WO2025111663A1

WO2025111663A1 - Multigene stack for rust resistance

Info

Publication number: WO2025111663A1
Application number: PCT/AU2024/051287
Authority: WO
Inventors: Ming Luo; Michael Ayliffe; Jian Chen; Dhara BHATT; Brenton BROOKS; Aihua Wang
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 2023-11-29
Filing date: 2024-11-29
Publication date: 2025-06-05
Anticipated expiration: 2026-05-29
Also published as: WO2025111662A1

Abstract

The present invention relates to a DNA construct and methods of making a multigene construct comprising five genes which confer upon a plant enhanced resistance to a wide variety of fungal pathogens. Also provided are transfer DNAs which can be used to produce a genetically modified plant that only has DNA from that species of plant. The present invention also relates to genetically modified plants with a transfer DNA of the invention which has been processed during integration and methods of making such plants.

Description

MULTI GENE STACK FOR RUST RESISTANCE

FIELD OF THE INVENTION

The present invention relates to transfer DNAs which can be used to produce a genetically modified plant that only has DNA from that species of plant. The present invention also relates to genetically modified plants with a transfer DNA of the invention which has been processed during integration and methods of making such plants. Also provided is a DNA construct and methods of making a multigene construct comprising five genes which confer upon a plant enhanced resistance to a wide variety of fungal pathogens.

BACKGROUND OF THE INVENTION

Agrobacterium tumefacians is a soil plant pathogen that infects by introducing a specific transferred DNA molecule (T-DNA) encoded on a Ti (tumour-inducing) plasmid into the recipient plant cell to cause crown gall disease. Encoded on the transferred T- DNA sequence are oncogenes that when expressed in the plant cause neoplastic tumourlike plant cell growth that is essential for disease manifestation. Agrobacterium strains are usually grouped by the opine compounds produced by their plant tumours, which are often either nopaline or octopine. These opines provide a carbon and nitrogen source for the bacterium and are produced by bacterial genes encoded on the respective T-DNAs that are transferred into the plant cells. In both cases the bacterium transfers theses T- DNAs using a type IV secretion system that consists of a T-pillus and membrane associate transporter complex. Biotechnology has exploited this natural gene transfer process to introduce genes of choice into plant cells by removing the oncogenes present in the T-DNA region (disarming the Ti plasmid) and replacing the oncogenes with genes of interest during vector construction.

Central to the success of the T-DNA transfer process are the Agrobacteriunf s two 25 bp repeat sequences that flank the T-DNA sequence, known as right border (RB) and left border (LB) sequences, respectively. These sequences delineate the T-DNA region that is transferred to the plant cell and they are present on both octopine and nopaline type Ti plasmids, although some sequence variation exits between these border repeat sequences. T-DNA transfer is initiated by the cleavage of one DNA strand of the RB sequence between the third and fourth base (indicated by the

in Figure 1) by the enzymes VirDl and VirD2 and subsequent transfer of a single strand of T-DNA sequence. The left border sequence is similarly cleaved such the resulting single strand transferred T-DNA encodes 21 bases of the LB repeat sequence and 3 bases of the RB repeat.

When the T-DNA sequences integrate into the plant genome the border sequences are also integrated, resulting in bacterial DNA being present in the target plant cell. Although frequently these genome integrations include only part, or sometimes none, of these border sequence repeats (Kim et al., 2003; Zhu et al., 2013; Kleinboelting et al., 2015; Gong et al., 2021) the screening of plants to identify the resulting lines without Agrobacterium derived sequences is difficult and expensive.

Cisgenic and/or intragenic plant engineering seeks to develop plants for improved products with reduced regulatory hurdle burden. Furthermore, these engineering approaches are attractive for developing and introducing new traits because it is not hampered by linkage drag problems associated with conventional breeding and can more rapidly introduce desired traits into preferred germplasm. To produce cisgenic plants it would be preferable to have modified LB and RB sequences that are derived from the target plant of interest to remove the opportunity for bacterium sequences to be integrated into the target plant.

There is a need for polynucleotides and associated methods which can be used to produce genetically modified plants via Agrobacterium transformation that do not have any DNA which is not from that species of plant.

SUMMARY OF THE INVENTION

Given the changing regulatory acceptance of cisgenic transformation events, the inventors endeavoured to substitute bacterial border sequences with plant genomic sequences to develop transgenic plants that contain no non-endogenous plant DNA sequences.

In an aspect, the present invention provides a transfer DNA (T-DNA) for Agrobacterium mediated gene transfer (AMGT), the T-DNA comprising a first component and a second component, wherein i) the first component is a plant transfer DNA (PT-DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT-DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the PT-DNA comprises part of a right border for integration into the genome, and ii) the second component is within the first component, wherein the second component is a transposable element comprising a polynucleotide encoding a selectable marker. In an aspect the present invention provides a plant having a plant transfer DNA (PT-DNA) integrated into the genome of the plant, wherein the integrated PT-DNA only has DNA from a plant, and wherein the PT-DNA comprises a polynucleotide of interest.

In another aspect, the present invention provides a wheat plant having a wheat transfer DNA (WT-DNA) integrated into the genome of the plant, wherein the integrated WT-DNA only has wheat DNA, and wherein the WT-DNA comprises a polynucleotide of interest.

In another aspect, the present invention provides a soybean plant having a soybean transfer DNA (ST-DNA) integrated into the genome of the plant, wherein the integrated ST-DNA only has soybean DNA, and wherein the ST-DNA comprises a polynucleotide of interest.

Also provided is a plant, or part thereof, having a T-DNA of the invention.

In a further aspect, the present invention provides a plant, or part thereof, having a plant transfer DNA (PT-DNA) integrated into the genome of the plant or part thereof, wherein the integrated PT-DNA only has DNA from a plant, and wherein the PT-DNA comprises a polynucleotide of interest, and wherein i) the plant is wheat and PT-DNA has a processed the left border which comprises SEQ ID NO: 1310, SEQ ID NO:797, SEQ ID NO:798, SEQ ID NO:799, SEQ ID NO:800, SEQ ID NO:801, SEQ ID NO:802, SEQ ID NO:803, SEQ ID NO:804, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 807, SEQ ID NO:808, SEQ ID NO: 809, SEQ ID NO:810, SEQ ID NO:811, SEQ ID NO: 1495, SEQ ID NO:812, SEQ ID NO:816, SEQ ID NO:819, SEQ ID NO:822, SEQ ID NO:823, SEQ ID NO:824, SEQ ID NO:827, SEQ ID NO:345, SEQ ID NO:832, SEQ ID NO:836, SEQ ID NO:839, SEQ ID NO:842, SEQ ID NO:845, SEQ ID NO:850, SEQ ID NO:853, or SEQ ID NO:860, or ii) the plant is soybean and PT-DNA has a processed the left border which comprises SEQ ID NO:939, SEQ ID NO:936, SEQ ID NO:946, SEQ ID NO:949, SEQ ID NO:952, SEQ ID NO:956, SEQ ID NO:959, SEQ ID NO:962, SEQ ID NO: 152, SEQ ID NO:941, SEQ ID NO:345, SEQ ID NO:385, SEQ ID NO:954, SEQ ID NO:314, or SEQ ID NO:303, or iii) the plant is barley and PT-DNA has a processed the left border which comprises SEQ ID NO:251, SEQ ID NO:249, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, or SEQ ID NO:291, or iv) the plant is sugarcane and PT-DNA has a processed the left border which comprises SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:230, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:211, SEQ ID NO: 1007, SEQ ID NO: 1008, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371, or SEQ ID NO:373.

In a further aspect, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT), wherein the Ti plasmid comprises a plant transfer DNA (PT-DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT-DNA comprises part of a left border for integration into the plant genome, and the 3’ end of the PT-DNA comprises part of a right border for integration into the plant genome.

In another aspect, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT), wherein the Ti plasmid comprises a soybean transfer DNA (ST-DNA) for integration into the genome of a soybean plant, wherein the ST-DNA comprises a polynucleotide of interest and only has soybean DNA, wherein the 5 ’ end of the ST-DNA comprises part of a left border for integration into the genome, and the 3’ end of the ST-DNA comprises part of a right border for integration into the genome.

In a further aspect, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT) comprising a T-DNA of the invention, or a PT-DNA of the invention.

In a further aspect, the present invention provides a plant transfer DNA (PT-DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT- DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the PT-DNA comprises part of a right border for integration into the genome.

In a further aspect, the present invention provides a wheat transfer DNA (WT- DNA) for integration into the genome of a wheat plant, wherein the WT-DNA comprises a polynucleotide of interest and only has wheat DNA, wherein the 5’ end of the WT- DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the WT-DNA comprises part of a right border for integration into the genome.

In a further aspect, the present invention provides a soybean transfer DNA (ST- DNA) for integration into the genome of a soybean plant, wherein the ST-DNA comprises a polynucleotide of interest and only has soybean DNA, wherein the 5 ’ end of the ST-DNA comprises part of, or has, a left border for integration into the genome, and the 3’ end of the ST-DNA comprises part of a right border for integration into the genome.

In another aspect, the present invention provides A plant transfer DNA (PT-DNA) for Agrobacterium mediated gene transfer (AMGT) integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT-DNA comprises part of, or has, a left border for integration into the genome, and the 3’ end of the PT-DNA comprises part of a right border for integration into the genome, and wherein, i) the PT-DNA is a wheat transfer DNA (WT-DNA), and wherein the left border comprises SEQ ID NO: 1340, SEQ ID NO: 1450, SEQ ID NO:877, SEQ ID NO: 1451, SEQ ID NO:880, SEQ ID NO: 1452, SEQ ID NO:882, SEQ ID NO:883, SEQ ID NO:884, SEQ ID NO:885, SEQ ID NO:887, SEQ ID NO: 1453, SEQ ID NO:890, SEQ ID NO: 1454, SEQ ID NO: 893, SEQ ID NO: 1455, SEQ ID NO: 896, SEQ ID NO: 1456, SEQ ID NO:899, SEQ ID NO: 1457, SEQ ID NO:902, SEQ ID NO: 1458, SEQ ID NO:905, SEQ ID NO: 1459, SEQ ID NO:908, SEQ ID NO: 1460, SEQ ID NO:911, SEQ ID NO: 1461, SEQ ID NO:914, SEQ ID NO: 1462, SEQ ID NO:917, SEQ ID NO: 1463, SEQ ID NO:874, SEQ ID NO: 1464, SEQ ID NO:922 or SEQ ID NO: 1465, or ii) the PT-DNA is a soybean transfer DNA (ST-DNA), and wherein the left border comprises SEQ ID NO:977, SEQ ID NO: 1466, SEQ ID NO:974, SEQ ID NO: 1467, SEQ ID NO:982, SEQ ID NO: 1468, SEQ ID NO:985, SEQ ID NO: 1469, SEQ ID NO:988, SEQ ID NO: 1470, SEQ ID NO:991, SEQ ID NO: 1471, SEQ ID NO:994, SEQ ID NO: 1472, SEQ ID NO:997 or SEQ ID NO: 1473, or iii) the plant is barley, and the left border comprises SEQ ID NO: 1500, SEQ ID NO:607, SEQ ID NO: 1474, SEQ ID NO:612, SEQ ID NO: 1475, SEQ ID NO:615, SEQ ID NO: 1476, SEQ ID NO:618, SEQ ID NO: 1477, SEQ ID NO:621, SEQ ID NO: 1478, SEQ ID NO:624, SEQ ID NO: 1479, SEQ ID NO:627, SEQ ID NO: 1480, SEQ ID NO:630, SEQ ID NO: 1481, SEQ ID NO:624, SEQ ID NO: 1482, SEQ ID NO:635, SEQ ID NO: 1483, SEQ ID NO:638 or SEQ ID NO: 1484, or iv) the plant is sugarcane, and the left border comprises SEQ ID NO:683, SEQ ID NO: 1485, SEQ ID NO:686, SEQ ID NO: 1486, SEQ ID NO:689, SEQ ID NO: 1487, SEQ ID NO:692, SEQ ID NO: 1488, SEQ ID NO:695, SEQ ID NO: 1489, SEQ ID NO:698, SEQ ID NO: 1490, SEQ ID NO: 1011, SEQ ID NO: 1491, SEQ ID NO:703, SEQ ID NO: 1492, SEQ ID NO:706, SEQ ID NO: 1493, SEQ ID NO:709 or SEQ ID NO: 1494.

In an embodiment, the WT-DNA left border comprises 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtg gtctagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaaga gaagagtcgcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgagaattcctcatttggtttcacattcttaag catgattactccattcttaattgggcctataactcatagcagagcatacacatttgaatttctttattatcttcttgaacaattactttttg tcaattcatattccatag-3’ (SEQ ID NO: 1311), or a 5’ fragment thereof comprising 5’- tggcaggatatattgtggtgtaaact-3’ (SEQ ID NO: 1340).

In an embodiment, the WT-DNA left border comprises 5’- cggcaggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtg gtctagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaaga gaagagtcgcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgagaattcctcatttggtttcacattcttaag catgattactccattcttaattgggcctataactcatagcagagcatacacatttgaatttctttattatcttcttgaacaattactttttg tcaattcatattccatag-3’ (SEQ ID NO: 1496), or a 5’ fragment thereof comprising 5’- cggcaggatatattgtggtgtaaac-3’ (SEQ ID NO: 1450).

In an embodiment, the WT-DNA left border comprises 5’- tggtaggatatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacaga aaagaaaagaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgca caaaggcccagcccacgtcggccttccact-3’ (SEQ ID NO: 1497) or a 5’ fragment thereof comprising 5’-tggtaggatatattgtggtgtgatt-3’ (SEQ ID NO:874).

In an embodiment, the WT-DNA left border comprises 5’- cggtaggatatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacag aaaagaaaagaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgc acaaaggcccagcccacgtcggccttccact-3’ (SEQ ID NO: 1498) or a 5’ fragment thereof comprising 5’-cggtaggatatattgtggtgtgatt-3’ (SEQ ID NO: 1464).

In another aspect, the present invention provides a polynucleotide comprising a left border and/or a right border of the invention.

In another aspect, the present invention provides a method of producing a genetically modified plant, the method comprising contacting a plant cell with an Agrobacterium sp. comprising a Ti plasmid of the invention.

In an embodiment, the cell is a cell of a wounded plant tissue explant.

In an embodiment, following being contacted with the Agrobacterium sp. the explant is cultured to produce a callus.

In an embodiment, the method further comprises producing a plant from the callus.

In an embodiment, the method further comprises screening the plant for the WT- DNA of the invention, the ST-DNA of the invention, or the PT-DNA of the invention.

In an embodiment, the method further comprises screening the plant for the WT- DNA, ST-DNA or PT-DNA in germline cells of the plant. In an embodiment, the method further comprises producing progeny plants from a plant having the WT-DNA, ST-DNA the PT-DNA in germline cells of the plant.

In a further aspect, the present invention a plant produced by the method of the invention, or having the WT-DNA of the invention, the ST-DNA of the invention, or the PT-DNA of the invention.

In another aspect, the present invention provides a method of producing a genetically modified plant, the method comprising the steps of i) crossing two parental plants, wherein at least one plant is a plant of the invention, ii) screening one or more progeny plants from the cross in i) for the presence or absence of the WT-DNA of the invention, the ST-DNA of the invention, or the PT-DNA of the invention, and iii) selecting a progeny plant comprising the WT-DNA of the invention, the ST- DNA of the invention, or the PT-DNA of the invention, thereby producing the plant.

In a further aspect, the present invention provides a method of producing a genetically modified plant, the method comprising the steps of i) crossing two parental plants, wherein at least one plant is a plant of the invention, ii) screening one or more progeny plants from the cross in i) for the presence or absence of the integrated PT-DNA, and iii) selecting a progeny plant comprising the integrated PT-DNA, thereby producing the plant.

In an embodiment, one parental plant is a plant having a T-DNA of the invention and the other parental plant is a transgenic plant expressing a transposase, and step ii) comprises screening the one or more progeny plants from the cross for the presence or absence of the second component, and step iii) comprises selecting a progeny lacking the second component.

In an embodiment, step ii) comprises analysing a sample comprising DNA from the plant for the WT-DNA, the ST-DNA or the PT-DNA.

In an embodiment, step iii) comprises selecting progeny plants which are homozygous for the WT-DNA, the ST-DNA or the PT-DNA.

In an embodiment, the method further comprises: iv) backcrossing the progeny of the cross of step i) with plants of the same genotype as a first parent plant which lacked the WT-DNA or the ST-DNA for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising the WT-DNA or the ST-DNA, and v) selecting a progeny plant which has the WT-DNA or the ST-DNA.

Also provided is a plant produced using a method of the invention.

In another aspect, the present invention provides a method of identifying a plant of the invention, the method comprising: i) obtaining a sample from a plant comprising genomic DNA, ii) screening the sample for the presence or absence of the WT-DNA of the invention, the ST-DNA of the invention, or the PT-DNA of the invention.

In another aspect, the present invention provides a method of identifying a plant of the invention, the method comprising i) obtaining a sample from a plant comprising genomic DNA, ii) screening the sample for the presence or absence of the integrated PT-DNA.

In an embodiment, the plant is a plant having a T-DNA of the invention, and the method further comprises screening the sample for the absence of the second component.

In an embodiment, the plant is a wheat plant and step ii) comprises analysing the DNA for the presence of the WT-DNA.

In an embodiment, the plant is a barley plant and step ii) comprises analysing the DNA for the presence of the barley PT-DNA.

In an embodiment, the plant is a sugarcane plant and step ii) comprises analysing the DNA for the presence of the sugarcane PT-DNA.

In an embodiment, the plant is a soybean plant and step ii) comprises analysing the DNA for the presence of the ST-DNA.

In an embodiment, step ii) comprises amplifying a region of the genome comprising the WT-DNA or ST-DNA using the polymerase chain reaction.

In an embodiment, the amplification is achieved using an oligonucleotide primer comprising a sequence of nucleotides provided as any one of SEQ ID NOs: 1 to 12, or a variant thereof which can be used to amplify the same region of the genome.

In an embodiment, the amplification is achieved using an oligonucleotide primer comprising a sequence of nucleotides provided as any one of SEQ ID NOs: 1031 to 1044, or a variant thereof which can be used to amplify the same region of the genome.

In an embodiment, step ii) comprises sequencing an amplicon produced from the genomic DNA.

