SEQ ID NO: 2 or a functional variant or homologue thereof. This is REF1.

[0113] In one embodiment, the functional variant or homologue is defined by a conserved motif. The conserved motif allows REF1 to bind to RER. In one embodiment, the conserved motif is (SEQ ID NO: 79) GXPPXXNN, wherein X is any amino acid. In another embodiment, the conserved motif is (SEQ ID NO: 80) SSGXXGXXN, wherein X is any amino acid. In another embodiment, the conserved motif is (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid. Therefore, in one embodiment, the functional variant or homologue may comprise one of these conserved motifs.

[0114] In a further embodiment, the homologue comprises or consists of a sequence as defined in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20. Also included within the scope of the present invention are functional variants and homologues of these sequences - e.g., SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 and 20.

[0115] In one embodiment, binding of REF1 to RER regulates regeneration via a kinase receptor, preferably a somatic embryogenesis receptor kinase (SERK), more preferably SERK3a and SERK3b. In one embodiment, REF1 is a SERK agonist.

[0116] In one embodiment, REF1 promotes wound-induced regeneration by activating the expression of WOUND-INDUCEDDEDIFFERENTIATION 1 (WIND1).

[0117] In one embodiment, WIND1 activates the expression of PROREF1.

[0118] The tissue regeneration factor can be exogenously applied to a plant, part thereof or one or more plant cells alone or as part of a plant tissue culture medium or plant growth medium (the terms are used interchangeably herein). In another aspect of the invention there is provided a plant tissue culture medium comprising the tissue regeneration factor of the invention.

[0119] The composition of plant growth or plant tissue culture media is well known in the art. For example, the culture media may contain micronutrients and macronutrients, vitamins, organic supplements, and plant growth regulators necessary for the growth and proliferation of plant cells, tissues, and organs in vitro. There are a variety of plant tissue culture media available on the market for different applications, and their composition can be changed according to the needs of the user.

[0120] In one embodiment, the plant tissue culture medium is callus induction medium (CIM).

[0121] In another embodiment, the plant tissue culture medium is Shoot Induction Medium (SIM).

[0122] In another embodiment, the plant tissue culture medium is root induction medium (RIM).

[0123] Preferably, the plant tissue culture medium comprises auxin and / or cytokinin. In a preferred embodiment, the plant tissue culture medium is a medium rich in auxin. It is noteworthy that the ratio of auxin to cytokinin in the plant tissue culture medium determines the fate of the regenerated organ. In one embodiment, a lower auxin / cytokinin ratio promotes shoot regeneration. In another embodiment, a higher auxin to cytokinin ratio promotes root regeneration.

[0124] Typically, plant tissue culture medium contains a basal medium, such as Murashige & Skoog basal medium, having one or more vitamins, glucose, phytol agar, and auxins and cytokinins in varying concentrations or ratios depending on the intended purpose of the medium, as described above.

[0125] Thus, in one aspect of the invention, the plant tissue culture medium comprises Murashige & Skoog basal medium with one or more vitamins, glucose, phytol agar, REF1, and auxins and cytokinins. In a preferred aspect of the invention, the plant tissue culture medium comprises Murashige & Skoog basal medium with one or more vitamins, 3% glucose, 0.8% phytol agar, REF1, and auxins and cytokinins. The skilled person will be able to modify the concentrations of REF1, auxins, and cytokinins (and other medium components) according to the intended use (e.g., callus induction, shoot induction, or root induction).

[0126] In one embodiment, callus induction medium (CIM) comprises Murashige & Skoog basal medium, phytol agar, sucrose, zeatin riboside (ZR), indole-3-acetic acid (IAA), and REF1.

[0127] In a preferred embodiment, callus induction medium (CIM) may include 4.43 g / L Murashige & Skoog basal medium, 8 g / L phytol agar, 30 g / L sucrose, 0.1 mg / L zeatin riboside (ZR), 0.4 mg / L indole-3-acetic acid (IAA), and 1 nmol / L REF1. In one embodiment, CIM having this composition can be used to culture tomato plants. In another embodiment, a technician can change the composition of CIM, which is optimized for inducing callus formation in a selected plant, wherein the selected plant is a monocot or a dicot.

[0128] In one embodiment, shoot induction medium (SIM) comprises Murashige & Skoog basal medium, sucrose, phytol agar, indole-3-acetic acid (IAA), zeatin riboside (ZR), and REF1.

[0129] In a preferred embodiment, the shoot induction medium (SIM) comprises 4.43 g / L Murashige & Skoog basal medium, 30 g / L sucrose, 8 g / L phytol agar, 0.1 mg / L indole-3-acetic acid (IAA), 0.4 mg / L zeatin riboside (ZR) and 1 nmol / L REF1. In one embodiment, a SIM having this composition can be used to cultivate tomato plants. In another embodiment, a technician can change the composition of a SIM that is optimized for inducing shoot formation in a selected plant, wherein the selected plant is a monocot or a dicot.

[0130] In one embodiment, root induction medium (RIM) comprises Murashige & Skoog basal medium, sucrose, phytol agar, and REF1.

[0131] In a preferred embodiment, the root induction medium (RIM) comprises 4.43 g / L Murashige & Skoog basal medium, 30 g / L sucrose, 8 g / L phytol agar, and 1 nmol / L REF1. In one embodiment, the RIM having this composition can be used to culture tomato plants. In another embodiment, the technician can change the composition of the RIM, which is optimized for inducing root formation in a selected plant, wherein the selected plant is a monocot or a dicot.

[0132] Plant growth medium may also refer to the material in which the plant grows. In one embodiment, the plant growth medium optionally comprises at least one of the following ingredients: soil, peat moss, perlite, vermiculite, zeolite, compost, water, wood bark, or sand.

[0133] In one embodiment, the tissue regeneration factor is incorporated into the plant tissue culture medium or plant growth medium at a concentration between 0.00001 and 1 mM, more preferably between 0.001 and 800 nM, even more preferably at about 0.1 nM, 1 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM or 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM and 800 nM. Even more preferably, the plant tissue culture medium comprises the tissue regeneration factor at a concentration of 0.1 nM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM.

[0134] In another embodiment, the tissue regeneration factor or plant tissue culture medium can be directly applied to the plant, its part or one or more plant cells. Preferably, the plant part includes, but is not limited to, the organ, tissue, seed and cell of the plant. Preferably, the plant part is an explant. An explant is a plant part cut from a whole plant and used to initiate culture in a plant tissue culture medium. Examples of explants particularly suitable for plant regeneration include, but are not limited to, shoots, stems and leaves.

[0135] In another aspect of the present invention, a nucleic acid construct is provided, comprising at least one nucleic acid sequence encoding at least one of tissue regeneration factor (REF1), tissue regeneration factor precursor (PRR) and tissue regeneration factor receptor (RER).

[0136] In one embodiment, a nucleic acid construct is provided, comprising at least one nucleic acid sequence encoding a tissue regeneration factor (REF1), wherein the tissue regeneration factor is operably linked to a regulatory sequence.

[0137] In another embodiment, a nucleic acid construct is provided, comprising at least one nucleic acid sequence encoding a tissue regeneration factor precursor (PRR), wherein the tissue regeneration factor precursor is operably linked to a regulatory sequence.

[0138] In another embodiment, a nucleic acid construct is provided, comprising at least one nucleic acid sequence encoding a tissue regeneration factor receptor (RER), wherein the tissue regeneration factor precursor is operably linked to a regulatory sequence.

[0139] In one embodiment, a nucleic acid construct is provided, comprising at least one nucleic acid sequence encoding a tissue regeneration factor (RER1), and a nucleic acid sequence encoding a tissue regeneration factor precursor (PRR) or a tissue regeneration factor receptor (RER), wherein the nucleic acid sequence is operably linked to one or more regulatory sequences, wherein REF1 and PRR or RER are operably linked to the same or different regulatory sequences.

[0140] In one embodiment, a nucleic acid construct is provided, the nucleic acid construct comprising at least one nucleic acid sequence encoding a tissue regeneration factor precursor (PRR), and a nucleic acid sequence encoding a tissue regeneration factor receptor (RER), wherein the nucleic acid sequence is operably linked to one or more regulatory sequences, wherein the PRR and RER are operably linked to the same or different regulatory sequences. Examples of such nucleic acid constructs are Fig.38 As shown. Fig.38 As shown, the use of the construct ("PRR+RER-OE") significantly increased the regeneration efficiency in tomato by up to 28-fold compared to the control. Fig.39 It was also shown that the construct significantly increased the regeneration frequency of recalcitrant soybean by up to 12-fold compared to the control.

[0141] The nucleic acid sequence may encode a tissue regeneration factor as defined in SEQ ID NO: 2 or a functional variant or homologue thereof. In one embodiment, the homologue encodes a tissue regeneration factor as defined in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homologue thereof. In one embodiment, the nucleic acid sequence homologue comprises a nucleic acid sequence as defined in SEQ ID NO: 1 or a functional variant or homologue thereof or consists of a nucleic acid sequence as defined in SEQ ID NO: 1 or a functional variant or homologue thereof. In one embodiment, the homologue comprises a nucleic acid sequence as defined in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 90 or 19 or a functional variant or homologue thereof or consists of a nucleic acid sequence as defined in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 90 or 19 or a functional variant or homologue thereof.

[0142] The nucleic acid sequence may encode a tissue regeneration factor as defined in SEQ ID NO: 22 or a functional variant or homologue thereof. In one embodiment, the homologue encodes a tissue regeneration factor precursor as defined in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof. In one embodiment, the nucleic acid sequence comprises a nucleic acid sequence as defined in SEQ ID NO: 21 or a functional variant or homologue thereof or consists of a nucleic acid sequence as defined in SEQ ID NO: 21 or a functional variant or homologue thereof. In one embodiment, the homologue comprises a nucleic acid sequence as defined in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 92 or 39 or a functional variant or homologue thereof or consists of a nucleic acid sequence as defined in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 92 or 39 or a functional variant or homologue thereof.

[0143] The nucleic acid sequence may encode a tissue regeneration factor receptor as defined in SEQ ID NO: 42 or a functional variant or homologue thereof. In one embodiment, the homologue encodes a tissue regeneration factor receptor as defined in SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homologue thereof. In one embodiment, the nucleic acid sequence comprises a nucleic acid sequence as defined in SEQ ID NO: 41 or a functional variant or homologue thereof or consists of a nucleic acid sequence as defined in SEQ ID NO: 41 or a functional variant or homologue thereof. In one embodiment, the homologue comprises a nucleic acid sequence as defined in SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 94 or 77 or a functional variant or homologue thereof.

