Methods for improving genome engineering and regeneration in plants II

Abstract

This document relates to methods and materials for genome engineering in eukaryotic cells, and in particular to methods for increasing the efficiency of genome engineering (i.e., transformation or genome editing) by co-delivering a combination of one or more enhancer polypeptides and enhancer genes with genome engineering components.

Description

Technical Field

[0001] Described herein are novel combinations of regenerative booster genes and polypeptides, as well as methods and materials for genome engineering in eukaryotic cells, and, in particular, methods for increasing the efficiency of genome engineering (i.e., transformation or genome editing) by co-delivering booster polypeptides and booster genes with genome engineering components. Background of the Invention

[0003] Traditional breeding has provided domesticated plants and animals, while modern biotechnology (especially genome engineering) is expanding breeding capabilities and achieving improvements that cannot be achieved using traditional hybridization of close species alone. Using biotechnology, a variety of traits such as high yield, herbicide tolerance and insect resistance have been introduced into crops, leading to great advances in global agriculture and food security. However, the presence of foreign DNA in such biotechnology products raises biosafety and environmental issues.

[0004] By isolating any integrated DNA, genome editing techniques can be used to generate site-specific modifications of the target genome without the presence of foreign DNA in the final plant. In addition, genome editing involves transient editing activity through transient expression to produce site-specific modifications without DNA integration at any point in the process. Genome-edited plants, particularly those derived from transient activity, are significantly different from conventional genome-modified plants and may not be regulated as genetically modified (GM) plants. Genome editing techniques (particularly through transient editing methods) can provide highly accurate, safe and powerful plant breeding and development tools in agriculture.

[0005] However, genome engineering based on transient activity faces more challenges. Compared with stable transformation, transient engineering usually leads to fewer modified cells. In the absence of integrated selectable markers, it is highly challenging to identify engineered cells and achieve homogeneous (homogenous) modifications in regenerated plants. These challenges hinder the conventional implementation of transient gene editing as a breeding tool for improved plants. Therefore, new methods and materials that enhance the efficiency of genome engineering are highly desirable. Summary of the invention

[0006] In a first aspect, nucleic acids encoding a first enhancer polypeptide and a second enhancer polypeptide are provided, wherein the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence at least 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% identical to SEQ ID NO:2; and wherein the second enhancer polypeptide comprises the amino acid sequence of any one of SEQ ID NO:4, 6 or 8, or an amino acid sequence at least 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% identical to SEQ ID NO: Any of NO:4, 6 or 8 has an amino acid sequence that is at least 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% identical.

[0007] In one embodiment of the first aspect of the invention, the nucleic acid encoding the first enhancer polypeptide comprises a coding sequence selected from the group consisting of:

[0008] (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1;

[0009] (ii) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1; and

[0010] (iii) a nucleic acid that hybridizes under stringent hybridization conditions to the complementary strand of the nucleic acid defined in (i) or (ii); and

[0011] The nucleic acid encoding the second enhancer polypeptide comprises a coding sequence selected from the group consisting of:

[0012] (I) a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 3, 5 or 7;

[0013] (II) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to any one of SEQ ID NOs: 3, 5, or 7; and

[0014] (III) a nucleic acid that hybridizes under stringent hybridization conditions to the complementary strand of the nucleic acid defined in (I) or (II). In a second aspect, a nucleic acid encoding a third enhancer polypeptide and a second enhancer polypeptide is provided, wherein the third enhancer polypeptide comprises an amino acid sequence of SEQ ID NO: 16, 18 or 20, or an amino acid sequence having at least 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% identity to SEQ ID NO: 16, 18 or 20; and wherein the second enhancer polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 4, 6 or 8, or an amino acid sequence having at least 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% identity to SEQ ID NO: 4, 6 or 8. NO:4, 6 or 8 has an amino acid sequence that is at least 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% identical, wherein expression of the first enhancer polypeptide is activated after translation of the third enhancer polypeptide; wherein the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:2 or is identical to SEQ ID NO:3. NO:2 has an amino acid sequence that is at least 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% identical.

[0015] In one embodiment of the second aspect of the invention, the nucleic acid encoding the third enhancer polypeptide comprises a coding sequence selected from the group consisting of:

[0016] (i) a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 15, 17 or 19;

[0017] (ii) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to any one of SEQ ID NOs: 15, 17 or 19; and

[0018] (iii) a nucleic acid that hybridizes under stringent hybridization conditions to the complementary strand of the nucleic acid defined in (i) or (ii); and

[0019] The nucleic acid encoding the second enhancer polypeptide comprises a coding sequence selected from the group consisting of:

[0020] (I) a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 3, 5 or 7;

[0021] (II) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to any one of SEQ ID NOs: 3, 5, or 7; and

[0022] (III) A nucleic acid that hybridizes to a complementary strand of the nucleic acid defined in (I) or (II) under stringent hybridization conditions.

[0023] In another aspect, there is provided a recombinant gene comprising a nucleic acid as defined above in the first and / or second aspect.

[0024] In some embodiments of the invention, the nucleic acid encoding the first enhancer polypeptide is operably linked to a native promoter or a first heterologous promoter, and the nucleic acid encoding the second enhancer polypeptide is operably linked to a native promoter or a second heterologous promoter; wherein the nucleic acid encoding the first enhancer polypeptide and the nucleic acid encoding the second enhancer polypeptide are operably linked to one heterologous promoter.

[0025] In some embodiments of the invention, the nucleic acid encoding the second enhancer polypeptide is operably linked to the native promoter or the first heterologous promoter, and the nucleic acid encoding the third enhancer polypeptide is operably linked to the native promoter or the second heterologous promoter; or the nucleic acid encoding the second enhancer polypeptide and the nucleic acid encoding the third enhancer polypeptide are operably linked to a heterologous promoter.

[0026] In some embodiments of the invention, the heterologous promoter can be a strong constitutive promoter, a tissue-specific promoter, a developmental-specific promoter, or an inducible promoter.

[0027] In another aspect, a DNA construct, preferably a vector, comprising the above-mentioned nucleic acid or recombinant gene is provided.

[0028] In another aspect, a plant cell is provided, comprising a nucleic acid, a recombinant gene or a DNA construct, in particular as a transgene or as a heterologous polypeptide or a heterologous nucleic acid.

[0029] Another aspect of the present invention is a plant, a part thereof or a seed comprising the above-mentioned plant cell.

[0030] In another aspect of the present invention, a method for genetic modification in plant cells is provided, the method comprising

[0031] (a) Introduce the following into plant cells

[0032] (i) a component selected from the group consisting of:

[0033] (ia) the above-mentioned nucleic acids, recombinant genes and / or DNA constructs; or

[0034] (i.b1) a nucleic acid encoding a first enhancer polypeptide; a recombinant gene comprising a nucleic acid encoding a first enhancer polypeptide, which is preferably operably linked to a promoter as defined above; a DNA construct, preferably a vector, comprising a nucleic acid encoding a first enhancer polypeptide as defined above, or the first enhancer polypeptide as defined above; and

[0035] (i.b2) a nucleic acid encoding the second enhancer polypeptide as defined above; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, which is preferably operably linked to a promoter as defined above, or a DNA construct, preferably a vector, comprising a nucleic acid encoding the second enhancer polypeptide, or the second enhancer polypeptide as defined above; or

[0036] (i.c1) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the first enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the first enhancer polypeptide; wherein the nucleic acid encoding the first enhancer polypeptide is an endogenous nucleic acid; and

[0037] (i.c2) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide; wherein the nucleic acid encoding the second enhancer polypeptide is an endogenous nucleic acid; or

[0038] (id) a combination of (i.b1) and (i.c2), or a combination of (i.b2) and (i.c1); and

[0039] (ii) transgenic and / or genome engineered components of interest;

[0040] (b) optionally, culturing the plant cell under conditions permissive for translation of the first and second enhancer polypeptides, preferably wherein translation is increased compared to a plant cell into which (ia), (i.b1) and (i.b2), (i.c1) and (i.c2), or (id) has not been introduced; and

[0041] (c) optionally, culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of a transgene of interest or by activity of a genome engineering component in the presence of the enhancer polypeptide.

[0042] Optionally, a method for genetic modification in a plant cell is provided, the method comprising:

[0043] (a) Introduce the following into plant cells

[0044] (i) a component selected from the group consisting of:

[0045] (ia) the above-mentioned nucleic acids, recombinant genes and / or DNA constructs; or

[0046] (i.b1) a nucleic acid encoding a third enhancer polypeptide; a recombinant gene comprising a nucleic acid encoding a third enhancer polypeptide, which is preferably operably linked to a promoter as defined above, a DNA construct, preferably a vector, comprising a nucleic acid encoding a third enhancer polypeptide as defined above, or the third enhancer polypeptide as defined above; and

[0047] (i.b2) a nucleic acid encoding the second enhancer polypeptide as defined above; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, which is preferably operably linked to a promoter as defined above, or a DNA construct, preferably a vector, comprising a nucleic acid encoding the second enhancer polypeptide, or the second enhancer polypeptide as defined above, or

[0048] (i.c1) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the third enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the third enhancer polypeptide; wherein the nucleic acid encoding the third enhancer polypeptide is an endogenous nucleic acid; and

[0049] (i.c2) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide; wherein the nucleic acid encoding the second enhancer polypeptide is an endogenous nucleic acid; or

[0050] (id) a combination of (i.b1) and (i.c2), or a combination of (i.b2) and (i.c1); and

[0051] (ii) transgenic and / or genome engineered components of interest;

[0052] (b) optionally, culturing the plant cell under conditions permissive for translation of the third and second enhancer polypeptides, preferably wherein translation is increased compared to a plant cell into which (ia), (i.b1) and (i.b2), (i.c1) and (i.c2), or (id) has not been introduced; and

[0053] (c) optionally, culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of a transgene of interest or by activity of a genome engineering component in the presence of the enhancer polypeptide.

[0054] In one embodiment of the above method, the first enhancer polypeptide and the second enhancer polypeptide, or the third enhancer polypeptide and the second enhancer polypeptide from the components of step (i) are transiently present, transiently active or transiently expressed in the plant cell or wherein component (i) is transiently present, transiently active or transiently expressed in the plant cell.

[0055] In another embodiment of the above method, step (i) of introducing components into plant cells additionally comprises introducing into the plant cells a PLT5 polypeptide, a KWS-RBP1 polypeptide, a KWS-RPB2 polypeptide, a RKD4 polypeptide and a RKD2 polypeptide, and / or one or more nucleic acids selected from the group consisting of nucleic acids encoding a PLT5 polypeptide, a KWS-RBP1 polypeptide, a KWS-RPB2 polypeptide, a RKD4 polypeptide and a RKD2 polypeptide, and / or one or more site-directed transcriptional activators suitable for transiently increasing the expression of an endogenous PLT5 polypeptide, an endogenous RKD4 polypeptide or an endogenous RKD2 polypeptide, and / or a nucleic acid encoding such a site-directed transcriptional activator.

[0056] In another embodiment of the above method, the PLT5 polypeptide, KWS-RBP1 polypeptide, KWS-RPB2 polypeptide, RKD4 polypeptide and RKD2 polypeptide are transiently present, transiently active or transiently expressed in the plant cell, or the nucleic acids encoding the PLT5 polypeptide, KWS-RBP1 polypeptide, KWS-RPB2 polypeptide, RKD4 polypeptide and RKD2 polypeptide are transiently present, transiently active or transiently expressed in the plant cell.

[0057] In another embodiment of the above method, the first and second enhancer polypeptides and the PLT5 polypeptide are introduced into the plant cell and optionally transiently co-expressed; and / or the first and second enhancer polypeptides and the KWS-RBP1 polypeptide are introduced into the plant cell and optionally transiently co-expressed; and / or the first and second enhancer polypeptides and the RKD4 polypeptide are introduced into the plant cell and optionally transiently co-expressed, and / or the first and second enhancer polypeptides and the RKD2 polypeptide are introduced into the plant cell and optionally transiently co-expressed.

[0058] In another embodiment of the above method, the third and second enhancer polypeptides and the PLT5 polypeptide are introduced into the plant cell and optionally transiently co-expressed; and / or the third and second enhancer polypeptides and the KWS-RBP1 polypeptide are introduced into the plant cell and optionally transiently co-expressed; and / or the third and second enhancer polypeptides and the KWS-RBP2 polypeptide are introduced into the plant cell and optionally transiently co-expressed; and / or the third and second enhancer polypeptides and the RKD4 polypeptide are introduced into the plant cell and optionally transiently co-expressed, and / or the third and second enhancer polypeptides and the RKD2 polypeptide are introduced into the plant cell and optionally transiently co-expressed.

[0059] In another embodiment of the above method, the PLT5 polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or 12, or an amino acid sequence that is at least 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% identical to SEQ ID NO: 10 or 12; or the nucleic acid encoding the PLT5 polypeptide encodes the amino acid sequence of SEQ ID NO: 10 or 12, or encodes an amino acid sequence that is at least 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% identical to SEQ ID NO: 10 or 12. NO: 10 or 12 having an amino acid sequence that is at least 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% identical; or a KWS-RBP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 14, or having an amino acid sequence that is at least 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% identical. ID NO: 14 has an amino acid sequence that is at least 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% identical; or a nucleic acid encoding a KWS-RBP1 polypeptide encodes an amino acid sequence of SEQ ID NO: 14, or encodes an amino acid sequence that is at least 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% identical. ID NO: 14 has an amino acid sequence that is at least 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% identical; or a KWS-RBP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or is identical to SEQ ID NO: 51. NO:50 has an amino acid sequence that is at least 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% identical; or a nucleic acid encoding a KWS-RBP1 polypeptide encodes the amino acid sequence of SEQ ID NO:50, or encodes an amino acid sequence identical to SEQ ID NO:50. NO:50 has an amino acid sequence that is at least 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% identical;or the RKD4 polypeptide comprises the amino acid sequence of SEQ ID NO:16, 18 or 20, or an amino acid sequence that is at least 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% identical to SEQ ID NO:16, 18 or 20; or the nucleic acid encoding the RKD4 polypeptide encodes the amino acid sequence of SEQ ID NO:16, 18 or 20, or encodes an amino acid sequence that is at least 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% identical to SEQ ID NO:16, 18 or 20. NO: 16, 18 or 20 having an amino acid sequence that is at least 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% identical; or the RKD2 polypeptide comprises the amino acid sequence of SEQ ID NO: 22, 24 or 26, or is identical to SEQ ID NO: NO: 22, 24 or 26 has an amino acid sequence that is at least 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% identical; or the nucleic acid encoding the RKD2 polypeptide encodes the amino acid sequence of SEQ ID NO: 22, 24 or 26, or encodes an amino acid sequence that is at least 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% identical. NO: 22, 24 or 26 has an amino acid sequence that is at least 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% identical. ;

[0060] In another embodiment of the above method, the nucleic acid encoding the PLT5 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of:

[0061] (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9 or 11;

[0062] (ii) comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 9 or 11;

[0063] (iii) a nucleic acid that hybridizes under stringent hybridization conditions to the complementary strand of the nucleic acid defined in (i) or (ii);

[0064] The nucleic acid encoding the KWS-RBP1 polypeptide comprises a nucleic acid having a coding sequence selected from the group consisting of:

[0065] (I) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 13;

[0066] (II) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO:13;

[0067] (III) a nucleic acid that hybridizes to the complementary strand of the nucleic acid defined in (I) or (III) under stringent hybridization conditions;

[0068] Nucleic acids encoding KWS-RBP2 polypeptides include nucleic acids having a coding sequence selected from the group consisting of:

[0069] (A) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 49;

[0070] (B) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO:49;

[0071] (C) a nucleic acid that hybridizes to the complementary strand of the nucleic acid defined in (A) or (B) under stringent hybridization conditions;

[0072] Nucleic acids encoding RKD4 polypeptides include nucleic acids having a coding sequence selected from the group consisting of:

[0073] (1) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19;

[0074] (2) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19; and

[0075] (3) a nucleic acid that hybridizes to the complementary strand of the nucleic acid defined in (1) or (2) under stringent hybridization conditions;

[0076] Nucleic acids encoding RKD2 polypeptides include nucleic acids having a coding sequence selected from the group consisting of:

[0077] a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 21, 23 or 25;

[0078] b) a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 21, 23 or 25; and

[0079] c) A nucleic acid that hybridizes under stringent hybridization conditions to the complementary strand of a nucleic acid as defined in a) or b).

[0080] In another embodiment of the above method, the genome engineering component comprises

[0081] a) an enzyme that induces a double strand break (DSB) or a nucleic acid encoding the same, wherein the DSB inducing enzyme optionally recognizes a predetermined site in the genome of said cell, and wherein the DSB inducing enzyme optionally is a repair nucleic acid molecule;

[0082] b) an enzyme that induces a single strand break (SSB) or a nucleic acid encoding the same, wherein the SSB inducing enzyme optionally recognizes a predetermined site in the genome of said cell, and wherein the SSB inducing enzyme optionally is a repair nucleic acid molecule;

[0083] c) a base editor enzyme, optionally fused to a disarmed DSB-inducing enzyme or a disarmed SSB-inducing enzyme, wherein the base editor enzyme preferentially recognizes a predetermined site in the genome of said cell; or

[0084] d) an enzyme that affects DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination, which is optionally fused to a disarmed DSB-inducing enzyme or a disarmed SSB-inducing enzyme, wherein the enzyme optionally recognizes a predetermined site in the genome of the cell.

[0085] In another embodiment of the above method, the genome engineering component comprises a DSB-inducing enzyme or a SSB-inducing enzyme or a variant thereof, which is a CRISPR / Cas endonuclease, a CRISPR / Cas9 endonuclease, a CRISPR / Cpf1 endonuclease, a CRISPR / Csm1 endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease or a TAL effector nuclease.

[0086] In another embodiment of the above method, the activity of the genome engineering component comprises inducing one or more double-strand breaks in the genome of the plant cell, inducing one or more single-strand breaks in the genome of the plant cell, inducing one or more base editing events in the genome of the plant cell, or inducing one or more DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination in the genome of the plant cell. The induction of one or more double-strand breaks or one or more single-strand breaks is followed by non-homologous end joining (NHEJ) and / or homology-directed repair of the breaks by homologous recombination mechanism (HDR).

[0087] In another embodiment of the above method, the transgene is selected from genes encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphorus deficiency, salt stress or waterlogging, genes encoding herbicide resistance, including resistance to glyphosate, glufosinate / phosphinothricin, hygromycin, protoporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors and Dicamba, genes encoding resistance or tolerance to biotic stress, including viral resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes, or genes encoding yield-related traits, including lodging resistance, flowering time, shattering resistance, seed color, endosperm composition or nutritional content.

[0088] In another embodiment of the above method, the modification of the genome is selected from i) substitution of at least one nucleotide; ii) deletion of at least one nucleotide; iii) insertion of at least one nucleotide; iv) change of DNA methylation; v) change of histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histidine SUMOylation, histone ribosylation or histone citrullination; and vi) any combination of i)-v).

[0089] In some embodiments, the method is effective to promote cell proliferation or cell regeneration, preferably after genetic modification / genomic modification, or the method is effective to increase the regeneration efficiency of a transgenic plant, a gene-edited plant, or a base-edited plant.

[0090] In some embodiments, the methods are effective to induce embryogenesis, directly or indirectly, from a single cell (preferably an embryonic cell, a somatic cell or a protoplast) or from a callus cell, preferably following genetic / genomic modification.

[0091] In some embodiments, the methods are effective to increase the efficiency of stable transformation of a transgene into a plant cell or to increase the efficiency of generation of a transgenic plant.