In a further aspect, the present invention provides a method of producing a left border for a T-DNA for Agrobacterium mediated gene transfer (AMGT), the method comprising: i) searching the genome of a plant species of interest for a polynucleotide which is 22 to 25 bases in length and which shares at least 16 nucleotides with an Agrobacterium sp. Ti plasmid left border, wherein if the polynucleotide is a) 22 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, b) 23 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 23 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, c) 24 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 24 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, ii) producing a T-DNA having a left border sequence, wherein the left border of the T-DNA a) comprises the polynucleotide, wherein the polynucleotide is 25 bases in length, or b) if the polynucleotide does not have a 5’-tgg or a 5 ’-egg and/or is not 25 bases in length, the polynucleotide is modified such that it is 25 bases in length and has a 5’-tgg or a 5 ’-egg to produce the left border sequence, and iii) determining if the T-DNA can be used to transform the plant species of interest using AMGT.

In an embodiment, if a plant is transformed, determining if the part of the left border integrated into the genome of the plant lacks the 5’-tgg or 5 ’-egg.

In a further aspect, the present invention provides a method of producing a left border for a T-DNA for Agrobacterium mediated gene transfer (AMGT), the method comprising: i) searching the genome of a plant species of interest for a polynucleotide which is 22 to 25 bases in length and which shares at least 16 nucleotides with an Agrobacterium sp. Ti plasmid left border, wherein if the polynucleotide is a) 22 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, b) 23 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 23 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, c) 24 bases in length the genome is searched for a polynucleotide which shares at least 16 nucleotides with the 24 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border, ii) producing a T-DNA having a left border sequence, wherein if the polynucleotide does not have a 5’-tggcagga or a 5’-cggcagga and/or is not 25 bases in length, the polynucleotide is modified such that it is 25 bases in length and has a 5’- tggcagga or a 5’-cggcaggato produce the left border, and iii) determining if the T-DNA can be used to transform the plant species of interest using AMGT.

In an embodiment, if a plant is transformed, determining if the part of the left border integrated into the genome of the plant lacks the 5’-tggcagga or 5’-cggcagga.

In an embodiment, step i) comprises searching the genome of a plant species of interest for a polynucleotide which is 22 to 25 bases in length and which shares 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides with an Agrobacterium sp. Ti plasmid left border.

In an embodiment, step i) comprises searching the genome of a plant species of interest for a polynucleotide which is 22 bases in length and which shares at least 16 nucleotides with the 22 3’ nucleotides of an Agrobacterium sp. Ti plasmid left border.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTGTGGTGTAAAC-3’ SEQ ID NO:769.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTGAATTGTAAAT-3’ SEQ ID NO: 765.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTCAATTGTAAAC-3’ SEQ ID NO: 766.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTTAGTTGTAAAA-3’ SEQ ID NO: 767.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTGTGATGTAAAC-3’ SEQ ID NO:768.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATCAAAGTGTAAGT-3’ SEQ ID NO:770.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATCGAGGTGTAAAA-3’ SEQ ID NO: 771.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATGTATTGTCATGTAAAA-3’ SEQ ID NO:772.

In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATCGTTATGTAATC-3’ SEQ ID NO:773. In an embodiment, the 22 3’ nucleotides of the Agrobacterium sp. Ti plasmid left border are 5’-CAGGATATATTGGTGTGTAAAC-3’ SEQ ID NO:774.

In an embodiment, the Ti plasmid used in step iii) is a Ti plasmid of the invention.

In an embodiment, step iv) comprises determining if the T-DNA integrated into the genome of the plant only has DNA of that plant Genus. In an embodiment, the Genus is Triticum.

In an embodiment, step iv) comprises determining if the T-DNA integrated into the genome of the plant only has DNA of that plant species.

In an embodiment, the plant is a cereal plant.

In an embodiment, the plant is wheat, soybean, rice, tomato, sugarcane, potato, banana, sorghum, grape or oats. In an embodiment, the plant is wheat, soybean, rice, tomato, sugarcane, potato, banana or sorghum.

Also provided is a plant produced by the method of the invention.

There is also a need for DNA constructs which can used to enhance in planta resistance to one or more fungal pathogen(s) and also be less prone to the pathogen(s) escaping the resistance. Whilst many proteins which confer enhanced resistance to one or more fungal pathogen(s) have been identified, many are overcome when deployed as single genes. The present inventors have identified a new combination of fungal resistance genes that provide a stable construct with can be used to confer widespread fungal resistance and reduce the chance of the pathogen(s) becoming resistant due to the polygenic nature of the in planta resistance.

Accordingly, in a further aspect the present invention provides a DNA construct comprising genes encoding: i) a first polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 13, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 13, ii) a second polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 14, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 14 iii) a third polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 15, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 15, iv) a fourth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 16, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 16, v) a fifth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 17, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 17, wherein each gene comprises a promoter that is capable of directing expression of the genes in a cell of a plant, and wherein each polypeptide confers enhanced resistance to one or more fungal pathog en(s). In an embodiment, the cell is a stem and/or a leaf cell.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 13, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 13.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 14, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 14.

In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 15 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 15.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 16, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 16.

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 17, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 17.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 13, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 13.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 14, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 14.

In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 15, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 15.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 16, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 16.

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 17, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 17.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 13.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 14. In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 15.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 16

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 17.

In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 19, or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 19.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:21, or a nucleotide sequence which is at least 80% identical to SEQ ID NO:21.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:23, or a nucleotide sequence which is at least 80% identical to SEQ ID NO:23.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:25 or a nucleotide sequence which is at least 80% identical to SEQ ID NO:25.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 27, or a nucleotide sequence which is at least 80% identical to SEQ ID NO:27.

In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 19, or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 19.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:21, or a nucleotide sequence which is at least 90% identical to SEQ ID NO:21.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:23, or a nucleotide sequence which is at least 90% identical to SEQ ID NO:23.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:25 or a nucleotide sequence which is at least 90% identical to SEQ ID NO:25.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 27, or a nucleotide sequence which is at least 90% identical to SEQ ID NO:27. In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 19, or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 19.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:21, or a nucleotide sequence which is at least 95% identical to SEQ ID NO:21.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:23, or a nucleotide sequence which is at least 95% identical to SEQ ID NO:23.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO:25 or a nucleotide sequence which is at least 95% identical to SEQ ID NO:25.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 27, or a nucleotide sequence which is at least 95% identical to SEQ ID NO:27.

In an embodiment, the gene encoding the first polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 18 or SEQ ID NO: 19.

In an embodiment, the gene encoding the second polypeptide comprises a sequence of nucleotides provided as SEQ ID NO:20 or SEQ ID NO:21.

In an embodiment, the gene encoding the third polypeptide comprises a sequence of nucleotides provided as SEQ ID NO:22 or SEQ ID NO:23.

In an embodiment, the gene encoding the fourth polypeptide comprises a sequence of nucleotides provided as SEQ ID NO:24 or SEQ ID NO:25.

In an embodiment, the gene encoding the fifth polypeptide comprises a sequence of nucleotides provided as SEQ ID NO:26 or SEQ ID NO:27.

In an embodiment, the DNA construct comprises a sequence of nucleotides provided as SEQ ID NO:28, or a nucleotide sequence which is at least 80% identical, at least 90% identical or at least 95% identical to SEQ ID NO:28.

In an embodiment, each polypeptide confers resistance to Puccinia sp. such as one or more or all of Puccinia graminis (stem rust), Puccinia striiformis (stripe rust), Puccinia triticina (leaf rust).

In a further aspect the present invention provides a DNA construct comprising genes encoding: i) a first polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1271, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1271, ii) a second polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1272, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1272, iii) a third polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1273, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1273, iv) a fourth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1274, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1274, v) a fifth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1275, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1275, wherein each gene comprises a promoter that is capable of directing expression of the genes in a cell of a plant, and wherein each polypeptide confers enhanced resistance to one or more fungal pathog en(s).

In an embodiment, the cell is a stem and/or a leaf cell.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1271, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 1271.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1272, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 1272.

In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1273 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 1273.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1274, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 1274.

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1275, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 1275.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1271, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 1271.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1272, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 1272. In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1273, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 1273.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1274, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 1274.

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1275, or an amino acid sequence which is at least 95% identical to SEQ ID NO: 1275.

In an embodiment, the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1271.

In an embodiment, the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1272.

In an embodiment, the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1273.

In an embodiment, the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1274

In an embodiment, the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 1275.

In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1276, or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1276.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1277, or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1277.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1278, or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1278.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1279 or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1279.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 1280, or a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1280. In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1276, or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 1276.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1277, or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 1277.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1278, or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 1278.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1279 or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 1279.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 1280, or a nucleotide sequence which is at least 90% identical to SEQ ID NO: 1280.

In an embodiment, the polynucleotide encoding the first polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1276, or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1276.

In an embodiment, the polynucleotide encoding the second polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1277, or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1277.

In an embodiment, the polynucleotide encoding the third polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1278, or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1278.

In an embodiment, the polynucleotide encoding the fourth polypeptide encodes a sequence of nucleotides provided as SEQ ID NO: 1279 or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1279.

In an embodiment, the polynucleotide encoding the fifth polypeptide encodes sequence of nucleotides provided as SEQ ID NO: 1280, or a nucleotide sequence which is at least 95% identical to SEQ ID NO: 1280.

In an embodiment, the gene encoding the first polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 1502 or SEQ ID NO: 1276.

In an embodiment, the gene encoding the second polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 1503 or SEQ ID NO: 1277.

In an embodiment, the gene encoding the third polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 1504 or SEQ ID NO: 1278. In an embodiment, the gene encoding the fourth polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 1505 or SEQ ID NO: 1279.

In an embodiment, the gene encoding the fifth polypeptide comprises a sequence of nucleotides provided as SEQ ID NO: 1506 or SEQ ID NO: 1280.

In an embodiment, the DNA construct comprises a sequence of nucleotides provided as SEQ ID NO: 1448, or a nucleotide sequence which is at least 80% identical, at least 90% identical or at least 95% identical to SEQ ID NO: 1448.

In an embodiment, one or more or all of the polypeptides confer resistance to Puccinia sp.. In an embodiment, one or more or all of the polypeptides confer resistance to Puccinia striiformis f. sp. Tritici (yellow wheat rust).

In a further aspect, the present invention provides a T-DNA comprising the DNA construct of the invention.

In an embodiment, the T-DNA is the WT-DNA of the invention, the ST-DNA of the invention, or the PT-DNA of the invention.

In a further aspect, the present invention provides a chimeric vector comprising the DNA construct of the invention. In a further aspect, the present invention provides a T-DNA comprising the DNA construct of the invention.

In an embodiment, the vector is a Ti plasmid of the invention.

In a further aspect, the present invention provides a recombinant cell comprising the DNA construct of the invention, the T-DNA of the invention, or the vector of the invention. In an embodiment, the recombinant cell is a bacterial cell.

In an embodiment, the recombinant cell is an Agrobacterium comprising a Ti plasmid of the invention.

In a further aspect, the present invention provides a method of producing the cell of the invention, the method comprising the step of introducing the DNA construct of the invention, the T-DNA of the invention, or the vector of the invention into a cell.

In another aspect, the present invention provides an Agrobacterium-mcAxeAcA transformation method for the insertion of a DNA into a plant cell, the method comprising: i) contacting a plant cell with Agrobacterium bacterium comprising a Ti plasmid of the invention under conditions that permit the Agrobacterium bacterium to infect the plant cell, thereby transforming the plant cell, ii) selecting and screening the transformed plant cells; and iii) regenerating whole transgenic plants from the selected and screened plant cells. In a further aspect, the present invention provides a method of producing the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide as defined herein, the method comprising expressing in a cell or cell free expression system the genes in the DNA construct of the invention, the T-DNA of the invention, or the vector of the invention.

In a further aspect, the present invention provides a genetically modified plant comprising the DNA construct of the invention.

In an embodiment, the plant has enhanced resistance to one or more fungal pathogen(s) when compared to a corresponding wild-type plant lacking the DNA construct.

In an embodiment, the one or more fungal pathogen(s) include one or more or all of Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolaris sp., Oidium sp., Gibberella sp., Cochliobolus sp., Exserohilum sp., Uredo sp. Microdochium sp., Helminthosporium sp., Monographella sp., Colletotrichum sp., Uromyces sp. or Erysiphe sp..

In an embodiment, the plant has enhanced resistance to one or more or all of stem rust, leaf rust or stripe rust, when compared to the corresponding wild-type plant lacking the DNA construct.

In an embodiment, the plant has enhanced resistance to stem rust when compared to the corresponding wild-type plant lacking the DNA construct.

In an embodiment, the plant is a cereal plant. Examples of cereal plants of the invention include, but are not limited to, wheat, oats, rye, barley, rice, com, sorghum or maize. In an embodiment, the plant is a wheat plant.

In an embodiment, the plant is a soybean or banana plant.

In an embodiment, the plant is homozygous for the DNA construct.

In an embodiment, the plant only comprises DNA found in a plant of the same species such as Triticum.

In an embodiment, the plant only comprises DNA found in a plant of the same species.

In a further aspect, the present invention provides a method of producing a plant with a DNA construct of the invention, the method comprising the steps of i) introducing a DNA construct of the invention to a plant cell, ii) regenerating a plant with the DNA construct from the cell, and iii) optionally harvesting seed from the plant, and/or iv) optionally producing one or more progeny plants, thereby producing the plant. In an embodiment, the DNA construct is in a T-DNA of a tumour-inducing (Ti) plasmid and is introduced using Agrobacterium mediated gene transfer (AMGT).

In a further aspect, the present invention provides a method of producing a plant with a DNA construct of the invention, the method comprising the steps of i) crossing two parental plants, wherein at least one plant comprises a DNA construct of the invention, ii) screening one or more progeny plants from the cross in i) for the presence or absence of the DNA construct, and iii) selecting a progeny plant comprising the DNA construct, thereby producing the plant.

In an embodiment, step ii) comprises analysing a sample comprising DNA from the plant for the DNA construct.

In an embodiment, step iii) comprises: i) selecting progeny plants which are homozygous for the DNA construct, and/or ii) analysing the plant or one or more progeny plants thereof for enhanced resistance to one or more fungal pathogen(s).

In an embodiment, the method further comprises: iv) backcrossing the progeny of the cross of step i) with plants of the same genotype as a first parent plant which lacked the DNA construct for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising the DNA construct, and v) selecting a progeny plant which has enhanced resistance to one or more fungal pathogen(s).

Also provided is plant produced using the method of the invention.

In a further aspect, the present invention provides a method for identifying a plant which has enhanced resistance to one or more fungal pathogen(s), the method comprising the steps of i) obtaining a sample from a plant, and ii) screening the sample for the presence or absence of a DNA construct of the invention.

In an embodiment, the screening comprises amplifying a region of the genome of the plant.

Also provided is the use of the Ti plasmid of the invention, the WT-DNA of the invention, the ST-DNA of the invention, the PT-DNA of the invention, the polynucleotide of the invention, or the DNA construct of the invention, to produce a recombinant cell and/or a genetically modified plant. In an embodiment, a plant of the invention is growing in a field.

In a further aspect, the present invention provides a population of at least 100 plants of the invention growing in a field.

In a further aspect, the present invention provides a plant part of the plant of the invention.

In an embodiment, the plant part is a seed that comprises WT-DNA of the invention, the ST-DNA of the invention, the PT-DNA of the invention, the T-DNA as described herein, or the DNA construct of the invention.

In a further aspect, the present invention provides a method of producing a plant part, the method comprising, a) growing a plant of the invention, and b) harvesting the plant part.

In an embodiment, the plant part is seed.

In a further aspect, the present invention provides a method of producing flour, wholemeal, starch, seedmeal, oil or other product obtained from seed, the method comprising: a) obtaining seed of the invention, and b) extracting the flour, wholemeal, starch, seedmeal, oil or other product.

In an embodiment, the other product is bran.

In a further aspect, the present invention provides a product produced from a plant of the invention and/or a plant part of the invention, wherein the product comprises the DNA construct, the integrated T-DNA or the integrated PT-DNA such as the integrated WT-DNA.

In an embodiment, wherein the part is a seed.

In an embodiment, the product is a food product or beverage product.

In an embodiment, the food product is selected from the group consisting of: flour, starch, leavened or unleavened breads, pasta, noodles, animal fodder, animal feed, breakfast cereals, snack foods, cakes, malt, pastries and foods containing flour-based sauces.

In an embodiment, the beverage product is beer, plant milk or malt.

In an embodiment, the product is a non-food product.

In a further aspect, the present invention provides a method of preparing a food product of the invention, the method comprising mixing seed, or flour, wholemeal, starch, seedmeal, or oil from the seed, with another food ingredient.

In a further aspect, the present invention provides a method of preparing malt, comprising the step of germinating seed of the invention. In a further aspect, the present invention provides the use of a plant of the invention, or part thereof, as animal feed, or to produce feed for animal consumption or food for human consumption.

In a further aspect, the present invention provides the use of a plant of the invention for controlling or limiting one or more fungal pathogen(s) in crop production.

In a further aspect, the present invention provides a composition comprising the Ti plasmid of the invention, the WT-DNA of the invention, the ST-DNA of the invention, the PT-DNA of the invention, the polynucleotide of the invention, or the DNA construct of the invention, and one or more acceptable carriers.

In a further aspect, the present invention provides a method of trading seed, comprising obtaining seed of the invention, and trading the obtained seed for pecuniary gain.

In an embodiment, obtaining the seed comprises cultivating the plant of the invention and/or harvesting the seed from the plants.

In an embodiment, obtaining the seed further comprises placing the seed in a container and/or storing the seed.

In an embodiment, obtaining the seed further comprises transporting the seed to a different location.

In an embodiment, the trading is conducted using electronic means such as a computer.

In a further aspect, the present invention provides a process of producing bins of seed comprising: a) swathing, windrowing and/or or reaping above-ground parts of plants comprising seed of the invention, b) threshing and/or winnowing the parts of the plants to separate the seed from the remainder of the plant parts, and c) sifting and/or sorting the seed separated in step b), and loading the sifted and/or sorted seed into bins, thereby producing bins of seed.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 - Example of Agrobacterium T-DNA’s and integration into the plant genome.

Figure 2 - Examples of Agrobacterium T-DNA’s (from Holsters et al., 1983).

Figure 3 - Alignment of RB sequences from 6 binary vectors. The 24 bp RB repeat is highlighted. Nucleotides 5’ of the RB sequence and conserved amongst five of the 6 plasmids are highlighted. The virD cleavage site is indicated with an arrow.