[0144] As used in any sequence described herein in its entirety, the term "variant" or "functional variant" refers to a variant gene sequence or a portion (e.g., fragment) of a gene sequence that retains the biological function of the complete non-variant sequence. Functional variants also include target gene variants that have sequence changes that do not affect function, such as in non-conserved residues. Variants that are substantially the same as the wild-type sequence shown herein, i.e., only have some sequence changes (e.g., in non-conserved residues), and have biological activity are also contemplated. Changes in nucleic acid sequences that result in different amino acids being produced at selected sites are known in the art, and the production of the amino acids does not affect the functional properties of the encoded polypeptide. For example, a codon for the amino acid alanine (a hydrophobic amino acid) can be replaced by a codon encoding another residue with poor hydrophobicity (e.g., glycine) or a residue with stronger hydrophobicity (e.g., valine, leucine, or isoleucine). Similarly, changes that result in one negatively charged residue replacing another negatively charged residue (e.g., aspartic acid replacing glutamic acid), or one positively charged residue replacing another positively charged residue (e.g., lysine replacing arginine) can also be expected to produce functionally equivalent products. Nucleotide changes that result in changes in the N-terminal and C-terminal portions of the polypeptide molecule are not expected to change the activity of the polypeptide. Each proposed modification is within the routine skill of the art, and it is also within the routine skill of the art to determine whether the biological activity of the encoded product is retained.

[0145] As used in any aspect of the invention, a "variant" or "functional variant" has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity.

[0146] When aligned for maximum correspondence as described below, two nucleic acid sequences or polypeptides are considered to be "identical" if the sequence of nucleotides or amino acid residues in the two sequences is identical, respectively. The term "identical" or percentage "identity", in the case of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When the percentage of sequence identity is used in reference to a protein or peptide, it is recognized that non-identical residue positions usually differ due to conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore do not change the functional properties of the molecule. When the conservative substitutions of the sequences are different, the percentage of sequence identity can be adjusted upward to correct for the conservative nature of the substitutions. Methods for making such adjustments are well known to those skilled in the art. For sequence comparison, usually one sequence is used as a reference sequence, and the test sequence is compared with it. When using a sequence comparison algorithm, a test sequence and a reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters. The non-limiting example of an algorithm that is suitable for determining sequence identity percentage and sequence similarity is BLAST and BLAST 2.0 algorithms.

[0147] Suitable homologues can be determined by sequence comparison and identification of conserved domains. Some predictors are available in the art to identify the sequence. As described herein, the function of a homologue can be determined, and the technician can therefore confirm its function, for example when overexpressing in a plant.

[0148] In one embodiment, by using SlPROPEP / PROREF1 or any known PROPEP sequence as an input sequence, REF1, PROREF1 and REF1 receptor orthologs can be identified in a given plant using sequence analysis and alignment tools such as hmmsearch search (HMMER v1.9; (Finn et al., 2011)) against sequence databases known in the art (e.g., NR, RefSeq, UniProtKB or any other known database). The inventors also found that the C-terminus of PROPEP comprising a Pep sequence (e.g., SEQ ID NO: 2) is strictly conserved. Therefore, SlPROPEP / PROREF1 orthologs of any plant species can be identified using this sequence. Similarly, using NCBI BLASTP / BLASTN, the RER sequence was used to identify new RER sequences in tomato.

[0149] Therefore, nucleotide sequence described herein and of the present invention can also be used for isolating corresponding sequence from other organisms, particularly other plants.In this way, methods such as PCR, hybridization can be used for identifying such sequences based on the sequence homology of it and sequence described herein.When identifying and separating homologues (homolog), the topological structure and characteristic structure domain structure of the sequence can be considered.The sequence can be separated based on the sequence identity of it and the whole sequence or its fragment.In hybridization techniques, all or part of the known nucleotide sequence is used as a probe, and the probe selectively hybridizes with other corresponding nucleotide sequences present in the cloned genomic DNA fragment or cDNA fragment group (i.e. genomic library or cDNA library) of selected plant.The hybridization probe can be a genomic DNA fragment, a cDNA fragment, an RNA fragment or other oligonucleotides, and can be marked with a detectable group or any other detectable marker. Methods for preparing probes for hybridization and for constructing cDNA and genomic libraries are known in the art and are disclosed in Sambrook et al. (1989), Molecular Cloning: A Library Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York.

[0150] In a further embodiment, a variant as used herein may comprise a nucleic acid sequence encoding a polypeptide as defined herein, said nucleic acid sequence being capable of hybridizing under stringent conditions as defined herein to any one of the SEQ ID NOs defined herein.

[0151] Hybridization of such sequences can be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refer to the expected conditions under which the hybridization of the probe to the target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times the background). Stringent conditions are sequence-dependent and will vary in different situations. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that lower similarities can be detected (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, preferably less than 500 nucleotides. Typically, stringent conditions refer to a salt concentration of less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). The duration of hybridization is generally less than about 24 hours, typically about 4-12 hours. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0152] As used herein, a regulatory sequence is a nucleic acid molecule fragment that can increase or decrease the expression of a specific gene in an organism. An example of a regulatory sequence is a "promoter," which refers to a nucleotide sequence that is usually located upstream (5') of its coding sequence and controls the expression of the coding sequence by providing recognition for RNA polymerase and other factors required for normal transcription. In one embodiment, the promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0153] "Constitutive promoter" refers to a promoter that directly directs gene expression in almost all tissues and at all times. Examples of constitutive promoters include, but are not limited to, CaMV 35S promoter or StUbi promoter.

[0154] An "inducible promoter" refers to a promoter that can be activated in one or more cell types by external stimuli (such as chemicals, light, hormones, stress, or pathogens). Examples of inducible promoters include, but are not limited to, the wound-inducible nopaline synthase promoter (NOSpro) and the dexamethasone-inducible p0p6 / LhGR promoter.

[0155] "Tissue-specific promoter" refers to a promoter that is active only in specific cell types. The use of tissue-specific promoters in nucleic acid construction can limit unnecessary transgene expression and promote sustained expression of transgenes.

[0156] As used herein, the term "operably linked" refers to a functional connection between a promoter sequence and a gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

[0157] Nucleic acid construct of the present invention may further comprise one or more reporter genes." reporter gene " refers to a gene whose product is easy to detect after transfection, which can be used as a marker for screening successful transfected cells, for studying the regulation of gene expression, or as a control for standardized transfection efficiency. Examples of reporter genes include, but are not limited to, GFP, luciferase and lacZ.

[0158] In another embodiment, the nucleic acid construct of the present invention may additionally comprise one or more selectable marker genes. A "selectable marker gene" is a gene introduced into a cell that confers a trait suitable for artificial selection and is used to indicate successful transformation of cells transformed with a vector containing the marker gene. Examples of selectable marker genes include, but are not limited to, NPTII, HPT, aadA, and DHPS.

[0159] Examples of nucleic acid constructs of the present invention are described in Fig. 22 and 35 -37 in.

[0160] In another aspect of the invention there is provided a host cell comprising and preferably expressing the nucleic acid construct of the invention. Preferably, the host cell is a plant cell.

[0161] In another aspect of the present invention, a transgenic plant, part thereof or one or more plant cells is provided, wherein the plant, part thereof or one or more plant cells is characterized by increased expression or protein level of one or more of the following:

[0162] (i) tissue regeneration factor (REF1) as described herein; and / or

[0163] (ii) a tissue regeneration factor precursor (PRR) as described herein; and / or

[0164] (iii) a tissue regeneration factor receptor (RER) as described herein.

[0165] In one embodiment, the increase of expression and / or protein level of (i) to (iii) above is achieved by introducing and expressing one of the nucleic acid constructs of the present invention in a plant, a part thereof or one or more plant cells. Therefore, in another aspect of the present invention, a transgenic plant, a part thereof or a plant cell comprising and expressing one or more nucleic acid constructs of the present invention is provided.

[0166] In another embodiment, the increase in expression and / or protein level of (i) to (iii) above is achieved by introducing one or more additional copies of genes encoding tissue regeneration factor and / or tissue regeneration factor precursor and / or tissue regeneration factor receptor into the plant genome using targeted genome editing such as CRISPR. Preferably, genome editing technology is used to introduce one or more additional copies of a gene at a certain position in the genome, so that one or more genes are under the control of a suitable regulatory sequence. Preferably, the regulatory sequence is a 35S promoter.

[0167] The gene encoding the tissue regeneration factor may contain SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 90 or its functional variants or homologs or consist of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 90 or its functional variants or homologs.

[0168] The gene encoding the tissue regeneration factor precursor may contain SEQ ID NO:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 92 or 40 or its functional variants or homologs or consist of SEQ ID NO:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 92 or 40 or its functional variants or homologs.

[0169] The gene encoding the tissue regeneration factor receptor may contain SEQ ID NO:41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 94 or its functional variants or homologs or consist of SEQ ID NO:41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 94 or its functional variants or homologs.

[0170] In another aspect of the present invention, there is provided use of the tissue regeneration factor of the present invention to increase or enhance the regeneration efficiency of a plant, a part thereof or one or more plant cells.

[0171] In another aspect of the present invention, there is provided use of the plant tissue culture medium of the present invention to increase or enhance the regeneration efficiency of a plant, a part thereof or one or more plant cells.

[0172] In another aspect of the present invention, there is provided use of one or more nucleic acid constructs of the present invention to increase or enhance the regeneration efficiency of a plant, a part thereof or one or more plant cells.

[0173] In another aspect of the present invention, there is provided a method for increasing the regeneration efficiency of a plant, a plant part thereof or one or more plant cells, the method comprising

[0174] i. applying the tissue regeneration factor of the present invention to the plant or part thereof or one or more plant cells; or

[0175] ii. applying the plant tissue culture medium of the present invention to the plant or part thereof or one or more plant cells; and / or

[0176] iii. introducing and expressing the nucleic acid construct of the invention in said plant or part thereof or in one or a plant cell.

[0177] "Regeneration efficiency" refers to the ability of a plant or plant part to regenerate after being injured or exposed to a regeneration stimulus. For example, increased regeneration capacity can be measured by at least one of increased hypocotyl number, stem cell formation, shoot regeneration capacity, callus formation capacity or callus area, taproot length, root meristem formation, and / or root hair formation.

[0178] The increase refers to an increase in regeneration capacity or efficiency compared to the regeneration amount of a control or wild-type plant (i.e., a plant to which the tissue regeneration factor or plant tissue culture medium of the present invention is not applied and / or a plant that does not express one or more nucleic acid constructs of the present invention). The increase may be between 2-50 times compared to a control or wild-type plant. Preferably, the increase may be at least 2 times, at least 3 times, at least 5 times, at least 7 times, at least 8 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times compared to the regeneration level of a control or wild-type plant. For example, Fig. 20 showed that 1 nM REF1 could increase the regeneration frequency of wild-type tomato by about 6-fold. Fig.23 showed that tomatoes overexpressing the REF1 receptor (RER) and REF1 precursor (PRR) grown in the presence of 1 nM REF1 had approximately 42-fold higher regeneration frequency than wild-type tomatoes grown in the absence of REF1.

[0179] In one embodiment, tissue regeneration factors or plant tissue culture medium are applied exogenously to the plant. In a further embodiment, tissue regeneration factors or plant tissue culture medium are applied to the transgenic plants of the present invention to further enhance the regeneration efficiency of the plants, such as Fig.23 shown.