[0092] In some embodiments, the methods are effective to increase the efficiency of genome engineering components to edit the genome of a plant cell or to increase the efficiency of generating a transgenic, gene-edited, or base-edited plant.

[0093] In some embodiments, the methods are effective to improve the regeneration efficiency of plants derived from recalcitrant genotypes, effective to improve the efficiency of regenerating plants from unconventional tissue types, or effective to accelerate the regeneration process, preferably following genetic modification / genomic modification.

[0094] In another embodiment of the above method, the site-directed transcriptional activator or the nucleic acid encoding it comprises at least one recognition domain and at least one activation domain, wherein the site-directed transcriptional activator is configured to increase the expression of a first enhancer polypeptide from an endogenous nucleic acid, a second enhancer polypeptide from an endogenous nucleic acid, a third enhancer polypeptide from an endogenous nucleic acid, an endogenous PLT5 polypeptide, an endogenous RKD4 polypeptide, or an endogenous RKD2 polypeptide, preferably by binding to a regulatory region located at a certain distance from the start codon of the first endogenous enhancer polypeptide, the second endogenous enhancer polypeptide, the third endogenous enhancer polypeptide, the endogenous PLT5 polypeptide, the endogenous RKD4 polypeptide, or the endogenous RKD2 polypeptide.

[0095] In another embodiment of the above method, at least one recognition domain is a molecule or fragment of a molecule selected from the group consisting of: at least one TAL effector, at least one disarmed CRISPR / nuclease system, at least one zinc finger domain, and at least one disarmed homing endonuclease, or any combination thereof.

[0096] In another embodiment of the above method, at least one disarmed CRISPR / nuclease system is selected from a CRISPR / dCas9 system, a CRISPR / dCpf1 system, a CRISPR / dCms1 system, a CRISPR / dCasX system or a CRISPR / dCasY system or any combination thereof, wherein at least one disarmed CRISPR / nuclease system comprises at least one guide RNA. In one embodiment of the above method, at least one activation domain is an acidic transcriptional activation domain, preferably at least one activation domain is a TAL effector gene from Xanthomonas oryzae, VP16 or tetrameric VP64 from herpes simplex, VPR, SAM, Scaffold, Suntag, P300, VP160 or any combination thereof.

[0097] Another aspect of the present invention is a method for improving plant regeneration efficiency or enhancing plant cell regeneration capacity, which comprises introducing the component (i), nucleic acid, recombinant gene or DNA construct defined above into plant cells.

[0098] In another aspect of the present invention, there is provided a genetically modified plant cell obtained or obtainable according to the above-mentioned method for genetic modification in a plant cell.

[0099] Another aspect is a plant or a plant part comprising a genetically modified plant cell as described above.

[0100] Another aspect is a microparticle coated with at least the components of (i) above described method for genetic modification in plant cells, a nucleic acid, a recombinant gene or a DNA construct as defined above.

[0101] In another embodiment, the microparticles are further coated with a genome engineering component.

[0102] Another aspect of the invention is a kit for genetically modifying the genome of a plant by microprojectile bombardment, said kit comprising (I) one or more microparticles as described above, and (II) means for coating the microparticles as defined above.

[0103] In one embodiment, the kit further comprises means for coating the microparticles with the genome engineering components.

[0104] In another aspect, the present invention provides a method for producing a genetically modified plant, comprising the steps of: (a) genetically modifying a plant cell according to any method for genetic modification in a plant cell as described above, and (b) regenerating a plant from the modified plant cell of step (a).

[0105] In one embodiment, the produced plant does not comprise any of the genome engineering components, enhancer genes and enhancer polypeptides introduced in step (a).

[0106] Another aspect is a genetically modified plant or part thereof obtained or obtainable by the above-described method for producing a genetically modified plant.

[0107] Another aspect of the present invention is the use of the component, nucleic acid, recombinant gene or DNA construct of (i) above for improving the efficiency of plant regeneration or enhancing the regeneration capacity of plant cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 A map of the enhanced gene expression vector pABM-BdEF1 (SEQ ID NO: 30) is shown. BdEF1 and nos-T define a strong constitutive promoter and nos terminator from the Brachypodium EF1 gene, respectively. BamHI and HindIII illustrate cloning sites.

[0110] Figure 2 A map of the maize WUS2 expression construct pABM-BdEF1_ZmWUS2 (SEQ ID NO: 28) is shown. The maize WUS2 gene (ZmWUS2) is driven by the strong constitutive EF1 promoter from Brachypodium (pBdEF1).

[0111] Figure 3 A map of the maize PLT7 expression construct pABM-BdEF1_ZmPLT7 (SEQ ID NO: 26) is shown. The maize PLT7 gene (ZmPLT7) is driven by the strong constitutive EF1 promoter from Brachypodium (pBdEF1).

[0112] Figure 4 A map of the KWS-RBP1 expression construct pABM-BdEF1-KWS-RBP1 (SEQ ID NO: 27) is shown. The KWS-RBP1 gene is driven by the strong constitutive EF1 promoter from Brachypodium (pBdEF1).

[0113] Figure 5 A map of the wheat RKD4 expression construct pABM-BdEF1-TaRKD4 (SEQ ID NO: 28) is shown. The wheat RKD4 (TaRKD4 gene is driven by the strong constitutive EF1 promoter from Brachypodium (pBdEF1).

[0114] Figure 6A map of the genome editing CRISPR Cpf1 expression construct pGEP359 (SEQ ID NO: 29) is shown. tDTomato defines the tdTomato gene (tDT). ZmLpCpf1 defines the maize codon-optimized CDS of the Lachnospiraceae bacterial CRISPR / Cpf1 (LbCpf1) gene.

[0115] Figure 7 A map of the genome editing CRISPR RNA construct pGEP324 (SEQ ID NO: 30) is shown. crGEP05 defines crRNA5 that targets the maize HMG13 gene. ZmUbil defines the promoter and introns from the maize ubiquitin 1 gene. Tnos defines the nos terminator.

[0116] Figure 8 A map of the maize PLT5 expression construct pABM-BdEF1_ZmPLT5 (SEQ ID NO: 25) is shown. The maize PLT5 gene (ZmPLT5) is driven by the strong constitutive EF1 promoter from Brachypodium (pBdEF1).

[0117] Fig. 9 Shown in the enhancer gene and pGEP359 ( Figure 6 ) and pGEP324( Figure 7 ) Fluorescence image of A188 immature embryos 18 hours after plasmid co-bombardment. The image was taken 18 hours after bombardment.

[0118] Fig.10 Transient co-expression of ZmWUS2 and ZmPLT7 is shown to promote embryogenesis in A188 immature embryos. Images show embryogenic structures induced from maize A188 embryos 1 month after co-bombardment with booster gene constructs and tDT constructs (B). Fig.10 A shows maize A188 embryos 1 month after co-bombardment with tDT construct alone.

[0119] Fig.11 Shown in A188 immature embryos promote the transient co-expression of ZmWUS2 and ZmPLT7 of embryogenesis.Image shows the embryogenesis structure induced from maize A188 embryo 7 days and 1 month after co-bombardment with booster gene construct.Red fluorescent image (asterisk) shows the structure of expression tDT produced from maize A188 embryo.Other images are obtained as reference by binocular microscope.

[0120] Fig.12 Shown is the quantification of embryonic structures formed with Tdt 1 month after bombardment with Tdt alone, with Tdt and PLT7, with Tdt and WUS2, and with Tdt, WUS2, and PLT7.

[0121] Fig.13 Shown are adult plants regenerated from stable transformation of the maize A188 genotype: (A) normal A188 plant (non-transgenic), (B) stable transgenic integration of ZmWUS2, and (C, D) stable transgenic integration of ZmWUS2 and ZmPLT7.

[0122] Fig.14 Transient expression of ZmPLT7 promotes stable transformation of the co-delivered tDT reporter gene in maize Hi II embryos. Red fluorescent images show stable tDT-expressing constructs generated from maize Hi II embryos 10 days after co-bombardment ( Fig.14 A and 14B). Fig.14 C shows that delivery of ZmPLT7 increased the stable transformation frequency of the tDT reporter gene in Hi II immature embryos by more than 20%. Results were obtained 10 days after bombardment.

[0123] Fig.15 Shown are adult plants regenerated from stable transformation of maize Hi II genotypes: (A) normal A188 plants (non-transgenic), (B) stable transgenic integration of ZmPLT7.

[0124] Fig.16 A map of the maize WUS2 (ZmWUS2) promoter reporter construct pAMK-ZmWUS2-tDT-nosT (SEQ ID NO: 29) is shown. tDToma defines a fluorescent tDT reporter gene driven by the maize WUSCHEL2 promoter (pZmWUS2).

[0125] Fig.17 The wheat TaRKD4 gene is shown to activate the maize WUS2 promoter by transient co-bombardment in maize immature embryos IE (upper panel) and leaves (lower panel). Fig.17 A shows the maize WUS2 promoter reporter construct only ( Fig.16 ; SEQ ID NO: 29) (reported only for pZmWUS2). Fig.17 B shows the maize WUS promoter reporter construct and the wheat RKD4 construct ( Figure 5 ) co-bombarded with pZmWUS2 reporter and TaRKD4. Images were taken 44 hours after bombardment. DETAILED DESCRIPTION OF THE INVENTION

[0127] definition

[0128] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0129] As used in the context of this application, the term "about" refers to + / -10% of the value, preferably + / -5% of the value. For example, about 100 nucleotides (nt) should be understood as a value between 90nt and 110nt, preferably between 95nt and 105nt.

[0130] As used herein, the terms "enhancer", "enhancer gene", "enhancer polypeptide", "enhancer polypeptide", "enhancer gene" and "enhancer factor" refer to proteins / peptides or (poly)nucleic acid fragments encoding proteins / polypeptides, which can lead to improved genome engineering and / or improved plant regeneration of transformed or gene-edited plant cells. Such proteins / polypeptides can improve the performance or ability of plant cells (preferably derived from somatic tissue, embryonic tissue, callus or protoplasts) to regenerate in whole plants (preferably in fertile plants). Therefore, they can regulate the formation of somatic embryos (somatic embryogenesis) and / or they can increase the proliferation rate of plant cells. Exemplary enhancer polypeptides include, but are not limited to, the first enhancer polypeptide (e.g., SEQ ID NO: 2) and the second enhancer polypeptide (SEQ ID NO: 4, 6 and 8) according to the present invention or the third enhancer polypeptide (e.g., SEQ ID NO: 16, 18 and 20) and the second enhancer polypeptide (SEQ ID NO: 4, 6 and 8) according to the present invention and variants thereof. The regeneration of transformed or gene-edited plant cells may include the process of somatic embryogenesis, which is an artificial process in which a plant or embryo is derived from a single somatic cell or a group of somatic cells. Somatic embryos are formed from plant cells (i.e., plant tissues such as buds, leaves, shoots, etc.) that are not usually involved in embryo development. The application of this process may include: cloning genetically uniform plant materials; eliminating viruses; providing source tissues for genetic transformation; generating complete plants from single cells (such as protoplasts); developing synthetic seed technology. Cells derived from competent source tissues can be cultured to form callus. Plant growth regulators (such as auxins or cytokinins) in tissue culture media can be manipulated to induce callus formation, and plant growth regulators (such as auxins or cytokinins) in tissue culture media can be subsequently changed to induce embryo formation from callus. Somatic embryogenesis has been described in two ways, directly or indirectly. Direct embryogenesis occurs when embryos produce identical clones directly from explant tissue. Indirect embryogenesis occurs when an explant gives rise to undifferentiated or partially differentiated cells (i.e., callus) that then maintain or differentiate into plant tissues such as leaves, stems, or roots.

[0131] The term "transgenic" used in accordance with the present disclosure refers to a plant, plant cell, tissue, organ or material comprising a gene or genetic construct, which comprises a "transgene" that has been transferred from another organism to a plant, plant cell, tissue organ or material by natural means or by means of transformation technology. The term "transgenic" comprises a nucleic acid sequence (including DNA or RNA) or an amino acid sequence, or a combination or mixture thereof. Therefore, the term "transgenic" is not limited to sequences that are usually identified as "genes", i.e., sequences that encode proteins. It can also refer to, for example, DNA or RNA sequences that do not encode proteins. Therefore, the term "transgenic" generally means that the corresponding nucleic acid or amino acid sequence is not naturally present in the corresponding target cell (including plants, plant cells, tissues, organs or materials). Therefore, the term "transgenic" or "transgenic" used herein refers to such a nucleic acid sequence or amino acid sequence that is obtained or synthesized from the genome of an organism and then introduced into another organism in a transient or stable manner by artificial techniques such as molecular biology, genetics, etc. As used herein, "plant material" refers to any material that can be obtained from a plant during any developmental stage. Plant material can be obtained in planta or from in vitro cultures of plants or their plant tissues or organs. Thus, the term encompasses plant cells, tissues and organs and developing plant structures and subcellular components, such as nucleic acids, polypeptides and all chemical plant substances or metabolites that can be found in plant cells or compartments and / or can be produced by plants or can be obtained from extracts of any plant cell, tissue or plant at any stage of development. The term also encompasses derivatives of plant material (e.g., protoplasts) derived from at least one plant cell comprised by the plant material. Thus, the term also encompasses meristem cells or meristems of plants.

[0132] The term "genome engineering" used herein refers to strategies and techniques for genetic modification of any genetic information or genome of plant cells, including genome transformation and genome editing. Therefore, "genome editing" refers to the technology for targeting, specific modification of any genetic information or genome of plant cells. Therefore, the term includes both gene editing of gene coding regions and editing of regions other than the gene coding regions of genomes. It also includes editing or engineering of the nucleus (if present) and other genetic information of plant cells. In addition, "genome engineering" also includes epigenetic editing or engineering, i.e. targeted modification, such as methylation, histone modification or targeted modification of non-coding RNA that may cause heritable changes in gene expression.

[0133] As used herein, the term "genome editing" refers to the strategies and techniques for targeting, specific modification of any genetic information or genome of plant cells. Therefore, these terms include both gene editing and the editing of regions other than the gene coding region of the genome, such as intron sequences, non-coding RNA, miRNA, regulatory element sequences (such as promoters, terminators, transcription activator binding sites, cis or trans-acting elements). Additionally, "genome editing" may include base editing for targeted substitution of single nuclear bases. It may further include the editing of the nuclear genome and other genetic information (i.e., mitochondrial genome or chloroplast genome and miRNA, pre-mRNA or mRNA) of plant cells. In addition, "genome editing" may include epigenetic editing or engineering that may cause heritable changes in gene expression, i.e., targeted modification, such as DNA methylation or histone modification (such as histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination). "Genome editing" may also encompass epigenetic editing or engineering of non-coding RNA that may result in heritable changes in gene expression.

[0134] As used herein, "base editor" refers to a protein or a fragment thereof having the same catalytic activity as the protein from which it is derived, which has the ability to mediate targeted base modification when provided alone or in a molecular complex (referred to herein as a base editing complex), that is, if the base conversion does not cause a silent mutation but causes a conversion of the amino acid encoded by the codon containing the position to be converted by the base editor to be used, the conversion of the base of interest leads to a point mutation of interest, which in turn can lead to a targeted mutation.

[0135] As used herein, "regulatory element" refers to a nucleotide sequence that is not a part of a nucleotide sequence encoding a protein, but mediates the expression of a nucleotide sequence encoding a protein. Regulatory elements include, for example, promoters, cis-regulatory elements, enhancers, introns, or terminators. Depending on the type of regulatory element, it is located on a nucleic acid molecule before (i.e., 5') or after (i.e., 3') the nucleotide sequence encoding the protein. Regulatory elements function in living plant cells. The term "operably linked" refers to a regulatory element that is connected to a nucleotide sequence encoding a protein in this manner, that is, it is positioned on, for example, a nucleic acid molecule relative to the nucleotide sequence encoding the protein in this manner so that expression of the nucleotide sequence encoding the protein under the control of the regulatory element can occur in living cells.

[0136] As used herein, "upstream" refers to a position on a nucleic acid molecule that is closer to the 5' end of the nucleic acid molecule. Similarly, the term "downstream" refers to a position on a nucleic acid molecule that is closer to the 3' end of the nucleic acid molecule. For the avoidance of doubt, nucleic acid molecules and their sequences are usually expressed in their 5' to 3' direction (from left to right).

[0137] As used herein, a "flanking region" is a region of a repair nucleic acid molecule having a nucleotide sequence that is homologous to a nucleotide sequence of a DNA region that flanks (ie, upstream or downstream) a preselected site.

[0138] As used herein, "transient expression" refers to a phenomenon in which transferred proteins / polypeptides and nucleic acid fragments encoding proteins / polypeptides are transiently expressed and / or transiently active in cells and are not soon shut down and / or weakened as the cells grow.

[0139] As used herein, a "double-strand DNA break-inducing enzyme," "double-strand break-inducing enzyme," or "DSBI enzyme" is an enzyme that can induce double-strand DNA breaks at a specific nucleotide sequence known as a "recognition site" or "predetermined site." Thus, a "single-strand DNA or RNA break-inducing enzyme," "single-strand break-inducing enzyme," or "SSBI enzyme" is an enzyme that can induce single-strand DNA or RNA breaks at a specific nucleotide sequence known as a "recognition site" or "predetermined site."

[0140] As used herein, "repair nucleic acid molecule" is a single-stranded or double-stranded DNA molecule or RNA molecule that is used as a template for modifying genomic DNA or RNA at a preselected site near or at the cleavage site. As used herein, "used as a template for modifying genomic DNA" refers to the replication or integration of a repair nucleic acid molecule at a predetermined site by homologous recombination between a flanking region and a corresponding homologous region in a target genome flanking a preselected site, optionally in combination with non-homologous end joining (NHEJ) at one of the two ends of the repair nucleic acid molecule (e.g., in the case of only one flanking region).

[0141] As used herein, "modification of a genome" means that the genome has been altered at least one nucleotide or by at least one epigenetic edit.

[0142] As used herein, "preselected site," "predetermined site," or "predefined site" refers to a specific nucleotide sequence in a genome (e.g., a nuclear genome or a chloroplast genome) at which insertion, substitution, and / or deletion of one or more nucleotides is desired.

[0143] As used herein, "plant hormone" or "plant growth regulator" refers to any naturally occurring or synthetic material and chemical substance that promotes plant cell division and / or plant morphogenesis. As used herein, "regeneration" refers to the process by which a single or multiple cells proliferate and develop into tissues, organs and ultimately the entire plant.

[0144] As used herein, the term "vector" or "plasmid" refers to a construct that particularly includes a plasmid or (plasmid) vector, a cosmid, an artificial yeast chromosome or a bacterial artificial chromosome (YAC and BAC), a phagemid, a bacterial phage-based vector, an expression cassette, an isolated single-stranded or double-stranded nucleic acid sequence (which includes a sequence in linear or circular form), or an amino acid sequence, a viral vector (including modified viruses), and combinations or mixtures thereof for introduction or transformation, transfection or transduction into any eukaryotic cell (including plants, plant cells, tissues, organs or materials according to the present disclosure).

[0145] " recombinant " under the background of recombinant gene can comprise regulating sequence and / or positioning sequence.Recombinant construct according to the present invention or DNA construct can be integrated into carrier (comprising plasmid vector) or can be carrier (comprising plasmid vector), and / or it can for example separate existence from carrier structure with the form of single-stranded or double-stranded nucleic acid.After it is imported, for example, by utilizing the conversion or transfection of biological or physical means, recombinant gene or DNA construct also can for example remain on extrachromosomal with the form of double-stranded or single-stranded DNA, promptly do not integrate into the genome of target cell.Alternatively, recombinant gene or DNA construct can stably integrate into the genome of target cell, comprise the nuclear genome or other genetic elements of target cell, comprise the genome of plastid (such as mitochondria or chloroplast).