Figure 4 - Wheat genomic sequences used as LB and RB substitutes in plasmid pMO. The upper LB sequence replacement sequence corresponds to nucleotides 631979550 - 631979773 of wheat chromosome 7D (IWGSC Chinese Spring v. 2.1), while the lower RB replacement corresponds to nucleotides 553784436 - 553784630 of wheat chromosome 5D (IWGSC Chinese Spring genome v2.1). Sequences underlined are identical to Agrobacterium LB and RB sequences. Bases boxed in yellow are not derived from wheat i.e., synthetic plasmid sequence. The position of virD cleavage sites are shown with arrows and sequence not transferred as part of the T-DNA indicated. Sequence boxed is a wheat sequence with identity to binary vector plasmid sequences in immediate juxtaposition to the RB (excluding nucleotides TGA).

Figure 5 - Transient Agrobacterium expression of RUBY in N. benthamiana of the plasmids depicted at top. The first panel shows leaves 2 days post infiltration while the lower panel shows leaves 5 days post-infiltration.

Figure 6 - Analysis of cleavage of the wheat substitute LB using transient Agrobacterium infiltration assays. Agrobacterium strains containing constructs 1-4 (above) were infiltrated into sites 1-4 on N. benthamiana leaves shown at bottom. Leaves on the right are the same leaves shown at left after clearing in ethanol. Figure 7 -Transformation of wheat with pMO. A) Betalain accumulation at cell foci on cultured wheat scutellum explant tissue transformed with pMO. B) Graph of frequency of Fielder calli showing betalain accumulation after transformation with pMO or pUbi- RUBY/BAR. C) Regeneration of a transgenic wheat shoot transformed with pMO, in the absence of selection. D) Red transgenic wheat plant transformed with pMO (left) with a control Fielder wheat plant shown at right.

Figure 8 - PCR analysis of cleavage of the wheat LB border repeat substitution sequence in pMO. DNA was extracted from three wild type Fielder calli (w) and five calli transformed with pMO and showing evidence of betalain accumulation (b). Approximate position of PCR amplification products are shown at right on a schematic of a small region of the pMO vector. The pMO vector was used as a positive control (+).

Figure 9 - PCR analysis of cleavage of the wheat RB border repeat substitution sequence in pMO. DNA was extracted from three wild type Fielder calli (w) and five calli transformed with pMO and showing evidence of betalain accumulation (b). Approximate position of PCR amplification products are shown at right on a schematic of a small region of the pMO vector. Black lines show non-transferred binary sequences while blue lines show T-DNA sequence. The pMO vector was used as a positive control (+).

Figure 10 - Binary constructs used for co-transformation of wheat cultivar Fielder. Each construct encoding RUBY was independently co-transformed with the wheat Sr gene stack shown below. Note constructs are not drawn to scale and the wheat Sr stack construct is significantly larger being approximately 40 kb in size.

Figure 11 - Wheat chromosome 5A (IWGS Chinese Spring version 2.1) 669141979 - 669142155 sequence (blue text) used in construct pM2. Sequence underlined at the 5’ end shows similarity to the Agrobacterium nopaline LB repeat sequence shown above (black sequence). Nucleotide differences are shown in red.

Figure 12 - Junction PCR results for wheat Sr stack construct in of transgenic plants produced by co transformation. Numbers show the number of independent transgenics positive for the primer pairs indicated for each of the three co-transformations.

Figure 13 - Soybean sequences used to substitute the Agrobacterium LB and RB sequences present in binary vector pCSIRO/RUBY (see Figure 5). Soybean sequence identical to the Agrobacterium LB or RB is highlighted. Bona fide Agrobacterium LB and RB sequences are shown in purple above and below the soybean sequences, respectively.

Figure 14 - Binary vector sequences produced using soybean LB and RB substitute sequences are shown at top. The N. benthamiana leaves shown below (left) were infiltrated with Agrobacterium ^xsaaAGLl cultures containing the constructs indicated. Shown at right are the same leaves after clearing in 100% ethanol.

Figure 15 - Map of the pMl-5Yr-PiggyBac-35SBar wheat T-DNA sequence.

Figure 16 - Structure of T-DNA insertion after excision of PiggyBac.

Figure 17 - HyPbase in pDONORl.

Figure 18 - 745 bp PCR product demonstrating that PiggyBac-35SBAR has excised.

Figure 19 - Barley LB sequences used in Example 11. The chromosome 6H nucleotide sequence 500797397 - 500797357 was used in construct pMBl. The chromosome 1H nucleotide sequence 151772483 - 151772512 was used in construct pMB2. Sequence underlined at the 5’ end shows similarity to the Agrobacterium nopaline LB repeat sequence shown above (black sequence).

Figure 20 - Barley chromosome 5H nucleotide sequence 50835791 - 508357886 was used in both constructs pMBl and pMB2 as a substitute RB sequence. Sequence underlined at the 3 ’ end shows similarity to sequences 5 ’ of the Agrobacterium nopaline RB repeat. The nopaline RB repeat sequence is bracketed above. Bases not derived from barley and are synthetic border sequence not transferred as part of the T-DNA. The position of virD cleavage sites is shown with a red arrow and sequence not transferred as part of the T-DNA indicated. Bases underlined on barley chr 5H sequence are identical to binary vector plasmid sequences in immediate juxtaposition to the RB, although nucleotides TGA are part of the RB sequence.

Figure 21 - Structure of the plant T-DNA region in binary plasmid pMX (upper) and the control standard binary vector pAM which encodes wild type Agrobacterium LB and RB sequences, a 35S-YFP gene and a phosphinothricin acetyltransferase selectable marker gene under the regulatory control of an Agrobacterium nopaline synthase promoter.

Figure 22 - At left is a Nicotiana benthamiana leaf 5 days post infiltration with Agrobacterium strain AGL1 carrying either vector; (1) pM/35S-YFP, (2) pMBl or (3) pMB2. Shown at right is the same leaf cleared in ethanol to further highlight betalain production.

KEY TO THE SEQUENCE LISTING

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, plant molecular biology, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 5%, or more preferably +/- 1%, of the designated value.

Transfer Nucleic Acids

Transfer nucleic acids can be used to deliver an exogenous polynucleotide to a cell and comprise one, preferably two, border sequences and a polynucleotide of interest. The transfer nucleic acid may or may not encode a selectable marker. Preferably, the transfer nucleic acid forms part of a binary vector in a bacterium, where the binary vector further comprises elements which allow replication of the vector in the bacterium, selection, or maintenance of bacterial cells containing the binary vector. Upon transfer to a eukaryotic cell, preferably a plant cell, the transfer nucleic acid component of the binary vector is capable of integration into the genome of the eukaryotic cell.

As used herein, the term "transfer nucleic acid" or T-DNA refers to a nucleic acid molecule that is capable of being transferred from a bacterium such as Agrobacterium sp., to a eukaryotic cell such as a plant cell. A T-DNA is a genetic element that is well- known as an element capable of being transferred, with the subsequent integration of a nucleotide sequence contained within its borders into the genome of the recipient cell. In this respect, a transfer nucleic acid is flanked, typically, by two "border" sequences, although in some instances a single border at one end can be used and the second end of the transferred nucleic acid is generated randomly in the transfer process. A polynucleotide of interest is typically positioned between the left border sequence and the right border sequence of a transfer nucleic acid. The polynucleotide contained within the transfer nucleic acid may be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, that is, transcription and/or translation of the polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. and man-made variants/mutants thereof are probably the best characterized examples of transfer nucleic acids.

As used herein, "T-DNA" refers to T-DNA of an Agrobacterium tumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid, or man-made variants thereof which function as T-DNA. The T-DNA may comprise an entire T-DNA including both right and left border sequences, but need only comprise the minimal sequences required in cis for transfer, that is, the right and T-DNA border sequence. The T-DNAs of the invention have inserted into them, anywhere between the right and left border sequences (if present), the polynucleotide of interest flanked by target sites for a site-specific recombinase. The sequences encoding factors required in trans for transfer of the T- DNA into a plant cell such as vir genes, may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or preferably are in trans on a compatible replicon in the Agrobacterium host. Such "binary vector systems" are well known in the art.

As used herein, a “plant transfer nucleic acid” (PT-DNA) refers to, depending on the context herein, a transfer nucleic acid which can be used to integrate only plant derived polynucleotide sequences into the genome of a plant, and/or a part of a PT-DNA which has been inserted into the plant genome, as a consequence of processing by the Agrobacterium host, which only has plant derived polynucleotide sequences (also referred herein as a “processed” border, or “part” of a border). Thus, a PT-DNA of the invention may have a left border and/or a right border which comprises at least one nonplant derived oligonucleotide sequence, but this oligonucleotide sequence is not integrated into the genome of the plant when used to transform a plant as described herein. Typically, any non-plant derived polynucleotide sequence is derived from a naturally occurring Agrobacterium T-DNA. However, a PT-DNA of the invention which has been processed, such as exemplified in Figure 1, during Agrobacterium mediated gene transfer (AMGT) and forms part of a plant, only comprises plant derived polynucleotide sequences . In another example, the PT-DNA comprises the border which during processing may be shortened by 1, 2, 3 or up to five nucleotides. In an embodiment, the plant derived polynucleotides sequences in the PT-DNA are from the same Genus of plant. In an embodiment, the plant derived polynucleotides sequences in the PT-DNA are from the same species of plant, or a sexually compatible species. The border sequence preferably shares at least 70%, at least 75%, at least 80%, at least 90% or at least 95%, but less than 100% sequence identity, with a T-DNA border sequence from an Agrobacterium sp. such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. Thus, PT-DNAs can be used instead of T-DNAs to transfer a nucleotide sequence contained within the PT-DNA from, for example Agrobacterium, to another cell. The PT-DNA is characterized in that it contains, at least a right border sequence and preferably also a left border sequence. In an embodiment, a plant of the invention lacks any portion of a PT-DNA right border and/or left PT-DNA border. More specifically, in some occasions, the PT-DNA is not inserted as depicted in Figure 1, with the entire right border and/or left PT-DNA border being deleted when inserted.

As used herein, a “wheat transfer nucleic acid” (WT-DNA) refers to, depending on the context herein, a transfer nucleic acid which can be used to integrate only wheat derived polynucleotide sequences into genome of wheat, and/or a part of a PT-DNA which has been inserted into the wheat genome which only has wheat derived polynucleotide sequences. Apart from the wheat derived polynucleotide sequences, a WT-DNA of the invention may have any feature outlined herein as described for a PT- DNA.

As used herein, a “soybean transfer nucleic acid” (ST-DNA) refers to, depending on the context herein, a transfer nucleic acid which can be used to integrate only soybean derived polynucleotide sequences into genome of soybean, and/or a part of a PT-DNA which has been inserted into the soybean genome which only has soybean derived polynucleotide sequences. Apart from the soybean derived polynucleotide sequences, a ST-DNA of the invention may have any feature outlined herein as described for a PT- DNA.

A similar nomenclature can be applied to other plant species such as banana (BT- DNA), rice (RT-DNA), sorghum (SOGT-DNA), tomato (TT-DNA) or potato (POT- DNA).

The border sequence promotes and facilitates the transfer of the polynucleotide to which it is linked and may facilitate its integration in the recipient cell genome. In an embodiment, a border-sequence is between 5-100 base pairs (bp) in length, 10-80 bp in length, 15-75 bp in length, 15-60 bp in length, 15-50 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp in length, 20-30 bp in length, 21-30 bp in length, 22-30 bp in length, 23-30 bp in length, 24-30 bp in length, 25-30 bp in length, or 26-30 bp in length. Border sequences from T-DNA from Agrobacterium sp. are well known in the art and include those described in Lacroix et al. (2008), Tzfira and Citovsky (2006) and Glevin (2003).

Whilst traditionally only Agrobacterium sp. have been used to transfer genes to plants cells, there are now a large number of systems which have been identified/developed which act in a similar manner to Agrobacterium sp. Several non- Agrobacterium species have recently been genetically modified to be competent for gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234, Sinorhizobium meliloti and Mezorhizobium loti. The bacteria are made competent for gene transfer by providing the bacteria with the machinery needed for the transformation process, that is, a set of virulence genes encoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing on a separate, small binary plasmid. Bacteria engineered in this way are capable of transforming different plant tissues (leaf disks, calli and oval tissue), monocots or dicots, and various different plant species (e.g., tobacco, rice).

As used herein, the terms "transfection", "transformation" and variations thereof are generally used interchangeably. "Transfected" or "transformed" cells may have been manipulated to introduce the polynucleotide(s) of interest, or may be progeny cells derived therefrom.

In an embodiment, the polynucleotide of interest is from a sexually compatible plant.

In an embodiment, the PT-DNA comprises one, two, three, four, five, six, seven, eight, nine, ten, or more polynucleotides of interest. In an embodiment, the PT-DNA comprises between 1 and 15, or between 1 and 10, or between 1 and 5, polynucleotides of interest.

In an embodiment, the polynucleotide of interest is at least lOkbp, at least 20kbp at least 30kbp, between lOkbp and 50kbp, between lOkbp and 40kbp, lOkbp and 30kbp, or lOkbp and 20kbp.

PT-DNA

The present invention provides plant transfer DNA (PT-DNA). The PT-DNA may be part of a Ti plasmid, or have been processed and be in the genome of a plant.

For example, the present invention provides a plant having a plant transfer DNA (PT-DNA) integrated into the genome of the plant, wherein the integrated PT-DNA only has DNA from a plant, and wherein the PT-DNA comprises a polynucleotide of interest.

In an embodiment, the integrated PT-DNA has a processed PT-DNA right border, or lacks any portion of a PT-DNA right border.

In an embodiment, the integrated PT-DNA has a processed PT-DNA left border, or lacks any portion of a PT-DNA left border.

In an embodiment, the integrated PT-DNA comprises processed PT-DNA right border, a processed PT-DNA left border and a polynucleotide of interest.

In an embodiment, the plant is wheat, soybean, maize, rice, tomato, sorghum, barley, banana, sugarcane or potato. In an embodiment, the polynucleotide of interest encodes a wheat protein, a soybean protein, a maize protein, a rice protein, a tomato protein, a sorghum protein, a barley protein, a banana protein, a sugarcane protein or a potato protein.

A polynucleotide of interest is not naturally present in an Agrobacterium Ti plasmid, or Agrobacterium T-DNA.

In an embodiment, the polynucleotide of interest encodes a protein which confers enhanced resistance to abiotic stress or biotic stress.

In an embodiment, the polynucleotide of interest encodes a protein which confers enhanced resistance to disease, enhanced tolerance to abiotic stress including salinity and metals, increased yield, increased grain quality and/or size, drought tolerance, or enhanced nutritional content such as modified protein or oil production and/or composition.

In an embodiment, the polynucleotide of interest encodes a protein which confers enhanced resistance to disease. In an embodiment, the disease is a fungal disease. In an embodiment, the fungal disease is a mildew, blight, rot, smut or rust. In an embodiment, the disease is a fungal disease. In an embodiment, the fungal disease is a mildew, blight, rot or rust. In an embodiment, the fungal disease is a rust.

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-atgtatatattgtggtgtaagatagaaaatgatagcaccccaacctcat-3’ (SEQ ID NO:29) or a 5’ fragment thereof comprising 5’-atgtatatattgtggtgtaaga-3’ (SEQ ID NO:30).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtggtgtaagatagaaaatgatagcaccccaacctcat-3’ (SEQ ID NO:31) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaga-3’ (SEQ ID NO:32).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-caggatatattgtatcttttttttaatcagttgggaggac-3’ (SEQ ID NO:33) or a 5’ fragment thereof comprising 5’-caggatatattgtatctttttt-3’ (SEQ ID NO:34).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtatcttttttttaatcagttgggaggac-3’ (SEQ ID NO:35) or a 5’ fragment thereof comprising 5’-tatattgtatctttttt-3’ (SEQ ID NO:36).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-aaggatatattgtggtttgaagacagattggtggttcctaa-3’ (SEQ ID NO:37) or a 5’ fragment thereof comprising 5’-aaggatatattgtggtttgaag-3’ (SEQ ID NO:38). In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtggtttgaagacagattggtggttcctaa-3’ (SEQ ID NO:39) or a 5’ fragment thereof comprising 5’-tatattgtggtttgaag-3’ (SEQ ID NO:40).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-caggatatatggtggtggtggtatgtatgtataatgatatgatataggtg-3’ (SEQ ID NO:41) or a 5’ fragment thereof comprising 5 ’-caggatatatggtggtggtggt-3’ (SEQ ID NO:42).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5 ’ -tatatggtggtggtggtatgtatgtataatgatatgatataggtg-3 ’ (SEQ ID NO:43) or a 5’ fragment thereof comprising 5’ -tatatggtggtggtggt-3 ’ (SEQ ID NO:44).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’ -ctatatatattgtggtgtaagatagaaaa-3’ (SEQ ID NO:45) or a 5’ fragment thereof comprising 5’-ctatatatattgtggtgtaaga-3’ (SEQ ID NO:46).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtggtgtaagatagaaaa-3’ (SEQ ID NO:47) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaga-3’ (SEQ ID NO:32).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’ -gaggatatattgtggtggacattt-3 ’ (SEQ ID NO:48) or a 5’ fragment thereof comprising 5’ -gaggatatattgtggtggacat-3 ’ (SEQ ID NO:49).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtggtggacattt-3’ (SEQ ID NO:50) or a 5’ fragment thereof comprising 5’-tatattgtggtggacat-3’ (SEQ ID NO:51).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-caggatatattgtggaaggttcat-3’ (SEQ ID NO:52) or a 5’ fragment thereof comprising 5’ -caggatatattgtggaaggttc-3’ (SEQ ID NO:53).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tatattgtggaaggttcat-3’ (SEQ ID NO:54) or a 5’ fragment thereof comprising 5’-tatattgtggaaggttc-3’ (SEQ ID NO:55).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-tcgataatattgtggtgaaaaccaagcaagtc-3’ (SEQ ID NO:56) or a 5’ fragment thereof comprising 5’ -tcgataatattgtggtgaaaac-3 ’ (SEQ ID NO:57).