[0180] In one embodiment, REF1 is exogenously applied to the plant in combination with at least one plant hormone, wherein preferably the one or more plant hormones are auxins and / or cytokinins. In another embodiment, REF1 is applied as part of a plant tissue culture medium, as described above. As described above, the balance between auxins and cytokinins determines the fate of the regenerated organ. For example, a medium rich in auxins or a high auxin: cytokinin ratio produces callus that is capable of organ regeneration. The technician will be able to adjust the concentration or ratio of the plant hormones according to the intended use.

[0181] In one embodiment, where REF1 is exogenously applied to a plant in a plant tissue culture medium matrix, the amount of plant tissue culture medium applied to the plant is adjusted according to the number and size of the explants to be cultured. For example, 1 nanomole / liter REF1 is equivalent to 2.5x10-3 mg / L, and 100 tomato hypocotyls are preferably cultured in 250 ml of culture medium. In another non-limiting example, 12-36 explants are preferably cultured in 100 ml of the plant tissue culture medium of the present invention.

[0182] In another embodiment, the time taken between application of REF1 or plant tissue culture medium of the present invention and when regeneration is observed varies depending on the nature of the plant - for example, but not limited to, the type of plant from which the explant is derived and the part of the plant from which the explant is taken. In one embodiment, the time taken between application and observation of regeneration is up to 10 days, up to 20 days, up to 30 days, up to 40 days, up to 50 days, up to 60 days or up to 70 days, promoting plant regeneration. In a preferred embodiment, the time taken between application and observation of regeneration is about 40 days.

[0183] In another embodiment, REF1 or tissue culture medium of the present invention is applied to the plant, preferably at the wound site. In a preferred embodiment, REF1 or tissue culture medium of the present invention is applied once. Alternatively, REF1 or tissue culture medium of the present invention is applied twice or more, and the intervals between applications vary depending on the plant type and the desired regeneration. If the plant is soybean, corn, sunflower, tomato or pepper, only one application is required. These plants can regenerate shoots directly from the wound site. Other plants require one or more applications. These plants (including many monocots) first form callus before regenerating shoots.

[0184] In one embodiment, the concentration of REF1 or plant tissue culture medium of the invention applied to a plant varies depending on the nature of the plant - for example, but not limited to, the type of plant from which the explant is derived and the part of the plant from which the explant is taken. In one embodiment, mature explants require a higher concentration of exogenously applied REF1 relative to immature explants. In one embodiment, REF1 is exogenously applied to mature explants at a concentration in the range of 1-500nmol / L, and REF1 is exogenously applied to immature explants at a concentration in the range of 0.01-1 nmol / L.

[0185] In a preferred embodiment, the method comprises administering a tissue regeneration factor or plant tissue culture medium, wherein the tissue regeneration factor is from the same plant to which the factor is administered - for example, tomato REF1 is administered to tomato. In other words, heterologous administration is not a preferred embodiment, i.e., rice REF1 is administered to tomato. The same applies equally to the introduction of the nucleic acid constructs of the present invention.

[0186] The nucleic acid construct may be introduced into plant cells using any suitable method known to the skilled person (the term "introduction" may be used interchangeably with "transformation" as described below).

[0187] Any nucleic acid construct described herein can be introduced into the plant by a process called transformation. The term "introduction" or "transformation" described herein comprises transferring exogenous polynucleotides into a host cell, regardless of the method used for the transfer. Plant tissues capable of subsequent clonal propagation, whether through organogenesis or embryogenesis, can be transformed by the genetic construct of the present invention, and whole plants can be regenerated therefrom. The selected specific tissue will be different according to the available and most suitable clonal propagation system for the specific species transformed. Exemplary tissue targets include leaf disks, pollen, embryos, microspores, anthers, ovule cotyledons, hypocotyls, megagametophytes, callus, existing meristems (such as apical meristems, axillary buds and root meristems) and induced meristems (such as cotyledon meristems and hypocotyl meristems). The polynucleotides can be transiently or stably introduced into a host cell and can, for example, be kept in a non-integrated state as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known to those skilled in the art.

[0188] Transferring foreign genes into the plant genome is called transformation. The transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods can be used to introduce the target gene into a suitable progenitor cell. The method for transforming and regenerating plants from plant tissues or plant cells can be used for transient or stable transformation. The transformation method includes the use of liposomes, electroporation, chemicals that increase free DNA uptake, direct injection of DNA into plants, particle gun bombardment, use of viruses or pollen transformation and microprojection. The method can be selected from the calcium / polyethylene glycol method of protoplasts, electroporation of protoplasts, microinjection into plant materials, bombardment of particles coated with DNA or RNA, infection (non-integration) viruses, etc. Transgenic plants, including transgenic crop plants, are preferably prepared by transformation mediated by Agrobacterium tumefaciens. According to the present invention, the nucleic acid is preferably stably integrated into the transgenic plant genome and the progeny of the plant therefore also includes the transgene.

[0189] In order to select the transformed plants, in certain embodiments, the plant material obtained in the transformation is subjected to selection conditions so that the transformed plants are distinguished from untransformed plants. For example, the seeds obtained in the above manner can be planted and, after the initial growth period, appropriately selected. A further possibility is to plant the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened to determine if there is a selectable marker.

[0190] Following DNA or nucleic acid transfer and regeneration, putatively transformed plants also can be evaluated, for example, using Southern Blot analysis.

[0191] The resulting transformed plants can be propagated by a variety of means, such as clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant can be selfed and homozygous second generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques. The resulting transformed organism may take a variety of forms. For example, it may be a chimera of transformed and non-transformed cells; a clonal transformant (e.g., all cells transformed to contain the expression cassette); a grafting of transformed and non-transformed tissues (e.g., in plants, a transformed rootstock grafted onto an untransformed scion).

[0192] In further embodiments of any of the methods described herein, the method may further include at least one or more of the following steps: evaluating the phenotype of the transgenic plant, evaluating the regeneration capacity of the plant by measuring at least one of shoot regeneration capacity (e.g., the frequency of explants with regenerated shoots) and / or callus formation capacity (e.g., the percentage of explants with regenerated embryonic callus) and / or taproot length, and comparing the phenotype to a wild-type or control plant to determine an increase in regeneration capacity compared to the wild-type or control plant. In other words, the method may involve a step of screening the plant for a desired phenotype.

[0193] In a further aspect of the present invention, there is provided a method for producing a plant, a plant part thereof or one or more plant cells having an improved regeneration efficiency as described above, the method comprising

[0194] i. applying the tissue regeneration factor of the present invention to the plant or part thereof or one or more plant cells; or

[0195] ii. applying the plant tissue culture medium of the present invention to the plant or part thereof or one or more plant cells; and / or

[0196] iii. introducing and expressing the above-mentioned nucleic acid construct of the present invention into said plant or part thereof or one or plant cell.

[0197] In a further aspect of the invention, a plant or plant part thereof or one or more plant cells obtained or obtainable by the above method is provided. Preferably, the plant is characterized by an increased regeneration capacity compared to a wild type or control plant. Preferably, the increased regeneration capacity can be measured by at least one of shoot regeneration capacity, callus formation capacity and / or taproot length.

[0198] Also included within the scope of the present invention are progeny of the plant of the present invention, wherein said progeny comprises the nucleic acid construct of the present invention. Another embodiment of the present invention is a transgenic plant obtained or obtainable by the above method for modifying the genome of a plant cell and regenerating a plant from said cell and its progeny or part, wherein said progeny or part comprises the modification introduced into the genome by the above modification method.

[0199] In one embodiment of the invention, there is provided a method for increasing the transformation efficiency of a plant, a plant part thereof or one or more plant cells, the method comprising

[0200] i. applying the tissue regeneration factor of the present invention to the plant or part thereof or one or more plant cells; or

[0201] ii. applying the plant tissue culture medium of the present invention to the plant or part thereof or one or more plant cells; and / or

[0202] iii. introducing and expressing the nucleic acid construct of the invention in said plant or part thereof or in one or a plant cell.

[0203] Also within the scope of the present invention is a plant, part thereof or plant cell obtained or obtainable by the above method, wherein said plant, part thereof or plant cell is characterized by an increased transformation efficiency.

[0204] "Transformation efficiency" refers to the ability of an organism to take up and integrate foreign DNA, such as plasmids, during the transformation process. Transformation efficiency is calculated by dividing the number of transformants by the total number of organisms that have undergone the transformation process, and multiplying by 100 to obtain a percentage.

[0205] In the context of the present invention, the increase in transformation efficiency is the transformation efficiency compared to a control or wild-type plant (i.e., a plant to which the tissue regeneration factor of the present invention or plant tissue culture medium is not applied and / or a plant that does not express one or more nucleic acid constructs of the present invention). The increase refers to an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 95% or more compared to a control or wild-type plant. Alternatively, the increase may be at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold or more compared to the transformation efficiency of a control or wild-type plant. For example, Fig.47 It was shown that GmREF1 improved the transformation efficiency of soybean.

[0206] In another aspect of the present invention, a recombinant expression vector (also referred to as "nucleic acid construct"), an expression cassette, a transgenic cell line or a recombinant strain containing the nucleic acid construct of the present invention is provided.

[0207] A recombinant expression vector comprising a protein, nucleic acid or DNA molecule as described herein, preferably SIPEP (REF1) and / or PROSIPEP (PRR) and / or SIPEPR1 (RER) can be constructed by using an existing expression vector. Preferably, the expression vector comprises a binary Agrobacterium tumefaciens vector and a vector for microprojectile bombardment. When a recombinant expression vector is constructed to comprise a protein, nucleic acid or DNA molecule as described herein, any enhanced, constitutive, tissue-specific or inducible promoter can be connected before the transcription start nucleotide, and the promoter can be used alone or in combination with other plant promoters. In addition, when a recombinant expression vector is constructed to comprise a protein, nucleic acid or DNA molecule as described herein, an enhancer can be included, including a translation enhancer or a transcription enhancer. These enhancer regions may be the ATG start codon or the start codon of an adjacent region, but need to be in frame with the coding sequence to ensure the correct translation of the entire sequence. Translation control signals and start codons are widely present and may be natural, or synthetic. The translation start region may come from a transcription start region or a structural gene. To facilitate the identification and screening of transgenic plants or transgenic microorganisms, the expression vectors used can be processed, for example, by adding genes that express enzymes or luminescent compounds that produce color changes in plants or microorganisms, antibiotic resistance markers, or chemical resistance marker genes. Considering the safety of transgenics, plants or microorganisms can be directly transformed by phenotypic selection without adding selectable marker genes.

[0208] The term androgenesis refers to plant regeneration directly from microspore culture under in vitro conditions. The basic principle of androgenesis is to arrest the development of pollen cells (which normally become sexual cells) and force them to develop directly into complete plants. The process inhibits typical gametophyte differentiation and instead allows cell division and regeneration to occur.

[0209] When gamete formation (microspore development) occurs, mature pollen grains are formed by mitosis. Since the developmental pathways are not yet determined during microgamete formation, there is an opportunity to interrupt the normal gametophytic pathway and induce sporophytic development. As a result of the sporophytic divisions, multicellular microspores develop within the anther. Differentiation of these multicellular units may lead to pollen embryos, which subsequently develop into haploid plants.