[0146] Enhancer polypeptide and nucleic acid encoding enhancer polypeptide

[0147] In a first aspect, nucleic acids encoding a first enhancer polypeptide and a second enhancer polypeptide are provided, wherein the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence at least 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% identical to SEQ ID NO:2; and wherein the second enhancer polypeptide comprises the amino acid sequence of any one of SEQ ID NO:4, 6 or 8, or an amino acid sequence at least 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% identical to SEQ ID NO: Any of NO:4, 6 or 8 has an amino acid sequence that is at least 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% identical.

[0148] In a second aspect, nucleic acids encoding a third enhancer polypeptide and a second enhancer polypeptide are provided, wherein the third enhancer polypeptide comprises an amino acid sequence of SEQ ID NO: 16, 18 or 20, or an amino acid sequence at least 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% identical to SEQ ID NO: 16, 18 or 20; and wherein the second enhancer polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 4, 6 or 8, or an amino acid sequence at least 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% identical to SEQ ID NO: NO:4, 6 or 8 has an amino acid sequence that is at least 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% identical, wherein expression of the first enhancer polypeptide is activated after translation of the third enhancer polypeptide; wherein the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:2 or is identical to SEQ ID NO:3. NO:2 has an amino acid sequence that is at least 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% identical.

[0149] The inventors show that the combination of the first and second enhancer polypeptides and the combination of the third and second enhancer polypeptides mediate unexpected strong enhancer effects. Through further combination with other enhancer polypeptides, even this effect can be further enhanced, especially in the early regeneration stage after delivering transgenic and / or genome engineering components. This enhanced effect will not damage plant development, and the regenerated plants demonstrate favorable plant growth and fertility in adulthood. Like this, the integration of enhancer genes or enhancer polypeptides can be isolated in the next generation by hybridization and selection.

[0150] In the various methods disclosed herein, any single enhancer polypeptide or combination of enhancer polypeptides can be transiently provided or co-expressed. The enhancer polypeptide itself can be introduced into the plant cell, or alternatively, a polynucleotide encoding the enhancer polypeptide can be introduced into the plant cell. Regarding combinations, one enhancer polypeptide can be introduced into the plant cell together with a nucleotide encoding another enhancer polypeptide or the same enhancer polypeptide. For example, a first enhancer polypeptide comprising the sequence of SEQ ID NO: 2 can be introduced into the plant cell together with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7 (which encodes a second enhancer polypeptide).

[0151] According to the first aspect, there is also provided a nucleic acid encoding a first enhancer polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a second enhancer polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8. Also provided is a nucleic acid encoding a first enhancer and a second enhancer polypeptide comprising the following, wherein the first enhancer polypeptide comprises an amino acid sequence having at least 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% identity to SEQ ID NO: 2 and wherein the second enhancer polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 4, 6 or 8 or a sequence identical to SEQ ID NO: 8. NO: 4, 6 or 8 have an amino acid sequence that is at least 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% identical. The nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 in combination with SEQ ID NO: 3, 5 or 7. The nucleic acid may comprise a nucleotide sequence encoding a first enhancer polypeptide at least 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% identical to SEQ ID NO: 1 and a nucleotide sequence encoding a second enhancer polypeptide at least 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% identical to SEQ ID NO: 3, 5 or 7.Alternatively, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1, and hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7, or hybridize under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: NO: 3, 5 or 7 hybridizes to the complementary strand of a nucleic acid having a nucleotide sequence that is at least 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% identical.

[0152] According to a second aspect, there is also provided a nucleic acid encoding a third enhancer polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 18 or 20 and a second enhancer polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8. Also provided is a nucleic acid encoding a third enhancer and a second enhancer polypeptide comprising the following, wherein the third enhancer polypeptide comprises an amino acid sequence having at least 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% identity to SEQ ID NO: 16, 18 or 20 and wherein the second enhancer polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 4, 6 or 8 or a sequence identical to any one of SEQ ID NO: 16, 18 or 20. The nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 in combination with SEQ ID NO: 3, 5 or 7. The nucleic acid may comprise a nucleotide sequence encoding a third enhancer polypeptide having at least 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% identity to SEQ ID NO: 15, 17 or 19 and a nucleotide sequence encoding a third enhancer polypeptide having at least 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% identity to SEQ ID NO: NO:3, 5 or 7 has a nucleotide sequence of a second enhancer polypeptide that is at least 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% identical.Alternatively, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19, or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19, and hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7, or hybridize under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: NO: 3, 5 or 7 hybridizes to the complementary strand of a nucleic acid having a nucleotide sequence that is at least 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% identical.

[0153] According to a first aspect, a recombinant gene comprising a nucleic acid encoding a first enhancer polypeptide and a second enhancer polypeptide is provided, wherein the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence at least 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% identical to SEQ ID NO:2, and the second enhancer polypeptide comprises the amino acid sequence of SEQ ID NO:4, 6 or 8, or an amino acid sequence at least 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% identical to SEQ ID NO:4, NO: 4, 6 or 8 has an amino acid sequence with at least 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% identity. The nucleic acid may be operably linked to one or more regulatory elements. The regulatory element may be a promoter, a cis-regulatory element, an enhancer, an intron or a terminator. The regulatory element may be 5' of the nucleic acid sequence. The regulatory element may be 3' of the nucleic acid sequence. The nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 in combination with the nucleotide sequence of SEQ ID NO: 3, 5 or 7. The nucleic acid may comprise a nucleotide sequence at least 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% identical to SEQ ID NO: 1 and a nucleotide sequence at least 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% identical to SEQ ID NO: 3, 5 or 7.The nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1, and the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7 or hybridize under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1. NO: 3, 5 or 7 hybridizes to the complementary strand of a nucleic acid having a nucleotide sequence that is at least 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% identical.

[0154] According to a second aspect, a recombinant gene comprising a nucleic acid encoding a third enhancer polypeptide and a second enhancer polypeptide is provided, wherein the third enhancer polypeptide comprises an amino acid sequence of SEQ ID NO: 16, 18 or 20, or an amino acid sequence at least 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% identical to SEQ ID NO: 16, 18 or 20, and the second enhancer polypeptide comprises an amino acid sequence of SEQ ID NO: 4, 6 or 8, or an amino acid sequence at least 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% identical to SEQ ID NO: NO: 4, 6 or 8 has an amino acid sequence that is at least 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% identical. The nucleic acid may be operably linked to one or more regulatory elements. The regulatory element may be a promoter, a cis-regulatory element, an enhancer, an intron or a terminator. The regulatory element may be 5' of the nucleic acid sequence. The regulatory element may be 3' of the nucleic acid sequence. The nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 in combination with the nucleotide sequence of SEQ ID NO: 3, 5 or 7. The nucleic acid may comprise a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19 and a nucleotide sequence that is at least 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% identical to SEQ ID NO: 3, 5 or 7.The nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19, and the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7 or hybridize under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: NO: 3, 5 or 7 hybridizes to the complementary strand of a nucleic acid having a nucleotide sequence that is at least 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% identical.

[0155] In some embodiments, the nucleic acid is operably linked to a heterologous promoter. The heterologous promoter can be a strong constitutive promoter (such as a double 35S promoter (d35S)), a tissue-specific promoter, a development-specific promoter, or an inducible promoter. The heterologous promoter can be a promoter from an EF1 gene (such as a Brachypodium EF1 gene (pBdEF1, SEQ ID NO: 30), a promoter from an ubiquitin 1 gene (such as a maize ubiquitin 1 gene), a WUSCHEL2 promoter (such as a maize WUSHCEL2 promoter (pZmWUS2)). The heterologous promoter can be a ubiquitin promoter described in U.S. Patent No. 6,528,701, which is incorporated herein by reference. Various tissue-specific promoters that can be used are described in U.S. Patent Nos. 7,763,774 and 7,767,801, each of which is incorporated herein by reference.

[0156] Also provided is a DNA construct, preferably a vector, comprising any of the above nucleic acids or recombinant genes. The nucleic acid may comprise a nucleic acid encoding a first enhancer polypeptide comprising the nucleotide sequence of SEQ ID NO: 1 and a nucleic acid encoding a second enhancer polypeptide comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7. The nucleic acid may comprise a nucleic acid encoding a third enhancer polypeptide comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 and a nucleic acid encoding a second enhancer polypeptide comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7. The nucleic acid encoding the first enhancer polypeptide may comprise a nucleotide sequence having at least 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% identity to SEQ ID NO: 1. The nucleic acid encoding the second enhancer polypeptide may comprise a nucleotide sequence that is at least 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% identical to SEQ ID NO:3, 5 or 7. The nucleic acid encoding the third enhancer polypeptide may comprise a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19. Alternatively, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1.In addition, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 3, 5 or 7. In addition, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19. In some embodiments, the DNA construct is a plasmid.

[0157] Plant Cells

[0158] In another aspect, a plant cell is provided, comprising the first and second enhancer polypeptides, the third and second enhancer polypeptides, nucleic acids, recombinant genes and DNA constructs described herein. In some embodiments, the first enhancer polypeptide comprises the amino acid sequence of SEQ ID NO: 2 and the second enhancer polypeptide comprises the amino acid sequence of SEQ ID NO: 4, 6 or 8. In some embodiments, the third enhancer polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 18 or 20 and the second enhancer polypeptide comprises the amino acid sequence of SEQ ID NO: 4, 6 or 8. In some embodiments, the first enhancer polypeptide comprises an amino acid sequence having at least 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% identity to SEQ ID NO: 2. In other embodiments, the second enhancer polypeptide comprises an amino acid sequence that is at least 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% identical to SEQ ID NO:4, 6 or 8. In some embodiments, the third enhancer polypeptide comprises an amino acid sequence that is at least 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% identical to SEQ ID NO: 16, 18 or 20. The nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7, or the nucleic acid may comprise a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7.The nucleic acid may comprise a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1 and a nucleotide sequence that is at least 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% identical to SEQ ID NO: 3, 5 or 7, or the nucleic acid may comprise a nucleotide sequence that is at least 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% identical to SEQ ID NO: NO:15, 17 or 19 having a nucleotide sequence at least 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% identical and a nucleotide sequence at least 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% identical to SEQ ID NO:3, 5 or 7. The nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 1. In addition, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3, 5 or 7 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 3, 5 or 7.In addition, the nucleic acid can hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19 or hybridize under stringent hybridization conditions to the complementary strand of a nucleic acid comprising a nucleotide sequence having at least 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% identity to SEQ ID NO: 15, 17 or 19. Also provided are plants, plant parts, seeds, embryos or callus tissue comprising plant cells.

[0159] The plant cell may be part of or derived from any type of plant material, preferably a bud, hypocotyl, cotyledon, stem, leaf, petiole, root, embryo, callus, flower, gametophyte or part thereof, or may be a protoplast or derived from a protoplast. Isolated plant cells as well as plant material, i.e. whole plants or parts of plants containing plant cells, may be used.

[0160] A part or parts of a plant may be attached to or separated from the entire intact plant. Such plant parts include, but are not limited to, organs, tissues and cells of the plant, and preferably seeds.

[0161] The plant cell, plant part or plant may be from any plant species, whether monocot or dicot. Preferably, the plant that can be subjected to the methods and uses of the present invention is a plant selected from the genera Hordeum, Sorghum, Saccharum, Zea, Setaria, Oryza, Triticum, Secale, Triticale, Malus, Brachypodium, Aegilops, Daucus, Beta, Eucalyptus, Nicotiana, Solanum, Coffea, Vitis, Erythrante, Genlisea, Cucumis, Marus, Arabidopsis. rabidopsis, Crucihimalaya, Cardamine, Lepidium, Capsella, Olmarabidopsis, Arabias, Brassica, Eruca, Raphanus, Citrus, Jatropha, Populus, Medicago, Cicer, Cajanus, Phaseolus, Glycine, Gossypium, Astragalus, Lotus, Torenia, Allium, or Helianthus. More preferably, the plant is selected from the group consisting of barley (Hordeum vulgare), bulbous barley (Hordeum bulbusom), sorghum (Sorghum bicolor), sugarcane (Saccharumofficinarium), Zea spp. including corn (Zea mays).), millet (Setariaitalica), small-grain rice (Oryza minuta), rice (Oryza sativa), Australian wild rice (Oryza australiensis), tall wild rice (Oryza alta), wheat (Triticum aestivum), durum wheat (Triticum durum), rye (Secale cereale), triticale (Triticale), apple (Malusdomestica), Brachypodium distachyon, seashore barley (Hordeum marinum), tauschii (Aegilops tauschii), Daucus glochidiatus, Beta spp. including Beta vulgaris), Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotianatomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Coffeacanephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii), Cardamine nexuosa, Lepidium virginicum, Capsella bursapastoris, Olmarabidopsis pumila, Arabis hirsute, Brassicanapus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicariasubsp.sativa), Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Jerusalem artichoke, tuberosus and / or leek (Allium tuberosum). Particularly preferred are sugar beet (Betavulgaris), corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), sunflower (Helianthus annuus), potato (Solanum tuberosum), bicolor sorghum (Sorghum bicolor), turnip (Brassica rapa), rapeseed (Brassica napus), mustard (Brassicajuncacea), cabbage (Brassica oleracea), radish (Raphanus sativus), rice (Oryza sativa), soybean (Glycine max) and / or cotton (Gossypium sp.). .

[0162] The genetically modified plant cell may be part of a whole plant or a part thereof. Therefore, the present invention also relates to a plant or plant part comprising the genetically modified plant cell described above.

[0163] Plant cells into which the genome engineering components have been (co-)introduced are cultured under conditions that allow genetic modification of the genome of said plant cells by integration of the transgene of interest and the activity of the genome engineering components in the presence of at least one enhancing factor.

[0164] Genetic modification of plant cells

[0165] A method for genetic modification in plant cells is also provided. The method comprises introducing into the plant cells (i) a component selected from the group consisting of:

[0166] (ia) the above-mentioned nucleic acids, recombinant genes and / or DNA constructs; or

[0167] (i.b1) a nucleic acid encoding a first enhancer polypeptide; a recombinant gene comprising a nucleic acid encoding a first enhancer polypeptide, which is preferably operably linked to a promoter as defined above, a DNA construct, preferably a vector, comprising a nucleic acid encoding a first enhancer polypeptide as defined above, or the first enhancer polypeptide as defined above; and

[0168] (i.b2) a nucleic acid encoding the second enhancer polypeptide as defined above; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, which is preferably operably linked to a promoter as defined above, or a DNA construct, preferably a vector, comprising a nucleic acid encoding the second enhancer polypeptide, or the second enhancer polypeptide as defined above; or

[0169] (i.c1) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the first enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the first enhancer polypeptide; wherein the nucleic acid encoding the first enhancer polypeptide is an endogenous nucleic acid; and

[0170] (i.c2) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide; wherein the nucleic acid encoding the second enhancer polypeptide is an endogenous nucleic acid; or

[0171] (id) a combination of (i.b1) and (i.c2), or a combination of (i.b2) and (i.c1); and

[0172] (ii) transgenic and / or genome engineering components. Plant cells can be cultured under conditions that allow the synthesis of a reinforcing sub-polypeptide from nucleic acid, recombinant gene or DNA construct. Plant cells can be cultured under conditions that allow the genome of the plant cells to be genetically modified by the activity of a genome engineering component when a reinforcing sub-polypeptide exists.

[0173] Alternatively, a method for genetic modification in a plant cell is provided. The method comprises introducing into the plant cell (i) a component selected from the group consisting of:

[0174] (ia) the nucleic acid, recombinant gene and / or DNA construct as described above; or

[0175] (i.b1) a nucleic acid encoding a third enhancer polypeptide; a recombinant gene comprising a nucleic acid encoding a third enhancer polypeptide, which is preferably operably linked to a promoter as defined above, a DNA construct, preferably a vector, comprising a nucleic acid encoding a third enhancer polypeptide as defined above, or the third enhancer polypeptide as defined above; and

[0176] (i.b2) a nucleic acid encoding the second enhancer polypeptide as defined above; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, which is preferably operably linked to a promoter as defined above, or a DNA construct, preferably a vector, comprising a nucleic acid encoding the second enhancer polypeptide, or the second enhancer polypeptide as defined above; or

[0177] (i.c1) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the third enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the third enhancer polypeptide; wherein the nucleic acid encoding the third enhancer polypeptide is an endogenous nucleic acid; and

[0178] (i.c2) a nucleic acid encoding a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide, or a site-directed transcriptional activator suitable for transiently increasing the expression of the second enhancer polypeptide; wherein the nucleic acid encoding the second enhancer polypeptide is an endogenous nucleic acid; or

[0179] (id) a combination of (i.b1) and (i.c2), or a combination of (i.b2) and (i.c1); and

[0180] (ii) transgenic and / or genome engineering components. Plant cells can be cultured under conditions that allow the synthesis of a reinforcing polypeptide from nucleic acid, recombinant gene or DNA construct. Plant cells can be cultured under conditions that allow the genome of the plant cell to be genetically modified by the activity of a genome engineering component in the presence of a reinforcing polypeptide.

[0181] Genome engineering components can be introduced as proteins and / or as nucleic acids encoding genome engineering components, particularly as DNA such as plasmid DNA, RNA, mRNA or RNP. Genome engineering can be used to prepare transgenic, gene-edited or base-edited plant materials.

[0182] For plant cells to be modified, transformation methods based on biological methods can be used, such as Agrobacterium transformation or viral vector-mediated plant transformation. A common biological means is transformation with Agrobacterium species, which has been used for various plant materials for decades. Viral vector-mediated plant transformation can also be used to import genetic material into cells of interest. Agrobacterium-mediated transformation refers to a method in which foreign DNA is delivered to plant cells using Agrobacterium tumefaciens (a soil bacterium that works as a natural genetic engineering vector). Agrobacterium tumefaciens can invade plants and transfer foreign DNA into a very wide range of plants.

[0183] Alternatively, transformation methods based on physical delivery methods, such as particle bombardment or microinjection, can be used. Particle bombardment includes biolistic transfection or microparticle-mediated gene transfer, which refers to a physical delivery method in which coated microparticles or nanoparticles containing nucleic acids or genetic constructs of interest are transferred into target cells or tissues. Physical introduction means are suitable for the introduction of nucleic acids, i.e., RNA and / or DNA, as well as proteins. Particle bombardment and microinjection have evolved as prominent techniques for the introduction of genetic material into plant cells or tissues of interest. Helenius et al., "Gene delivery into intact plants using the Helios TM Gene Gun", Plant Molecular Biology Reporter, 2000, 18 (3): 287-288 discloses a particle bombardment as a physical method for introducing material into plant cells. Thus, there are a variety of plant transformation methods to introduce genetic material in the form of genetic constructs into plant cells of interest, including biological and physical means known to those skilled in the field of plant biotechnology and that can be used to introduce at least one gene encoding at least one wall-associated kinase into at least one cell of at least one of a plant cell, tissue, organ or intact plant.