In an embodiment, the plant is maize and the processed left border of the PT-DNA in the maize genome comprises 5’-aatattgtggtgaaaaccaagcaagtc-3’ (SEQ ID NO: 58) or a 5’ fragment thereof comprising 5’-aatattgtggtgaaaac-3’ (SEQ ID NO:59).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-ctttaaattaaattttgtggctatcagtgtttga-3’ (SEQ ID NO:60) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO:61) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-caacctgatggttctcagatcagtgtttga-3’ (SEQ ID NO:63) or a 3’ fragment thereof comprising 5’-tctcagatcagtgtttga-3’ (SEQ ID NO:64) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-acattcataactaagttttgtggctatcagtgtttga-3’ (SEQ ID NO:65) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO:61) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-agttttgcaaaagtagccatcagtgtttga-3’ (SEQ ID NO:66) or a 3’ fragment thereof comprising 5’-gtagccatcagtgtttga-3’ (SEQ ID NO:67) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-aagctgaggcgtgctgcatatcagtgtttga-3’ (SEQ ID NO:68) or a 3’ fragment thereof comprising 5’-ctgcatatcagtgtttga-3’ (SEQ ID NO:69) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-tcataaccaagttttgtggctatcagtgtttga-3’ (SEQ ID NO:70) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO:61) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-agatatcaaatttttacctctatcagtgtttga-3’ (SEQ ID NO:71) or a 3’ fragment thereof comprising 5’-acctctatcagtgtttga-3’ (SEQ ID NO:72) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-attcacccccccctctaggcgactatcagtgtttga-3’ (SEQ IDNO:73) ora 3’ fragment thereof comprising 5’ -gcgactatcagtgtttga-3’ (SEQ IDNO:74) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is maize and the processed right border of the PT- DNA in the maize genome comprises 5’-cgctcatggaagcacttaaattatcagtgtttga-3’ (SEQ ID NO:75) or a 3’ fragment thereof comprising 5’-taaattatcagtgtttga-3’ (SEQ ID NO:76) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza saliva subsp. japonica. and the processed left border of the PT-DNA in the rice genome comprises 5’- aactatatattgtggtgtaaccagacatccacc-3’ (SEQ ID NO: 77) or a 5’ fragment thereof comprising 5’ -aactatatattgtggtgtaacc-3 ’ (SEQ ID NO:78).

In an embodiment, the plant is rice, preferably Oryza saliva subsp. japonica. and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgtaaccagacatccacc-3 ’ (SEQ ID NO:79) or a 5’ fragment thereof comprising 5’- tatattgtggtgtaacc-3 ’ (SEQ ID NO: 80).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- taggatatattgtggtgaaatgacgtgttatt-3’ (SEQ ID NO: 81) or a 5’ fragment thereof comprising 5’-taggatatattgtggtgaaatg-3’ (SEQ ID NO: 82).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgaaatgacgtgttatt-3 ’ (SEQ ID NO: 83) or a 5’ fragment thereof comprising 5’- tatattgtggtgaaatg-3’ (SEQ ID NO:84).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- caggatatattgtttggcaacaaat-3 ’ (SEQ ID NO: 85) or a 5’ fragment thereof comprising 5’- caggatatattgtttggcaaca-3’ (SEQ ID NO:86).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtttggcaacaaat-3’ (SEQ ID NO:87) or a 5’ fragment thereof comprising 5’- tatattgtttggcaaca-3’ (SEQ ID NO:88).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- taatatatattgtggtgtataatattttcctcga-3’ (SEQ ID NO:89) or a 5’ fragment thereof comprising 5’-taatatatattgtggtgtataa-3’ (SEQ ID NO:90).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgtataatattttcctcga-3’ (SEQ ID NO:91) or a 5’ fragment thereof comprising 5’- tatattgtggtgtataa-3 ’ (SEQ ID NO:92).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- ttggatatattgtggtgctttgaagagacaa-3’ (SEQ ID NO:93) or a 5’ fragment thereof comprising 5’-ttggatatattgtggtgctttg-3’ (SEQ ID NO: 94).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgctttgaagagacaa-3 ’ (SEQ ID NO:95) or a 5’ fragment thereof comprising 5’- tatattgtggtgctttg-3’ (SEQ ID NO:96).

In an embodiment, the plant is rice, preferably Oryza saliva subsp. japonica. and the processed left border of the PT-DNA in the rice genome comprises 5’- agatcggtattgtggtgtaaaccgggaagacgatttc-3’ (SEQ ID NO:97) or a 5’ fragment thereof comprising 5’ -agatcggtattgtggtgtaaac-3’ (SEQ ID NO:98).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- ggtattgtggtgtaaaccgggaagacgatttc-3’ (SEQ ID NO: 99) or a 5’ fragment thereof comprising 5’-ggtattgtggtgtaaac-3’ (SEQ ID NO: 100).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- taggatatattgtggttccgtatgatat-3 ’ (SEQ ID NO: 101) or a 5 ’ fragment thereof comprising 5 ’- taggatatattgtggttccgta-3’ (SEQ ID NO: 102).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggttccgtatgatat-3 ’ (SEQ ID NO: 103) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- aaggatatattgtggttccgtatgatattttcgt-3’ (SEQ ID NO: 105) or a 5’ fragment thereof comprising 5’-aaggatatattgtggttccgta-3’ (SEQ ID NO: 106).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggttccgtatgatattttcgt-3’ (SEQ ID NO: 107) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tcatatatattgtggtgcaaacatatttttggtggaatct-3’ (SEQ ID NO: 108) or a 5’ fragment thereof comprising 5’-tcatatatattgtggtgcaaac-3’ (SEQ ID NO: 109).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgcaaacatatttttggtggaatct-3’ (SEQ ID NO: 110) or a 5’ fragment thereof comprising 5’-tatattgtggtgcaaac-3’ (SEQ ID NO: 111).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tgggatatattgtgatgtaactatagcagttgttca-3’ (SEQ ID NO: 112) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113).

In an embodiment, the plant is rice, preferably Oryza saliva subsp. japonica. and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtgatgtaactatagcagttgttcact-3’ (SEQ ID NO: 114) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed right border of the PT-DNA in the rice genome comprises 5’- ttaaagccgtattctgcttttgaaactttcagtgtttga-3’ (SEQ ID NO: 116) or a 3’ fragment thereof comprising 5’-gaaactttcagtgtttga-3’ (SEQ ID NO: 117) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed right border of the PT-DNA in the rice genome comprises 5’- aatcacaacaatattcactcaactctcagtgtttga-3’ (SEQ ID NO: 118) or a 3’ fragment thereof comprising 5’-tcaactctcagtgtttga-3’ (SEQ ID NO: 119) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the processed right border of the PT-DNA in the rice genome comprises 5’- caatcacgacatgagaactatcagtgcttga-3’ (SEQ ID NO: 120) or a 3’ fragment thereof comprising 5’-agaactatcagtgcttga-3’ (SEQ ID NO: 121) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- caggatatattgtttggcaacaaatctactt-3 ’ (SEQ ID NO: 122) or a 5 ’ fragment thereof comprising 5’-caggatatattgtttggcaaca-3’ (SEQ ID NO:86).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtttggcaacaaatctactt-3 ’ (SEQ ID NO: 123) or a 5’ fragment thereof comprising 5’- tatattgtttggcaaca-3’ (SEQ ID NO:88).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- aactatatattgtggtgtaaccagacatccacctgagc-3’ (SEQ ID NO: 124) or a 5’ fragment thereof comprising 5’ -aactatatattgtggtgtaacc-3 ’ (SEQ ID NO:78).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgtaaccagacatccacctgagc-3’ (SEQ ID NO: 125) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaacc-3’ (SEQ ID NO:80).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- taatatatattgtggtgtataatattttcctcgagact-3’ (SEQ ID NO: 126) or a 5’ fragment thereof comprising 5’-taatatatattgtggtgtataa-3’ (SEQ ID NO: 90).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgtataatattttcctcgagact-3’ (SEQ ID NO: 127) or a 5’ fragment thereof comprising 5’-tatattgtggtgtataa-3’ (SEQ ID NO:92).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- aaggatatattgtggttccgtatgatatttccg-3’ (SEQ ID NO: 129) or a 5’ fragment thereof comprising 5’-aaggatatattgtggttccgta-3’ (SEQ ID NO: 106).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggttccgtatgatatttccg-3’ (SEQ ID NO: 129) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- ttggatatattgtggtgctttgaagagacaaaag-3’ (SEQ ID NO: 130) or a 5’ fragment thereof comprising 5’-ttggatatattgtggtgctttg-3’ (SEQ ID NO:94).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgctttgaagagacaaaag-3’ (SEQ ID NO: 131) or a 5’ fragment thereof comprising 5’-tatattgtggtgctttg-3’ (SEQ ID NO:96).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tgggatatattgtgatgtaactatagcagttgttcactg-3’ (SEQ ID NO: 132) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtgatgtaactatagcagttgttcactg-3’ (SEQ ID NO: 133) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tcatatatattgtggtgcaaacatatttttggtggaatcttg-3’ (SEQ ID NO: 134) or a 5’ fragment thereof comprising 5’-tcatatatattgtggtgcaaac-3’ (SEQ ID NO: 109).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtgcaaacatatttttggtggaatcttg-3’ (SEQ ID NO: 135) or a 5’ fragment thereof comprising 5’-tatattgtggtgcaaac-3’ (SEQ ID NO: 111).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the processed left border of the PT-DNA in the rice genome comprises 5’- tgggatatattgtgatgtaactatagcagatggtcactgat-3’ (SEQ ID NO: 136) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtgatgtaactatagcagatggtcactgat-3’ (SEQ ID NO: 137) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- gaggatatattggggtgtacacaataaaaattctttcgtct-3’ (SEQ ID NO: 138) or a 5’ fragment thereof comprising 5’ -gaggatatattggggtgtacac-3 ’ (SEQ ID NO: 139).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattggggtgtacacaataaaaattctttcgtct-3’ (SEQ ID NO: 140) or a 5’ fragment thereof comprising 5’-tatattggggtgtacac-3’ (SEQ ID NO: 141).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- gcttatatattgtggtataaaataaatctgaatcct-3’ (SEQ ID NO: 142) or a 5’ fragment thereof comprising 5’-gcttatatattgtggtataaaa-3’ (SEQ ID NO: 143).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed left border of the PT-DNA in the rice genome comprises 5’- tatattgtggtataaaataaatctgaatcct-3’ (SEQ ID NO: 144) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 145).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed right border of the PT-DNA in the rice genome comprises 5’- aagccgtattctgcttttgaaactttcagtgtttga-3’ (SEQ ID NO: 146) or a 3’ fragment thereof comprising 5’-gaaactttcagtgtttga-3’ (SEQ ID NO: 117) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed right border of the PT-DNA in the rice genome comprises 5’- tcacaacaatattcactcaactctcagtgtttga-3’ (SEQ ID NO: 147) or a 3’ fragment thereof comprising 5’-tcaactctcagtgtttga-3’ (SEQ ID NO: 119) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the processed right border of the PT-DNA in the rice genome comprises 5’- tcaatcacgacatgagaactatcagtgcttga-3’ (SEQ ID NO: 148) or a 3’ fragment thereof comprising 5’-agaactatcagtgcttga-3’ (SEQ ID NO: 121) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-gcaaatatattgtggtgtaaatctttttctt-3’ (SEQ ID NO: 149) or a 5’ fragment thereof comprising 5’-gcaaatatattgtggtgtaaat-3’ (SEQ ID NO: 150).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatattgtggtgtaaatctttttctt-3’ (SEQ ID NO: 151) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatcgtatattgtggtgtaaatatacacctaa-3’ (SEQ ID NO: 153) or a 5’ fragment thereof comprising 5’-tatcgtatattgtggtgtaaat-3’ (SEQ ID NO: 154).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatattgtggtgtaaatatacacctaa-3’ (SEQ ID NO: 155) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-aaggatatattgtggtcttcactccctct-3’ (SEQ ID NO: 156) or a 5’ fragment thereof comprising 5’-aaggatatattgtggtcttcac-3’ (SEQ ID NO: 157).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’ -tatattgtggtcttcactccctct-3 ’ (SEQ ID NO: 158) or a 5’ fragment thereof comprising 5’-tatattgtggtcttcac-3’ (SEQ ID NO: 159).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-caggatatattgggtcctctcgaaag-3’ (SEQ ID NO: 160) or a 5’ fragment thereof comprising 5’ -caggatatattgggtcctctcg-3 ’ (SEQ ID NO: 161).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’ -tatattgggtcctctcgaaag-3 ’ (SEQ ID NO: 162) or a 5’ fragment thereof comprising 5’-tatattgggtcctctcg-3’ (SEQ ID NO: 163).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-caggatatattgattcctctcaatgag-3’ (SEQ ID NO: 164) or a 5’ fragment thereof comprising 5’-caggatatattgattcctctca-3’ (SEQ ID NO: 165). In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatattgattcctctcaatgag-3’ (SEQ ID NO: 166) or a 5’ fragment thereof comprising 5’-tatattgattcctctca-3’ (SEQ ID NO: 167).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-caggatatattgggtcttctcgatggg-3’ (SEQ ID NO: 168) or a 5’ fragment thereof comprising 5’ -caggatatattgggtcttctcg-3’ (SEQ ID NO: 169).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’ -tatattgggtcttctcgatggg-3 ’ (SEQ ID NO: 170) or a 5’ fragment thereof comprising 5’-tatattgggtcttctcg-3’ (SEQ ID NO: 171).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-cataatattttgtggtgtaaaaatatagtctatgcaaga-3’ (SEQ ID NO: 172) or a 5’ fragment thereof comprising 5’-cataatattttgtggtgtaaaa-3’ (SEQ ID NO: 173).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tattttgtggtgtaaaaatatagtctatgcaaga-3’ (SEQ ID NO: 174) or a 5’ fragment thereof comprising 5’-tattttgtggtgtaaaa-3’ (SEQ ID NO: 175).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-atgactatattgtggtggaaactcactaagggttcctta-3’ (SEQ ID NO: 176) or a 5’ fragment thereof comprising 5’-atgactatattgtggtggaaac-3’ (SEQ ID NO: 177).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatattgtggtggaaactcactaagggttcctta-3’ (SEQ ID NO: 178) or a 5’ fragment thereof comprising 5’-tatattgtggtggaaac-3’ (SEQ ID NO: 179).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-acatatatcttgtggtgtaaaattatacataatttta-3’ (SEQ ID NO: 182) or a 5’ fragment thereof comprising 5’-acatatatcttgtggtgtaaaa-3’ (SEQ ID NO: 181).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatcttgtggtgtaaaattatacataatttta-3’ (SEQ ID NO: 182) or a 5 ’ fragment thereof comprising 5 ’-tatcttgtggtgtaaaa-3 ’ (SEQ ID NO: 183).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-aaggaaatattgtggtgttgatggggatcaaact-3’ (SEQ ID NO: 184) or a 5’ fragment thereof comprising 5’-aaggaaatattgtggtgttgat-3’ (SEQ ID NO: 185). In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-aatattgtggtgttgatggggatcaaact-3’ (SEQ ID NO: 186) or a 5’ fragment thereof comprising 5’-aatattgtggtgttgat-3’ (SEQ ID NO: 187).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-cagtatatattgtggttcatatgtttaagctat-3’ (SEQ ID NO: 188) or a 5’ fragment thereof comprising 5’-cagtatatattgtggttcatat-3’ (SEQ ID NO: 189).

In an embodiment, the plant is tomato and the processed left border of the PT- DNA in the tomato genome comprises 5’-tatattgtggttcatatgtttaagctat-3’ (SEQ ID NO: 190) or a 5’ fragment thereof comprising 5’-tatattgtggttcatat-3’ (SEQ ID NO: 191).

In an embodiment, the plant is tomato and the processed right border of the PT- DNA in the tomato genome comprises 5’-cacgcaaagagaaggaaaaactttcagtgtttga-3’ (SEQ ID NO: 192) or a 3’ fragment thereof comprising 5’-aaaactttcagtgtttga-3’ (SEQ ID NO: 193) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the processed right border of the PT- DNA in the tomato genome comprises 5’-aaagaaagacaacaaactatcagtttttga-3’ (SEQ ID NO: 194) or a 3’ fragment thereof comprising 5’-caaactatcagtttttga-3’ (SEQ ID NO: 195) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the processed right border of the PT- DNA in the tomato genome comprises 5’-ggccatttcaagtccaactatcagtgcttga-3’ (SEQ ID NO: 196) or a 3’ fragment thereof comprising 5’-ccaactatcagtgcttga-3’ (SEQ ID NO: 197) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the processed right border of the PT- DNA in the tomato genome comprises 5’-caatgttaggtatgaactatcagtgtttga-3’ (SEQ ID NO: 198) or a 3’ fragment thereof comprising 5’-tgaactatcagtgtttga-3’ (SEQ ID NO: 199) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-caagatatattgtggcacaactatgag-3’ (SEQ ID NO:200) or a 5’ fragment thereof comprising 5’-caagatatattgtggcacaact-3’ (SEQ ID NO:201).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’ -tatattgtggcacaactatgag-3 ’ (SEQ ID NO:202) or a 5’ fragment thereof comprising 5’-tatattgtggcacaact-3’ (SEQ ID NO:203).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-caggatatatgttggtgtaattgatcttg-3’ (SEQ ID NO:204) or a 5’ fragment thereof comprising 5’-caggatatatgttggtgtaatt-3’ (SEQ ID NO:205).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatatgttggtgtaattgatcttg-3’ (SEQ ID NO:206) or a 5’ fragment thereof comprising 5’-tatatgttggtgtaatt-3’ (SEQ ID NO: 207).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-aagcttatattgtggtttaaaaaccttattaagtggtt-3’ (SEQ ID NO:208) or a 5’ fragment thereof comprising 5’-aagcttatattgtggtttaaaa-3’ (SEQ ID NO:209).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatattgtggtttaaaaaccttattaagtggtt-3’ (SEQ ID NO:210) or a 5’ fragment thereof comprising 5’-tatattgtggtttaaaa-3’ (SEQ ID NO:211).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tggtttatattgtggtataaaatttaaact-3’ (SEQ ID NO:212) or a 5’ fragment thereof comprising 5’-tggtttatattgtggtataaaa-3’ (SEQ ID NO:213).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA inthe sorghum genome comprises 5’ -tatattgtggtataaaatttaaact-3 ’ (SEQ IDNO:214) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 145).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tttagcatattgtggtgtaaatgaagatgagctgatg-3’ (SEQ ID NO:215) or a 5’ fragment thereof comprising 5’-tttagcatattgtggtgtaaat-3’ (SEQ ID NO:216).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-catattgtggtgtaaatgaagatgagctgatg-3’ (SEQ ID NO:217) or a 5’ fragment thereof comprising 5’-catattgtggtgtaaat-3’ (SEQ ID NO:218).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-ggcgatatattgtggtgttcagtgtttagtcagaagtc-3’ (SEQ ID NO: 219) or a 5’ fragment thereof comprising 5’ -ggcgatatattgtggtgttcag-3 ’ (SEQ ID NO:220).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatattgtggtgttcagtgtttagtcagaagtc-3’ (SEQ ID NO:221) or a 5’ fragment thereof comprising 5’-tatattgtggtgttcag-3’ (SEQ ID NO:222).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-acctctatattgtggtgtaattttcttgtggcaac-3’ (SEQ ID NO:223) or a 5’ fragment thereof comprising 5’-acctctatattgtggtgtaatt-3’ (SEQ ID NO: 224).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatattgtggtgtaattttcttgtggcaac-3’ (SEQ ID NO:225) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaatt-3’ (SEQ ID NO:226).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tgatatatattgtggtgtaccacgcggcatgggt-3’ (SEQ ID NO:227) or a 5’ fragment thereof comprising 5’-tgatatatattgtggtgtacca-3’ (SEQ ID NO:228).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatattgtggtgtaccacgcggcatgggt-3’ (SEQ ID NO:229) or a 5’ fragment thereof comprising 5’-tatattgtggtgtacca-3’ (SEQ ID NO:230).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-aatatgatattgtggtgtaaataactgaatattgatattt-3’ (SEQ ID NO:231) or a 5’ fragment thereof comprising 5’-aatatgatattgtggtgtaaat-3’ (SEQ ID NO:232).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-gatattgtggtgtaaataactgaatattgatattt-3’ (SEQ ID NO:233) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaat-3’ (SEQ ID NO:234).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’- tggtatatattgtggtgtataactctgtataaccgaagcgtaattttcaacaccctagtaatggcgcaaactttggtatatattgtgg tgtataactccgtataaccgaagcaaga-3 ’ (SEQ ID NO:235) or a 5’ fragment thereof comprising 5’-tggtatatattgtggtgtataa-3’ (SEQ ID NO:236).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’- tatattgtggtgtataactctgtataaccgaagcgtaattttcaacaccctagtaatggcgcaaactttggtatatattgtggtgtat aactccgtataaccgaagcaaga-3 ’ (SEQ ID NO:237) or a 5’ fragment thereof comprising 5’- tatattgtggtgtataa-3 ’ (SEQ ID NO:92).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-gataatatattgtggtgtatatgattagaaaaaaaaaac-3’ (SEQ ID NO:238) or a 5’ fragment thereof comprising 5’-gataatatattgtggtgtatat-3’ (SEQ ID NO:239).