[0210] Under certain conditions, microspores may undergo sporophytic development rather than enter the gamete production pathway. Many microspores arrest and / or die, some develop into pollen-like structures before dying or arresting, and others develop into multinucleate, haploid callus-like structures. At the same time, other microspores directly participate in embryogenesis and undergo many changes at different levels to become microspore-derived embryos. As a result, microspores are significantly enlarged, the nucleus is relocated to the center of the cell, the cytoplasm is cleared, and the large vacuole splits into smaller fragments.

[0211] Included within the scope of the present invention are methods of inducing androgenesis mediated by exogenous administration of REF1 and / or overexpression of PRR and / or RER.

[0212] Also included within the scope of the present invention is a method of inducing organogenesis by exogenously administering REF1 and / or overexpression of PRR and / or RER. Organogenesis refers to the formation of organs from cultured explants and the subsequent regeneration of plants. In direct organogenesis, in vitro organs are induced directly from explant tissue; in indirect organogenesis, de novo organs are usually formed from intermediate tissues (i.e., callus tissue).

[0213] Methods for inducing somatic embryogenesis by exogenous administration of REF1 and / or overexpression of PRR and / or RER are within the scope of the present invention. In somatic embryogenesis, totipotent cells can form somatic embryos through the embryogenesis pathway, and the somatic embryos can regenerate the entire plant after growth. Somatic embryogenesis occurs directly when the embryo develops from a somatic cell; somatic embryogenesis occurs indirectly when the embryogenic structure is after the division of a non-embryonic cell.

[0214] Methods for enhancing the embryogenic response from primary callus induced by REF1 are also within the scope of the present invention. Plant regeneration refers to the process by which plants grow and develop again after suffering severe injury. The inventors have demonstrated that REF1 is derived from wounding to promote regeneration and root hair formation in parallel with the embryogenic response.

[0215] The plants according to all aspects of the invention described herein may be monocots or dicots.

[0216] The dicotyledonous plant may be selected from the following families, including but not limited to Asteraceae, Brassicaceae (such as Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant can be selected from tomato, soybean, lettuce, sunflower, Arabidopsis, broccoli, spinach, watermelon, pumpkin, cabbage, potato, yam, pepper, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, field (fava) bean, pea, lentil, peanut, chickpea, apricot, pear, peach, grapevine or citrus species.

[0217] For example, the monocotyledonous plant may be selected from the family Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop. Examples of crop plants include wheat, rice, barley, corn, oats, sorghum, rye, millet, buckwheat, turf grass, multiflora ryegrass, sugarcane or fescue, or crops such as onions, leeks, yams or bananas.

[0218] Preferably, the plant is selected from tomato, pepper, rice, wheat, barley, soybean, corn, sunflower, potato, cotton, cucumber or peach.

[0219] In one embodiment, when the plant is rice, the variety is selected from japonica rice, indica rice or wild allotetraploid rice, more preferably, ZH11 rice, 9311 rice or L-04 rice.

[0220] In one embodiment, when the plant is wheat, the variety is selected from Chinese Spring, JM22 or KN199.

[0221] In one embodiment, when the plant is corn, the variety is selected from B-104 or Chan 7-2

[0222] In one embodiment, the plant may be a species or cultivar that is, or was previously, recalcitrant to regeneration.

[0223] The term "plant" as used herein includes whole plants, ancestors and progeny of plants and plant parts, including seeds, fruits, branches, stems, pollen, microspores, anthers, ovules, leaves, roots (including tubers), flowers, tissues and organs, each of which comprises the nucleic acid constructs described herein. The term "plant" also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, each of which comprises the nucleic acid constructs or mutations described herein.

[0224] The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.

[0225] In a most preferred embodiment, the plant part or harvestable part is a seed or a fruit. Thus, in a further aspect of the present invention, there is provided a seed or a fruit produced by a transgenic plant as described herein.

[0226] According to all aspects of the invention, a control plant as used herein is a plant that has not been modified according to the method of the invention. Thus, in one embodiment, the control plant does not have one or more of the above constructs and / or has not been exogenously exposed to SIPEP (REF1). In one embodiment, the control plant is a wild-type plant. The control plant is generally of the same plant species, preferably of the same genetic background as the modified plant.

[0227] Although the foregoing disclosure provides a general description of the main contents contained within the scope of the present invention, including methods of making and using the present invention and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the present invention and provide a complete written description thereof. However, it will be understood by those skilled in the art that the specific details of these examples should not be construed as limitations of the present invention, the scope of which should be understood from the claims attached to this disclosure and their equivalents. In view of this disclosure, various further aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0228] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, equivalent to each being listed individually herein.

[0229] Unless the context dictates otherwise, the descriptions and definitions of the features described above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0230] The above-mentioned applications, and all documents and serial accession numbers cited herein or during the pendency of their applications ("Documents cited in the Application") and all documents cited or referenced in documents cited in the Application, and all documents cited or referenced in documents cited in the Application ("Documents cited in the Application"), and all documents cited or referenced in documents cited in the Application, are incorporated herein by reference, together with, and any manufacturer's instructions, descriptions, product specifications, and product instructions for any products described herein or incorporated by reference in any document herein, and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0231] The invention is now described in the following non-limiting examples.

[0232] In the context of this example, the measurements were performed as follows:

[0233] The relative callus area was determined by quantifying the area of ​​each callus using ImageJ software, calculating the mean value of each group, and then dividing the mean value of each group by the mean value of the control group.

[0234] The relative regeneration frequency was determined by counting the number of regenerated shoots and calculating the mean value for each group, which was then divided by the mean value of the control group.

[0235] The number of hypocotyls refers to explants with different numbers of regenerated shoots.

[0236] The shoot regeneration frequency was determined by calculating the average number of shoots. In the context of the present invention, the shoot regeneration capacity refers to the number of shoots per explant in each treatment.

[0237] Example

[0238] Example 1 - SIPEP (REF1) as a local wound signal in tomato

[0239] Green and Ryan (1972) found that PI-II (proteinase inhibitor II) is a marker of defense gene expression in plants, such as Figure 2 As shown, the expression of PI-II is increased locally at sites of injury, ie, resulting from insect attack, and is also increased systemically in uninjured sites.

[0240] Systemin is known to regulate the systemic wound response in plants. Systemic wound responses do not occur in wounded PROSYSTEMIN knockout plants (prs), as indicated by the lack of systemic PI-II expression in prs plants, whereas wounded wild-type plants show high expression of PI-II both systemically and locally compared to control unwounded tomato plants. PROSYSTEMIN (prr) knockouts express PI-II locally at moderate levels, but not systemically, suggesting the existence of systemin-independent signals that regulate the local wound response in plants.

[0241] Plant elicitor peptide (PEP) is a peptide signal known to regulate innate immunity in Arabidopsis thaliana. A PEP homolog has been identified in tomato, namely SIPep, also known as REF1, such as Figure 2 SIPEP is a 23 amino acid peptide cleaved from its precursor PROSIPEP, also known as PROREF1 and PRR (these terms are used interchangeably herein), as shown in Figure 3 shown.

[0242] Generation of PROSIPEP knockout Figure 3 B (prr-1 and prr-2), and the generation of PROSYSTEMIN knockouts (prs-1 and prs-2). The role of SIPEP (REF1) and systemin was investigated by measuring local (red) and systemin (blue) expression of PI-II in wounded tomato plants of wild type, PROSIPEP knockouts (prr-1 and prr-2), PROSYSTEMIN knockouts (prs-1 and prs-2), or PROSIPEP PROSYSTEMIN double knockouts (prr-1 prs-1). Figure 4showed that PI-II was highly expressed both locally and systemically in wounded wild-type tomato plants, suggesting that when both PROSIPEP and PROSYSTEMIN are expressed, thereby producing both SIPEP and systemin, both local and systemic wound responses occur. In PROSIPEP knockouts (prr-1 and prr-2), PI-II was expressed both locally at the wound site and systemically, although at lower levels than in wild-type plants. PROSYSTEMIN knockouts (prs-1 and prs-2) expressed PI-II locally at levels similar to those observed in PROSIPEP knockouts, however, they had little systemic PI-II expression, suggesting that systemin is a key regulator of the systemic wound response. Importantly, PROSIPEP PROSYSTEMIN double mutants exhibited a much lower local wound response (local PI-II expression) than the parental single mutants, suggesting that SIPEP (REF1) is a local wound signal that is a regulator of the local wound response in tomato.

[0243] Example 2 - SIPEP(REF1) rescues the defective callus formation in the PROSIPEP(prr) mutant

[0244] To further investigate the role of SIPEP as a local wound signal in tomato, the tomato cultivar AislaCraig (AC) was used. AC was analyzed in its wild-type form, PROSIPEP knockouts (prr-1 and prr2), and PROSYSTEMIN knockout (prs-1). Figure 5 A clearly shows that callus formed in both wild-type and prs-1 plants, indicating that callus can form in the absence of systemin and that systemin is not essential for local wound signaling in tomato. Figure 5 A shows that PROSIPEP knockouts (prr-1 and prr-2) only formed minimal callus, which was Figure 5 The relative callus area shown in B further demonstrates that, based on these data, it is apparent that deletion of the precursor gene (PRR) of SIPEP (REF1), but not deletion of systemin (PRS), abolishes callus formation in tomato tissue culture, supporting that SIPEP (REF1) is a wound signal that promotes regeneration.

[0245] To further test whether SIPEP(REF1) is a wound signal that promotes regeneration in tomato, wild-type, prr-1, and prr-2 Aisla Craig hypocotyl explants were cultured on callus induction medium in the presence or absence of 1 nM SIPEP(REF1). Callus phenotypes were then scored after 14 days of incubation ( Figure 6 ), it can be seen that the presence of 1nM REF1 in the culture medium increased the callus area in all tested explants. Crucially, when REF1 was present in the culture medium, the relative callus area of ​​prr-1 and prr-2 was restored. In addition, REF1 also induced a larger callus area in wild-type plants when added to callus induction medium. These data provide strong evidence that REF1 is a wound signal that promotes plant regeneration.

[0246] Example 3 - SIPEP(REF1) rescues defective shoot regeneration in PROSIPEP(prr) mutants

[0247] To further investigate the role of SIPEP in tomato regeneration, the cultivar Aisla Craig (AC) was used. AC was analyzed in its wild-type form, as well as in strains in which PROSIPEP was knocked out (prr-1 and prr2). Figure 7 A shows visually that the ability of shoot regeneration is completely abolished in PROSIPEP mutants (prr-1 and prr2). Figure 7 Quantified in B, where it can be seen that the relative regeneration frequency and hypocotyl number of prr-1 and prr-2 are greatly reduced compared to wild type (WT).

[0248] To test whether SIPEP(REF1) could rescue the shoot regeneration defect in prr mutants, the mutants were cultured in shoot induction medium (SIM) in the presence and absence of 1 nM REF1. Shoot regeneration was scored after 21 days. Figure 8 It was clearly shown that shoot regeneration was restored in prr-1 and prr-2 mutants and increased in wild-type plants when cultured with 1 nM REF1. Figure 8 B shows that REF-1 increases the relative regeneration frequency in WT, prr-1, and prr-2 mutants. Figure 8 C shows that REF-1 increases hypocotyl number in WT, prr-1, and prr-2 mutants. These data suggest that REF1 plays a role in plant regeneration.