[0184] As used herein, the term "particle bombardment", also known as "biolistics transfection" or "microparticle-mediated gene transfer", refers to a physical delivery method for transferring coated microparticles or nanoparticles containing booster genes, booster polypeptides, genome engineering components and / or transgenes into target cells or tissues. The micro- or nanoparticles act as projectiles and are fired at the target structure of interest under high pressure using a suitable device (commonly referred to as a gene gun). Transformation by particle bombardment uses metal microprojectiles coated with the construct of interest, which are then fired at the target cells using a device called a "gene gun" (Sandford et al., 1987) at a high speed (~1500 km / h) fast enough to penetrate the target tissue cell wall, but not harsh enough to cause cell death. For protoplasts, whose cell walls are completely removed, the conditions are theoretically different. The precipitated construct on at least one microprojectile is released into the cell after bombardment. Acceleration of the microprojectile is achieved by high-voltage discharge or compressed gas (helium). Regarding the metal particles used, they must be non-toxic, non-reactive, and have a diameter smaller than that of the target cell. Gold or tungsten are most commonly used. A great deal of information on their general uses is publicly available from manufacturers and suppliers of gene guns and related systems.

[0185] In a particularly preferred embodiment of microprojectile bombardment, one or more booster genes, booster polypeptides, genome engineering components and / or transgenes are co-delivered via a microcarrier comprising gold particles having a size in the range of 0.4-1.6 micrometers (μm), preferably in the range of 0.4-1.0 μm. In an exemplary method, 10-1000 μg of gold particles, preferably 50-300 μg, are used per bombardment.

[0186] The booster gene, booster polypeptide, genome engineering components and / or transgenes can be delivered into target cells, for example, using a Bio-Rad PDS-1000 / He particle gun or a handheld Helios gene gun system. When the PDS-1000 / He particle gun system is used, the bombardment rupture pressure is 450 psi to 2200 psi, preferably 450-1100 psi, while the Helios gene gun system has a rupture pressure of 100-600 psi. More than one chemical substance or construct can be co-delivered into the target cell simultaneously with the genome engineering components.

[0187] The above-mentioned delivery methods for conversion and transfection can be applied to the tool of importing the present invention simultaneously. Similarly, there are specific conversion or transfection methods for importing nucleic acid or amino acid constructs of interest into plant cells specifically, including electroporation, microinjection, nanoparticles and cell penetrating peptides (CPP). In addition, there are chemical-based transfection methods to import genetic constructs and / or nucleic acids and / or proteins, especially including calcium phosphate transfection, using liposomes (e.g., cationic liposomes) transfection or using cationic polymers (including DEAD-dextran or polyethyleneimine) transfection or its combination. The above delivery techniques (alone or in combination) can be used for in vivo (including plant original position (in planta)) or in vitro methods.

[0188] In some embodiments, the genome engineering components comprise:

[0189] a) an enzyme that induces a double strand break (DSB) or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the DSB inducing enzyme optionally recognizes a predetermined site in the genome of the cell;

[0190] b) an enzyme that induces single strand breaks (SSBs) or a nucleic acid encoding the same, and optionally a repair nucleic acid molecule, wherein the SSB inducing enzyme optionally recognizes a predetermined site in the genome of the cell;

[0191] c) a base editor enzyme, optionally fused to a disarmed DSB-inducing enzyme or SSB-inducing enzyme, wherein the base editor enzyme preferentially recognizes a predetermined site in the genome of said cell; or

[0192] d) an enzyme that affects DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination, optionally fused to a disarmed DSB-inducing enzyme or SSB-inducing enzyme, wherein the enzyme preferably recognizes a predetermined site in the genome of the cell.

[0193] In order to have a break at a predetermined target site, the enzyme preferably includes a binding / recognition domain and a cleavage domain. Specific enzymes that can induce double-stranded or single-stranded breaks are nucleases or nickases and variants thereof, including molecules that no longer contain nuclease or nickase functions but act as recognition molecules in combination with another enzyme. In recent years, many suitable nucleases have been developed, particularly customized endonucleases, including meganucleases, zinc finger nucleases, TALE nucleases, Argonaute nucleases (derived from, for example, Natronobacterium gregoryi) and CRISPR nucleases (including, for example, Cas9, Cpf1, Csm1, CasX or CasY nucleases as part of a clustered regularly interspaced short palindromic repeat (CRISPR) system). Therefore, in a preferred aspect of the present invention, the genome engineering component comprises a DSB-inducing enzyme or a SSB-inducing enzyme or a variant thereof, which is selected from CRISPR / Cas endonuclease, preferably CRISPR / Cas9 endonuclease, CRISPR / Cpf1 endonuclease, or CRISPR / Csm1 endonuclease, zinc finger nuclease (ZFN), homing endonuclease, large-range nuclease and TAL effector nuclease.

[0194] Rare-cleaving endonucleases are DSBI / SSBI enzymes with a recognition site of preferably about 14 to 70 continuous nucleotides, and therefore have a very low cutting frequency even in larger genomes (such as most plant genomes). Homing endonucleases, also known as meganucleases, constitute a family of such rare-cutting endonucleases. They can be encoded by introns, independent genes or intervening sequences, and present surprising structural and functional properties that distinguish them from more classical restriction enzymes, usually from bacterial restriction modification type II systems. Their recognition sites have a general asymmetry, which is contrary to the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns or inteins have been shown to promote their respective genetic elements to home to alleles without introns or intein sites. By making site-specific double-strand breaks in intronless or inteinless alleles, these nucleases generate recombinogenic ends that participate in the gene conversion process that replicates the coding sequence and results in the insertion of introns or intervening sequences at the DNA level. A list of other rare-cutting meganucleases and their respective recognition sites is provided in Table I (pages 17 to 20) of WO03 / 004659, which is incorporated herein by reference.

[0195] In addition, there are methods for designing customized rare-cutting endonucleases that recognize essentially any target nucleotide sequence of choice. In short, a hybrid between a zinc finger domain designed to recognize a specific nucleotide sequence and a nonspecific DNA cleavage domain from a natural restriction enzyme such as Fokl can be used to prepare a chimeric restriction enzyme. Such methods are described, for example, in WO03 / 080809, WO94 / 18313 or WO95 / 09233 and Isalan et al., (2001), A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nature biotechnology, 19 (7): 656; Liu et al., (1997), Design of polydactyl zinc-finger proteins for unique addressing within complex genomes, Proceedings of the National Academy of Sciences, 94 (11): 5525-5530.

[0196] Another example of a specially designed endonuclease includes TALE nucleases (TALENs), which are based on transcription activator-like effectors (TALEs) from the genus Xanthomonas bacteria fused to the catalytic domain of the nuclease (e.g., FokI or a variant thereof). The DNA binding specificity of these TALEs is defined by repeated variable di-residues (RVDs) of 34 / 35 amino acid repeat units arranged in tandem, so that one RVD specifically recognizes one nucleotide in the target DNA. Repeat units can be assembled to recognize essentially any target sequence, and repeat units can be fused to the catalytic domain of the nuclease to produce sequence-specific endonucleases (see, e.g., Boch et al., (2009), Breaking the code of DNA binding specificity of TAL-type III effectors, Science, 326 (5959), 509-1512; Moscou & Bogdanove (2009). A simple cipher governs DNA recognition by TAL effectors, Science, 326 (5959), 1501-1501; and WO2010 / 079430, WO2011 / 072246, WO2011 / 154393, WO2011 / 146121, WO2012 / 001527, WO2012 / 093833, WO2012 / 104729, WO2012 / 138927, WO2012 / 138939). WO2012 / 138927 also describes monomeric (compact) TALENs and TALEs with various catalytic domains and combinations thereof.

[0197] Recently, a new type of customizable endonuclease system has been described; the so-called CRISPR / Cas system. The CRISPR system in its natural environment describes a molecular complex comprising at least one small and separate non-coding RNA combined with a Cas nuclease or another CRISPR nuclease such as a Cpf1 nuclease or a Csm1 nuclease (Zetsche et al., "Cpf1 Is a Single RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System", Cell, 163, pp. 1-13, October 2015; US 2017 / 0233756A1) that can produce specific DNA double-strand breaks. Currently, CRISPR systems are divided into two categories, including five types of CRISPR systems, such as type II systems with Cas9 as effectors, and type V systems with Cpf1 as effector molecules (Makarova et al., Nature Rev. Microbiol., 2015). In artificial CRISPR systems, synthetic non-coding RNAs and CRISPR nucleases and / or optionally modified CRISPR nucleases (modified to act as nickases or lack any nuclease function) can be used in combination with at least one synthetic or artificial guide RNA or gRNA that combines the functions of crRNA and / or tracrRNA (Makarova et al., 2015, supra). CRISPR / Cas-mediated immune responses in natural systems require CRISPR-RNA (crRNA), in which the maturation of the guide RNA that controls the specific activation of CRISPR nucleases varies significantly between the various CRISPR systems characterized to date. First, the invading DNA (also called the spacer) is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. The type II CRISPR system encodes the Cas9 nuclease as a key enzyme in the interference step, and the system contains both crRNA and trans-activating RNA (tracrRNA) as a guide motif. These hybridize and form a double-stranded (ds) RNA region, which is recognized by RNAseIII and can be cut to form a mature crRNA. These in turn then associate with Cas molecules in order to guide the nuclease specifically to the target nucleic acid region. Recombinant gRNA molecules can contain both variable DNA recognition regions and also Cas interaction regions and can therefore be specifically designed independently of the specific target nucleic acid and the desired Cas nuclease.

[0198] As another safety mechanism, a PAM (motif adjacent to the pre-spacer region) must be present in the target nucleic acid region; these are DNA sequences directly linked to the DNA recognized by the Cas9 / RNA complex. The PAM sequence for Cas9 from Streptococcus pyogenes has been described as "NGG" or "NAG" (standard IUPAC nucleotide code) (Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science 2012, 337: 816-821). The PAM sequence for Cas9 from Staphylococcus aureus is "NNGRRT" or "NNGRR (N)". Other variant CRISPR / Cas9 systems are known. Thus, Neisseria meningitidis Cas9 cuts at the PAM sequence NNNNGATT. Streptococcus thermophilus Cas9 cuts at the PAM sequence NNAGAAW. Recently, another PAM motif NNNNRYAC (WO2016 / 021973A1) for the CRISPR system of Campylobacter has been described. For Cpf1 nuclease, it has been described that the Cpf1-crRNA complex without tracrRNA effectively recognizes and cuts the target DNA via a short T-rich PAM, in contrast to the G-rich PAM usually recognized by the Cas9 system (Zetsche et al., supra). In addition, by using modified CRISPR polypeptides, specific single-strand breaks can be obtained. The combined use of Cas nickase and various recombinant gRNAs can also induce highly specific DNA double-strand breaks by means of double DNA nicking. In addition, by using two gRNAs, the specificity of DNA binding can be optimized, and thus DNA cutting can be optimized. Meanwhile, other CRISPR effectors originally described for bacteria, such as CasX and CasY effectors are available and represent additional effectors that can be used for genome engineering purposes (Burstein et al., “New CRISPR-Cas systems from uncultivated microbes”, Nature, 2017, 542, 237-241).

[0199] The cleavage site of the DSBI / SSBI enzyme is related to the exact position of the induced break on the DNA or RNA. The cleavage site may or may not be included in the recognition site of the DSBI / SSBI enzyme (may overlap with the recognition site of the DSBI / SSBI enzyme or may not overlap with the recognition site of the DSBI / SSBI enzyme), and it is therefore said that the cleavage site of the DSBI / SSBI enzyme is located at or near its recognition site. The recognition site of the DSBI / SSBI enzyme, sometimes also referred to as the binding site, is a nucleotide sequence that is recognized by the DSBI / SSBI enzyme (specificity) and determines its binding specificity. For example, a TALEN or ZNF monomer has a recognition site determined by its RVD repeats or ZF repeats, respectively, and its cleavage site is determined by its nuclease domain (e.g., FokI) and is generally located outside the recognition site. In the case of a dimeric TALEN or ZFN, the cleavage site is located between the two recognition / binding sites of the corresponding monomer, and the intervening DNA or RNA region where the cleavage occurs is called the spacer region.

[0200] Those skilled in the art will be able to select DSBI / SSBI enzymes that recognize a specific recognition site and induce DSB or SSB at a cleavage site at or near a preselected / predetermined site, or engineer such DSBI / SSBI enzymes. Alternatively, a DSBI / SSBI enzyme recognition site can be introduced into the target genome using any conventional transformation method or by hybridization with an organism having a DSBI / SSBI enzyme recognition site in its genome, and then any desired nucleic acid can be introduced at or near the cleavage site of the DSBI / SSBI enzyme.

[0201] In various embodiments, the modification of the genome comprises one or more of the following: i) substitution of at least one nucleotide; ii) deletion of at least one nucleotide; iii) insertion of at least one nucleotide; iv) change in DNA methylation; and v) change in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination.

[0202] In some embodiments, the activity of the genome engineering component induces one or more double-strand breaks in the genome of the plant cell, induces one or more single-strand breaks in the genome of the plant cell, induces one or more base editing events in the genome of the plant cell, or one or more of DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation, or histone citrullination in the genome of the plant cell.

[0203] In some embodiments, induction of one or more double-strand breaks or one or more single-strand breaks is followed by non-homologous end joining (NHEJ) and / or homology-directed repair of the breaks by a homologous recombination mechanism (HDR). NHEJ and HDR are two major and unique pathways for repairing breaks. Homologous recombination requires the presence of a homologous sequence (e.g., a repair nucleic acid molecule or "donor") as a template to guide the cell repair process, and the results of the repair are error-free and predictable. When the template (repair nucleic acid molecule or "donor") sequence for homologous recombination is missing, the cell typically attempts to repair the break by a non-homologous end joining (NHEJ) process.

[0204] In the particularly preferred aspect of this embodiment, the repair nucleic acid molecule is additionally imported into the plant cell.The repair nucleic acid molecule is a single-stranded or double-stranded DNA molecule or an RNA molecule, which is used as a template for modifying the genomic DNA or RNA at or near the preselected site of the cleavage site.In some embodiments, the repair nucleic acid molecule is used as a template for modifying genomic DNA, wherein by homologous recombination between the flanking region and the corresponding homology region in the flanking target genome at the preselected site, optionally with the non-homologous end connection (NHEJ) combination at one end in the two ends of the repair nucleic acid molecule (for example, when only having a flanking region), and at a predetermined site, the repair nucleic acid molecule is replicated or integrated.Integration by homologous recombination allows the repair nucleic acid molecule to be accurately connected to the target genome until the nucleotide level, and NHEJ can cause the small insertion / deletion of the junction between the repair nucleic acid molecule and the genomic DNA.

[0205] In various embodiments of the aspects described herein, the modification of the genome occurs in the genome has been changed at least one nucleotide. The modification of the genome can occur by inserting a transgene, preferably an expression cassette comprising a transgene of interest, replacing at least one nucleotide and / or deleting at least one nucleotide and / or inserting at least one nucleotide, as long as the nucleotide sequence of the preselected genomic target site before modification results in a total change of at least one nucleotide, thereby allowing identification of the modification, for example by techniques such as sequencing or PCR analysis well known to those skilled in the art.

[0206] The modification of genome can occur at a preselected site, a predetermined site or a predefined site, i.e., a specific nucleotide sequence in a genome (e.g., a nuclear genome or a chloroplast genome), where it is desired to insert, replace and / or lack one or more nucleotides. For example, a preselected site, a predetermined site or a predefined site can be an endogenous locus or a specific nucleotide sequence in or connected to a previously introduced foreign DNA, RNA or transgenic. The preselected site can be a specific nucleotide position, where (after the position) it is intended to manufacture an insertion of one or more nucleotides. The preselected site can also include a sequence of one or more nucleotides to be exchanged (replaced) or lacking.

[0207] In various embodiments, the length and sequence identity percentage of the flanking region are selected to enable homologous recombination between the flanking region and their corresponding DNA regions upstream or downstream of the preselected site. One or more DNA regions that are located at the flanking of the preselected site and have homology with the flanking DNA regions of one or more repair nucleic acid molecules are also referred to as homologous regions in one or more genomic DNAs.

[0208] In order to have enough recombinant homology, the length of the flanking DNA region of the repair nucleic acid molecule can be different, and the length should be at least about 10nt, at least about 15nt, at least about 20nt, at least about 25nt, at least about 30nt, at least about 40nt or at least about 50nt. However, the flanking region can be as long as practical (for example, up to about 100-150kb, such as complete bacterial artificial chromosome (BAC)). Preferably, the flanking region will be about 50nt to about 2000nt, such as about 100nt, about 200nt, about 500nt or about 1000nt. In addition, the region positioned at the DNA flank of interest need not be identical with the homology region (the DNA region positioned at the flanking preselected site), and can have about 80% to about 100% sequence identity with the DNA region flanking the preselected site, preferably have about 95% to about 100% sequence identity. The longer the flanking region, the less stringent the requirement to homology. Furthermore, in order to achieve exchange of the target DNA sequence at the preselected site without changing the DNA sequence of the adjacent DNA sequences, the flanking DNA sequence should preferably be identical to the upstream and downstream DNA regions flanking the preselected site.

[0209] In order to modify the target sequence at the preselected site, the flanking regions must be selected so that the 3' end of the upstream flanking region and / or the 5' end of the downstream flanking region are aligned with the ends of the predefined site. In this way, the 3' end of the upstream flanking region determines the 5' end of the predefined site, while the 5' end of the downstream flanking region determines the 3' end of the predefined site.

[0210] The preselected site is located outside or away from the cleavage (and / or recognition) site, so that the site to be modified (preselected site) does not contain a cleavage site and / or a recognition site for a DSBI / SSBI enzyme, so that the preselected site does not overlap with the cleavage (and / or recognition) site. Therefore, outside / away from in this regard refers to upstream or downstream of the cleavage (and / or recognition) site.

[0211] In various embodiments, at least one base editor according to the present invention is temporarily or permanently connected to at least one site-specific DSBI / SSBI enzyme complex or at least one modified site-specific DSBI / SSBI enzyme complex or optionally to a component of the at least one site-specific DSBI / SSBI enzyme complex. The connection can be covalent and / or non-covalent. Any base editor or site-specific DSBI / SSBI enzyme complex disclosed herein or a catalytically active fragment thereof, or any component of a base editor complex or any component of a site-specific DSBI / SSBI enzyme complex can be introduced into a cell as a nucleic acid fragment representing or encoding a DNA, RNA or protein effector, or it can be introduced as DNA, RNA and / or protein or any combination thereof.

[0212] Base editors are proteins or fragments thereof that have the ability to mediate targeted base modifications (i.e., conversion of bases of interest resulting in point mutations of interest). Preferably, in the context of the present invention, at least one base editor is temporarily or permanently fused to at least one DSBI / SSBI enzyme, or optionally fused to a component of at least one DSBI / SSBI. Fusion can be covalent and / or non-covalent. Multiple publications have shown targeted base conversions (mainly from cytosine (C) to thymine (T)) using CRISPR / Cas9 nickases or non-functional nucleases connected to cytidine deaminase domains (apolipoprotein B mRNA editing catalytic polypeptide (APOBEC1) (e.g., APOBEC from rats)). The deamination of cytosine (C) is catalyzed by cytosine deaminase, and uracil (U) with base pairing properties of thymine (T) is produced. Most known cytidine deaminases act on RNA, and a few examples of known acceptance of DNA require single-stranded (ss) DNA. Studies on dCas9-target DNA complexes reveal that after forming the Cas9-guide RNA-DNA "R-loop" complex, at least nine nucleotides (nt) of the replaced DNA strand are unpaired (Jore et al., Nat. Struct. Mol. Biol., 18, 529-536 (2011)). Indeed, in the structure of the Cas9 R-loop complex, the first 11 nt of the pre-spacer sequence on the replaced DNA strand are disordered, indicating that their movement is not highly restricted. It has also been speculated that the mutations in the cytosine of the non-template strand induced by the Cas9 nickase may be caused by the accessibility of the cellular cytosine deaminase to them. It is reasonable to believe that a subset of this ssDNA fragment in the R-loop can be used as an effective substrate for the cytidine deaminase connected by dCas9- to achieve direct programmable conversion of C to U in DNA (Komor et al., supra). Recently, Goudelli et al., Programmable base editing of A·T to G·C ingenomic DNA without DNA cleavage, Nature, 2017, 551(7681), 464, described an adenine base editor (ABE) that mediates A·T to G·C conversion in genomic DNA.