In an embodiment, the plant is sorghum and the processed left border of the PT- DNA in the sorghum genome comprises 5’-tatattgtggtgtatatgattagaaaaaaaaaac-3’ (SEQ ID NO:240) or a 5’ fragment thereof comprising 5’-tatattgtggtgtatat-3’ (SEQ ID NO:241).

In an embodiment, the plant is sorghum and the processed right border of the PT- DNA in the sorghum genome comprises 5’-cctgtcttcctccaaggaactgtcagtgtttga-3’ (SEQ ID NO:242) or a 3’ fragment thereof comprising 5’-ggaactgtcagtgtttga-3’ (SEQ ID NO: 243) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sorghum and the processed right border of the PT- DNA in the sorghum genome comprises 5’-ttaaactaaagttaacactatcagtgtttga-3’ (SEQ ID NO:243) or a 3’ fragment thereof comprising 5’-aacactatcagtgtttga-3’ (SEQ ID NO:245) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sorghum and the processed right border of the PT- DNA in the sorghum genome comprises 5’-tttttaataatgcctctatcagtgtttga-3’ (SEQ ID NO:246) or a 3’ fragment thereof comprising 5’-gcctctatcagtgtttga-3’ (SEQ ID NO:247) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gtggatatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO:248) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaaac-3’ (SEQ ID NO: 249).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO:250) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaac-3’ (SEQ ID NO:251).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gtggatatattgtgatgtaaaccttgtagtaatatgt-3’ (SEQ ID NO:252) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaaac-3’ (SEQ ID NO: 249).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’ -tatattgtgatgtaaaccttgtagtaatatgt-3’ (SEQ ID NO:253) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaac-3’ (SEQ ID NO:251).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-catgatatattgtggtgcatgtctctt-3’ (SEQ ID NO:254) or a 5’ fragment thereof comprising 5’ -catgatatattgtggtgcatgt-3 ’ (SEQ ID NO:255).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’ -tatattgtggtgcatgtctctt-3 ’ (SEQ ID NO:256) or a 5’ fragment thereof comprising 5’-tatattgtggtgcatgt-3’ (SEQ ID NO:257).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gtggatatattgtggcgtaaaccttgcagta-3’ (SEQ ID NO:258) or a 5’ fragment thereof comprising 5’ -gtggatatattgtggcgtaaac-3’ (SEQ ID NO:259).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5 ’-tatattgtggcgtaaaccttgcagta-3 ’ (SEQ ID NO:260) or a 5’ fragment thereof comprising 5’-tatattgtggcgtaaac-3’ (SEQ ID NO:261).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-caggatatattatggtgtagattataaaaacta-3’ (SEQ ID NO:262) or a 5’ fragment thereof comprising 5’-caggatatattatggtgtagat-3’ (SEQ ID NO:263).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattatggtgtagattataaaaacta-3’ (SEQ ID NO:264) or a 5’ fragment thereof comprising 5’-tatattatggtgtagat-3’ (SEQ ID NO:265).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-catgatatattgtggtgaaaacatgcaagaaccta-3’ (SEQ ID NO:266) or a 5’ fragment thereof comprising 5’-catgatatattgtggtgaaaac-3’ (SEQ ID NO:267).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtggtgaaaacatgcaagaaccta-3’ (SEQ ID NO:268) or a 5’ fragment thereof comprising 5’-tatattgtggtgaaaac-3’ (SEQ ID NO269).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gtggatatattgtgatgtaatccttgcagtaata-3’ (SEQ ID NO:270) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaatc-3’ (SEQ ID NO:271).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtgatgtaatccttgcagtaata-3’ (SEQ ID NO:272) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaatc-3’ (SEQ ID NO:273).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-catgatatattgtgttgtatgcctctagtgtc-3’ (SEQ ID NO:274) or a 5’ fragment thereof comprising 5’-catgatatattgtgttgtatgc-3’ (SEQ ID NO:275).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtgttgtatgcctctagtgtc-3’ (SEQ ID NO:276) or a 5’ fragment thereof comprising 5’-tatattgtgttgtatgc-3’ (SEQ ID NO: 277).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-catgatatattgtgttgtatgtttcttgtgatgt-3’ (SEQ ID NO:278) or a 5’ fragment thereof comprising 5’-catgatatattgtgttgtatgt-3’ (SEQ ID NO:279).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’ -tatattgtgttgtatgtttcttgtgatgt-3’ (SEQ ID NO:280) or a 5’ fragment thereof comprising 5’-tatattgtgttgtatgt-3’ (SEQ ID NO:281).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gtggatatattgtgatgtaatccttgcggaaatat-3’ (SEQ ID NO:282) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaatc-3’ (SEQ ID NO:271).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtgatgtaatccttgcggaaatat-3’ (SEQ ID NO:283) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaatc-3’ (SEQ ID NO:273).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-gatcatatattgtggtctaaaccctaatgacgtagacatt-3’ (SEQ ID NO: 284) or a 5’ fragment thereof comprising 5’ -gatcatatattgtggtctaaac-3 ’ (SEQ ID NO:285).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtggtctaaaccctaatgacgtagacatt-3’ (SEQ ID NO:286) or a 5’ fragment thereof comprising 5’-tatattgtggtctaaac-3’ (SEQ ID NO:287).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-caagatatattgtgatgtaaatgccatcgtcact-3’ (SEQ ID NO:288) or a 5’ fragment thereof comprising 5’ -caagatatattgtgatgtaaat-3 ’ (SEQ ID NO:289).

In an embodiment, the plant is barley and the processed left border of the PT- DNA in the barley genome comprises 5’-tatattgtgatgtaaatgccatcgtcact-3’ (SEQ ID NO:290) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaat-3’ (SEQ ID NO:291).

In an embodiment, the plant is barley and the processed right border of the PT- DNA in the barley genome comprises 5’-ctttgcttatcgcagaaactatcagtgtttga-3’ (SEQ ID NO:292) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO:293) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the processed right border of the PT- DNA in the barley genome comprises 5’ -ctttgcttatcacagaaactatcagtgtttga-3’ (SEQ ID NO:294) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO:291) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the processed right border of the PT- DNA in the barley genome comprises 5’ -ctttgcttatggtagaaactatcagtgtttga-3’ (SEQ ID NO:295) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO:291) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the processed right border of the PT- DNA in the barley genome comprises 5’-gccgccatagttaccaactatcagtgtttga-3’ (SEQ ID NO:296) or a 3’ fragment thereof comprising 5’-ccaactatcagtgtttga-3’ (SEQ ID NO:297) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the processed right border of the PT- DNA in the barley genome comprises 5’-gatccccctggcggagaactttcagtgtttga-3’ (SEQ ID NO:298) or a 3’ fragment thereof comprising 5’-agaactttcagtgtttga-3’ (SEQ ID NO:299) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’ -cagcatatattgtggtgtaaaggatgtccacttccaa-3’ (SEQ ID NO:300) or a 5’ fragment thereof comprising 5’-cagcatatattgtggtgtaaag-3’ (SEQ ID NO:301).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtggtgtaaaggatgtccacttccaa-3’ (SEQ ID NO:302) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaag-3’ (SEQ ID NO:303).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caccttatattgtggtgtaaatgagttgagtgtac-3’ (SEQ ID NO: 304) or a 5’ fragment thereof comprising 5’-caccttatattgtggtgtaaat-3’ (SEQ ID NO:305).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtggtgtaaatgagttgagtgtac-3’ (SEQ ID NO:306) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 150).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caggatatattgtggtttttgtaatcc-3’ (SEQ ID NO:307) or a 5’ fragment thereof comprising 5’-caggatatattgtggtttttgt-3’ (SEQ ID NO:308).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtggtttttgtaatcc-3’ (SEQ ID NO: 309) or a 5’ fragment thereof comprising 5’-tatattgtggtttttgt-3’ (SEQ ID NO:310).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’ -ttgtatatattgtggtgtaacttcattatagaa-3’ (SEQ ID NO:311) or a 5’ fragment thereof comprising 5’-ttgtatatattgtggtgtaact-3’ (SEQ ID NO:312). In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtggtgtaacttcattatagaa-3’ (SEQ ID NO:313) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaact-3’ (SEQ ID NO:314).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caagatatattgtgatgtaaaatagactttgacttgccca-3’ (SEQ ID NO:315) ora 5’ fragment thereof comprising 5’-caagatatattgtgatgtaaaa-3’ (SEQ ID NO:316).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtgatgtaaaatagactttgacttgccca-3’ (SEQ ID NO:317) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaaa-3’ (SEQ ID NO:318).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caggatatattctggtgatgctattatgtgctct-3’ (SEQ ID NO:319) or a 5’ fragment thereof comprising 5’ -caggatatattctggtgatgct-3’ (SEQ ID NO:320).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattctggtgatgctattatgtgctct-3’ (SEQ ID NO:321) or a 5’ fragment thereof comprising 5’-tatattctggtgatgct-3’ (SEQ ID NO:322).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-gaatatatattgtggtataaaattggatattaa-3’ (SEQ ID NO:323) or a 5’ fragment thereof comprising 5’ -gaatatatattgtggtataaaa-3’ (SEQ ID NO:324).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattgtggtataaaattggatattaa-3’ (SEQ ID NO:325) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 145).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caggatattttgtgttagcactgtgtata-3’ (SEQ ID NO:326) or a 5’ fragment thereof comprising 5’ -caggatattttgtgttagcact-3 ’ (SEQ ID NO:327).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tattttgtgttagcactgtgtata-3’ (SEQ ID NO:328) or a 5’ fragment thereof comprising 5’-tattttgtgttagcact-3’ (SEQ ID NO:329).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-caggatatattctggtgataacaccacaatggcat-3’ (SEQ ID NO:330) or a 5’ fragment thereof comprising 5’-caggatatattctggtgataac-3’ (SEQ ID NO:331). In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-tatattctggtgataacaccacaatggcat-3’ (SEQ ID NO:332) or a 5’ fragment thereof comprising 5’-tatattctggtgataac-3’ (SEQ ID NO:333).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-gaggacatattgtggtgttcatagctagattcttctaaacttattt- 3’ (SEQ ID NO:334) or a 5’ fragment thereof comprising 5’ -gaggacatattgtggtgttcat-3 ’ (SEQ ID NO:335).

In an embodiment, the plant is banana and the processed left border of the PT- DNA in the banana genome comprises 5’-catattgtggtgttcatagctagattcttctaaacttattt-3’ (SEQ ID NO:336) or a 5’ fragment thereof comprising 5’-catattgtggtgttcat-3’ (SEQ ID NO:337).

In an embodiment, the plant is banana and the processed right border of the PT- DNA in the banana genome comprises 5’-aagatagatttattgataaattatcagtgtttga-3’ (SEQ ID NO:338) or a 3’ fragment thereof comprising 5’-taaattatcagtgtttga-3’ (SEQ ID NO:76) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is banana and the processed right border of the PT- DNA in the banana genome comprises 5’-tgatcctctcaagggtaaactgtcagtgtttga-3’ (SEQ ID NO:339) or a 3’ fragment thereof comprising 5’-taaactgtcagtgtttga-3’ (SEQ ID NO:340) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is banana and the processed right border of the PT- DNA in the banana genome comprises 5’-cttcttcagaacaaaaactttcagtgtttga-3’ (SEQ ID NO:341) or a 3’ fragment thereof comprising 5’-aaaactttcagtgtttga-3’ (SEQ ID NO: 193) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tctgatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:342) or a 5’ fragment thereof comprising 5’-tctgatatattgtggtgtaaaa-3’ (SEQ ID NO:343).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:344) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tctaatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:346) or a 5’ fragment thereof comprising 5’-tctaatatattgtggtgtaaaa-3’ (SEQ ID NO:347).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’ -attgatatattgtggtgtaccacgcggcatgcat-3’ (SEQ ID NO:348) or a 5’ fragment thereof comprising 5’-attgatatattgtggtgtacca-3’ (SEQ ID NO:349).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtgtaccacgcggcatgcat-3’ (SEQ ID NO:350) or a 5’ fragment thereof comprising 5’-tatattgtggtgtacca-3’ (SEQ ID NO:230).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’ -atggatatattgtggtataaatttcttggtca-3’ (SEQ ID NO:351) or a 5’ fragment thereof comprising 5’-atggatatattgtggtataaat-3’ (SEQ ID NO:352).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtataaatttcttggtca-3’ (SEQ ID NO:353) or a 5’ fragment thereof comprising 5’-tatattgtggtataaat-3’ (SEQ ID NO:354).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-ttggatttattgtggtgtaacctgtcagttcggatttt-3’ (SEQ ID NO:355) or a 5’ fragment thereof comprising 5’-ttggatttattgtggtgtaacc-3’ (SEQ ID NO:356).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tttattgtggtgtaacctgtcagttcggatttt-3’ (SEQ ID NO:357) or a 5’ fragment thereof comprising 5’-tttattgtggtgtaacc-3’ (SEQ ID NO:358).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-agagatatattgtggtttaaaagatcagcata-3’ (SEQ ID NO:359) or a 5’ fragment thereof comprising 5’-agagatatattgtggtttaaaa-3’ (SEQ ID NO:360).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtttaaaagatcagcata-3’ (SEQ ID NO:361) or a 5’ fragment thereof comprising 5’-tatattgtggtttaaaa-3’ (SEQ ID NO:211).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5 ’-caggatatattgtcgtgtcttcacttcattgat-3 ’ (SEQ ID NO:362) or a 5’ fragment thereof comprising 5’ -caggatatattgtcgtgtcttc-3 ’ (SEQ ID NO: 1007).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtcgtgtcttcacttcattgat-3’ (SEQ ID NO:363) or a 5’ fragment thereof comprising 5’-tatattgtcgtgtcttc-3’ (SEQ ID NO: 1008).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-accaatatattgtggtataaactactgcaaatcttgg-3’ (SEQ ID NO: 364) or a 5 ’ fragment thereof comprising 5 ’-accaatatattgtggtataaac-3 ’ (SEQ ID NO:365).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’ -tatattgtggtataaactactgcaaatcttgg-3’ (SEQ ID NO:366) or a 5’ fragment thereof comprising 5’-tatattgtggtataaac-3’ (SEQ ID NO:367).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-ttctatatattgtggtctaaaatatgatggaacatg-3’ (SEQ ID NO:368) or a 5’ fragment thereof comprising 5’-ttctatatattgtggtctaaaa-3’ (SEQ ID NO:369).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtctaaaatatgatggaacatg-3’ (SEQ ID NO:370) or a 5’ fragment thereof comprising 5’-tatattgtggtctaaaa-3’ (SEQ ID NO:371).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-atgaatatattgtggtataaattacatggtcattgct-3’ (SEQ ID NO:372) or a 5’ fragment thereof comprising 5’-atgaatatattgtggtataaat-3’ (SEQ ID NO:373).

In an embodiment, the plant is sugarcane and the processed left border of the PT- DNA in the sugarcane genome comprises 5’-tatattgtggtataaattacatggtcattgct-3’ (SEQ ID NO:374) or a 5’ fragment thereof comprising 5’-tatattgtggtataaat-3’ (SEQ ID NO:354).