[0249] The relative regeneration frequency was determined by counting the number of regenerated shoots and calculating the mean value of each group, which was then divided by the mean value of the WT-Mock group.

[0250] “Hypocotyl number” refers to explants with different numbers of regenerated shoots (0, 1, 2, 3, and >4) in each genotype (WT, prr-1, prr-2).

[0251] Example 4 - SIPEP (REF1) dose-dependently promotes callus formation

[0252] To test the effect of SIPEP(REF1) dosage on callus formation, WT (Ailsa Craig) hypocotyl explants were cultured in Fig. 9 The indicated concentrations were expressed on callus induction medium (CIM) (0 nM for Mock, 10 -4 , 10 -3 , 10 2 , 10 1 , 1 and 10 nM), and the callus phenotype was scored after 14 days of incubation. The box plots represent the projected callus area distribution. As can be seen from the figure, the relative callus area increased in a dose-dependent manner, with the highest mean at 1 nM and a sharp decrease at 10 nM. This suggests that for WT Ailsa Craig explants, 1 nM of REF1 is the optimal concentration to be applied to the culture medium to maximize callus formation.

[0253] Example 5—Effects of overexpressing PROSIPEP (PRR)

[0254] The effect of overexpression of SIPEP on both callus formation and shoot regeneration was tested.

[0255] Fig.10 The effect of SIPEP (PRR) overexpression on callus formation is shown. Fig.10 A shows visually that callus formation was enhanced in SIPEP (PRR) overexpressing plants (PRR-OE-1 and PRR-OE-9) compared with wild type and SIPEP (PRR) knockout plants (prr-1 and prr-2). Fig.10 Quantification was performed in B. Fig.10 B shows that the relative callus areas of PRR-OE-1 and PRR-OE-9 were increased compared with WT and prr-1 and prr-2.

[0256] Fig.11 Shown is the effect of SIPEP (PRR) overexpression on shoot regeneration. Fig.10 A shows visually that shoot regeneration was enhanced in SIPEP (PRR) overexpressing plants (PRR-OE-1 and PRR-OE-9) compared with wild type and SIPEP (PRR) knockout plants (prr-1 and prr-2). Fig.10 Quantification was performed in B. Fig.10B shows that the relative regeneration frequencies of PRR-OE-1 and PRR-OE-9 increased relative to WT and prr-1 and prr-2, with PRR-OE-1 and PRR-OE-9 increasing by ~4.2-fold and ~4-fold relative to WT, respectively. Fig.10 C shows that the number of hypocotyls in PRR-OE-1 and PRR-OE-9 were increased compared with WT and prr-1 and prr-2.

[0257] The data show that increased expression of PROSIPEP (PRR) improves the regeneration capacity of plants.

[0258] Example 6 - SIPEPR1 (RER) is a receptor for SIPEP (REF1)

[0259] SIPEPR1 (RER) was identified as the tomato ortholog of the Arabidopsis PEP receptor, as Fig.12 Therefore, RER was investigated to determine whether it is the receptor for SIPEP (REF1). REF consists of an extracellular LRR domain, a transmembrane domain, and a cytoplasmic kinase domain.

[0260] A study was conducted to determine whether systemin could trigger autophosphorylation of the SIPEP (REF1) receptor (RER).

[0261] Fig.13 A shows a pull-down experiment between the HA-Flag-tagged ectodomain of RER (purified from tobacco leaves) and biotinylated REF1. As can be seen, RER1 binds to SIPEP(REF1), but systemin does not.

[0262] Fig.13 B shows the expression Fig.13 Autophosphorylation analysis of tobacco leaves of the nucleic acid constructs shown in C, which were treated with 100 nM REF1 for 20 minutes prior to the autophosphorylation analysis. This shows that SIPEP(REF1) triggers autophosphorylation of RER in vivo, whereas systemin does not.

[0263] Receptor activation was examined by using the expression of FRK1 as a marker gene. Protoplasts were transfected with pFRK1::LUC and the indicated constructs and induced with 100 nM of REF1 or systemin for 3 h. Data are mean ± SD, n = 3 replicates. ***P < 0.001 (Student's t-test); ns, not significant. The expression of FRK-luciferase was used as a marker to detect REF1 binding-mediated activation of its receptor. Based on these assays, the EC50 (half maximal stimulation) of REF1 receptor binding was calculated to assess the binding affinity of REF1 to its receptor RER. In these assays, systemin and its two receptors SYR1 and SYR2 were used as controls. Fig.14 a shows that REF1 binds to RER, indicating that RER is the receptor of REF1. Fig.14 b shows that RER binds REF1 with high affinity, providing further evidence that RER is the receptor for REF1.

[0264] Example 7 - SIPEPR1 (RER) is involved in determining the regeneration capacity of plants

[0265] To determine whether SIPEPR1 (RER) is directly involved in determining plant regeneration capacity, RER knockout mutants (rer-1 and rer-3) were generated, as Fig.15 A. RER overexpressing plants (RER-OE-3 and RER-OE-5) were also generated. Fig.15 B shows the Western blot results and revealed that the RER expression levels of RER-OE-3 and RER-OE-5 were higher than that of wild-type (WT) plants.

[0266] Callus formation in RER knockout mutants (rer-1 and rer-3) was determined in comparison with wild-type tomato plants (Ailsa Craig) in the presence and absence of SIPEP(REF1). Fig.16 A visually shows that callus formation in rer-1 and rer-3 plants was very minimal compared to WT in the presence and absence of REF1. Fig.16 B shows the relative callus area of ​​each plant. It can be seen that the callus area of ​​WT plants cultured with REF1 is the largest, because REF1 induces callus formation, as shown in Examples 2 and 4. Regardless of the presence of REF1, callus formation in rer-1 and rer-3 plants is very small, and REF1 does not lead to any increase in relative callus area. This indicates that REF1-induced callus formation does not occur if RER is missing, i.e., rer knockout is defective in REF1-induced callus formation.

[0267] Shoot regeneration of RER knockout mutants (rer-1 and rer-3) was determined in the presence and absence of SIPEP(REF1) compared to wild-type tomato plants (Ailsa Craig). Fig.19 A shows visually that shoot regeneration in rer-1 and rer-3 plants is very small compared to WT in the presence and absence of REF1. Fig.17 As shown in B and C, the relative regeneration frequency and number of hypocotyls of WT plants cultured with REF1 were the highest, since REF1 induced shoot regeneration as shown in Example 3. In rer-1 and rer-3 knockouts, the regeneration frequency of shoots and the number of hypocotyls were very small, and growth in the presence of REF1 did not result in any increase in the regeneration frequency of shoots and the number of hypocotyls. This suggests that REF1-induced shoot regeneration does not occur if RER is absent, i.e., rer knockouts are defective in REF1-induced shoot regeneration.

[0268] Overexpression of RER was tested to investigate whether it could enhance the regeneration capacity of plants, as measured by callus formation and shoot regeneration. Fig.18 As shown in Figure 2, callus formation in RER-OE-3 and RER-OE-5 plants was much greater than that in WT and RER knockout plants, indicating that increased levels of RER enhanced the regeneration capacity of the plants. Fig.19 As shown, the shoot regeneration capacity of RER-OE-3 and RER-OE-5 plants was much higher than that of WT, rer-1, and rer-3, among which the relative regeneration frequency of RER-OE-3 and RER-OE-5 plants was ~3.5-fold and ~3-fold higher than that of WT plants, respectively.

[0269] Together these data suggest that RER enhances plant regeneration capacity.

[0270] Example 8 - SIPEP (REF1) enhances the regeneration capacity of recalcitrant wild tomato

[0271] The tomato varieties Ailsa Craig (AC) used in Examples 1-7 are known in the art to have high transformation efficiency. Therefore, the inventors studied the effect of SIPEP (REF1) technology on recalcitrant tomatoes.

[0272] The effect of exogenous application of REF1 in shoot-inducing growth medium was studied in LA1777, an accession of wild tomato S. habrochaites with very low transformation efficiency, by growing LA1777 tomatoes in the presence and absence of 1 nM REF1. Intuitively, Fig. 20As shown in A, shoot regeneration in LA1777 was better when grown in the presence of 1 nM REF1 than when REF1 was absent from the growth medium. Fig. 20 As shown in B, the relative regeneration frequency of LA1777 was 6-fold higher in the presence of REF1 compared to the absence of REF1, and similarly, the number of hypocotyls was higher in the presence of REF1. These data indicate that REF1 increases the regeneration capacity of LA1777 tomato.

[0273] The effect of exogenous application of REF1 in shoot induction growth medium was studied in PI126944, a wild tomato strain S. peruvianum with very low transformation efficiency, by growing PI126944 in the presence and absence of 1 nM REF1. Fig.21 A shows that shoot regeneration in PI126944 was better when grown in the presence of 1 nM REF1 than when REF1 was absent from the growth medium. Fig.21 As shown in B, the relative regeneration frequency of PI126944 was 5.5-fold higher in the presence of REF1 than in the absence of REF1, and similarly, the number of hypocotyls was higher in the presence of REF1. These data indicate that REF1 increases the regeneration capacity of PI126944 tomato.

[0274] Currently, these data indicate that exogenous application of REF1 is able to increase the regeneration capacity of recalcitrant tomato. Next, it will be necessary to determine whether the regeneration capacity of recalcitrant tomato can also be improved by plant transformation and / or genome editing. It has been previously demonstrated that expression of PROSIPEP (PRR) genes and / or expression of the SIPEP receptor SIPEPR1 gene (RER) are effective in increasing the regeneration capacity of AC tomato. Therefore, constructs have been designed to induce the expression of PRRs and RERs in tomato and other plants. Fig. 22 As shown in A, the overexpression vector (pCeGFP) designed for plant transformation is referred to as PRR+RER-OE construct in the present invention, and as Fig. 22 Figure B shows the CRISPR-Cas9 vector (pTX041) designed for genome editing, referred to as the PRR+RER construct.

[0275] PI126944 tomatoes were transformed with the PRR+RER-OE construct as well as an equivalent construct containing only PRR and another construct containing only RER. Plants expressing the constructs were grown for 19 days with wild-type controls in the presence or absence of REF1. Fig.23As shown, shoot regeneration was increased in plants transformed with constructs containing both PRR and RER, respectively, with the relative regeneration frequency in each case increasing by approximately 25-fold compared to wild-type, untransformed plants. The greatest increase in regeneration capacity was observed in PI126944 tomatoes transformed with PRR+RER-OE, in which case the relative regeneration frequency increased by approximately 42-fold compared to wild-type plants without REF1. Importantly, however, the PRR+RER-OE construct also resulted in a very large increase in the regeneration frequency of PI126944 tomatoes in the absence of REF1. Collectively, these data indicate that overexpression of PRR and / or RER increases the regeneration capacity of plants in the presence or absence of exogenous REF1, and that exogenous exposure to REF1 even further enhances the regeneration capacity of plants.

[0276] Example 9 - The pepper ortholog of SIPEP / REF1 (CaREF1) promotes regeneration in pepper.