[0213] Enzymes and histone modifying enzymes that affect DNA methylation have been identified in the art. Histone post-translational modification plays an important role in regulating chromatin structure and gene expression. For example, in Sterner DE, Berger SL (June 2000): "Acetylation of histones and transcription-related factors", Microbiol.Mol.Biol.Rev.64 (2): 435–59, described enzymes for histone acetylation. Enzymes that affect histone methylation are described in Zhang Y., Reinberg D (2001): "Transcription regulation by histonemethylation: interplay between different covalent modifications of the corehistone tails", Genes Dev.15 (18): 2343–60. Ubiquitination of histones is described in Shilatifard A (2006): "Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression", Annu.Rev.Biochem.75: 243–69. Enzymes for histone phosphorylation are described in Nowak SJ., Corces VG. (April 2004): "Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation", Trends genet. 20(4): 214–20. Enzymes for histone SUMOylation are described in Nathan D., Ingvarsdottir K., Sterner DE. et al., (April 2006): "Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications", Genes Dev. 20(8): 966–76.Enzymes for histone ribosylation are described in Hassa PO., Haenni SS., Elser M, Hottiger MO. (September 2006): "Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going?", Microbiol. Mol. Biol. Rev. 70(3): 789–829. Histone citrullination is catalyzed by an enzyme, for example, called peptidyl arginine deiminase 4 (PAD4, also known as PADI4), which converts both histone arginine (Arg) and monomethylarginine residues to citrulline.

[0214] Enzymes affecting DNA methylation and histone modifying enzymes may be fused to disarming DSB or SSB inducing enzymes, which preferably recognize predetermined sites in the genome of the cell.

[0215] Exemplary transgenes

[0216] In various embodiments of the method of genetic modification in a plant cell, the transgene is selected from a gene encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphorus deficiency, salt stress or waterlogging, a gene encoding herbicide resistance, including resistance to glyphosate, glufosinate / phosphinothricin, hygromycin, protoporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors and Dicamba, a gene encoding resistance or tolerance to biotic stress, including viral resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes, or a gene encoding a yield-related trait, including lodging resistance, flowering time, pod dehiscence resistance, seed color, endosperm composition or nutritional content.

[0217] In various embodiments of the method for genetic modification in plant cells, the method effectively promotes cell proliferation or cell regeneration, or effectively improves the regeneration efficiency of transgenic, gene-edited or base-edited plants. The method is preferably effective after genetic modification / genomic modification. In various embodiments of the method for genetic modification in plant cells, the method effectively induces direct or indirect (somatic) embryogenesis from a single cell (preferably embryonic cells, somatic cells or protoplasts) or from callus cells or from callus cells. The method is preferably effective after genetic modification / genomic modification. In various embodiments, the method effectively improves the efficiency of stable transformation of transgenic into plant cells, or effectively improves the efficiency of producing transgenic plants. In various embodiments, the method effectively improves the efficiency of genome engineering component editing the genome of plant cells, or effectively improves the efficiency of producing transgenic, gene-edited or base-edited plants.

[0218] In some embodiments, the methods are effective to improve the efficiency of regeneration of plants derived from recalcitrant genotypes, effective to improve the efficiency of regeneration of plants from unconventional tissue types, or effective to accelerate the regeneration process, preferably following genetic modification / genomic modification.

[0219] Enhancer polypeptides and transient expression of enhanced genes

[0220] Also provided are methods for transiently expressing first and second enhancer polypeptides and / or nucleic acids encoding the first and second enhancer polypeptides and third and second enhancer polypeptides and / or nucleic acids encoding the third and second enhancer polypeptides in plant cells. The method comprises introducing into a plant cell: (i) the components of step (i) described in the context of a method for genetic modification in a plant cell, the first and second enhancer polypeptides, the third and second enhancer polypeptides and / or nucleic acids, recombinant genes or DNA constructs described herein; and (ii) a transgenic and / or genome engineered component.

[0221] In some embodiments, one or more enhancer polypeptides and enhancer genes are transiently co-expressed. Co-expression may effectively promote cell proliferation. This co-expression may effectively promote cell regeneration. Co-expression can effectively induce embryogenesis from a single cell, and therefore provides the ability to regenerate homogeneous plants without selection. Co-expression can improve genome editing efficiency by co-delivery with genome editing components. Co-expression can include instantaneous co-introduction of the first and second enhancer polypeptides, the third and second enhancer polypeptides, the components of step (i) described in the context of the method for genetic modification in plant cells, nucleic acids as described herein, recombinant genes or DNA constructs, and one or more nucleic acids encoding another enhancer polypeptide (e.g., PLT5 (SEQ ID NO: 9-12), KWS-RBP-1 (SEQ ID NO: 13 and 14), KWS-RBP-2 (SEQ ID NO: 49 and 50), RKD4 (SEQ ID NO: 15-20), and RKD2 (SEQ ID NO: 21-26)).

[0222] Transient co-delivery of first and second enhancer polypeptides / third and second enhancer polypeptides with one or more enhancer genes can be performed as described in U.S. Provisional Application No. 62 / 685,626 (incorporated herein by reference in its entirety).

[0223] In various embodiments, other enhancing factors such as chemical HDACi and plant hormones may be delivered as described in US Provisional Application No. 62 / 685,626.

[0224] In some embodiments, the enhancer polypeptide is transiently present, transiently active and / or transiently expressed in the plant cell. In some embodiments, the nucleic acid encoding the enhancer polypeptide is transiently present, transiently active and / or transiently expressed in the plant cell. In addition to the first and second enhancer polypeptides, nucleic acids, recombinant genes or DNA constructs described herein, one or more polypeptides selected from the group consisting of PLT5 polypeptides, KWS-RBP-1 polypeptides, KWS-RPB-2 polypeptides, RKD4 polypeptides and RKD2 polypeptides and / or one or more nucleic acids selected from the group consisting of nucleic acids encoding PLT5 polypeptides, KWS-RBP-1 polypeptides, KWS-RPB-2 polypeptides, RKD4 polypeptides and RKD2 polypeptides and / or one or more site-directed transcriptional activators suitable for transiently increasing the expression of endogenous PLT5 polypeptides, endogenous RKD4 polypeptides or endogenous RKD2 polypeptides, and / or nucleic acids encoding such site-directed transcriptional activators may be introduced into the plant cell.

[0225] Transient expression can be performed by transient transformation / transfection of enhancer proteins / polypeptides or nucleic acid fragments encoding proteins / polypeptides, preferably expressed under a strong constitutive promoter. Transient expression of nucleic acids encoding first and second enhancer polypeptides, nucleic acids encoding third and second enhancer polypeptides, nucleic acids encoding PLT5 polypeptides, nucleic acids encoding KWS-RBP1 polypeptides, nucleic acids encoding KWS-RBP2 polypeptides and / or transient expression of one or more site-directed transcriptional activators suitable for transiently increasing expression of first and second enhancer polypeptides, third and second enhancer polypeptides, endogenous PLT5 polypeptides, endogenous RKD4 polypeptides or endogenous RKD2 polypeptides can also be achieved by stable transformation of enhancer genes under the control of tissue and development-specific promoters or inducible promoters. Enhancer genes can be expressed and then transiently active. Then, when plant cell development changes or inducing conditions are eliminated, the enhancer genes can be quickly turned off and weakened. For example, the strong constitutive promoter pBdEF1 (SEQ ID NO: 30) from the Brachypodium EF1 gene can be used to drive enhancer genes for transient transformation (see, e.g., Example 1).

[0226] Transient expression can be produced from any of transient transfection, transient transformation and stable transformation." transient transformation " and " transient transfection " comprise transferring foreign material [i.e. nucleic acid fragment, protein, ribonucleoprotein (RNP) etc.] into host cell, resulting in gene expression and / or activity without integration and stable inheritance foreign material. Foreign components are not permanently incorporated into the cell genome, but provide temporary effects that result in genome modification. Transient transformation events may not be passed to the next generation, and are therefore non-heritable. " stable transformation " refers to that the nucleic acid fragment transferred is integrated into the genome (including nuclear and organelle genomes) of the host cell, resulting in stable inheritance of the nucleic acid fragment.

[0227] For example, transient expression can be used for transient genome editing. The transient activity and / or transient presence of genome engineering components in plant cells can result in the introduction of one or more double-strand breaks in the genome of plant cells, the introduction of one or more single-strand breaks in the genome of plant cells, the introduction of one or more base editing events in the genome of plant cells, or the introduction of one or more of DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination in the genome of plant cells. The modification produced in the genome of plant cells can be, for example, selected from the substitution of at least one nucleotide, the deletion of at least one nucleotide, the insertion of at least one nucleotide, the change of DNA methylation, the change of histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination, or a combination thereof.

[0228] Site-directed transcriptional activators refer to synthetic transcription factors described in U.S. Provisional Application No. 62 / 609,508 (which is incorporated herein by reference). The synthetic transcription factor may comprise at least one recognition domain and at least one gene expression regulation domain, in particular an activation domain, wherein the synthetic transcription factor is configured to regulate the expression of an endogenous gene in the genome of a plant or plant cell. Such an endogenous gene is preferably a (natural) morphogenic gene that encodes a polypeptide involved in plant development processes such as root formation or bud formation. In some embodiments, the endogenous morphogenic gene is selected from an endogenous nucleic acid encoding a first enhancer polypeptide and an endogenous nucleic acid encoding a second enhancer polypeptide, and an endogenous nucleic acid encoding a PLT5 polypeptide, an endogenous nucleic acid encoding a RKD4 polypeptide, and an endogenous nucleic acid encoding a RKD2 polypeptide. In some embodiments, at least one recognition domain is a molecule selected from the following or a fragment of the following molecule: at least one TAL effector, at least one disarmed CRISPR / nuclease system, at least one zinc finger domain, and at least one disarmed homing endonuclease, or any combination thereof.

[0229] In some embodiments, at least one disarmed CRISPR / nuclease system is a CRISPR / dCas9 system, a CRISPR / dCpf1 system, a CRISPR / dCsm1 system, a CRISPR / dCasX system, or a CRISPR / dCasY system, or any combination thereof, and wherein at least one disarmed CRISPR / nuclease system comprises at least one guide RNA.

[0230] In some embodiments, at least one activation domain is selected from an acidic transcriptional activation domain, preferably, wherein at least one activation domain is from a TAL effector gene of Xanthomonas oryzae, VP16 or tetrameric VP64 from herpes simplex, VPR, SAM, Scaffold, Suntag, P300, VP160 or any combination thereof. In some embodiments, the activation domain is VP64.

[0231] In some embodiments, the synthetic transcription factor is configured to regulate the expression of the morphogenic gene, preferably the transcription of the morphogenic gene, by binding to a regulatory region located at a distance from the start codon. In a preferred embodiment, the synthetic transcription factor is configured to increase the expression of the morphogenic gene, preferably the transcription of the morphogenic gene, by binding to a regulatory region located at a distance from the start codon.

[0232] In some embodiments, the site-directed transcriptional activator / synthetic transcription factor or a nucleic acid encoding the same comprises at least one recognition domain and at least one activation domain, wherein the site-directed transcriptional activator is configured to increase the expression of the first and second enhancer polypeptides or the third and second enhancer polypeptides. In addition, the site-directed transcriptional activator can be configured to increase the expression of an endogenous PLT5 polypeptide, an endogenous RKD4 polypeptide, or an endogenous RKD2 polypeptide. The site-directed transcriptional activator / synthetic transcription factor can preferably bind to a regulatory region located at a distance from the start codon of an endogenous nucleic acid encoding the first and second enhancer polypeptides or the third and second enhancer polypeptides, the endogenous PLT5 polypeptide, the endogenous RKD4 polypeptide, or the endogenous RKD2 polypeptide.

[0233] As used herein, "regulatory region" refers to the binding site of at least one recognition domain to a target sequence in the genome at or near a morphogenetic gene. Depending on the nature of the at least one activation domain and at least one recognition domain further disclosed herein, there may be two separate regulatory regions, or there may be overlapping regulatory regions, wherein the different domains of the synthetic transcription factor may be assembled in a modular manner.

[0234] In certain embodiments, at least one recognition domain can target at least one sequence (recognition site) relative to the start codon of the gene of interest, and the sequence can be at least 1000bp upstream (-) or downstream (+), -700bp to +700bp, -550bp to +500bp or -550bp to +425bp relative to the start codon of the gene of interest. In certain embodiments, the recognition domain near the recognition promoter may be preferred, and this represents the advantages of the following specific synthetic transcription factors: the targeting range of the synthetic transcription factor is highly extended relative to conventional or naturally occurring transcription factors. Because the recognition and / or activation domain can be specially designed and constructed to specifically recognize and target the hot spots of regulation.

[0235] In certain embodiments, at least one recognition site can be -169 bp to -4 bp, -101 bp to -48 bp, -104 to -42 bp, or -175 to +450 bp (upstream (-) or downstream (+), respectively) relative to the start codon of the gene of interest to provide an optimal spatial binding environment to allow optimal regulatory (preferably transcriptional activation) activity. In particular, for CRISPR-based synthetic transcription factors that work with a guide RNA as a recognition moiety, the binding site can also be located within the coding region of the gene of interest (downstream of the start codon of the gene of interest).

[0236] In other embodiments, the recognition domain of the synthetic transcription factor can be bound to the 5' and / or 3' untranslated region (UTR) of the gene of interest. In embodiments, in the case of using different recognition domains, at least two recognition domains can be bound to different target regions of the morphogenic gene, including 5' and / or 3' UTR, but they can also be bound outside the gene region, but still within a specific distance of up to 1 to 1500bp. A preferred region that the recognition domain can bind to is located about -4bp to about -300bp upstream of the start codon of the morphogenic gene of interest, preferably about -40bp to about -170bp. In addition, the length of the recognition domain and the length of the corresponding recognition site in the genome of interest can vary depending on the properties of the synthetic transcription factor and the recognition domain applied. Based on the molecular characteristics of at least one recognition domain, this will also determine the length of the corresponding at least one recognition site. For example, a single zinc finger can be about 8bp to about 20bp, wherein an array between three to six zinc finger motifs may be preferred, in which case a single TALE recognition site can be about 11 to about 30bp, or more. The recognition site of the gRNA of a CRISPR-based synthetic transcription factor contains a targeting sequence or "spacer" sequence of the gRNA that hybridizes to the genomic region of interest, while the gRNA contains other domains, including a domain that interacts with the disarmed CRISPR effector. The recognition site of a synthetic transcription factor based on a disarmed CRISPR effector will contain a PAM motif, because the PAM sequence is necessary for target binding of any CRISPR effector, and the exact sequence depends on the species of CRISPR effector (i.e., disarmed CRISPR effector).

[0237] Enhanced gene and peptide introduction

[0238] Enhancers and / or genome engineering components can be introduced as a protein / polypeptide or as a nucleic acid encoding a protein / polypeptide, in particular as a protein / polypeptide or a DNA such as plasmid DNA, RNA, mRNA or RNP.

[0239] The reinforcing factor can be delivered together with one or more genome engineering components. As used herein, "delivery together" or "delivery together" and "import together" or "import together" are used interchangeably. For the purposes of the present invention, "import together" refers to such a process in which at least two different components are delivered into the same plant cell simultaneously. Therefore, the genome engineering components and the reinforcing factor are imported into the same plant cell together. Preferably, two types of components, the reinforcing factor and the gene of interest, are imported by separate constructs. Can be imported together into plant cells by particle bombardment, microinjection, Agrobacterium-mediated transformation, electroporation, electrofusion, Agrobacterium infiltration or vacuum infiltration.

[0240] Regeneration enhancing gene

[0241] It is believed that the reproducibility of the transformed cells is lower than that of wild-type cells. Due to the presence of foreign DNA in the cells, the transformed cells are easily subjected to programmed cell death. The stress caused by delivery (such as bombardment) may also cause cell death. Therefore, promoting cell division is essential for the regeneration of modified cells. In addition, genome engineering efficiency is controlled by the host cell state to a great extent. The cells (like those in plant meristems) that experience rapid cell division are the most suitable receptors for genome engineering. Promoting cell division will likely improve the DNA integration or modification in DNA replication and fission process, and thus improve genome engineering efficiency.

[0242] Designed nucleic acid of the first and second encode strengthener polypeptides, nucleic acid of the third and second encode strengthener polypeptides, recombinant gene as herein described or DNA construct to improve the activity of genome engineering component.When the first and second encode strengthener polypeptides or the third and second encode strengthener polypeptides are imported into plant cells or in plant cells, their amount is improved together with transgenic, the encode strengthener polypeptide can improve the expression of transgenic and transgenic encoded polypeptide.When the encode strengthener polypeptide and genome engineering component are imported into plant cells together with transgenic, the activity of genome engineering component may be improved.This raising can cause transgenic to be integrated into the genome of plant cells more effectively.One or more encode strengthener genes can be co-expressed with the first and second encode strengthener polypeptides or with the third and second encode strengthener polypeptides.One or more encode strengthener genes can be co-transfected with the first and second encode strengthener polypeptides or with the third and second encode strengthener polypeptides.

[0243] Such additional strengthening genes are selected based on their function in promoting cell division and plant morphogenesis. Each candidate gene is cloned and driven by a strong constitutive promoter and evaluated by transient expression in corn cells without selection. Examples of additional strengthening genes are PLT5 (PLETHORA5; SEQ ID NOs: 9 and 11), KWS-RBP1 (SEQ ID NO: 13), KWS-RBP2 (SEQ ID NO: 49), RKD4 (SEQ ID NOs: 15, 17 and 19) and RKD2 (SEQ ID NOs: 21, 23 and 25).

[0244] PLT (PLETHORA), also known as AIL (AINTEGUMENT-LIKE) gene, is a member of the transcriptional regulator AP2 family. Transcription factor AP2 family members play an important role in plant cell proliferation and embryogenesis (El Ouakfaoui, S., Schnell, J., Abdeen, A., Colville, A., Labbé, H., Han, S., Baum, B., Laberge, S., Miki, B (2010) Control of somatic embryogenesis and embryo development by AP2 transcription factors. PLANT MOLECULAR BIOLOGY 74 (4-5): 313-326.). The PLT gene is mainly expressed in the developmental tissues of buds and roots, and is necessary for stem cell homeostasis, cell division and regeneration, and organ primordium patterning.

[0245] The PLT family contains the AP2 subclade with six members. The four PLT members, PLT1 / AIL3, PLT2 / AIL4, PLT3 / AIL6 and BBM / PLT4 / AIL2, are partially overlappingly expressed in the root apical meristem (RAM) and are required for the expression of QC (quiescent center) markers in the correct location within the stem cell niche. These genes play redundant roles in maintaining cell division and preventing cell differentiation in the root apical meristem.