In an embodiment, the plant is sugarcane and the processed right border of the PT-DNA in the sugarcane genome comprises 5’-ttgtgaattttaagtttaaaatatcagtgtttga-3’ (SEQ ID NO:375) or a 3’ fragment thereof comprising 5’-taaaatatcagtgtttga-3’ (SEQ ID NO: 376) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sugarcane and the processed right border of the PT-DNA in the sugarcane genome comprises 5’-ctggtggtccaatgcaactatcagtgtttga-3’ (SEQ ID NO:377) or a 3’ fragment thereof comprising 5’-gcaactatcagtgtttga-3’ (SEQ ID NO:378) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is sugarcane and the processed right border of the PT-DNA in the sugarcane genome comprises 5’-gttcaggtgcatcaactatcagtgtttga-3’ (SEQ ID NO: 379) or a 3’ fragment thereof comprising 5’-tcaactatcagtgtttga-3’ (SEQ ID NO:380) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is sugarcane and the processed right border of the PT-DNA in the sugarcane genome comprises 5’-cctacctggcgcgccaactatcagtgtttga-3’ (SEQ ID NO:381) or a 3’ fragment thereof comprising 5’-ccaactatcagtgtttga-3’ (SEQ ID NO: 297) or 5’-ttga-3’ (SEQ ID NO: 62). In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’- ttttgaatattgtggtgtaaacctaaactccttgtgtgagtgttgaaagcttg-3’ (SEQ ID NO:382) or a 5’ fragment thereof comprising 5’-ttttgaatattgtggtgtaaac-3’ (SEQ ID NO:383).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’- aatattgtggtgtaaacctaaactccttgtgtgagtgttgaaagcttg-3’ (SEQ ID NO:384) or a 5’ fragment thereof comprising 5’-aatattgtggtgtaaac-3’ (SEQ ID NO:385).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-caggatatattgttataacctatttgcataa-3’ (SEQ ID NO:386) or a 5’ fragment thereof comprising 5’-caggatatattgttataaccta-3’ (SEQ ID NO: 1009).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgttataacctatttgcataa-3’ (SEQ ID NO:387) or a 5’ fragment thereof comprising 5’-tatattgttataaccta-3’ (SEQ ID NO: 1010).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-caggatatattttggggtaggtaaagtctgcttgctg-3’ (SEQ ID NO:388) or a 5’ fragment thereof comprising 5’ -caggatatattttggggtaggt-3 ’ (SEQ ID NO:389).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattttggggtaggtaaagtctgcttgctg-3’ (SEQ ID NO:390) or a 5’ fragment thereof comprising 5’-tatattttggggtaggt-3’ (SEQ ID NO:391).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-caggatatatttttgtgtaggggccaataagctacttttctaa-3’ (SEQ ID NO: 392) or a 5’ fragment thereof comprising 5’-caggatatatttttgtgtaggg-3’ (SEQ ID NO:393).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatatttttgtgtaggggccaataagctacttttctaa-3’ (SEQ ID NO:394) or a 5’ fragment thereof comprising 5’-tatatttttgtgtaggg-3’ (SEQ ID NO:395).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-gaggatatattgtggtggacaggagatg-3’ (SEQ ID NO:396) or a 5’ fragment thereof comprising 5’ -gaggatatattgtggtggacag-3 ’ (SEQ ID NO:397). In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’ -tatattgtggtggacaggagatg-3 ’ (SEQ ID NO:398) or a 5’ fragment thereof comprising 5’-tatattgtggtggacag-3’ (SEQ ID NO:399).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-caggatatattgcagtttaggaactctg-3’ (SEQ ID NO:400) or a 5’ fragment thereof comprising 5’-caggatatattgcagtttagga-3’ (SEQ ID NO:401).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgcagtttaggaactctg-3’ (SEQ ID NO:402) or a 5’ fragment thereof comprising 5’-tatattgcagtttagga-3’ (SEQ ID NO:403).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-gaggatatattgttgtgttaggatgatatttggatc-3’ (SEQ ID NO:404) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgttgtgttaggatgatatttggatc-3’ (SEQ ID NO:406) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-atatctatattgtggtggaaactcttttttgaaactgg-3’ (SEQ ID NO:408) or a 5’ fragment thereof comprising 5’-atatctatattgtggtggaaac-3’ (SEQ ID NO:409).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgtggtggaaactcttttttgaaactgg-3’ (SEQ ID NO:410) or a 5’ fragment thereof comprising 5’-tatattgtggtggaaac-3’ (SEQ ID NO: 179).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-gaggatatattgttgtgttaggctgatagttggatcatttt-3’ (SEQ ID NO:411) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgttgtgttaggctgatagttggatcatttt-3’ (SEQ ID NO:412) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-gaggatatattgttgtgttaggctgatatttgga-3’ (SEQ ID NO:413) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405). In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgttgtgttaggctgatatttgga-3’ (SEQ ID NO:414) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-gaggatatattgttgtgttagtctgattgtgg-3’ (SEQ ID NO:415) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagt-3’ (SEQ ID NO:416).

In an embodiment, the plant is potato and the processed left border of the PT- DNA in the potato genome comprises 5’-tatattgttgtgttagtctgattgtgg-3’ (SEQ ID NO:417) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagt-3’ (SEQ ID NO:418).

In an embodiment, the plant is potato and the processed right border of the PT- DNA in the potato genome comprises 5’-agtatgatgctgtgcataaactatcagtatttga-3’ (SEQ ID NO:419) or a 3’ fragment thereof comprising 5’-taaactatcagtatttga-3’ (SEQ ID NO:420) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is potato and the processed right border of the PT- DNA in the potato genome comprises 5’-gtatgtaatttcttatttgattcaaactatcagtttttga-3’ (SEQ ID NO:748) or a 3’ fragment thereof comprising 5’-caaactatcagtttttga-3’ (SEQ ID NO: 195) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is potato and the processed right border of the PT- DNA in the potato genome comprises 5’-gaaaatgtatgaagagaggaaaaacttccagtgtttga-3’ (SEQ ID NO:421) or a 3’ fragment thereof comprising 5’-aaaacttccagtgtttga-3’ (SEQ ID NO: 422) or 5’-ttga-3’ (SEQ ID NO: 62).

As another example, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT), wherein the Ti plasmid comprises a plant transfer DNA (PT-DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT-DNA comprises part of a left border for integration into the plant genome, and the 3’ end of the PT-DNA comprises part of a right border for integration into the plant genome.

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- atgtatatattgtggtgtaagatagaaaatgatagcaccccaacctcat-3’ (SEQ ID NO:29) or a 5’ fragment thereof comprising 5’-atgtatatattgtggtgtaaga-3’ (SEQ ID NO:30).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- tatattgtggtgtaagatagaaaatgatagcaccccaacctcat-3’ (SEQ ID NO:31) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaga-3’ (SEQ ID NO:32).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- caggatatattgtatcttttttttaatcagttgggaggac-3’ (SEQ ID NO:33) or a 5’ fragment thereof comprising 5’-caggatatattgtatctttttt-3’ (SEQ ID NO:34).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtatcttttttttaatcagttgggaggac-3’ (SEQ ID NO:35) or a 5’ fragment thereof comprising 5’-tatattgtatctttttt-3’ (SEQ ID NO:36).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- aaggatatattgtggtttgaagacagattggtggttcctaa-3’ (SEQ ID NO:37) or a 5’ fragment thereof comprising 5’ -aaggatatattgtggtttgaag-3 ’ (SEQ ID NO:38).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtttgaagacagattggtggttcctaa- 3’ (SEQ ID NO:39) or a 5’ fragment thereof comprising 5’-tatattgtggtttgaag-3’ (SEQ ID NO:40).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- caggatatatggtggtggtggtatgtatgtataatgatatgatataggtg-3’ (SEQ ID NO:41) or a 5’ fragment thereof comprising 5’ -caggatatatggtggtggtggt-3 ’ (SEQ ID NO:42).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’- tatatggtggtggtggtatgtatgtataatgatatgatataggtg-3’ (SEQ ID NO:43) or a 5’ fragment thereof comprising 5’ -tatatggtggtggtggt-3 ’ (SEQ ID NO:44).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-ctatatatattgtggtgtaagatagaaaa-3’ (SEQ ID NO:45) or a 5’ fragment thereof comprising 5’-ctatatatattgtggtgtaaga-3’ (SEQ ID NO:46).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtgtaagatagaaaa-3 ’ (SEQ ID NO:47) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaga-3’ (SEQ ID NO:32).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaggatatattgtggtggacattt-3’ (SEQ ID NO:48) or a 5’ fragment thereof comprising 5’ -gaggatatattgtggtggacat-3 ’ (SEQ ID NO:49).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtggacattt-3’ (SEQ ID NO:50) or a 5’ fragment thereof comprising 5’-tatattgtggtggacat-3’ (SEQ ID NO:51).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgtggaaggttcat-3’ (SEQ ID NO:52) or a 5’ fragment thereof comprising 5’-caggatatattgtggaaggttc-3’ (SEQ ID NO:53).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggaaggttcat-3’ (SEQ ID NO:54) or a 5’ fragment thereof comprising 5’-tatattgtggaaggttc-3’ (SEQ ID NO:55).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tcgataatattgtggtgaaaaccaagcaagtc-3’ (SEQ ID NO:56) or a 5’ fragment thereof comprising 5’ -tcgataatattgtggtgaaaac-3 ’ (SEQ ID NO:57).

In an embodiment, the plant is maize and the part of the left border of the PT- DNA for integration into the genome comprises 5’-aatattgtggtgaaaaccaagcaagtc-3’ (SEQ ID NO:58) ora 5’ fragment thereof comprising 5 ’ -aatattgtggtgaaaac-3 ’ (SEQ ID NO : 59) .

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggatgtatatattgtggtgtaagatagaaaatgatagcaccccaacctcat-3’ (SEQ ID NO:423) or a 5’ fragment thereof comprising 5’-tggatgtatatattgtggtgtaaga-3’ (SEQ ID NO:424), or wherein the 5’ t is replaced with a c (SEQ ID NO:425).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggcaggatatattgtatcttttttttaatcagttgggaggac-3’ (SEQ ID NO:426) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtatctttttt-3 ’ (SEQ ID NO:427), or wherein the 5’ t is replaced with a c (SEQ ID NO:428).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggaaggatatattgtggtttgaagacagattggtggttcctaa-3’ (SEQ ID NO:429) or a 5’ fragment thereof comprising 5’-tggaaggatatattgtggtttgaag-3’ (SEQ ID NO:430), or wherein the 5’ t is replaced with a c (SEQ ID NO:431).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggcaggatatatggtggtggtggtatgtatgtataatgatatgatataggtg-3’ (SEQ ID NO:432) or a 5’ fragment thereof comprising 5’ -tggcaggatatatggtggtggtggt-3 ’ (SEQ ID NO:433), or wherein the 5’ t is replaced with a c (SEQ ID NO:434). In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggctatatatattgtggtgtaagatagaaaa-3’ (SEQ ID NO:435) or a 5’ fragment thereof comprising 5’-tggctatatatattgtggtgtaaga-3’ (SEQ ID NO:436), or wherein the 5’ t is replaced with a c (SEQ ID NO: 437).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tgggaggatatattgtggtggacattt-3’ (SEQ ID NO:438) or a 5’ fragment thereof comprising 5 ’-tgggaggatatattgtggtggacat-3 ’ (SEQ ID NO:439), or wherein the 5 ’ t is replaced with a c (SEQ ID NO: 440).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggcaggatatattgtggaaggttcat-3’ (SEQ ID NO:441) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtggaaggttc-3’ (SEQ ID NO:442), or wherein the 5’ t is replaced with a c (SEQ ID NO:443).