[0277] SIPEP, PROSIPEP, and SIPEP receptor / SIPEPR1 (REF1, PRR, and RER) are largely conserved in the plant kingdom and have orthologs in most species. Here, we demonstrate that the role of SIPEP (REF1) is redundant in different plants.

[0278] The pepper SIPEP (REF1) ortholog (CaREF1) was studied to determine whether CaREF1 could enhance the regeneration capacity of pepper (Capsicum annuum). The results showed that CaREF1 promoted the formation of root hairs, such as Fig.24 shown.

[0279] To further support a role for CaREF1 in pepper in enhancing its regeneration capacity, we showed that callus formation was also increased in plants grown in the presence of CaREF1 ( Fig.25 ).

[0280] Example 10 - The soybean ortholog of SIPEP / REF1 (GmREF1) promotes regeneration in soybean.

[0281] The soybean SIPEP (REF1) ortholog (GmREF1) was investigated to determine whether GmREF1 could enhance the regeneration capacity of soybean (Glycine max).

[0282] Soybean explants were cultured in medium containing the indicated concentrations of REF1. Shoot regeneration was scored after 21 days of cultivation. Fig. 27As shown, GmREF1 increased soybean regeneration capacity in plants grown in the presence of GmREF1. Compared to plants grown in the absence of GmREF1, ~2.1, ~2.8, and ~3.8-fold increases were observed in plants grown in the presence of 10, 20, and 50 nM GmREF1, respectively ( Fig.26 ).

[0283] The tomato PRR+RER-OE construct was also found to greatly improve transformation efficiency in recalcitrant soybean, showing a ~12-fold increase in regeneration frequency in recalcitrant soybean grown in the presence of 100 nM GmREF1 compared to control recalcitrant soybean grown in the absence of GmREF1.

[0284] Example 11 - The rice ortholog of SIPEP / REF1 (OsREF1) promotes regeneration in rice.

[0285] The rice SIPEP (REF1) ortholog (OsREF1) was investigated to determine whether it could enhance the regeneration capacity of rice (Oryza sativa).

[0286] Mature seeds of ZH11 were cultured in CIM containing the indicated concentrations of OsREF1 (1 nM and 10 nM). Fig. 27 As shown in A, calli formed in the presence of OsREF1 were larger than those formed in the absence of OsREF1. Fig. 27 As shown in Figure B, the callus formation ability of ZH11 grown in the presence of 1 nM and 10 nM OsREF1 increased by ~2-fold and ~3-fold, respectively, compared with that of ZH11 grown in the absence of OsREF1. These data indicate that OsREF1 promotes callus formation in japonica rice.

[0287] Shoot regeneration was studied in ZH11, Nipponbare japonica, and 9311 indica rice varieties. OsREF1 promoted shoot production in all three rice varieties. A ~4-fold increase in shoot regeneration was observed in ZH11 and Nipponbare rice varieties grown in the presence of 10 nM OsREF1 compared to rice grown in the absence of OsREF1 ( Fig.28 and Fig.29 ), while the shoot regeneration capacity of 9311 rice increased by ~3.5 times ( Fig.30 ).

[0288] Since this particular rice variety showed a low transformation efficiency, a wild allotetraploid rice (L-04) was also tested. We found that OsREF1 promoted the growth of the wild allotetraploid rice (L-04). Fig.31As shown in Figure 2, the taproot length of wild allotetraploid rice grown in the presence of 10 nM OsREF1 was significantly increased. OsREF1 was also shown to promote shoot regeneration in wild allotetraploid rice (L-04), with the shoot regeneration frequency of L-04 rice grown in the presence of 10 nM OsREF1 increasing by ~12.2-fold compared to L-04 rice grown in the absence of OsREF1 ( Fig.43 ).

[0289] OsREF1 also promotes callus formation in wild allotetraploid rice. Seeds of wild allotetraploid rice L-04 were cultured on CIM containing indicated concentrations of OsREF1 (1 nM and 10 nM) for 20 days. Fig.32 As shown, wild allotetraploid rice L-04 grown in the presence of 1 and 10 nM OsREF1 for 20 days showed ~7.4-fold and ~8.9-fold enhancement in callus formation ability, respectively, compared with wild allotetraploid rice (L-04) grown in the absence of OsREF1.

[0290] These data suggest that the SIPEP (REF1) ortholog (OsREF1) can enhance the regeneration capacity of rice, including japonica, indica, and wild allotetraploid rice.

[0291] Example 12 - The wheat ortholog of SIPEP / REF1 (TaREF1) promotes root and shoot growth in wheat.

[0292] The wheat SIPEP (REF1) ortholog (TaREF1) was investigated to determine whether REF1 could enhance wheat regeneration capacity. Wheat grown in the presence of 100 nM TaREF1 was observed to show increased root and shoot length compared to wheat grown in the absence of TaREF1.

[0293] When applied exogenously, TaREF1 has also been shown to increase the yield of Chinese spring wheat ( Fig.43 ) and JM22 wheat ( Fig.54 ) callus formation ability. When applied exogenously, TaREF1 also promoted the callus formation of Chinese spring wheat ( Fig.53 ), Kn199 wheat ( Fig.44 ) and JM22 wheat ( Fig.55 ) of shoot regeneration.

[0294] This suggests that REF1 can improve wheat regeneration capacity in a range of wheat varieties.

[0295] ImageJ software was used to quantify the area of ​​each callus and the mean of each group was calculated, which was then divided by the mean of WT-Mock.

[0296] The shoot regeneration frequency was determined by calculating the average number of shoots. In the context of the present invention, the shoot regeneration capacity refers to the number of shoots per explant under each condition.

[0297] Example 13 - The peach ortholog of SIPEP / REF1 (PpREF1) promotes callus formation in peach

[0298] The peach SIPEP (REF1) ortholog (GaREF1) was studied to determine its effect on the regenerative capacity of peach.

[0299] Leaves of peach cv. GF677 were cultured on CIM containing the indicated concentrations of PpREF1, and callus phenotype was scored after 24 days of incubation on CIM. Callus formation capacity was expressed as the percentage of leaf explants with regenerated embryonic callus. Data are mean ± SD of three independent experiments. Callus formation capacity of peach cv. GF677 grown in the presence of 100 nM PpREF1 was increased 10-fold compared to leaves grown in the absence of PpREF1.

[0300] The data suggest that PpREF1 improves peach regeneration capacity ( Fig.34 ).

[0301] Example 14 - The sunflower ortholog of SIPEP / REF1 (HanPEP1) promotes shoot regeneration in sunflower.

[0302] The sunflower SIPEP (REF1) ortholog HanREF1 was studied to determine its effect on the regeneration capacity of sunflower. The results showed that the shoot regeneration frequency of sunflower explants grown in the presence of HanREF1 was increased compared to that in the absence of HanREF1, with an increase of ~2.1-fold in the presence of 1 nM HanREF1 and ~4.5-fold in the presence of 10 nM HanREF1.

[0303] These data suggest that HanREF1 increases the regeneration capacity of sunflower ( Fig.40 ).

[0304] For the regeneration protocol, we refer to Radonic et al., 2015. The explants used for the sunflower regeneration assay were cotyledons.

[0305] The shoot regeneration frequency was determined by calculating the average number of shoots. In the context of the present invention, the shoot regeneration capacity refers to the number of shoots per explant under each treatment.

[0306] Example 15 - The cotton ortholog of SIPEP / REF1 (GaPEP1) promotes callus formation in cotton

[0307] The cotton SIPEP (REF1) ortholog (GaREF1) was investigated to determine its effect on the regeneration capacity of cotton. The results showed that the relative callus area of ​​cotton hypocotyl explants grown in the presence of GaREF1 increased compared to that in its absence, and the relative callus area increased with increasing concentrations of GaREF1 (0.1–500 nM).

[0308] The data suggest that GaREF1 improves cotton's regeneration capacity ( Fig.41 ).

[0309] Example 16 - MsREF1 promotes callus formation in alfalfa (Medicago sativa)

[0310] Leaf explants were cultured on callus induction medium (CIM) containing the indicated concentrations of MsREF1, and callus phenotypes were scored after 14 days of incubation. Fig.45 It can be seen that the callus formation of alfalfa treated with 500 nM MsREF1 was significantly increased.

[0311] Example 17 - CsREF1 promotes shoot regeneration in cucumber

[0312] Leaf explants were cultured on medium containing 10 nM CsREF1. Compared with control explants without CsREF1 treatment, CsREF1-treated explants showed significantly enhanced shoot regeneration ability, as shown in Figure 2. Fig.46 shown.

[0313] Example 18 - GmREF1 improves soybean transformation efficiency

[0314] Soybean gene A was introduced into Williams 82 soybean variety and treated with 100 nM GmREF1 or control. Gene A is a transcription factor gene that was used as a reporter gene for transformation efficiency. Fig.47 The GmREF1-treated plants were shown to be visually healthier (in a representative image of the experiment) and had higher transformation efficiency. The data obtained from these experiments are as follows:

[0315]

[0316] Example 19 - The tomato "PRP+RER-OE" construct improves shoot regeneration in recalcitrant rice.

[0317] Recalcitrant rice was transformed with an empty vector or with the PROSIPEP (PRP) + RER–OE construct. Fig.48 It was shown that the shoot regeneration ability of rice ZH11 and 9311 transformed with PROPEP (PRP)+RER-OE construct was improved.

[0318] Example 20 - ZmREF1 promotes shoot regeneration

[0319] The young embryos of B104 and Chang7-2 were treated with 0, 0.1 and 1 nM ZmREF1, respectively. When ZmREF1 was applied, the shoot regeneration frequency of B-104 and Chan 7-2 increased, with the maximum regeneration frequency at 1 nM for B-104 and at 0.1 nM for Chan 7-2. Fig.52 shown.

[0320] Example 21 - REF1-RER regulates regeneration via the SERK co-receptor.

[0321] Somatic embryogenesis receptor kinase (SERK) is a co-receptor for multiple LRR-RLKs (Ma et al., 2016; Peng and Kaloshian, 2014). Co-immunoprecipitation (co-IP) assays showed that REF1 can mimic the complex formation of RER with three SERK members in tomato ( Fig.49 A). The callus formation and shoot organogenesis abilities of serk3a-1 or serk3b-1 single mutants were slightly reduced, while those of serk3a-1 and serk3b-1 double mutants were greatly reduced ( Fig.49 B-49E). In addition, REF1 was unable to rescue the regeneration defects of the above-mentioned serk mutants ( Fig.49 B-49E). These results suggest that REF1-RER regulates regeneration through the co-receptors SERK3a and SERK3b.