[0246] Three PLT genes, PLT3 / AIL6, PLT5 / AIL5, and PLT7 / AIL7, are expressed in the shoot apical meristem (SAM), where they play redundant roles in the positioning and outgrowth of lateral organs. PLT3, PLT5, and PLT7 regulate de novo shoot regeneration in Arabidopsis by controlling two distinct developmental events. PLT3, PLT5, and PLT7 are required to maintain high levels of PIN1 expression at the periphery of the meristem and regulate localized auxin production in the central region of the SAM, which underlies the phyllotactic transition. Cumulative loss of function of these three genes results in an intermediate cell mass, the callus, that fails to form shoot progenitors, whereas induction of PLT5 or PLT7 allows shoot regeneration in a hormone-independent manner. PLT3, PLT5, and PLT7 regulate and require the shoot-promoting factor CUP-SHAPEDCOTYLEDON2 (CUC2) to complete the shoot formation program. PLT3, PLT5 and PLT7 are also expressed in lateral root founder cells, where they redundantly activate the expression of PLT1 and PLT2 and subsequently regulate lateral root formation.

[0247] Additional strengthening genes can be from any number of plants known in the art. Such plants include, but are not limited to, corn, Arabidopsis thaliana, and wheat. In some embodiments, the strengthening gene is corn PLT5. In some embodiments, the strengthening gene is Arabidopsis thaliana PLT5. In some embodiments, the additional strengthening gene can be an artificially synthesized KWS-RBP1 or KWS-RBP2. In some embodiments, the strengthening gene is wheat RKD4. In some embodiments, the strengthening gene is Arabidopsis thaliana RKD4. In some embodiments, the strengthening gene is corn RKD4. In some embodiments, the strengthening gene is wheat RKD2. In some embodiments, the strengthening gene is Arabidopsis thaliana RKD2. In some embodiments, the strengthening gene is corn RKD2.

[0248] In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and PLT5 polypeptides (encoded by PLT5 enhancer genes) are introduced into plant cells and are optionally transiently co-expressed. In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and KWS-RBP1 polypeptides (encoded by KWS-RBP1 polynucleotides) are introduced into plant cells and are optionally transiently co-expressed. In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and KWS-RBP2 polypeptides (encoded by KWS-RBP2 polynucleotides) are introduced into plant cells and are optionally transiently co-expressed. In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and RKD4 polypeptides (encoded by RKD4 polynucleotides) are introduced into plant cells and are optionally transiently co-expressed. In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and RKD2 polypeptides (encoded by RKD2 polynucleotides) are introduced into plant cells and are optionally transiently co-expressed. In some embodiments, nucleic acids encoding the first / third and second enhancer polypeptides according to the present invention and a PLT5 polypeptide (encoded by a PLT5 enhancer gene), a KWS-RBP1 polypeptide (encoded by a KWS-RBP1 polynucleotide), a RKD4 polypeptide (encoded by a RKD4 polynucleotide) and / or a RKD2 polypeptide (encoded by a RKD2 polynucleotide) are introduced into a plant cell and optionally transiently co-expressed. The polypeptide encoded by the PLT5 enhancer gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 10. The polypeptide encoded by the PLT5 enhancer gene may comprise the sequence of SEQ ID NO: 10. The polypeptide encoded by the PLT5 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 12. The polypeptide encoded by the PLT5 booster gene may comprise the sequence of SEQ ID NO: 12.

[0249] The polypeptide encoded by the maize PLT5 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 10. The polypeptide encoded by the maize PLT5 enhancing gene may comprise the sequence of SEQ ID NO: 10.

[0250] The polypeptide encoded by the Arabidopsis thaliana PLT5 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 12. The polypeptide encoded by the Arabidopsis thaliana PLT5 enhancing gene may comprise the sequence of SEQ ID NO: 12.

[0251] The polypeptide encoded by the KWS-RBP1 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 14. The polypeptide encoded by the KWS-RBP1 enhancing gene may comprise the sequence of SEQ ID NO: 14.

[0252] The polypeptide encoded by the KWS-RBP2 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 50. The polypeptide encoded by the KWS-RBP2 enhancing gene may comprise the sequence of SEQ ID NO: 50.

[0253] The polypeptide encoded by the RKD4 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 16. The polypeptide encoded by the RKD4 booster gene may comprise the sequence of SEQ ID NO: 16. The polypeptide encoded by the RKD4 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 18. The polypeptide encoded by the RKD4 booster gene may comprise the sequence of SEQ ID NO: 18. The polypeptide encoded by the RKD4 booster gene may comprise an amino acid sequence having at least 60%, 65%, 70%, 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% identity to SEQ ID NO: 20. The polypeptide encoded by the RKD4 booster gene may comprise the sequence of SEQ ID NO: 20.

[0254] The polypeptide encoded by the wheat RKD4 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 16. The polypeptide encoded by the wheat RKD4 enhancing gene may comprise the sequence of SEQ ID NO: 16.

[0255] The polypeptide encoded by the Arabidopsis thaliana RKD4 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 18. The polypeptide encoded by the Arabidopsis thaliana RKD4 enhancing gene may comprise the sequence of SEQ ID NO: 18.

[0256] The polypeptide encoded by the maize RKD4 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 20. The polypeptide encoded by the maize RKD4 enhancing gene may comprise the sequence of SEQ ID NO: 20.

[0257] The polypeptide encoded by the RKD2 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 22. The polypeptide encoded by the RKD2 booster gene may comprise the sequence of SEQ ID NO: 22. The polypeptide encoded by the RKD2 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 24. The polypeptide encoded by the RKD2 booster gene may comprise the sequence of SEQ ID NO: 24. The polypeptide encoded by the RKD2 booster gene may comprise an amino acid sequence that is at least 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% identical to SEQ ID NO: 26. The polypeptide encoded by the RKD2 booster gene may comprise the sequence of SEQ ID NO: 26.

[0258] The polypeptide encoded by the wheat RKD2 enhancing gene may comprise an amino acid sequence having at least 60%, 65%, 70%, 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% identity to SEQ ID NO: 22. The polypeptide encoded by the wheat RKD2 enhancing gene may comprise the sequence of SEQ ID NO: 22.

[0259] The polypeptide encoded by the Arabidopsis thaliana RKD2 enhancing gene may comprise an amino acid sequence having at least 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% identity to SEQ ID NO: 24. The polypeptide encoded by the Arabidopsis thaliana RKD2 enhancing gene may comprise the sequence of SEQ ID NO: 24.

[0260] The polypeptide encoded by the maize RKD2 enhancing gene may comprise an amino acid sequence having at least 60%, 65%, 70%, 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% identity to SEQ ID NO: 26. The polypeptide encoded by the maize RKD2 enhancing gene may comprise the sequence of SEQ ID NO: 26.

[0261] In some embodiments, the nucleic acid encoding the PLT5 polypeptide comprises a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 9 or 11. In some embodiments, the nucleic acid encoding the PLT5 polypeptide comprises a nucleic acid comprising a nucleotide sequence having at least 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% identity to SEQ ID NO: 9 or 11. In some embodiments, the nucleic acid encoding the PLT5 polypeptide comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 9 or 11, or comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 9 or 11.

[0262] In some embodiments, the nucleic acid encoding the KWS-RBP1 polypeptide comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding the KWS-RBP1 polypeptide comprises a nucleic acid comprising a nucleotide sequence having at least 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% identity to SEQ ID NO: 13. In some embodiments, the nucleic acid encoding a KWS-RBP1 polypeptide comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 13, or comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 13.

[0263] In some embodiments, the nucleic acid encoding the KWS-RBP2 polypeptide comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 49. In some embodiments, the nucleic acid encoding the KWS-RBP2 polypeptide comprises a nucleic acid comprising a nucleotide sequence having at least 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% identity to SEQ ID NO: 49. In some embodiments, the nucleic acid encoding a KWS-RBP2 polypeptide comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO:49, or comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:49.

[0264] In some embodiments, the nucleic acid encoding the RKD4 polypeptide comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15, 17 or 19. In some embodiments, the nucleic acid encoding the RKD4 polypeptide comprises a nucleic acid comprising a nucleotide sequence at least 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% identical to SEQ ID NO: 15, 17 or 19. In some embodiments, the nucleic acid encoding the RKD4 polypeptide comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 15, 17 or 19, or comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 15, 17 or 19.

[0265] In some embodiments, the nucleic acid encoding the RKD2 polypeptide comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 21, 23, or 25. In some embodiments, the nucleic acid encoding the RKD2 polypeptide comprises a nucleic acid comprising a nucleotide sequence at least 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% identical to SEQ ID NO: 21, 23, or 25. In some embodiments, the nucleic acid encoding the RKD2 polypeptide comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence that is at least 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% identical to SEQ ID NO: 21, 23 or 25, or comprises a nucleic acid that hybridizes to the complementary strand of a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 21, 23 or 25.

[0266] For purposes of the present invention, " sequence identity " (expressed as percentage) of two related nucleotides or amino acid sequences refers to the number of positions in two optimally aligned sequences with identical residues (x100) divided by the number of positions compared. A gap, i.e., a position in the alignment of a residue that exists in a sequence and does not exist in another sequence, is considered to be a position with non-identical residues. The comparison of two sequences is carried out by Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The above computer-assisted sequence alignment can be easily carried out using standard software programs such as the NEEDLE program implemented in European Molecular Biology Open Software Suite (EMBOSS) as version 6.3.1.2 (Trends in genetics 16 (6), 276 (2000)) (its default parameters are for example for protein matrix=EBLOSUM62, gapopen=10.0 and gapextend=0.5).

[0267] As used herein, the term "hybridization" refers to the formation of a hybrid between two nucleic acid molecules by base pairing of complementary nucleotides. The term "hybridization under stringent conditions" refers to hybridization under specific conditions. An example of such conditions includes conditions under which substantially complementary chains (i.e., chains consisting of nucleotide sequences having at least 80% complementarity) hybridize with a given chain, while lower complementary chains do not hybridize. Alternatively, these conditions refer to specific hybridization conditions of sodium salt concentration, temperature, and washing conditions. For example, highly stringent conditions are included in 42°C, 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate, 5x Denhardt solution, 10x dextran sulfate, 20mg / ml sheared salmon sperm DNA incubated and washed in 0.2x SSC (SSC stands for 0.15M sodium chloride and 0.015M trisodium citrate buffer) at about 65°C. Alternatively, highly stringent conditions may mean hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDDS, 1 mM EDTA and 1% BSA for 16 hours, and washing twice with 2× SSC and 0.1% SDDS at 68° C. Further alternatively, highly stringent hybridization conditions are, for example, hybridization at 65° C. in 4× SSC, and then washing multiple times at 65° C. in 0.1× SSC for a total of about 1 hour, or hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours, and then washing twice with 2× SSC and 0.1% SDS at 68° C.

[0268] Epigenetic regulatory chemicals

[0269] Epigenetic regulatory chemicals, such as protein deacetylase inhibitors (ii.1) can be co-introduced with the genome engineering components. Exemplary epigenetic regulatory chemicals used according to the present invention include, but are not limited to, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and DNA methyltransferase inhibitors.

[0270] It is hypothesized that the co-delivered epigenetic regulatory chemicals (ii.1) (particularly HDACi) relax the chromatin structure of the plant, promoting DNA accessibility to genome engineering components in the bombarded cells, and thus subsequently promoting genome engineering (i.e., transformation and genome editing) efficiency. The reason for this hypothesis is that the basic structural and functional unit of genetic material is the nucleosome, in which negatively charged DNA is wrapped around a positively charged histone octamer and associated linker histones. The nucleosomal unit is further folded and stacked into chromatin (Andrews, AJ, and Luger, K. (2011). Nucleosome structure (s) and stability: Variations on a theme. Annu. Rev. Biophys. 40: 99–117). DNA accessibility depends largely on the compactness of nucleosomes and chromatin. Chromatin remodeling enzymes can dynamically modify the lysine or other amino acids of histones, which leads to changes in their charge and interactions with DNA and other proteins, and causes chromatin folding or unfolding (Bannister AJ., Kouzarides T. (2011) Regulation of chromatin by histone modifications. Cell Res 21: 381–95). Acetylation and deacetylation of lysine residues in histones are often involved in the reversible regulation of chromatin structure in eukaryotes by adding or removing acetyl groups, and mediate chromatin accessibility and gene expression regulation. Histone deacetylase (HDAC) is an enzyme that removes acetyl groups from lysine residues located at the N-terminal tail of histones, which makes histones more positively charged and thus allows histones to wrap DNA more tightly. Inhibiting HDAC may help chromatin unfolding and make DNA more accessible.

[0271] Chromatin remodeling and other epigenetic modifications undoubtedly play an important role in regulating cell totipotency and regeneration (Zhang, H., and Ogas, J. (2009). An epigenetic perspective on developmental regulation of seed genes. Mol. Plant 2: 610–627). It has been shown that inhibition of histone deacetylase (HDAC) activity is associated with plant regeneration and microspore embryogenesis (Miguel, C., and L. Marum, 2011. Anepigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J. Exp. Bot. 62: 3713–3725., Li Hui et al. (2014) The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte PLANTCELL, 26: 195–209). Inhibition of HDAC activity or downstream HDAC-mediated pathways plays a major role in initiating stress-induced haploid embryogenesis. One such HDACi is trichostatin A (TSA). TSA has been shown to induce a large number of embryogenic cell proliferations in the male gametophytes of Brassica napus. TSA treatment results in a high frequency of sporophytic cell divisions in cultured microspores and pollen.

[0272] A variety of methods can be used to further improve genome engineering efficiency in the presence of one or more epigenetic regulatory chemicals, such as protein deacetylase inhibitors, particularly HDACi. Such HDACi can be trichostatin A (TSA), N-hydroxy-7-(4-dimethylaminobenzoyl)-aminoheptylamide (M344), suberoylanilide hydroxamic acid (SAHA) or other. These HDACIs are selected from chemicals based on hydroxamic acid (HA), and their target is zinc-dependent HDAC.

[0273] Plant hormones

[0274] In various embodiments, one or more plant hormones, such as auxins and cytokinins, such as 2,4-D, 6-benzylaminopurine (6-BA), and zeatin, are co-delivered with one or more enhancer genes, enhancer polypeptides, genome engineering components, and transgenes.

[0275] Plant somatic cells can resume cell division and regenerate into whole plants in vitro through somatic embryogenesis or organogenesis, which mainly depends on plant hormones such as auxin and cytokinin. In the present invention, it is found that plant hormones promote cell proliferation, increase the sensitivity of plant cells to genome engineering, and thus improve the efficiency of genome engineering (i.e., transformation and genome editing).

[0276] One of the auxins is 2,4-dichlorophenoxyacetic acid (2,4-D), which is almost essential for somatic embryogenesis and cell regeneration in monocots such as corn and wheat. At the same time, cytokinins such as 6-benzylaminopurine (6-BA) or zeatin are essential for plant organogenesis and the initiation and development of shoot meristems. Methods for improving genome engineering efficiency can include delivering one or more plant hormones (2,4-D, 6-BA, zeatin, etc.) together with genome engineering components.

[0277] The genome engineering component and at least one epigenetic regulatory chemical and a plant hormone can be co-introduced into a plant cell.

[0278] As used herein, "co-delivery" or "co-delivery" and "co-introduction" or "co-introduction" are used interchangeably. For the purposes of the present invention, "co-introduction" refers to a process in which at least two different components are delivered into the same plant cell at the same time. Thus, the genome engineering component and at least one epigenetic regulatory chemical and plant hormone can be introduced into the same plant cell together.

[0279] Co-introduction into plant cells can be performed by particle bombardment, microinjection, Agrobacterium-mediated transformation, electroporation, agroinfiltration or vacuum infiltration. According to the present invention, methods based on physical delivery (such as particle bombardment, microinjection, electroporation, nanoparticles and cell penetrating peptides (CPP)) are particularly preferred for co-introduction of booster genes, booster polypeptides, genome engineering components and / or transgenes. Co-introduction by particle bombardment is particularly preferred.

[0280] Plant cells regenerate into whole plants

[0281] According to another aspect of the present invention, the genetically modified plant cells can be regenerated into complete (fertile) plants. Therefore, in a preferred aspect of the present invention, the genetic modification of the plant cells is followed by a step of regenerating the plant. Therefore, the present invention provides a method for producing a genetically modified plant, comprising the following steps:

[0282] a) genetically modifying a plant cell according to any of the methods described above for genetic modification in a plant cell, and

[0283] b) regenerating a plant from the modified plant cell of step a),

[0284] Single cells or multiple cells proliferate and develop into tissues, organs, and ultimately whole plants. In some embodiments, the resulting plant does not include any genome engineering components, enhanced genes, and enhancer polypeptides introduced or co-introduced in step a). Step b) of regenerating the plant can, for example, include culturing the genetically modified plant cells from step a) on a regeneration medium.

[0285] The efficiency of plant regeneration or the efficiency of increasing the regeneration capacity of plant cells can be improved by introducing into plant cells the components of step (i) described in the context of the method for genetic modification in plant cells, the first and second enhancer polypeptides, the third and second enhancer polypeptides, or nucleic acids encoding the same, in combination with any enhancer polypeptide, enhancer gene, nucleic acid, recombinant gene and DNA construct described herein.

[0286] Producing genetically modified plants

[0287] The present invention also provides a genetically modified plant obtained or obtainable by the above method for producing a genetically modified plant or a progeny plant thereof.The genetically modified plant may comprise any genetically modified plant cell described herein.

[0288] In various embodiments, the plants produced do not comprise any genome engineering components, enhancing genes, and enhancer polypeptides introduced or co-introduced into the plant cells used to produce the plants produced.

[0289] The present invention also provides plants or seeds derived from the above genetically modified cells without conventional selection. As used herein, "conventional selection" refers to any method of selecting and purifying transformed cells from wild-type cells by using integrated selection markers such as antibiotics (e.g., kanamycin, hygromycin) or herbicides (e.g., phosphinothricin, glyphosate) resistance genes. Without conventional selection, such plants or seeds may not have any integrated genome engineering components, and thus result in genetically modified plants without transgenes.

[0290] Genetic modification can be a permanent and heritable change in the plant cell genome. Plant tissue culture and genome engineering can be carried out using currently available methods containing microprojectile bombardment, Agrobacterium transformation, electroporation, etc. Visible reporter genes, such as red fluorescence tDTomato gene (tDT) (it encodes abnormally bright red fluorescent protein, excitation maximum of 554nm, and emission maximum of 581nm) can be used to monitor transformation and transgenic expression. Genome editing efficiency can be analyzed, for example, by next generation sequencing (NGS), qPCR, labeled capillary electrophoresis analysis and Droplet Digital PCR. Site-specific modification is further confirmed by Sanger sequencing.

[0291] Cultivation steps

[0292] Plant cells into which enhanced genes, enhancer polypeptides, genome engineering components and / or transgenes have been introduced or co-introduced can be cultured under conditions that allow genetic modification of the genome of the plant cells through the activity of the genome engineering components in the presence of the enhancer gene, enhancer polypeptide and one or more transgenes.

[0293] As used herein, "genetic modification of a genome" includes any type of manipulation such that endogenous nucleotides have been altered to include mutations such as deletions, insertions, transitions, transversions, or combinations thereof. For example, an endogenous coding region may be deleted. Such mutations may result in a polypeptide having an amino acid sequence that is different from the amino acid sequence encoded by the endogenous polynucleotide. Another example of a genetic modification is a change in a regulatory sequence such as a promoter to result in increased or decreased expression of an operably linked endogenous coding region.

[0294] "Suitable" conditions for genetic modification of the plant genome to occur, such as cleavage of polynucleotides, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions allow, enhance, promote and / or facilitate the event. Depending on the individual genome engineering components (i), these conditions may be different.