In an embodiment, the plant is maize and the left border of the PT-DNA comprises 5’-tggtcgataatattgtggtgaaaaccaagcaagtc-3’ (SEQ ID NO:444) or a 5’ fragment thereof comprising 5’-tggtcgataatattgtggtgaaaac-3’ (SEQ ID NO: 445), or wherein the 5’ t is replaced with a c (SEQ ID NO:446).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-ctttaaattaaattttgtggctatcagtgtttga-3’ (SEQ ID NO: 60) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO:61) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-caacctgatggttctcagatcagtgtttga-3’ (SEQ ID NO:63) or a 3’ fragment thereof comprising 5’-tctcagatcagtgtttga-3’ (SEQ ID NO:64) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-acattcataactaagttttgtggctatcagtgtttga- 3’ (SEQ ID NO:65) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO: 61) or 5 ’-ttga-3 ’ (SEQ ID NO: 62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-agttttgcaaaagtagccatcagtgtttga-3’ (SEQ ID NO:66) or a 3’ fragment thereof comprising 5’-gtagccatcagtgtttga-3’ (SEQ ID NO:67) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-aagctgaggcgtgctgcatatcagtgtttga-3’ (SEQ ID NO:68) or a 3’ fragment thereof comprising 5’-ctgcatatcagtgtttga-3’ (SEQ ID NO:69) or 5’-ttga-3’ (SEQ ID NO:62). In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’ -tcataaccaagttttgtggctatcagtgtttga-3’ (SEQ ID NO: 70) or a 3’ fragment thereof comprising 5’-gtggctatcagtgtttga-3’ (SEQ ID NO:61) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-agatatcaaatttttacctctatcagtgtttga-3’ (SEQ ID NO:71) or a 3’ fragment thereof comprising 5’-acctctatcagtgtttga-3’ (SEQ ID NO:72) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5 ’-attcacccccccctctaggcgactatcagtgtttga- 3’ (SEQ ID NO:73) or a 3’ fragment thereof comprising 5’-gcgactatcagtgtttga-3’ (SEQ ID NO: 74) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is maize and the part of the right border of the PT- DNA for integration into the genome comprises 5’-cgctcatggaagcacttaaattatcagtgtttga-3’ (SEQ ID NO:75) or a 3’ fragment thereof comprising 5’-taaattatcagtgtttga-3’ (SEQ ID NO:76) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-ctttaaattaaattttgtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:447) or a 3’ fragment thereof comprising 5’- gtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:448) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-caacctgatggttctcagatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:450) or a 3’ fragment thereof comprising 5’- tctcagatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:451) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-acattcataactaagttttgtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:452) or a 3’ fragment thereof comprising 5’- gtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:448) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-agttttgcaaaagtagccatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:453) or a 3’ fragment thereof comprising 5’- gtagccatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:454) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449). In an embodiment, the plant is maize the PT-DNA has a right border comprising 5’-aagctgaggcgtgctgcatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:455) or a 3’ fragment thereof comprising 5’-ctgcatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:456) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-tcataaccaagttttgtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:457) or a 3’ fragment thereof comprising 5’- gtggctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:448) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-agatatcaaatttttacctctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:458) or a 3’ fragment thereof comprising 5’- acctctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:459) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-attcacccccccctctaggcgactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:460) or a 3’ fragment thereof comprising 5’- gcgactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:461) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is maize and the PT-DNA has a right border comprising 5’-cgctcatggaagcacttaaattatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:462) or a 3’ fragment thereof comprising 5’- taaattatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:463) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza saliva subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- aactatatattgtggtgtaaccagacatccacc-3’ (SEQ ID NO: 77) or a 5’ fragment thereof comprising 5’ -aactatatattgtggtgtaacc-3 ’ (SEQ ID NO:78).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgtaaccagacatccacc-3 ’ (SEQ ID NO:79) or a 5’ fragment thereof comprising 5’- tatattgtggtgtaacc-3 ’ (SEQ ID NO: 80).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- taggatatattgtggtgaaatgacgtgttatt-3’ (SEQ ID NO: 81) or a 5’ fragment thereof comprising 5’-taggatatattgtggtgaaatg-3’ (SEQ ID NO: 82). In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica. and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgaaatgacgtgttatt-3 ’ (SEQ ID NO: 83) or a 5’ fragment thereof comprising 5’- tatattgtggtgaaatg-3’ (SEQ ID NO:84).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- caggatatattgtttggcaacaaat-3 ’ (SEQ ID NO: 85) or a 5’ fragment thereof comprising 5’- caggatatattgtttggcaaca-3’ (SEQ ID NO:86).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtttggcaacaaat-3’ (SEQ ID NO:87) or a 5’ fragment thereof comprising 5’- tatattgtttggcaaca-3’ (SEQ ID NO:88).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- taatatatattgtggtgtataatattttcctcga-3’ (SEQ ID NO:89) or a 5’ fragment thereof comprising 5’-taatatatattgtggtgtataa-3’ (SEQ ID NO:90).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgtataatattttcctcga-3’ (SEQ ID NO:91) or a 5’ fragment thereof comprising 5’- tatattgtggtgtataa-3 ’ (SEQ ID NO:92).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- ttggatatattgtggtgctttgaagagacaa-3’ (SEQ ID NO:93) or a 5’ fragment thereof comprising 5’-ttggatatattgtggtgctttg-3’ (SEQ ID NO: 94).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgctttgaagagacaa-3 ’ (SEQ ID NO:95) or a 5’ fragment thereof comprising 5’- tatattgtggtgctttg-3’ (SEQ ID NO:96).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- agatcggtattgtggtgtaaaccgggaagacgatttc-3’ (SEQ ID NO:97) or a 5’ fragment thereof comprising 5’ -agatcggtattgtggtgtaaac-3’ (SEQ ID NO:98).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- ggtattgtggtgtaaaccgggaagacgatttc-3’ (SEQ ID NO: 99) or a 5’ fragment thereof comprising 5’-ggtattgtggtgtaaac-3’ (SEQ ID NO: 100). In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica. and the part of the left border of the PT-DNA for integration into the genome comprises 5’- taggatatattgtggttccgtatgatat-3 ’ (SEQ ID NO: 101) or a 5 ’ fragment thereof comprising 5 taggatatattgtggttccgta-3’ (SEQ ID NO: 102).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggttccgtatgatat-3 ’ (SEQ ID NO: 103) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- aaggatatattgtggttccgtatgatattttcgt-3’ (SEQ ID NO: 105) or a 5’ fragment thereof comprising 5’-aaggatatattgtggttccgta-3’ (SEQ ID NO: 106).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggttccgtatgatattttcgt-3’ (SEQ ID NO: 107) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tcatatatattgtggtgcaaacatatttttggtggaatct-3’ (SEQ ID NO: 108) or a 5’ fragment thereof comprising 5’-tcatatatattgtggtgcaaac-3’ (SEQ ID NO: 109).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgcaaacatatttttggtggaatct-3’ (SEQ ID NO: 110) or a 5’ fragment thereof comprising 5’-tatattgtggtgcaaac-3’ (SEQ ID NO: 111).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tgggatatattgtgatgtaactatagcagttgttca-3’ (SEQ ID NO: 112) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtgatgtaactatagcagttgttcact-3’ (SEQ ID NO: 114) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggaactatatattgtggtgtaaccagacatccacc-3’ (SEQ ID NO:464) or a 5’ fragment thereof comprising 5’-tggaactatatattgtggtgtaacc-3’ (SEQ ID NO:465), or wherein the 5’ t is replaced with a c (SEQ ID NO:466). In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica. and the left border comprises 5’-tggtaggatatattgtggtgaaatgacgtgttatt-3’ (SEQ ID NO:467) or a 5’ fragment thereof comprising 5’-tggtaggatatattgtggtgaaatg-3’ (SEQ ID NO:468), or wherein the 5 ’ t is replaced with a c (SEQ ID NO:469).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggcaggatatattgtttggcaacaaat-3’ (SEQ ID NO:470) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtttggcaaca-3’ (SEQ ID NO:471), or wherein the 5’ t is replaced with a c (SEQ ID NO:472).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggtaatatatattgtggtgtataatattttcctcga-3’ (SEQ ID NO:473) or a 5’ fragment thereof comprising 5’-tggtaatatatattgtggtgtataa-3’ (SEQ ID NO:474), or wherein the 5’ t is replaced with a c (SEQ ID NO:475).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggttggatatattgtggtgctttgaagagacaa-3’ (SEQ ID NO: 476) or a 5’ fragment thereof comprising 5’-tggttggatatattgtggtgctttg-3’ (SEQ ID NO:477), or wherein the 5’ t is replaced with a c (SEQ ID NO:478).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggagatcggtattgtggtgtaaaccgggaagacgatttc-3’ (SEQ ID NO:479) or a 5’ fragment thereof comprising 5’-tggagatcggtattgtggtgtaaac-3’ (SEQ ID NO:480), or wherein the 5’ t is replaced with a c (SEQ ID NO:481).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggtaggatatattgtggttccgtatgatat-3’ (SEQ ID NO:482) or a 5’ fragment thereof comprising 5’-tggtaggatatattgtggttccgta-3’ (SEQ ID NO:483), or wherein the 5’ t is replaced with a c (SEQ ID NO:484).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggaaggatatattgtggttccgtatgatattttcgt-3’ (SEQ ID NO:485) or a 5’ fragment thereof comprising 5’-tggaaggatatattgtggttccgta-3’ (SEQ ID NO:486), or wherein the 5’ t is replaced with a c (SEQ ID NO:487).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggtcatatatattgtggtgcaaacatatttttggtggaatct-3’ (SEQ ID NO:488) or a 5’ fragment thereof comprising 5’ -tggtcatatatattgtggtgcaaac-3’ (SEQ ID NO:489), or wherein the 5’ t is replaced with a c (SEQ ID NO:490).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the left border comprises 5’-tggtgggatatattgtgatgtaactatagcagttgttca-3’ (SEQ ID NO: 491) or a 5’ fragment thereof comprising 5’ -tggtgggatatattgtgatgtaact-3 ’ (SEQ ID NO:492), or wherein the 5’ t is replaced with a c (SEQ ID NO:493). In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica. and the left border comprises 5’-ttaaagccgtattctgcttttgaaactttcagtgtttga-3’ (SEQ ID NO: 116) or a 3’ fragment thereof comprising 5’-gaaactttcagtgtttga-3’ (SEQ ID NO: 117) or 5’- ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the right border of the PT-DNA for integration into the genome comprises 5 aatcacaacaatattcactcaactctcagtgtttga-3’ (SEQ ID NO: 118) or a 3’ fragment thereof comprising 5’-tcaactctcagtgtttga-3’ (SEQ ID NO: 119) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the part of the right border of the PT-DNA for integration into the genome comprises 5 caatcacgacatgagaactatcagtgcttga-3’ (SEQ ID NO: 120) or a 3’ fragment thereof comprising 5’-agaactatcagtgcttga-3’ (SEQ ID NO: 121) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the right border of the PT-DNA comprises 5’- ttaaagccgtattctgcttttgaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:494) or a 3’ fragment thereof comprising 5’-gaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:495) or 5’ -ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the right border of the PT-DNA comprises 5’- aatcacaacaatattcactcaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:496) or a 3’ fragment thereof comprising 5’-tcaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:497) or 5 ’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. japonica, and the right border of the PT-DNA comprises 5’- caatcacgacatgagaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:498) or a 3’ fragment thereof comprising 5’-agaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:499) or 5 ’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- caggatatattgtttggcaacaaatctactt-3 ’ (SEQ ID NO: 122) or a 5 ’ fragment thereof comprising 5’-caggatatattgtttggcaaca-3’ (SEQ ID NO:86).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtttggcaacaaatctactt-3 ’ (SEQ ID NO: 123) or a 5’ fragment thereof comprising 5’- tatattgtttggcaaca-3’ (SEQ ID NO:88). In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the part of the left border of the PT-DNA for integration into the genome comprises 5’- aactatatattgtggtgtaaccagacatccacctgagc-3’ (SEQ ID NO: 124) or a 5’ fragment thereof comprising 5’ -aactatatattgtggtgtaacc-3 ’ (SEQ ID NO:78).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgtaaccagacatccacctgagc-3’ (SEQ ID NO: 125) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaacc-3’ (SEQ ID NO:80).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- taatatatattgtggtgtataatattttcctcgagact-3’ (SEQ ID NO: 126) or a 5’ fragment thereof comprising 5’-taatatatattgtggtgtataa-3’ (SEQ ID NO: 90).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgtataatattttcctcgagact-3’ (SEQ ID NO: 127) or a 5’ fragment thereof comprising 5’-tatattgtggtgtataa-3’ (SEQ ID NO:92).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- aaggatatattgtggttccgtatgatatttccg-3’ (SEQ ID NO: 128) or a 5’ fragment thereof comprising 5’-aaggatatattgtggttccgta-3’ (SEQ ID NO: 106).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggttccgtatgatatttccg-3’ (SEQ ID NO: 129) or a 5’ fragment thereof comprising 5’- tatattgtggttccgta-3 ’ (SEQ ID NO: 104).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- ttggatatattgtggtgctttgaagagacaaaag-3’ (SEQ ID NO: 130) or a 5’ fragment thereof comprising 5’-ttggatatattgtggtgctttg-3’ (SEQ ID NO:94).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgctttgaagagacaaaag-3’ (SEQ ID NO: 131) or a 5’ fragment thereof comprising 5’-tatattgtggtgctttg-3’ (SEQ ID NO:96).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tgggatatattgtgatgtaactatagcagttgttcactg-3’ (SEQ ID NO: 132) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113). In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtgatgtaactatagcagttgttcactg-3’ (SEQ ID NO: 133) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tcatatatattgtggtgcaaacatatttttggtggaatcttg-3’ (SEQ ID NO: 134) or a 5’ fragment thereof comprising 5’-tcatatatattgtggtgcaaac-3’ (SEQ ID NO: 109).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtgcaaacatatttttggtggaatcttg-3’ (SEQ ID NO: 135) or a 5’ fragment thereof comprising 5’-tatattgtggtgcaaac-3’ (SEQ ID NO: 111).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tgggatatattgtgatgtaactatagcagatggtcactgat-3’ (SEQ ID NO: 136) or a 5’ fragment thereof comprising 5’-tgggatatattgtgatgtaact-3’ (SEQ ID NO: 113).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtgatgtaactatagcagatggtcactgat-3’ (SEQ ID NO: 137) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaact-3’ (SEQ ID NO: 115).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- gaggatatattggggtgtacacaataaaaattctttcgtct-3’ (SEQ ID NO: 138) or a 5’ fragment thereof comprising 5’ -gaggatatattggggtgtacac-3 ’ (SEQ ID NO: 139).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattggggtgtacacaataaaaattctttcgtct-3’ (SEQ ID NO: 140) or a 5’ fragment thereof comprising 5’-tatattggggtgtacac-3’ (SEQ ID NO: 141).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- gcttatatattgtggtataaaataaatctgaatcct-3’ (SEQ ID NO: 142) or a 5’ fragment thereof comprising 5’-gcttatatattgtggtataaaa-3’ (SEQ ID NO: 143).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the left border of the PT-DNA for integration into the genome comprises 5’- tatattgtggtataaaataaatctgaatcct-3’ (SEQ ID NO: 144) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 145). In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the left border comprises 5’-tggcaggatatattgtttggcaacaaatctactt-3’ (SEQ ID NO: 500) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtttggcaaca-3’ (SEQ ID NO:471), or wherein the 5’ t is replaced with a c (SEQ ID NO:501).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggaactatatattgtggtgtaaccagacatccacctgagc-3’ (SEQ IDNO:502) or a 5’ fragment thereof comprising 5’-tggaactatatattgtggtgtaacc-3’ (SEQ ID NO:465), or wherein the 5’ t is replaced with a c (SEQ ID NO:503).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggtaatatatattgtggtgtataatattttcctcgagact-3’ (SEQ ID NO:504) or a 5’ fragment thereof comprising 5’-tggtaatatatattgtggtgtataa-3’ (SEQ ID NO:474), or wherein the 5’ t is replaced with a c (SEQ ID NO:505).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggaaggatatattgtggttccgtatgatatttccg-3’ (SEQ ID NO:506) or a 5’ fragment thereof comprising 5’-tggaaggatatattgtggttccgta-3’ (SEQ ID NO:486), or wherein the 5’ t is replaced with a c (SEQ ID NO:507).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggttggatatattgtggtgctttgaagagacaaaag-3’ (SEQ ID NO:508) or a 5’ fragment thereof comprising 5’-tggttggatatattgtggtgctttg-3’ (SEQ ID NO:477), or wherein the 5’ t is replaced with a c (SEQ ID NO:509).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggtgggatatattgtgatgtaactatagcagttgttcactg-3’ (SEQ ID NO:510) or a 5’ fragment thereof comprising 5’ -tggtgggatatattgtgatgtaact-3’ (SEQ ID NO:492), or wherein the 5’ t is replaced with a c (SEQ ID NO:511).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggtcatatatattgtggtgcaaacatatttttggtggaatcttg-3’ (SEQ ID NO:512) or a 5’ fragment thereof comprising 5’ -tggtcatatatattgtggtgcaaac-3’ (SEQ ID NO:489), or wherein the 5’ t is replaced with a c (SEQ ID NO:513).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tggtgggatatattgtgatgtaactatagcagatggtcactgat-3’ (SEQ ID NO:514) or a 5’ fragment thereof comprising 5 ’-tggtgggatatattgtgatgtaact-3’ (SEQ ID NO:492), or wherein the 5’ t is replaced with a c (SEQ ID NO:515).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the left border comprises 5’-tgggaggatatattggggtgtacacaataaaaattctttcgtct-3’ (SEQ ID NO:516) or a 5’ fragment thereof comprising 5’ -tgggaggatatattggggtgtacac-3’ (SEQ ID NO:517), or wherein the 5’ t is replaced with a c (SEQ ID NO:518). In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica. and the left border comprises 5’-tgggcttatatattgtggtataaaataaatctgaatcct-3’ (SEQ ID NO:519) or a 5’ fragment thereof comprising 5’ -tgggcttatatattgtggtataaaa-3 ’ (SEQ ID NO:520), or wherein the 5’ t is replaced with a c (SEQ ID NO:521).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the right border of the PT-DNA for integration into the genome comprises 5’- aagccgtattctgcttttgaaactttcagtgtttga-3’ (SEQ ID NO: 146) or a 3’ fragment thereof comprising 5’-gaaactttcagtgtttga-3’ (SEQ ID NO: 117) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the right border of the PT-DNA for integration into the genome comprises 5’- tcacaacaatattcactcaactctcagtgtttga-3’ (SEQ ID NO: 147) or a 3’ fragment thereof comprising 5’-tcaactctcagtgtttga-3’ (SEQ ID NO: 119) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the part of the right border of the PT-DNA for integration into the genome comprises 5’- tcaatcacgacatgagaactatcagtgcttga-3’ (SEQ ID NO: 148) or a 3’ fragment thereof comprising 5’-agaactatcagtgcttga-3’ (SEQ ID NO: 121) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the right border comprises 5’-aagccgtattctgcttttgaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:522) or a 3’ fragment thereof comprising 5’- gaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:495) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the right border comprises 5’-tcacaacaatattcactcaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:523) or a 3’ fragment thereof comprising 5’- tcaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:497) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is rice, preferably Oryza sativa subsp. indica, and the right border comprises 5’-tcaatcacgacatgagaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:524) or a 3’ fragment thereof comprising 5’- agaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:499) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gcaaatatattgtggtgtaaatctttttctt-3’ (SEQ ID NO: 149) or a 5’ fragment thereof comprising 5’-gcaaatatattgtggtgtaaat-3’ (SEQ ID NO: 150). In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaaatctttttctt-3’ (SEQ ID NO: 151) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatcgtatattgtggtgtaaatatacacctaa-3’ (SEQ ID NO: 153) or a 5’ fragment thereof comprising 5’-tatcgtatattgtggtgtaaat-3’ (SEQ ID NO: 154).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaaatatacacctaa-3’ (SEQ ID NO: 155) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-aaggatatattgtggtcttcactccctct-3’ (SEQ ID NO: 156) or a 5’ fragment thereof comprising 5’-aaggatatattgtggtcttcac-3’ (SEQ ID NO: 157).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtcttcactccctct-3 ’ (SEQ ID NO: 158) or a 5’ fragment thereof comprising 5’-tatattgtggtcttcac-3’ (SEQ ID NO: 159).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -caggatatattgggtcctctcgaaag-3’ (SEQ ID NO: 160) or a 5’ fragment thereof comprising 5’ -caggatatattgggtcctctcg-3’ (SEQ ID NO: 161).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgggtcctctcgaaag-3 ’ (SEQ ID NO: 162) or a 5’ fragment thereof comprising 5’-tatattgggtcctctcg-3’ (SEQ ID NO: 163).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgattcctctcaatgag-3’ (SEQ ID NO: 164) or a 5’ fragment thereof comprising 5’-caggatatattgattcctctca-3’ (SEQ ID NO: 165).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgattcctctcaatgag-3’ (SEQ ID NO: 166) or a 5’ fragment thereof comprising 5’-tatattgattcctctca-3’ (SEQ ID NO: 167).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgggtcttctcgatggg-3’ (SEQ ID NO: 168) or a 5’ fragment thereof comprising 5’ -caggatatattgggtcttctcg-3’ (SEQ ID NO: 169). In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgggtcttctcgatggg-3 ’ (SEQ ID NO: 170) or a 5’ fragment thereof comprising 5’-tatattgggtcttctcg-3’ (SEQ ID NO: 171).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- cataatattttgtggtgtaaaaatatagtctatgcaaga-3’ (SEQ ID NO: 172) or a 5’ fragment thereof comprising 5’-cataatattttgtggtgtaaaa-3’ (SEQ ID NO: 173).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tattttgtggtgtaaaaatatagtctatgcaaga-3’ (SEQ ID NO: 174) or a 5’ fragment thereof comprising 5’-tattttgtggtgtaaaa-3’ (SEQ ID NO: 175).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- atgactatattgtggtggaaactcactaagggttcctta-3’ (SEQ ID NO: 176) or a 5’ fragment thereof comprising 5’-atgactatattgtggtggaaac-3’ (SEQ ID NO: 177).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtggaaactcactaagggttcctta-3’ (SEQ ID NO: 178) or a 5’ fragment thereof comprising 5’-tatattgtggtggaaac-3’ (SEQ ID NO: 179).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-acatatatcttgtggtgtaaaattatacataatttta- 3’ (SEQ ID NO: 180) or a 5’ fragment thereof comprising 5’-acatatatcttgtggtgtaaaa-3’ (SEQ ID NO: 181).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatcttgtggtgtaaaattatacataatttta-3’ (SEQ ID NO: 182) or a 5’ fragment thereof comprising 5’-tatcttgtggtgtaaaa-3’ (SEQ ID NO: 183).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-aaggaaatattgtggtgttgatggggatcaaact- 3’ (SEQ ID NO: 184) or a 5’ fragment thereof comprising 5’-aaggaaatattgtggtgttgat-3’ (SEQ ID NO: 185).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-aatattgtggtgttgatggggatcaaact-3’ (SEQ ID NO: 186) or a 5’ fragment thereof comprising 5’-aatattgtggtgttgat-3’ (SEQ ID NO: 187). In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-cagtatatattgtggttcatatgtttaagctat-3’ (SEQ ID NO: 188) or a 5’ fragment thereof comprising 5’-cagtatatattgtggttcatat-3’ (SEQ ID NO: 189).

In an embodiment, the plant is tomato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggttcatatgtttaagctat-3’ (SEQ ID NO: 190) or a 5’ fragment thereof comprising 5’-tatattgtggttcatat-3’ (SEQ ID NO: 191).

In an embodiment, the plant is tomato and the left border comprises 5’- tgggcaaatatattgtggtgtaaatctttttctt-3’ (SEQ ID NO:525) or a 5’ fragment thereof comprising 5’-tgggcaaatatattgtggtgtaaat-3’ (SEQ ID NO:526), or wherein the 5’ t is replaced with a c (SEQ ID NO: 527).

In an embodiment, the plant is tomato and the left border comprises 5’- tggtatcgtatattgtggtgtaaatatacacctaa-3’ (SEQ ID NO:528) or a 5’ fragment thereof comprising 5’ -tggtatcgtatattgtggtgtaaat-3 ’ (SEQ ID NO:529), or wherein the 5’ t is replaced with a c (SEQ ID NO: 530).

In an embodiment, the plant is tomato and the left border comprises 5’- tggaaggatatattgtggtcttcactccctct-3’ (SEQ ID NO:531) or a 5’ fragment thereof comprising 5’-tggaaggatatattgtggtcttcac-3’ (SEQ ID NO:532), or wherein the 5’ t is replaced with a c (SEQ ID NO: 533).

In an embodiment, the plant is tomato and the left border comprises 5’- tggcaggatatattgggtcctctcgaaag-3’ (SEQ ID NO:534) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgggtcctctcg-3 ’ (SEQ ID NO:535), or wherein the 5’ t is replaced with a c (SEQ ID NO:536).

In an embodiment, the plant is tomato and the left border comprises 5’- tggcaggatatattgattcctctcaatgag-3’ (SEQ ID NO:537) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgattcctctca-3’ (SEQ ID NO:538), or wherein the 5’ t is replaced with a c (SEQ ID NO:539).

In an embodiment, the plant is tomato and the left border comprises 5’- tggcaggatatattgggtcttctcgatggg-3 ’ (SEQ ID NO:540) or a 5 ’ fragment thereof comprising 5’ -tggcaggatatattgggtcttctcg-3 ’ (SEQ ID NO:541), or wherein the 5’ t is replaced with a c (SEQ ID NO:542).

In an embodiment, the plant is tomato and the left border comprises 5’- tggcataatattttgtggtgtaaaaatatagtctatgcaaga-3’ (SEQ ID NO:543) or a 5’ fragment thereof comprising 5’-tggcataatattttgtggtgtaaaa-3’ (SEQ ID NO:544), or wherein the 5’ t is replaced with a c (SEQ ID NO: 545). In an embodiment, the plant is tomato and the left border comprises 5’- tggatgactatattgtggtggaaactcactaagggttcctta-3’ (SEQ ID NO:546) or a 5’ fragment thereof comprising 5’-tggatgactatattgtggtggaaac-3’ (SEQ ID NO:547), or wherein the 5’ t is replaced with a c (SEQ ID NO: 548).

In an embodiment, the plant is tomato and the left border comprises 5’- tggacatatatcttgtggtgtaaaattatacataatttta-3’ (SEQ ID NO: 549) or a 5’ fragment thereof comprising 5’-tggacatatatcttgtggtgtaaaa-3’ (SEQ ID NO:550), or wherein the 5’ t is replaced with a c (SEQ ID NO: 551).