[0322] Example 22 - REF1 regulates regeneration by promoting the expression of SWIND1

[0323] To understand the mechanism by which REF1 regulates organ regeneration, the inventors investigated whether REF1 induces the tomato homolog of WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), an AP2 / ERF transcription factor in Arabidopsis that functions as a central regulator of wound-induced cellular reprogramming (Iwase et al., 2011). Using transgenic plants carrying a SlWIND1 promoter-driven β-glucuronidase (pSlWIND1::GUS) fusion, the inventors found that SlWIND1 promoter activity was induced upon hypocotyl cutting, and this induction was enhanced upon REF1 treatment ( Fig.50 A). Significantly, reverse transcription quantitative PCR (RT-qPCR) analysis revealed that wound-induced SlWIND1 expression was disrupted in prp-2 and rer1-3 mutants ( Fig.50 B), indicating that the REF1-RER1 module is important for wound-induced activation of SlWIND1 expression.

[0324] To determine the function of SlWIND1 in plant regeneration, we generated SlWIND1 null mutant (slwind1) and overexpressing plants (SlWIND1-OE) and examined their regeneration phenotypes in conventional tissue culture. The results showed that when the slwind1 mutant exhibited severely impaired callus formation and shoot organogenesis compared with WT ( Fig.50 C-50F), SlWIND1-OE plants showed enhanced callus formation and shoot organogenesis ( Fig.50 G-50J), indicating that SlWIND1 plays a key role in the acquisition of regenerative capacity in tissue culture. In addition, the impaired regenerative capacity of slwind1 mutants cannot be rescued by REF1 ( Fig.50 C-50F). Taken together, these results support that REF1 promotes wound-induced regeneration by activating the expression of SlWIND1.

[0325] Example 23 - SlWIND1 activates PRP expression in response to trauma

[0326] Since we have established that REF1 promotes regeneration by activating the principal cell reprogramming regulator SlWIND1, we wondered whether and how activated SlWIND1 could further amplify REF1 signaling. In fact, the promoter activity of the REF1 precursor gene is induced by wounding during hypocotyl cutting ( Fig.51 A), as assessed in a pPRP::GUS reporter assay. Furthermore, wound- and REF1-mediated induction of PRP expression was impaired in the slwind1-3 mutant compared with WT ( Fig.51B and 51C), indicating that SlWIND1 plays an important role in the induction of wound-triggered PRP expression. To understand the transcriptional mechanism by which SlWIND1 regulates wound-induced PRP expression, we detected putative SlWIND1 binding motifs in the PRP promoter and identified eight vasculature-specific and wound-responsive cis-element (VWRE)-like motifs within a 2,000 bp region of the PRP promoter (Iwase et al., 2017; Sasaki et al., 2002; Sasaki et al., 2006) ( Fig.51 D). Importantly, the wound-responsive VWRE was also present in the promoters of SlWIND1, RER1, PRS, and the systemin receptor genes SYR1 and SYR2, reinforcing the idea that these genes are involved in plant wound response. Consistent with the observation that WIND family proteins preferentially bind to VWRE (Iwase et al., 2017; Sasaki et al., 2007), electrophoretic mobility shift assays (EMSA) showed that SlWIND1-His recombinant protein bound to a DNA probe containing the VWRE (5'-AAATTT-3') motif, but failed to bind to a DNA probe in which the VWRE motif was mutated ( Fig.51 E). Consistently, chromatin immunoprecipitation-PCR (ChIP-qPCR) assays using SlWIND1-GFP plants and GFP antibodies (Du et al., 2017) revealed that SlWIND1-GFP was enriched in the PRP promoter ( Fig.51 D and 51F). Taken together, these results suggest that SlWIND1 binds to VWRE within the PRP promoter and activates its expression. Thus, SlWIND1 activation initiated by REF1 positively feeds back to the REF1 precursor gene to amplify the primary wound signal ( Fig.51 G).

[0327] Sequence Listing

[0328] SEQ ID NO: 1 - SIPEP (REF1) tomato DNA sequence (SiREF1)

[0329]

[0330] SEQ ID NO: 2 - SIPEP (REF1) tomato amino acid sequence (SiREF1)

[0331]

[0332] SEQ ID NO: 3 - SIPEP (REF1) pepper DNA sequence (CaREF1)

[0333]

[0334] SEQ ID NO: 4 - SIPEP (REF1) Capsicum annuum amino acid sequence (CaREF1)

[0335]

[0336] SEQ ID NO: 5 - SIPEP (REF1) soybean DNA sequence (GmREF1)

[0337]

[0338] SEQ ID NO: 6 - SIPEP (REF1) soybean amino acid sequence (GmREF1)

[0339]

[0340] SEQ ID NO: 7 - SIPEP (REF1) sunflower DNA sequence (HanREF1)

[0341]

[0342] SEQ ID NO: 8 - SIPEP (REF1) sunflower amino acid sequence (HanREF1)

[0343]

[0344] SEQ ID NO: 9 - SIPEP (REF1) cotton DNA sequence (GaREF1)

[0345]

[0346] SEQ ID NO: 10 - SIPEP (REF1) cotton amino acid sequence (GaREF1)

[0347]

[0348] SEQ ID NO: 11 - SIPEP (REF1) rice DNA sequence (OsREF1)

[0349]

[0350] SEQ ID NO: 12 - SIPEP (REF1) rice amino acid sequence (OsREF1)

[0351]

[0352] SEQ ID NO: 13 - SIPEP (REF1) maize DNA sequence (ZmREF1)

[0353]

[0354] SEQ ID NO: 14 - SIPEP (REF1) maize amino acid sequence (ZmREF1)

[0355]

[0356] SEQ ID NO: 15 - SIPEP (REF1) wheat DNA sequence (TaeREF1)

[0357]

[0358] SEQ ID NO: 16 - SIPEP (REF1) wheat amino acid sequence (TaeREF1)

[0359]

[0360] SEQ ID NO: 17 - SIPEP (REF1) cucumber DNA sequence (CsaREF1)

[0361]

[0362] SEQ ID NO: 18 - SIPEP (REF1) cucumber amino acid sequence (CsaREF1)

[0363]

[0364] SEQ ID NO: 19 - SIPEP (REF1) peach DNA sequence (PpREF1)

[0365]

[0366] SEQ ID NO: 20 - SIPEP (REF1) peach amino acid sequence (PpREF1)

[0367]

[0368] SEQ ID NO: 21 - PROSIPEP(PROREF1)-(PRR) tomato DNA sequence (SiPRR)

[0369]

[0370] SEQ ID NO: 22 - PROSIPEP(PROREF1) - (PRR) Tomato amino acid sequence (SiPRR)

[0371]

[0372] SEQ ID NO: 23 - PROSIPEP (PROREF1) - (PRR) Pepper DNA sequence (CaPRR)

[0373]

[0374] SEQ ID NO: 24 - PROSIPEP (PROREF1) Capsicum annuum amino acid sequence (CaPRR)

[0375]

[0376] SEQ ID NO: 25 - PROSIPEP (PROREF1) - (PRR) soybean DNA sequence (GmPRR)

[0377]

[0378] SEQ ID NO: 26 - PROSIPEP(PROREF1) - (PRR) soybean amino acid sequence (GmPRR)

[0379]

[0380] SEQ ID NO: 27 - PROSIPEP (PROREF1) sunflower DNA sequence (HanPRR)

[0381]

[0382] SEQ ID NO: 28 - PROSIPEP (PROREF1) sunflower amino acid sequence (HanPRR)

[0383]

[0384] SEQ ID NO: 29 - PROSIPEP(PROREF1) - (PRR) Cotton DNA sequence (GaPRR)

[0385]

[0386] SEQ ID NO: 30 - PROSIPEP (PROREF1) - (PRR) Cotton amino acid sequence (GaPRR)

[0387]

[0388] SEQ ID NO: 31 - PROSIPEP (PROREF1) - (PRR) Rice DNA sequence (OsPRR)

[0389]

[0390] SEQ ID NO: 32 - PROSIPEP (PROREF1) - (PRR) Rice amino acid sequence (OsPRR)

[0391]

[0392] SEQ ID NO: 33 - PROSIPEP (PROREF1) - (PRR) Maize DNA sequence (ZmPRR)

[0393]

[0394] SEQ ID NO: 34 - PROSIPEP (PROREF1) - (PRR) Maize amino acid sequence (ZmPRR)

[0395]

[0396] SEQ ID NO: 35 - PROSIPEP (PROREF1) - (PRR) wheat DNA sequence (TaePRR)

[0397]

[0398] SEQ ID NO: 36 - PROSIPEP (PROREF1) - (PRR) wheat amino acid sequence (TaePRR)

[0399]

[0400] SEQ ID NO: 37 - PROSIPEP (PROREF1) - (PRR) Cucumber DNA sequence (CsaPRR)

[0401]

[0402] SEQ ID NO: 38 - PROSIPEP (PROREF1) - (PRR) Cucumber amino acid sequence (CsaPRR)

[0403]

[0404] SEQ ID NO: 39 - PROSIPEP (PROREF1) - (PRR) peach DNA sequence (PpPRR)

[0405]

[0406] SEQ ID NO: 40 - PROSIPEP (PROREF1) - (PRR) Peach amino acid sequence (PpPRR)

[0407]

[0408] SEQ ID NO: 41 - SIPEPR1 (RER) Tomato DNA sequence (SiRER)

[0409]

[0410] SEQ ID NO: 42 - SIPEPR1 (RER) Tomato amino acid sequence (SiRER)

[0411]

[0412] SEQ ID NO: 43 - SIPEPR1 (RER) Pepper DNA sequence (CaRER)

[0413]

[0414] SEQ ID NO: 44 - SIPEPR1 (RER) Capsicum annuum amino acid sequence (CaRER)

[0415]

[0416] SEQ ID NO: 45 - SIPEPR1 (RER) soybean DNA sequence (GmRER)

[0417]

[0418] SEQ ID NO: 46 - SIPEPR1 (RER) soybean amino acid sequence (GmRER)

[0419]

[0420] SEQ ID NO: 47 - SIPEPR1 LIKE1 (RER-LIKE1) soybean DNA sequence (GmRER-LIKE1)

[0421]

[0422] SEQ ID NO: 48 - SIPEPR1 LIKE1 (RER-LIKE1) soybean amino acid sequence (GmRER-LIKE1)

[0423]

[0424] SEQ ID NO: 49 - SIPEPR1 LIKE2 (RER-LIKE1) soybean DNA sequence (GmRER-LIKE2)

[0425]

[0426] SEQ ID NO: 50 - SIPEPR1 LIKE2 (RER-LIKE1) soybean amino acid sequence (GmRER-LIKE2)

[0427]

[0428] SEQ ID NO: 51 - SIPEPR1 (RER) sunflower DNA sequence (HanRER)

[0429]

[0430] SEQ ID NO: 52 - SIPEPR1 (RER) sunflower amino acid sequence (HanRER)

[0431]

[0432] SEQ ID NO: 53 - SIPEPR1 (RER) cotton DNA sequence (GaRER)

[0433]

[0434] SEQ ID NO: 54 - SIPEPR1 (RER) cotton amino acid sequence (GaRER)

[0435]

[0436] SEQ ID NO: 55 - SIPEPR1 (RER) Rice DNA sequence (OsRER)

[0437]

[0438] SEQ ID NO: 56 - SIPEPR1 (RER) Rice amino acid sequence (OsRER)

[0439]

[0440] SEQ ID NO: 57 - SIPEPR1-LIKE1 (RER-LIKE1) Rice DNA sequence (OsRER-LIKE1)