[0295] In the method of the present invention, preferably the plant cell is transiently transformed with the genome engineering component (i) and at least one compound (ii). As used herein, "transient transformation" refers to the transfer of foreign materials [i.e., nucleic acid fragments, proteins, ribonucleoproteins (RNPs), etc.] into host cells to cause gene expression and / or activity without integrating and stabilizing genetic foreign materials. Therefore, the genome engineering component (i) is transiently active and / or transiently present in the plant cell. The genome engineering component is not permanently incorporated into the cell genome but provides a temporary effect to cause the modification of the genome. For example, the transient activity and / or transient presence of the genome engineering component in the plant cell can lead to the introduction of one or more double-strand breaks in the genome of the plant cell, the introduction of one or more single-strand breaks in the genome of the plant cell, the introduction of one or more base editing events in the genome of the plant cell, or the introduction of one or more of the following in the genome of the plant cell: DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination.

[0296] The introduction of the double strand break(s) or single strand break(s) is preferably followed by non-homologous end joining (NHEJ) and / or by homology directed repair (HDR) of the break(s) by homologous recombination mechanisms.

[0297] The resulting modification in the genome of the plant cell can be, for example, selected from the insertion of a transgene (preferably an expression cassette comprising a gene of interest), a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a change in DNA methylation, a change in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination, or a combination thereof. According to a particularly preferred aspect of the present invention, no exogenous genetic material associated with the applied gene editing mechanism / system is stably integrated into the genome of the plant cell.

[0298] A genetic modification may be a permanent and heritable change in the genome of a plant cell.

[0299] Optional pretreatment

[0300] In various embodiments, pretreatment of the plant material with one or more chemicals described in U.S. Provisional Application No. 62 / 685,626 (incorporated herein by reference) may be included. Thus, the method for genetic modification in plant cells may further comprise a step of pretreatment of the plant cells, the pretreatment comprising culturing the plant cells or plant material comprising the same in a culture medium containing (1) an epigenetic regulatory chemical or an active derivative thereof, in particular a histone deacetylase inhibitor (HDACi) or a DNA methyltransferase inhibitor, or (2) a plant hormone or an active derivative thereof, or any combination thereof.

[0301] After the pretreatment step, the treated plant cells are extracted from the culture medium containing at least one of compounds (1) and (2) and used for co-introduction.

[0302] As an example, for the histone deacetylase inhibitor TSA, the duration of HDACi pretreatment is 10 minutes to 2 days, preferably 2.0 hours to 24 hours. The TSA concentration used for pretreatment is 1.0nM to 1000nM, preferably 10nM to 100nM. Thereafter, the treated plant material is transferred to a medium without HDACi and immediately used for TSA co-introduction (extended TSA pretreatment may cause non-selective enhancement of cell regeneration, which may increase the difficulty of recovering bombarded and modified cells).

[0303] Similar pretreatment conditions can be applied to all types of compounds (1) and (2). Plant tissue culture and genome engineering can be carried out using currently available methods. Transient transformation and transgenic expression can be monitored using a red fluorescence reporter gene tdTomato (which encodes an abnormally bright red fluorescent protein with an excitation maximum of 554nm and an emission maximum of 581nm) or a green fluorescence reporter gene mNeonGreen (which encodes the brightest monomeric green or yellow fluorescent protein with a maximum excitation wavelength of 506nm and a maximum emission wavelength of 517nm). Genome editing efficiency can be analyzed, for example, by next generation sequencing (NGS).

[0304] particle

[0305] In another aspect, there is provided a microparticle coated with at least the components of step (i) described in the context of a method for genetic modification in a plant cell, the first and second enhancer polypeptides, the third and second enhancer polypeptides, the above nucleic acids, recombinant genes and DNA constructs. In some embodiments, the microparticle is further coated with a genome engineering component.

[0306] In another aspect, a kit for genetically modifying a plant genome by microprojectile bombardment is provided, comprising

[0307] (I) one or more microparticles, and

[0308] (II) Means for coating microparticles.

[0309] In some embodiments, the kit further comprises means for coating the microparticles with the genome engineering components.

[0310] In various embodiments, the microparticles are coated with at least:

[0311] (i) the components of step (i), the first and second enhancer polypeptides, the third and second enhancer polypeptides, or nucleic acids encoding the first and second enhancer polypeptides described in the context of a method for genetic modification in a plant cell;

[0312] (ii) transgenic; and / or genome engineered components.

[0313] In a particularly preferred embodiment of microprojectile bombardment, the components of step (i) described in the context of the method for genetic modification in plant cells, the first and second booster polypeptides or the third and second booster polypeptides, and / or one or more booster genes can be delivered together with the genome engineering components by microcarriers containing gold particles having a size in the range of 0.4-1.6 micrometers (μm), preferably in the range of 0.4-1.0 μm. In an exemplary method, each bombardment uses 10 ng-10 μg DNA, preferably 50-1000 ng DNA, coated on 10-1000 μg gold particles, preferably 50-300 μg gold particles. Up to 10 bombardments (shots), preferably 1-4 shots, per sample plate can be used to deliver foreign molecules into plant cells.

[0314] The boosting factor (e.g., a boosting polypeptide or a polynucleotide encoding such a boosting polypeptide) and the genome engineering components can be delivered into the target cells, for example, using a Bio-Rad PDS-1000 / He particle gun or a handheld Helios gene gun system. When using the PDS-1000 / He particle gun system, the bombardment rupture pressure is 450 psi to 2200 psi, preferably 450 psi to 1100 psi, while the Helios gene gun system has a rupture pressure of 100 psi to 600 psi. More than one chemical substance or construct can be co-delivered into the cell with the genome engineering components at the same time.

[0315] The microparticle coating may further comprise one or more coating layers. For example, the microparticle may contain a first coating layer comprising a boost factor and a second coating layer comprising a genome engineering component and a transgene. Alternatively, the microparticle may contain a coating layer comprising a first / third and second boost factor and a transgene or genome engineering component.

[0316] In addition, the present invention provides a kit for genetically modifying plant genomes by microprojectile bombardment, comprising

[0317] (I) one or more microparticles, and

[0318] (II) means for coating microparticles with at least a genome engineering component and (1) an epigenetic regulatory chemical, such as a DNA methyltransferase inhibitor or a protein deacetylase inhibitor or an active derivative thereof, in particular a histone deacetylase inhibitor (HDACi), and / or (2) a plant hormone or an active derivative thereof.

[0319] Another aspect of the invention is the use of microparticles as described above for biolistic transformation of plant cells.

[0320] The subject of the present invention is also a plant cell obtained or obtainable by the above method.Therefore, one embodiment of the present invention is a plant cell obtained or obtainable through genetic modification by the above method for genetic modification in plant cells.Compared with the original plant cell, the genetic modification in these plant cells can for example include the insertion of a transgenic (preferably an expression cassette comprising a gene of interest), the replacement of at least one nucleotide, the disappearance of at least one nucleotide, the insertion of at least one nucleotide, the change of DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination, or a combination thereof.Preferably, the plant cell through genetic modification does not include any exogenous genetic material that is stably integrated into the plant cell genome.

[0321] The genetically modified plant cell may be part of a whole plant or a part thereof. Therefore, the present invention also relates to a plant or plant part comprising the genetically modified plant cell described above.

[0322] According to another aspect of the present invention, the genetically modified plant cells can be regenerated into complete (fertile) plants. Therefore, in a preferred aspect of the present invention, the genetic modification of the plant cells is followed by a step of regenerating the plant. Therefore, the present invention provides a method for producing a genetically modified plant, comprising the following steps:

[0323] a) genetically modifying a plant cell according to the above method for genetic modification in a plant cell, and

[0324] b) regenerating a plant from the modified plant cell of step a).

[0325] Step b) of regenerating the plant may for example comprise culturing the genetically modified plant cells from step a) on a regeneration medium.

[0326] Regeneration techniques rely on the treatment of certain plant hormones in tissue culture growth media, and occasionally on biocide and / or herbicide markers that can be introduced. Regeneration can be obtained from plant somatic cells, callus cells or embryonic cells and protoplasts derived from different explants (e.g., callus, immature or mature embryos, leaves, buds, roots, flowers, microspores, embryonic tissues, meristems, organs or any part thereof). Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38: 467-486. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. To obtain a complete plant from the transformed or gene-edited cells, the cells are grown in a range of media containing nutrients and hormones under controlled environmental conditions, a process known as tissue culture. Once a complete plant is generated and produces seeds, the progeny are evaluated.

[0327] The present invention also provides a genetically modified plant obtained or obtainable by the above-mentioned method for producing a genetically modified plant or a progeny plant thereof.

[0328] Another subject of the present invention are plant cells or seeds derived from the above-described genetically modified plants.

[0329] Another subject of the present invention is the plant, plant cell or seed derived from the above-mentioned genetically modified cell without the need for selection based on marker genes. As used herein, "selection based on marker genes" refers to selecting from wild-type cells by using integrated selection markers (genes), such as antibiotic resistance genes (such as kanamycin resistance genes, hygromycin resistance genes) or herbicide resistance genes (such as phosphinothricin resistance genes, glyphosate resistance genes), identifying and / or purifying modified cells, particularly any process of cells transformed, gene-edited or base-edited. Without such selection, such plants, plant cells or seeds may not have any integrated genome engineering components, and this can produce (i) genetically modified plants without transgenics or (ii) only plants with the modification of the transgenics of interest integrated.

[0330] Unless otherwise indicated in the examples, all recombinant DNA techniques were performed according to standard protocols as described in Sambrook et al. (1989), (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY; and Ausubel et al. (1994), Current Protocols in Molecular Biology, Volumes 1 and 2 of Current Protocols, USA. Standard materials and methods for plant molecular studies are described in Plant Molecular Biology Labfax (1993) by RDD Ray, jointly published by BIOS Scientific Publications (UK) and Blackwell Scientific Publications, UK. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax, Second Edition, Volumes I and II of Academic Press (UK). Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al., (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0331] sequence

[0332] SEQ ID NO: Description

[0333] 1 ZmWUS2 cDNA

[0334] 2 ZmWUS2 protein

[0335] 3 cDNA of ZmPLT7 (genotype A188)

[0336] 4 Protein of ZmPLT7 (genotype A188)

[0337] 5 cDNA of ZmPLT7 (genotype B73)

[0338] 6 Protein of ZmPLT7 (genotype B73)

[0339] 7 AtPLT7 cDNA

[0340] 8 Proteins of AtPLT7

[0341] 9 ZmPLT5 cDNA

[0342] 10 ZmPLT5 Protein

[0343] 11 AtPLT5 cDNA

[0344] 12 Proteins of AtPLT5

[0345] 13 KWS-RBP1 cDNA

[0346] 14 KWS-RBP1 protein

[0347] 15 TaRKD4 cDNA

[0348] 16 TaRKD4 protein

[0349] 17 AtRKD4 cDNA

[0350] 18 Proteins of AtRKD4

[0351] 19 ZmRKD4 cDNA

[0352] 20 ZmRKD4 protein

[0353] 21 TaRKD2 cDNA

[0354] 22 TaRKD2 protein

[0355] 23 cDNA of AtRKD2

[0356] 24 AtRKD2 protein

[0357] 25 ZmRKD2 cDNA

[0358] 26 ZmRKD2 protein

[0359] 27 pABM-BdEF1_ZmPLT7

[0360] 28 pABM-BdEF1_WUS2

[0361] 29 pAMK-ZmWUS2-tDT-nosT

[0362] 30 pABM-BdEF1

[0363] 31 BdEF1 promoter

[0364] 32 pABM-BdEF1_ZmPLT5

[0365] 33 pABM-BdEF1_KWS-RBP1

[0366] 34 pABM-BdEF1_TaRKD4

[0367] 35 pGEP359

[0368] 36 pGEP324

[0369] 37 BdEF1::ZmPLT5_expression cassette

[0370] 38 BdEF1::ZmPLT7_expression cassette

[0371] 39 BdEF1::KWS-RBP1_expression cassette

[0372] 40 BdEF1::TaRKD4_expression cassette

[0373] 41 BdEF1::ZmWUS2_expression cassette

[0374] 42 pUbi::LpCpf1_expression cassette

[0375] 43 pUbi::crRNA5_expression cassette

[0376] 44 LbCpf1 cDNA

[0377] 45 LbCpf1 protein

[0378] 46 crRNA5_target_HMG13

[0379] 47 crRNA5_target_sequence

[0380] 48 pZmWUS2::tDT-nosT expression cassette 49 KWS-RBP2

[0381] 50 KWS-RBP2 protein

[0382] 51 pABM-BdEF1_KWS-RBP2

[0383] 52 BdEF1::KWS-RBP2_expression_cassette

[0384] All patents, patent applications, and publications or public disclosures (including publications on the internet) cited or referenced herein are incorporated by reference in their entirety. Example

[0385] The present invention is further illustrated by the following examples. However, it should be understood that the present invention is not limited to these examples. The use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the present invention or any exemplary term. Likewise, the present invention is not limited to any particular preferred embodiment described herein. In fact, after reading this specification, many modifications and variations of the present invention will be apparent to those skilled in the art, and such variations may be made without departing from the spirit or scope of the present invention. Therefore, the present invention is limited only by the terms of the appended claims and the full scope of equivalents given by those claims.

[0386] Example 1. Transient co-expression of booster genes and gene of interest (GOI) by co-bombardment.

[0387] Gene cloning and construct preparation

[0388] Maize WUS2 (ZmWUS2) and PLT7 (ZmPLT7) genes were cloned by RT-PCR using total RNA isolated from maize immature embryos of genotype A188. Wheat RKD4 and RKD2 and KWS-RBP1 genes were codon-optimized from their protein sequences and synthesized by Integrated DNA Technologies (IDT, San Diego, CA, USA). The enhanced gene fragments were cloned into the expression vector pABM-BdEF1 ( Figure 1 ) and expressed under the control of the BdEF1 promoter (pBdFE1) and nos terminator (nos-T). pBdFE1 is a strong constitutive promoter from Brachypodium. The sequence-confirmed construct map is shown in Figures 2 to 5 and 8.

[0389] Preparation of immature maize embryos for bombardment

[0390] 9-12 days after pollination, harvest the corn ears (i.e. A188 or Hi II) with immature embryos of 0.8-1.8 mm, preferably 1.0-1.5 mm in size. The ears are sterilized with 70% ethanol for 10-15 minutes. After short air drying in a laminar hood, remove the upper ~ 1 / 3 of the corn kernels from the ears with a shark scalpel, and then carefully remove the immature embryos from the corn kernels with a spatula. The freshly separated embryos are placed on the bombardment target area in the infiltration culture plate (see below) with the embryo cotyledon facing upward. Before bombardment, the plate is wrapped with parafilm and incubated in the dark at 25°C for 4 hours.

[0391] Particle co-bombardment

[0392] DNA was delivered into the embryonic cotyledon cells of maize immature embryos using a particle bombardment gun and gold particles with a size of 0.4 or 0.6 micrometers (μm). The booster plasmid was premixed with the gene of interest (GOI), for example, the genome editing construct pGEP359 ( Figure 6 ), and pGEP324 containing CRISPR guide RNA crRNA5 targeting HMG13 ( Figure 7 ). For 10 shots, 1 mg of gold particles in 50% (v / v) glycerol in a total volume of 100 microliters (μl) (100 μg of gold particles per shot) were pipetted into a clear low-retention microcentrifuge tube. The mixture was sonicated for 15 seconds to suspend the gold particles. While vortexing at low speed, the following were added to each 100 μl of gold particles in sequence: (a) up to 10 μl of DNA (1.0-10.0 μg of premixed total DNA, 100-1000 ng per shot), (b) 100 μl of 2.5 M CaCl2 (precooled on ice), and (c) 40 μl of 0.1 M cold spermidine.

[0393] Cover the lid and vortex the tube at 0-10°C for 2-30 minutes, and spin down the DNA-coated gold particles. After washing twice with 500μl 100% ethanol, the pellet is resuspended in 120μl of 100% ethanol. During low-speed vortexing, 10μl of the co-coated gold particles are evenly pipetted from the tube to the center of the macrocarrier with a wide-mouthed 20μl pipette tip. Since the particles tend to form clumps at this time, the gold particles are placed on the macrocarrier as soon as possible and then air-dried. Bombardment is performed using a Bio-Rad PDS-1000 / He particle gun. Bombardment conditions are: 28mm / Hg vacuum, 450 or 650psi rupture disc, 6mm gap distance, sample platform at a second position 60mm away from the bottom of the chamber, and three shots per sample (maize immature embryo) plate.

[0394] Post-bombardment observation and embryo culture

[0395] After bombardment, embryos were kept on the osmotic medium for another 16 hours. 16-20 hours after bombardment, transient transformation was checked for tDT expression using a fluorescence microscope at an excitation maximum of 554 nm and an emission maximum of 581 nm. Embryos with dense fluorescent signals under a fluorescence microscope were selected ( Fig. 9 ) and were transferred from N6OSM to N6-5Ag plates (~15 embryos per plate) with the embryonic cotyledons facing up to induce callus (see below).

[0396] Osmotic medium: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 0.7 g / L L-proline, 0.2 M mannitol (36.4 g / L), 0.2 M sorbitol (36.4 g / L), 20 g / L sucrose, 15 g / L bacterial agar, pH 5.8.

[0397] N6-5Ag: N6 salts, N6 vitamins, 1.0 mg / L 2,4-D, 100 mg / L casein, 2.9 g / L L-proline, 20 g / L sucrose, 5 g / L glucose, 5 mg / L AgNO3, 8 g / L agar, pH 5.8.

[0398] Example 2. Transient co-expression of a combination of ZmWUS2 and ZmPLT7 in maize A188 immature embryos promotes early embryogenesis and regeneration.

[0399] Transient co-delivery, embryo preparation and culture are described in Example 1 above. For each bombardment, four premixed DNA plasmids were coated on 100 μg of 0.4 μm gold particles and co-introduced into the embryonic cotyledon cells of A188 immature embryos at a bursting pressure of 650 psi. For one bombardment, the four plasmids were premixed as follows:

[0400] -100ng boost ZmPLT5 or ZmPLT7 ( Figure 2 and Figure 3 )

[0401] -200ng KWS-RBP1( Figure 4 )

[0402] -100ng pGEP359( Figure 6 )

[0403] -150ng pGEP324( Figure 7 )

[0404] Select embryos with dense fluorescent signals under a fluorescence microscope ( Fig. 9) and transferred from N6OSM to N6-5Ag to induce embryonic callus. The selected embryos were cultured in N6-5Ag plates with the embryo cotyledon side facing up (about 15 embryos per plate) at 27°C in the dark for 14 h in the dark. The induction of embryogenic callus was monitored by observation under a dissecting microscope. Specifically, the enhancement of cell division and regeneration was measured by visual observation under a fluorescent microscope through the ability to induce embryo formation 5-7 days after bombardment.

[0405] Fig.10 It was shown that co-expression of ZmWUS2 and ZmPLT7 by microprojectile bombardment significantly promoted embryogenic structural induction and accelerated tissue growth in maize A188 immature embryos. Fig.11 As can be seen, multiple embryonic structures were formed and emerged 7 days after particle bombardment (A) and 1 month after particle bombardment (B).

[0406] Fig.12 Quantification of embryonic structures formed with Tdt is shown. It has been demonstrated that transient expression of PLT7 alone or WUS2 alone resulted in a slight increase in the number / development of embryonic structures per embryo, but co-expression of ZmWUS2 and ZmPLT7 induced the generation of more embryonic structures compared to the single enhancer genes.

[0407] This strong strengthening effect on the level of the embryo is also effective for the regeneration efficiency of the entire transgenic plant. Stable integration of the construct pBdEF1-ZmPLT7 (SEQ ID NO: 27) including Pat-Tdt in the corn genotype A188 was not possible and transgenic plants have not been successfully generated. Stable integration of the construct pBdEF1-ZmWUS2 (SEQ ID NO: 28) including Pat-Tdt in the corn genotype A188 resulted in a low level of efficiency in transgenic plants. In contrast, stable integration of pBdEF1-ZmPLT7 (SEQ ID NO: 27) and pBdEF1-ZmWUS2 (SEQ ID NO: 28) including Pat-Tdt in the corn genotype A188 significantly improved the efficiency of transgenic plant regeneration.

[0408] The resulting transgenic plants with a single ZmWUS2 construct, as well as the co-transformation with ZmWUS2 and PLT7 resulted in plants without any deleterious phenotypes. They were comparable to normal A188 maize plants (see Fig.13 ).

[0409] Example 3. Transient expression of ZmPLT7 in maize Hi immature embryos improves stable transformation of co-delivered reporter genes

[0410] Examples 1 and 2 describe the preparation of maize embryos, transient bombardment, and induction of embryogenic callus. The embryos were cultured in N6-5Ag medium at 27°C in the dark for 14 days. tDT fluorescence was used to monitor the induction and stable transformation of embryogenic callus by observation under a fluorescence microscope. Specifically, the enhancement was measured by the ability to increase the transformation frequency (TF) of the tDT reporter gene 12 days after bombardment without selection.

[0411] From Fig.14 The strong and uniform tDT fluorescence signal of the newly generated embryonic structures in the blastocysts indicated the integration and stable transformation of the tDT gene. The stable transformation frequency was defined as the number of embryos induced with at least one stable tDT fluorescent structure from the 100 embryos initially used. The stable transformation frequency was measured 10 days after bombardment.

[0412] Transient expression of the ZmPLT7 gene (SEQ ID NO: 27) resulted in a 23% increase in the transformation frequency of the tDT gene compared to controls without the enhancer. Fig.14 The results showed that transient expression of ZmPLT7 promoted the stable transformation frequency in maize Hi II immature embryos.

[0413] The ZmPLT7 gene was successfully stably transformed into the Hi II genotype. Transgenic plants with a single ZmPLT7 construct showed an appearance comparable to normal A188 maize plants (see Fig.15 ).

[0414] Example 4. Wheat RKD4 activates maize WUSCHEL (WUS) expression.

[0415] The homeobox domain transcription factor WUSCHEL (WUS) plays an important role in establishing and maintaining shoot meristems. To identify enhancers that promote endogenous WUS2 expression, a maize WUSCHEL 2 promoter reporter construct (pAMK-ZmWUS2-tDT-noT) (SEQ ID NO: 29; Fig.16 ) is used to demonstrate the activity of the maize WUS2 promoter. The maize WUS2 promoter (pZmWUS2) drives the expression of the tDT reporter gene in this reporter construct ( Fig.16 ). The WUS2 promoter reporter construct was co-bombarded with the booster factor in maize immature embryos and leaf segments, respectively.

[0416] Fresh leaf segments of 1-2 cm in length were prepared from 10-14 day old maize A188 seedlings cultured in vitro and placed adaxially upward on the osmotic medium for 4 hours. For co-bombardment, two plasmids (100 ng ZmWUS2 promoter reporter ( Fig.16 ) and 100 ng of booster construct (e.g. TaRKD4) ( Figure 5 )) were premixed and coated on 100 μg of 0.4 μm gold particles. Immature embryo preparation, bombardment and post-bombardment culture were performed as described in Examples 1 and 2. Red fluorescence indicating tDT expression was monitored using a fluorescence microscope starting 16 hours after bombardment.

[0417] WUS is specifically transcribed in the organizing center (OC) of the plant shoot apical meristem (SAM) and controls stem cell identity in the SAM.

[0418] Bombardment with the ZmWUS2 promoter reporter alone (pZmWUS2 reporter alone) did not result in any tDT fluorescence signal from the bombarded leaf samples at any time during the post-bombardment culture period (16 hours to 7 days). However, when co-bombarded with the wheat RKD4 construct ( Figure 5 ), tDT signals were detected in leaf segments approximately 36 hours after bombardment and reached a peak approximately 44 hours after bombardment ( Fig.17 B). Compared to control bombardments using only the WUS promoter reporter, only a weak tDT signal was observed in immature embryos ( Fig.17 A), an abnormally strong red fluorescent signal was observed in embryos co-bombarded with the WUS promoter reporter and the wheat RKD4 construct ( Fig.17 B, upper panel). These results indicate that wheat RKD4 strongly activates the maize WUS2 gene. Images were taken 44 hours after bombardment.

[0419] This observed effect of RKD4 can be combined with transient expression of, for example, ZmPLT7 to result in co-expression of ZmWUS2 and ZmPLT7 according to the present invention.

[0420] Example 5. Co-expression of ZmWUS2 and ZmPLT7 with genome editing components promotes transient genome editing in maize.

[0421] For embryo preparation, bombardment and post-bombardment embryo culture, the processes described in Examples 1 and 2 were performed. After 14 days (Hi II) or 18 days (A188) of callus induction in N6-5Ag medium, rapidly growing embryogenic callus was selected from the surface of the embryo cotyledons of the bombarded embryos and transferred to MRM1 medium (see below) for embryo maturation. After about two weeks of cultivation in MRM1 medium at 25° C. in the dark, the mature embryos were moved to MSO medium (see below) to germinate the embryos in a plant tray under the illumination of 25° C. After about 10 days of cultivation in MSO medium, the regenerated plantlets were ready for molecular analysis and transferred to soil. Leaf tips of approximately 5 mm were collected from all leaves of the regenerated plantlets for DNA extraction. Site-specific genomic modifications were screened from the regenerated plants by Taqman qPCR, marker capillary electrophoresis, and confirmed by Digital PCR, next generation sequencing (NGS) and Sanger sequencing. DNA integration was checked by qPCR.

[0422] Without enhancers, genome editing using Cpf1 (pGEP359) and crRNA5 (pGEP324) did not result in any detectable editing events by transient expression with selection (GE only). However, by co-expression with ZmWUS2 and ZmPLT7, transient genome editing efficiencies of up to 1% could be achieved. These results suggest that co-expression of ZmWUS2 and ZmPLT7 improves transient genome editing.

[0423] Culture medium

[0424] MRM1: MS salt + MS vitamins + 100 mg / L inositol + 6% sucrose + 9 g / L bacterial agar, pH 5.8

[0425] MS0: MS salt + MS vitamin + 2g / L inositol + 2% sucrose + 8g / L bacterial agar, pH 5.8

[0426] ***

[0427] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all values ​​are approximate and are provided for description.

[0428] Throughout this application, patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. A nucleic acid encoding a first enhancer polypeptide and a second enhancer polypeptide, characterized in that: wherein the amino acid sequence of the first enhancer polypeptide is shown in SEQ ID NO: 2; and wherein the amino acid sequence of the second enhancer polypeptide is shown in any one of SEQ ID NO: 4, 6 or 8; or The nucleic acid encoding the third enhancer polypeptide and the second enhancer polypeptide is characterized in that wherein the amino acid sequence of the third enhancer polypeptide is as shown in any one of SEQ ID NO: 16, 18 or 20; and The amino acid sequence of the second enhancer polypeptide is as shown in any one of SEQ ID NO: 4, 6 or 8, wherein after the translation of the third enhancer polypeptide, the expression of the first enhancer polypeptide is activated; wherein the amino acid sequence of the first enhancer polypeptide is as shown in SEQ ID NO: 2, The enhancer polypeptide is a polypeptide that results in improved genome engineering and / or improved plant regeneration of transformed or gene-edited plant cells.

2. The nucleic acid according to claim 1, characterized in that The nucleotide sequence of the nucleic acid encoding the first enhancer polypeptide is shown in SEQ ID NO: 1; and The nucleotide sequence of the nucleic acid encoding the second enhancer polypeptide is shown in any one of SEQ ID NO: 3, 5 or 7, The nucleotide sequence of the nucleic acid encoding the third enhancer polypeptide is shown in any one of SEQ ID NO: 15, 17 or 19.

3. A recombinant gene comprising the nucleic acid of claim 1 or 2.

4. The recombinant gene according to claim 3, characterized in that The nucleic acid encoding the first enhancer polypeptide is operably linked to a native promoter or a first heterologous promoter, and the nucleic acid encoding the second enhancer polypeptide is operably linked to a native promoter or a second heterologous promoter; or the nucleic acid encoding the first enhancer polypeptide and the nucleic acid encoding the second enhancer polypeptide are operably linked to one heterologous promoter; or The nucleic acid encoding the second enhancer polypeptide is operably linked to a native promoter or a first heterologous promoter, and the nucleic acid encoding the third enhancer polypeptide is operably linked to a native promoter or a second heterologous promoter; or the nucleic acid encoding the second enhancer polypeptide and the nucleic acid encoding the third enhancer polypeptide are operably linked to one heterologous promoter.

5. The recombinant gene according to claim 4, characterized in that The heterologous promoter is a strong constitutive promoter, a tissue-specific promoter, a developmental-specific promoter or an inducible promoter.

6. A DNA construct comprising the nucleic acid of claim 1 or 2 or the recombinant gene of any one of claims 3 to 5.

7. The DNA construct according to claim 6, characterized in that The DNA construct is a vector.

8. A method for genetic modification in plant cells, characterized in that The method comprises (1-a) Introducing the following into the plant cells (i) a component selected from the group consisting of: (i.a1) a nucleic acid encoding the first enhancer polypeptide defined in claim 1; a recombinant gene comprising a nucleic acid encoding the first enhancer polypeptide; a DNA construct comprising a nucleic acid encoding the first enhancer polypeptide defined in claim 1, or a first enhancer polypeptide defined in claim 1; and (i.a2) a nucleic acid encoding the second enhancer polypeptide defined in claim 1; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, or a DNA construct comprising a nucleic acid encoding the second enhancer polypeptide defined in claim 1, or a second enhancer polypeptide defined in claim 1; and (ii) transgenic and / or genome engineered components of interest; or The method comprises (2-a) Introducing the following into the plant cells (i) a component selected from the group consisting of: (i.a1) a nucleic acid encoding the third enhancer polypeptide defined in claim 1; a recombinant gene comprising a nucleic acid encoding the third enhancer polypeptide, a DNA construct comprising a nucleic acid encoding the third enhancer polypeptide defined in claim 1, or a third enhancer polypeptide defined in claim 1; and (i.a2) a nucleic acid encoding the second enhancer polypeptide defined in claim 1; a recombinant gene comprising a nucleic acid encoding the second enhancer polypeptide, or a DNA construct comprising a nucleic acid encoding the second enhancer polypeptide defined in claim 1, or a second enhancer polypeptide defined in claim 1; and (ii) transgenic and / or genome engineered components of interest; or The method comprises (3-a) Introducing the following into the plant cells (i) a component selected from the group consisting of: The nucleic acid of claim 1 or 2, the recombinant gene of any one of claims 3 to 5 or the DNA construct of claim 6; and (ii) transgenic and / or genome engineered components of interest, The genome engineering component is a component used to genetically modify the genetic information or genome of plant cells.

9. The method of claim 8, characterized in that The nucleic acid encoding the first enhancer polypeptide contained in the recombinant gene as defined in (1-a)(i.a1), the nucleic acid encoding the second enhancer polypeptide contained in the recombinant gene as defined in (1-a)(i.a2), the nucleic acid encoding the third enhancer polypeptide contained in the recombinant gene as defined in (2-a)(i.a1), or the nucleic acid encoding the second enhancer polypeptide contained in the recombinant gene as defined in (2-a)(i.a2) is operably linked to the promoter defined in claim 4 or 5.

10. The method of claim 8, characterized in that The DNA construct defined in (1-a)(i.a1), (1-a)(i.a2), (2-a)(i.a1) and / or (2-a)(i.a2) is a vector.

11. The method of claim 8, characterized in that After step (1-a), the method further comprises (1-b) culturing the plant cell under conditions that allow translation of the first enhancer polypeptide and the second enhancer polypeptide.

12. The method of claim 8, characterized in that After step (1-a), the method further comprises (1-c) culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of the transgene of interest or by the activity of the genome engineering component in the presence of the enhancer polypeptide.

13. The method of claim 8, characterized in that After step (2-a), the method further comprises (2-b) culturing the plant cell under conditions that allow translation of the third enhancer polypeptide and the second enhancer polypeptide.

14. The method of claim 8, characterized in that After step (2-a), the method further comprises (2-c) culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of the transgene of interest or by the activity of the genome engineering component in the presence of the enhancer polypeptide.

15. The method of claim 8, characterized in that After step (3-a), the method further comprises (3-b) culturing the plant cell under conditions that allow translation of the enhancer polypeptide.

16. The method of claim 8, characterized in that After step (3-a), the method further comprises (3-c) culturing the plant cell under conditions that allow genetic modification of the genome of the plant cell by integration of the transgene of interest or by activity of the genome engineering component in the presence of the enhancer polypeptide.

17. The method of claim 11, 13 or 15, characterized in that The translation level is increased compared to a plant cell into which (1-a)(i.a1) and (i.a2), (2-a)(i.a1) and (i.a2), or (3-a)(i) has not been introduced.

18. The method of claim 8, characterized in that The first and second enhancer polypeptides or the second and third enhancer polypeptides from component (i) are transiently present, transiently active or transiently expressed in the plant cell, or wherein component (i) is transiently present, transiently active or transiently expressed in the plant cell.

19. The method according to any one of claims 8 to 16 and 18, characterized in that The genome engineering components include a) an enzyme that induces a double-strand break (DSB) or a nucleic acid encoding the same; b) an enzyme that induces single-strand breaks (SSBs) or a nucleic acid encoding the same; c) a base editor enzyme; or d) enzymes that affect DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination.

20. The method of claim 19, characterized in that The DSB-inducing enzyme recognizes a predetermined site in the genome of the cell.

21. The method of claim 19, characterized in that The DSB-inducing enzyme is a repair nucleic acid molecule, wherein the repair nucleic acid molecule serves as a template for modifying the genomic DNA or RNA at a predetermined site near or at the cleavage site.

22. The method of claim 19, characterized in that The SSB-inducing enzyme recognizes a predetermined site in the genome of the cell.

23. The method of claim 19, characterized in that The SSB-inducing enzyme is a repair nucleic acid molecule, wherein the repair nucleic acid molecule serves as a template for modifying the genomic DNA or RNA at a predetermined site near or at the cleavage site.

24. The method of claim 19, characterized in that The base editor enzyme is fused to a disarmed DSB-inducing enzyme or a disarmed SSB-inducing enzyme.

25. The method of claim 19, characterized in that The base editor enzyme recognizes a predetermined site in the genome of the cell.

26. The method of claim 19, characterized in that The enzyme affecting DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination is fused to the disarmed DSB-inducing enzyme or the disarmed SSB-inducing enzyme.

27. The method of claim 19, characterized in that The enzyme affecting DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination recognizes a predetermined site in the genome of the cell.

28. The method according to any one of claims 8 to 16 and 18, characterized in that The genome engineering component comprising a DSB-inducing enzyme or a SSB-inducing enzyme or a variant thereof is a CRISPR / Cas endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease or a TAL effector nuclease.

29. The method of claim 28, characterized in that The genome engineering component comprising an enzyme that induces DSB or an enzyme that induces SSB or a variant thereof is CRISPR / Cas9 endonuclease, CRISPR / Cpf1 endonuclease or CRISPR / Csm1 endonuclease.

30. The method of any one of claims 12, 14 and 16, characterized in that The activity of the genome engineering component in step (1-c), (2-c) or (3-c) includes inducing one or more double-strand breaks in the genome of the plant cell, inducing one or more single-strand breaks in the genome of the plant cell, inducing one or more base editing events in the genome of the plant cell, or inducing one or more DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination in the genome of the plant cell.

31. The method of claim 30, characterized in that The induction of one or more double-strand breaks or one or more single-strand breaks is followed by non-homologous end joining (NHEJ) or homology-directed repair of the breaks by homologous recombination mechanisms (HDR).

32. The method according to any one of claims 8 to 16 and 18, characterized in that The transgene in step (1-a)(ii), (2-a)(ii) or (3-a)(ii) is selected from a gene encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphorus deficiency, salt stress or waterlogging, a gene encoding herbicide resistance, including resistance to glyphosate, glufosinate / phosphinothricin, hygromycin, protoporphyrinogen oxidase (PPO) inhibitors, ALS inhibitors and Dicamba, a gene encoding resistance or tolerance to biotic stress, including virus resistance genes, fungal resistance genes, bacterial resistance genes, insect resistance genes, or a gene encoding a yield-related trait, including lodging resistance, flowering time, pod dehiscence resistance, seed color, endosperm components or nutritional content.

33. The method of any one of claims 12, 14 and 16, characterized in that In step (1-c), (2-c) or (3-c), the modification of the genome is selected from i) replacing at least one nucleotide; ii) lack of at least one nucleotide; iii) inserting at least one nucleotide; iv) changes in DNA methylation; v) changes in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone SUMOylation, histone ribosylation or histone citrullination; and vi) Any combination of i)-v).

34. The method according to any one of claims 8 to 16 and 18, characterized in that The method effectively promotes cell proliferation or cell regeneration.

35. The method according to any one of claims 8 to 16 and 18, characterized in that The method effectively induces embryogenesis from a single cell.

36. The method according to any one of claims 8 to 16 and 18, characterized in that The method effectively improves the efficiency of stable transformation of transgenes into the plant cells.

37. The method according to any one of claims 8 to 16 and 18, characterized in that The method effectively increases the efficiency of the genome engineering component in editing the genome of the plant cell.

38. A method for improving the efficiency of plant regeneration or enhancing the regeneration capacity of plant cells, which comprises introducing the components of (1-a)(i) or (2-a)(i) defined in claim 8, the nucleic acid of claim 1 or 2, the recombinant gene of any one of claims 3 to 5, or the DNA construct of claim 6 into the plant cells.

39. A microparticle coated with at least the component (1-a)(i) or (2-a)(i) defined in claim 8, the nucleic acid of claim 1 or 2, the recombinant gene of any one of claims 3 to 4, or the DNA construct of claim 6.

40. Microparticles according to claim 39, characterized in that The microparticles are further coated with a genome engineering component.

41. A kit for genetically modifying a plant genome by microprojectile bombardment, characterized in that: The kit comprises (I) one or more microparticles according to claim 39 or 40, and (II) means for coating the microparticles.

42. The kit of claim 41, further comprising means for coating the microparticles with genome engineering components.

43. A method for producing a genetically modified plant comprising the steps of: (a) genetically modifying a plant cell according to a method according to any one of claims 8 to 37, and (b) regenerating a plant from the modified plant cell of step (1-a), (2-a) or (3-a).

44. The method of claim 43, characterized in that The produced plant does not contain any of the genome engineering components, the nucleic acid encoding the enhancer polypeptide, and the enhancer polypeptide introduced in step (1-a), (2-a) or (3-a).

45. Use of the component of (1-a)(i) or (2-a)(i) defined in claim 8, the nucleic acid of claim 1 or 2, the recombinant gene of any one of claims 3 to 5 or the DNA construct of claim 6 for improving the efficiency of plant regeneration or enhancing the regeneration capacity of plant cells.

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