[0441]

[0442] SEQ ID NO: 58 - SIPEPR1-LIKE1 (RER-LIKE1) Rice amino acid sequence (OsRER-LIKE1)

[0443]

[0444] SEQ ID NO: 59 - SIPEPR1 (RER) Maize DNA sequence (ZmRER)

[0445]

[0446] SEQ ID NO: 60 - SIPEPR1 (RER) Maize amino acid sequence (ZmRER)

[0447]

[0448] SEQ ID NO: 61 - SIPEPR1-LIKE1 (RER-LIKE1) Maize DNA sequence (ZmRER-LIKE1)

[0449]

[0450] SEQ ID NO: 62 - SIPEPR1-LIKE1 (RER-LIKE1) Maize amino acid sequence (ZmRER-LIKE1)

[0451]

[0452] SEQ ID NO: 63 - SIPEPR1 (RER) wheat DNA sequence (TaeRER)

[0453]

[0454]

[0455] SEQ ID NO: 64 - SIPEPR1 (RER) wheat amino acid sequence (TaeRER)

[0456]

[0457] SEQ ID NO: 65 - SIPEPR1-LIKE1 (RER-LIKE1) wheat DNA sequence (TaeRER-LIKE1)

[0458]

[0459] SEQ ID NO: 66 - SIPEPR1-LIKE1 (RER-LIKE1) wheat amino acid sequence (TaeRER-LIKE1)

[0460]

[0461] SEQ ID NO: 67 - SIPEPR1-LIKE2 (RER-LIKE2) wheat DNA sequence (TaeRER-LIKE2)

[0462]

[0463] SEQ ID NO: 68 - SIPEPR1-LIKE2 (RER-LIKE2) wheat amino acid sequence (TaeRER-LIKE2)

[0464]

[0465] SEQ ID NO: 69 - SIPEPR1-LIKE3 (RER-LIKE3) wheat DNA sequence (TaeRER-LIKE3)

[0466]

[0467]

[0468] SEQ ID NO: 70 - SIPEPR1-LIKE3 (RER-LIKE3) wheat amino acid sequence (TaeRER-LIKE3)

[0469]

[0470] SEQ ID NO: 71 - SIPEPR1-LIKE4 (RER-LIKE4) wheat DNA sequence (TaeRER-LIKE4)

[0471]

[0472] SEQ ID NO: 72 - SIPEPR1-LIKE4 (RER-LIKE4) wheat amino acid sequence (TaeRER-LIKE4)

[0473]

[0474] SEQ ID NO: 73 - SIPEPR1 (RER) cucumber DNA sequence (CsaRER)

[0475]

[0476] SEQ ID NO: 74 - SIPEPR1 (RER) cucumber amino acid sequence (CsaRER)

[0477]

[0478] SEQ ID NO: 75 - SIPEPR1 (RER) peach DNA sequence (PpRER)

[0479]

[0480]

[0481] SEQ ID NO: 76 - SIPEPR1 (RER) peach amino acid sequence (PpRER)

[0482]

[0483] SEQ ID NO: 77 - SIPEPR1-LIKE1 (RER-LIKE1) peach DNA sequence (PpRER-LIKE1)

[0484]

[0485] SEQ ID NO: 78 - SIPEPR1-LIKE1 (RER-LIKE1) peach amino acid sequence (PpRER-LIKE1)

[0486]

[0487] SEQ ID NO: 89 - SIPEP (REF1) barley amino acid sequence (HvREF1)

[0488]

[0489] SEQ ID NO: 90 - SIPEP (REF1) barley DNA sequence (HvREF1)

[0490]

[0491] SEQ ID NO: 91 - PROSIPEP (PROREF1) - (PRR) Barley amino acid sequence (HvPRR)

[0492]

[0493] SEQ ID NO: 92 - PROSIPEP (PROREF1) - (PRR) Barley DNA sequence (HvPRR)

[0494]

[0495]

[0496] SEQ ID NO: 93 - SIPEPR1 (RER) barley amino acid sequence (HvRER)

[0497]

[0498] SEQ ID NO: 94 - SIPEPR1 (RER) barley DNA sequence (HvRER)

[0499]

Claims

1. A tissue regeneration factor comprising an amino acid sequence as defined in SEQ ID NO: 2 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20, or a functional variant or homologue thereof of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20.

2. The tissue regeneration factor according to claim 1, wherein the homologue or functional variant comprises a conserved motif, wherein the sequence of the conserved motif is selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

3. A plant tissue culture medium comprising the tissue regeneration factor according to claim 1 or 2. The plant tissue culture medium according to claim 3 , wherein the tissue culture medium is a callus induction medium, a shoot induction medium or a root induction medium.

5. The plant tissue culture medium according to claim 3 or 4, wherein the plant tissue culture medium further comprises an auxin and / or a cytokinin.

6. The plant tissue culture medium according to claims 3 to 5, wherein the plant tissue culture medium comprises tissue regeneration factors at a concentration between 0.00001 nM and 100 nM.

7. The plant tissue culture medium of claim 6, wherein the plant tissue culture medium comprises the tissue regeneration factor at a concentration of between about 0.1 nM, 1 nM, 10 nM, 50 nM or 100 nM.

8. A nucleic acid construct comprising a nucleic acid sequence encoding at least one of a tissue regeneration factor, a tissue regeneration factor precursor, and a tissue regeneration factor receptor; wherein the tissue regeneration factor is selected from SEQ ID NO: 2 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homologue thereof; and wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof; and wherein the tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

9. The nucleic acid construct of claim 8, wherein the homologue or functional variant of the tissue regeneration factor or tissue regeneration factor precursor comprises a conserved motif, wherein the sequence of the conserved motif is selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

10. The nucleic acid construct according to claim 8 or 9, wherein the nucleic acid construct comprises a nucleic acid sequence encoding a tissue regeneration factor precursor or a tissue regeneration factor receptor.

11. The nucleic acid construct according to claim 8 or 9, wherein the nucleic acid construct comprises a nucleic acid sequence encoding a tissue regeneration factor precursor and a tissue regeneration factor receptor.

12. according to the nucleic acid construct described in any one in claim 8 to 11, the nucleic acid sequence of at least one of wherein said coding tissue regeneration factor, tissue regeneration factor precursor and tissue regeneration factor receptor is operably connected to one or more regulating and controlling sequences.

13. Nucleic acid construct according to claim 12, wherein the regulatory sequence is a constitutive or strong promoter or an inducible promoter, wherein the inducible promoter is a nopaline synthase promoter.

14. A host cell comprising the nucleic acid construct of any one of claims 8 to 13.

15. A transgenic plant, plant part or plant cell thereof, wherein said plant, part or plant cell thereof is characterized by increased expression and / or level of at least one of the following i. tissue regeneration factors; and / or ii. tissue regeneration factor precursor; and / or iii. Tissue regeneration factor receptor wherein the tissue regeneration factor is selected from SEQ ID NO: 2 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 89 or 20 or a functional variant or homologue thereof; and wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof; and wherein the tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

16. The transgenic plant according to claim 15, wherein the homologue or functional variant of the tissue regeneration factor or tissue regeneration factor precursor comprises a conserved motif, wherein the sequence of the conserved motif is selected from (SEQ ID NO: 79) GXPPXXNN, (SEQ ID NO: 80) SSGXXGXXN and (SEQ ID NO: 81) SGGXGGXHX, wherein X is any amino acid.

17. The transgenic plant, part thereof or plant cell according to claim 15 or 16, wherein the plant, part thereof or plant cell comprises the nucleic acid construct according to any one of claims 8 to 13.

18. The transgenic plant according to any one of claims 15 to 17, wherein the nucleic acid construct is stably incorporated into the plant genome.

19. A transgenic plant, plant part thereof or plant cell, wherein the plant is characterized by one or more mutations in the plant genome, wherein the mutation is the insertion of at least one additional copy of a nucleic acid sequence encoding a tissue regeneration factor precursor and / or at least one additional copy of a nucleic acid sequence encoding a tissue regeneration factor receptor, such that the one or more nucleic acid sequences are operably linked to a regulatory sequence, wherein the mutation is preferably introduced using targeted genome editing; wherein the tissue regeneration factor precursor is selected from SEQ ID NO: 22 or a functional variant or homologue thereof, wherein the homologue is selected from SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 91 or 40 or a functional variant or homologue thereof; and wherein the tissue regeneration factor receptor is selected from SEQ ID NO: 42 or a functional variant or homolog thereof, wherein the homolog is selected from SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 93 or 78 or a functional variant or homolog thereof.

20. Use of the tissue regeneration factor of claim 1, the plant tissue culture medium of any one of claims 2 to 8, or the nucleic acid construct of any one of claims 9 to 14 to increase the regeneration capacity of a plant or a part thereof.

21. The use according to claim 20, wherein the plant, part thereof or plant cell is a monocotyledonous plant or a dicotyledonous plant.

22. A method for increasing the regeneration efficiency of a plant, a plant part thereof or one or more plant cells, the method comprising i. applying the tissue regeneration factor according to claim 1 to the plant or part thereof or one or more plant cells; or ii. applying the plant tissue culture medium according to any one of claims 2 to 7 to the plant or part thereof or one or more plant cells; and / or iii. Introducing and expressing the nucleic acid construct according to any one of claims 8 to 13 in said plant or part thereof or in one or plant cells.

23. A method for producing a plant, a plant part thereof or one or more plant cells having increased regeneration efficiency, the method comprising i. applying the tissue regeneration factor according to claim 1 to the plant or part thereof or one or more plant cells; or ii. applying the plant tissue culture medium according to any one of claims 2 to 9 to the plant or part thereof or one or more plant cells; and / or iii. Introducing and expressing the nucleic acid construct according to any one of claims 8 to 13 in said plant or part thereof or in one or plant cells.

24. The method of claim 23, further comprising producing a plant from the one or more plant cells.

25. The method according to any one of claims 22 to 24, wherein 0.1-500 nmol / L of tissue regeneration factor is exogenously applied to the plant, part thereof or plant cell.

26. The method of any one of claims 22 to 24, wherein 0.1 to 0.5 mL of plant tissue culture medium is applied exogenously to the plant, part thereof or plant cell.

27. The method of any one of claims 22 to 26, wherein the method increases one or more of callus formation, shoot regeneration.

28. The method according to claim 27, wherein the plant part is an explant, wherein preferably, the explant is selected from the group consisting of a node, an apical meristem, a root, a cotyledon, an embryo, a leaf disk, a leaf blade, a pedicel, a petiole, pollen, a microspore, anther, an ovary, a hypocotyl.

29. The method of any one of claims 22 to 28, wherein the plant is a monocot or a dicot.

30. A method for increasing transformation efficiency in a plant, the method comprising: i. applying the tissue regeneration factor according to claim 1 to the plant or part thereof or one or more plant cells; or ii. applying the plant tissue culture medium according to any one of claims 2 to 9 to the plant or part thereof or one or more plant cells; and / or iii. Introducing and expressing the nucleic acid construct according to any one of claims 8 to 13 in said plant or part thereof or in one or plant cells.

Images