In an embodiment, the plant is tomato and the left border comprises 5’- tggaaggaaatattgtggtgttgatggggatcaaact-3’ (SEQ ID NO: 552) or a 5’ fragment thereof comprising 5’-tggaaggaaatattgtggtgttgat-3’ (SEQ ID NO:553), or wherein the 5’ t is replaced with a c (SEQ ID NO: 554).

In an embodiment, the plant is tomato and the left border comprises 5’- tggcagtatatattgtggttcatatgtttaagctat-3’ (SEQ ID NO:555) or a 5’ fragment thereof comprising 5’-tggcagtatatattgtggttcatat-3’ (SEQ ID NO: 556) or wherein the 5’ t is replaced with a c (SEQ ID NO: 557).

In an embodiment, the plant is tomato and the part of the right border of the PT- DNA for integration into the genome comprises 5’ -cacgcaaagagaaggaaaaactttcagtgtttga- 3’ (SEQ ID NO: 192) or a 3’ fragment thereof comprising 5’-aaaactttcagtgtttga-3’ (SEQ ID NO: 193) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the part of the right border of the PT- DNA for integration into the genome comprises 5’-aaagaaagacaacaaactatcagtttttga-3’ (SEQ ID NO: 194) or a 3’ fragment thereof comprising 5’-caaactatcagtttttga-3’ (SEQ ID NO: 195) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the part of the right border of the PT- DNA for integration into the genome comprises 5’-ggccatttcaagtccaactatcagtgcttga-3’ (SEQ ID NO: 196) or a 3’ fragment thereof comprising 5’-ccaactatcagtgcttga-3’ (SEQ ID NO: 197) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the part of the right border of the PT- DNA for integration into the genome comprises 5’ -caatgttaggtatgaactatcagtgtttga-3’ (SEQ ID NO: 198) or a 3’ fragment thereof comprising 5’-tgaactatcagtgtttga-3’ (SEQ ID NO: 199) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is tomato and the right border comprises 5’- cacgcaaagagaaggaaaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:558) or a 3’ fragment thereof comprising 5’-aaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:559) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449). In an embodiment, the plant is tomato and the right border comprises 5’- aaagaaagacaacaaactatcagtttttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 560) or a 3’ fragment thereof comprising 5’-caaactatcagtttttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:561) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is tomato and the right border comprises 5’- ggccatttcaagtccaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:562) or a 3’ fragment thereof comprising 5’-ccaactatcagtgcttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:563) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is tomato and the right border comprises 5’- caatgttaggtatgaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:564) or a 3’ fragment thereof comprising 5’-tgaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:565) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caagatatattgtggcacaactatgag-3’ (SEQ ID NO:200) or a 5’ fragment thereof comprising 5’-caagatatattgtggcacaact-3’ (SEQ ID NO:201).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggcacaactatgag-3’ (SEQ ID NO:202) or a 5’ fragment thereof comprising 5’-tatattgtggcacaact-3’ (SEQ ID NO:203).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatatgttggtgtaattgatcttg-3’ (SEQ ID NO: 204) or a 5’ fragment thereof comprising 5’-caggatatatgttggtgtaatt-3’ (SEQ ID NO:205).

In an embodiment, the plant is sorghum the part of the left border of the PT-DNA for integration into the genome comprises 5’-tatatgttggtgtaattgatcttg-3’ (SEQ ID NO:206) or a 5’ fragment thereof comprising 5’-tatatgttggtgtaatt-3’ (SEQ ID NO:207).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -aagcttatattgtggtttaaaaaccttattaagtggtt- 3’ (SEQ ID NO:208) or a 5’ fragment thereof comprising 5’-aagcttatattgtggtttaaaa-3’ (SEQ ID NO:209).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtttaaaaaccttattaagtggtt-3’ (SEQ ID NO:210) or a 5’ fragment thereof comprising 5’-tatattgtggtttaaaa-3’ (SEQ ID NO:211).

In an embodiment, the plant is sorghum the part of the left border of the PT-DNA for integration into the genome comprises 5’-tggtttatattgtggtataaaatttaaact-3’ (SEQ ID I l l

NO:212) or a 5’ fragment thereof comprising 5’-tggtttatattgtggtataaaa-3’ (SEQ ID NO:213).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtataaaatttaaact-3’ (SEQ ID NO:214) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 145).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’- tttagcatattgtggtgtaaatgaagatgagctgatg-3’ (SEQ ID NO:215) or a 5’ fragment thereof comprising 5’-tttagcatattgtggtgtaaat-3’ (SEQ ID NO:215).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-catattgtggtgtaaatgaagatgagctgatg-3’ (SEQ ID NO:217) or a 5’ fragment thereof comprising 5’-catattgtggtgtaaat-3’ (SEQ ID NO:218).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’- ggcgatatattgtggtgttcagtgtttagtcagaagtc-3’ (SEQ ID NO:219) or a 5’ fragment thereof comprising 5’-ggcgatatattgtggtgttcag-3’ (SEQ ID NO:220).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgttcagtgtttagtcagaagtc-3’ (SEQ ID NO:221) or a 5’ fragment thereof comprising 5’-tatattgtggtgttcag-3’ (SEQ ID NO: 222).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-acctctatattgtggtgtaattttcttgtggcaac-3’ (SEQ ID NO:223) or a 5 ’ fragment thereof comprising 5 ’-acctctatattgtggtgtaatt-3 ’ (SEQ ID NO:224).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5 ’ -tatattgtggtgtaattttcttgtggcaac-3 ’ (SEQ ID NO:225) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaatt-3’ (SEQ ID NO: 226).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tgatatatattgtggtgtaccacgcggcatgggt- 3’ (SEQ ID NO:227) or a 5’ fragment thereof comprising 5’-tgatatatattgtggtgtacca-3’ (SEQ ID NO:228).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaccacgcggcatgggt-3’ (SEQ ID NO:229) or a 5’ fragment thereof comprising 5’-tatattgtggtgtacca-3’ (SEQ ID NO:230).

In an embodiment, the plant is sorghum the part of the left border of the PT-DNA for integration into the genome comprises 5’-aatatgatattgtggtgtaaataactgaatattgatattt-3’ (SEQ ID NO:231) or a 5’ fragment thereof comprising 5’-aatatgatattgtggtgtaaat-3’ (SEQ ID NO:232).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gatattgtggtgtaaataactgaatattgatattt-3’ (SEQ ID NO:233) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaat-3’ (SEQ ID NO:234).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’- tggtatatattgtggtgtataactctgtataaccgaagcgtaattttcaacaccctagtaatggcgcaaactttggtatatattgtgg tgtataactccgtataaccgaagcaaga-3 ’ (SEQ ID NO:235) or a 5’ fragment thereof comprising 5’-tggtatatattgtggtgtataa-3’ (SEQ ID NO:236).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’- tatattgtggtgtataactctgtataaccgaagcgtaattttcaacaccctagtaatggcgcaaactttggtatatattgtggtgtat aactccgtataaccgaagcaaga-3 ’ (SEQ ID NO:236) or a 5’ fragment thereof comprising 5’- tatattgtggtgtataa-3 ’ (SEQ ID NO:92).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’- gataatatattgtggtgtatatgattagaaaaaaaaaac-3’ (SEQ ID NO:238) or a 5’ fragment thereof comprising 5’-gataatatattgtggtgtatat-3’ (SEQ ID NO:239).

In an embodiment, the plant is sorghum and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtatatgattagaaaaaaaaaac-3’ (SEQ ID NO:240) or a 5’ fragment thereof comprising 5’-tatattgtggtgtatat-3’ (SEQ ID NO:241).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggcaagatatattgtggcacaactatgag-3’ (SEQ ID NO:566) or a 5’ fragment thereof comprising 5’ -tggcaagatatattgtggcacaact-3 ’ (SEQ ID NO:567), or wherein the 5’ t is replaced with a c (SEQ ID NO:568).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggcaggatatatgttggtgtaattgatcttg-3’ (SEQ ID NO:569) or a 5’ fragment thereof comprising 5’ -tggcaggatatatgttggtgtaatt-3 ’ (SEQ ID NO:570), or wherein the 5’ t is replaced with a c (SEQ ID NO:571). In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggaagcttatattgtggtttaaaaaccttattaagtggtt-3’ (SEQ ID NO:572) or a 5’ fragment thereof comprising 5’-tggaagcttatattgtggtttaaaa-3’ (SEQ ID NO:573), or wherein the 5’ t is replaced with a c (SEQ ID NO:574).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggtggtttatattgtggtataaaatttaaact-3’ (SEQ ID NO:575) or a 5’ fragment thereof comprising 5’ -tggtggtttatattgtggtataaaa-3 ’ (SEQ ID NO: 576), or wherein the 5’ t is replaced with a c (SEQ ID NO:577).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggtttagcatattgtggtgtaaatgaagatgagctgatg-3’ (SEQ ID NO:578) or a 5’ fragment thereof comprising 5’-tggtttagcatattgtggtgtaaat-3’ (SEQ ID NO:579), or wherein the 5’ t is replaced with a c (SEQ ID NO:580).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggggcgatatattgtggtgttcagtgtttagtcagaagtc-3’ (SEQ ID NO:581) or a 5’ fragment thereof comprising 5’ -tggggcgatatattgtggtgttcag-3’ (SEQ ID NO:582), or wherein the 5’ t is replaced with a c (SEQ ID NO:583).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5 ’-tggacctctatattgtggtgtaattttcttgtggcaac-3’ (SEQ ID NO:584) ora 5’ fragment thereof comprising 5’-tggacctctatattgtggtgtaatt-3’ (SEQ ID NO:585), or wherein the 5’ t is replaced with a c (SEQ ID NO:586).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggtgatatatattgtggtgtaccacgcggcatgggt-3’ (SEQ ID NO:587) or a 5’ fragment thereof comprising 5’-tggtgatatatattgtggtgtacca-3’ (SEQ ID NO:588), or wherein the 5’ t is replaced with a c (SEQ ID NO:589).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggaatatgatattgtggtgtaaataactgaatattgatattt-3’ (SEQ ID NO:590) or a 5’ fragment thereof comprising 5’-tggaatatgatattgtggtgtaaat-3’ (SEQ ID NO:591), or wherein the 5’ t is replaced with a c (SEQ ID NO:592).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’- tggtggtatatattgtggtgtataactctgtataaccgaagcgtaattttcaacaccctagtaatggcgcaaactttggtatatattg tggtgtataactccgtataaccgaagcaaga-3’ (SEQ ID NO:593) or a 5’ fragment thereof comprising 5’-tggtggtatatattgtggtgtataa-3’ (SEQ ID NO:594), or wherein the 5’ t is replaced with a c (SEQ ID NO: 595).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tgggataatatattgtggtgtatatgattagaaaaaaaaaac-3’ (SEQ ID NO:596) or a 5’ fragment thereof comprising 5’-tgggataatatattgtggtgtatat-3’ (SEQ ID NO:597), or wherein the 5’ t is replaced with a c (SEQ ID NO:598).

In an embodiment, the plant is sorghum and the left border of the PT-DNA comprises 5’-tggcctgtcttcctccaaggaactgtcagtgtttga-3’ (SEQ ID NO:599) or a 3’ fragment thereof comprising 5’ -tggggaactgtcagtgtttga-3 ’ (SEQ ID NO:600) or 5’-ttga-3’ (SEQ ID NO:62), or wherein the 5’ t is replaced with a c (SEQ ID NO:601).

In an embodiment, the plant is sorghum and the part of the right border of the PT- DNA for integration into the genome comprises 5’-ttaaactaaagttaacactatcagtgtttga-3’ (SEQ ID NO:244) or a 3’ fragment thereof comprising 5’-aacactatcagtgtttga-3’ (SEQ ID NO: 245) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sorghum and the part of the right border of the PT- DNA for integration into the genome comprises 5’-tttttaataatgcctctatcagtgtttga-3’ (SEQ ID NO:246) or a 3’ fragment thereof comprising 5’-gcctctatcagtgtttga-3’ (SEQ ID NO: 247) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sorghum and the right border of the PT-DNA comprises 5’-ttaaactaaagttaacactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:602) or a 3’ fragment thereof comprising 5’- aacactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:603) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is sorghum and the right border of the PT-DNA comprises 5’-tttttaataatgcctctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:604) or a 3’ fragment thereof comprising 5’-gcctctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:605) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’- gtggatatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO:248) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaaac-3’ (SEQ ID NO:249).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO:250) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaac-3’ (SEQ ID NO:251).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gtggatatattgtgatgtaaaccttgtagtaatatgt- 3’ (SEQ ID NO:252) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaaac-3’ (SEQ ID NO:249). In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgatgtaaaccttgtagtaatatgt-3’ (SEQ ID NO:253) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaac-3’ (SEQ ID NO: 249).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -catgatatattgtggtgcatgtctctt-3’ (SEQ ID NO:254) or a 5’ fragment thereof comprising 5’-catgatatattgtggtgcatgt-3’ (SEQ ID NO:255).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgcatgtctctt-3’ (SEQ ID NO:256) or a 5’ fragment thereof comprising 5’-tatattgtggtgcatgt-3’ (SEQ ID NO:257).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gtggatatattgtggcgtaaaccttgcagta-3’ (SEQ ID NO:258) or a 5’ fragment thereof comprising 5’ -gtggatatattgtggcgtaaac-3’ (SEQ ID NO:259).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggcgtaaaccttgcagta-3’ (SEQ ID NO:260) or a 5’ fragment thereof comprising 5’-tatattgtggcgtaaac-3’ (SEQ ID NO:261).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattatggtgtagattataaaaacta-3’ (SEQ ID NO: 262) ora 5’ fragment thereof comprising 5’-caggatatattatggtgtagat-3’ (SEQ ID NO:263).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattatggtgtagattataaaaacta-3’ (SEQ ID NO:264) or a 5’ fragment thereof comprising 5’-tatattatggtgtagat-3’ (SEQ ID NO:265).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-catgatatattgtggtgaaaacatgcaagaaccta- 3’ (SEQ ID NO:266) or a 5’ fragment thereof comprising 5’-catgatatattgtggtgaaaac-3’ (SEQ ID NO:267).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtgaaaacatgcaagaaccta-3’ (SEQ ID NO:268) or a 5’ fragment thereof comprising 5’-tatattgtggtgaaaac-3’ (SEQ ID NO:269). In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gtggatatattgtgatgtaatccttgcagtaata-3’ (SEQ ID NO:270) or a 5’ fragment thereof comprising 5’-gtggatatattgtgatgtaatc-3’ (SEQ ID NO:271).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgatgtaatccttgcagtaata-3’ (SEQ ID NO:272) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaatc-3’ (SEQ ID NO:273).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-catgatatattgtgttgtatgcctctagtgtc-3’ (SEQ ID NO:274) or a 5’ fragment thereof comprising 5’-catgatatattgtgttgtatgc-3’ (SEQ ID NO:275).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgttgtatgcctctagtgtc-3’ (SEQ ID NO:276) or a 5’ fragment thereof comprising 5’-tatattgtgttgtatgc-3’ (SEQ ID NO:277).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-catgatatattgtgttgtatgtttcttgtgatgt-3’ (SEQ ID NO:278) or a 5’ fragment thereof comprising 5’-catgatatattgtgttgtatgt-3’ (SEQ ID NO:279).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtgttgtatgtttcttgtgatgt-3’ (SEQ ID NO:280) or a 5’ fragment thereof comprising 5’-tatattgtgttgtatgt-3’ (SEQ ID NO:281).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -gtggatatattgtgatgtaatccttgcggaaatat- 3’ (SEQ ID NO:282) or a 5’ fragment thereof comprising 5’ -gtggatatattgtgatgtaatc-3’ (SEQ ID NO:271).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgatgtaatccttgcggaaatat-3’ (SEQ ID NO:283) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaatc-3’ (SEQ ID NO:273).

In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’- gatcatatattgtggtctaaaccctaatgacgtagacatt-3’ (SEQ ID NO:284) or a 5’ fragment thereof comprising 5’ -gatcatatattgtggtctaaac-3 ’ (SEQ ID NO:285). In an embodiment, the plant is barley and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtctaaaccctaatgacgtagacatt- 3’ (SEQ ID NO:286) or a 5’ fragment thereof comprising 5’-tatattgtggtctaaac-3’ (SEQ ID NO:287).

In an embodiment, the plant is barley the part of the left border of the PT-DNA for integration into the genome comprises 5’-caagatatattgtgatgtaaatgccatcgtcact-3’ (SEQ ID NO:288) or a 5’ fragment thereof comprising 5’-caagatatattgtgatgtaaat-3’ (SEQ ID NO:289).

In an embodiment, the plant is barley the part of the left border of the PT-DNA for integration into the genome comprises 5’-tatattgtgatgtaaatgccatcgtcact-3’ (SEQ ID NO:290) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaat-3’ (SEQ ID NO:291).

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggtggatatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO:606) or a 5’ fragment thereof comprising 5’-tgggtggatatattgtgatgtaaac-3’ (SEQ ID NO: 607), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NO:608 and 1474.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-ttggtggatatattgtgatgtaaaccttgcagtaatatgta-3’ (SEQ ID NO: 1501) or a 5’ fragment thereof comprising 5’-ttggtggatatattgtgatgtaaac-3’ (SEQ ID NO: 1500).

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggtggatatattgtgatgtaaaccttgtagtaatatgt-3’ (SEQ ID NO:609) or a 5’ fragment thereof comprising 5’-tgggtggatatattgtgatgtaaac-3’ (SEQ ID NO: 607), or wherein the 5’ t is replaced with a c (SEQ ID NO:610).

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcatgatatattgtggtgcatgtctctt-3’ (SEQ ID NO:611) or a 5’ fragment thereof comprising 5’-tggcatgatatattgtggtgcatgt-3’ (SEQ ID NO:612), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:613 and 1475.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggtggatatattgtggcgtaaaccttgcagta-3’ (SEQ ID NOAM) or a 5’ fragment thereof comprising 5’ -tgggtggatatattgtggcgtaaac-3’ (SEQ ID NOAM), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:616 and 1476.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcaggatatattatggtgtagattataaaaacta-3’ (SEQ ID NOAM) or a 5’ fragment thereof comprising 5’-tggcaggatatattatggtgtagat-3’ (SEQ ID NO:618). or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:619 and 1477. In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcatgatatattgtggtgaaaacatgcaagaaccta-3’ (SEQ ID NO:620) or a 5’ fragment thereof comprising 5’-tggcatgatatattgtggtgaaaac-3’ (SEQ ID NO:621), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs: 622 and 1478.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggtggatatattgtgatgtaatccttgcagtaata-3’ (SEQ ID NO:623) or a 5’ fragment thereof comprising 5’ -tgggtggatatattgtgatgtaatc-3’ (SEQ ID NO:624), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:625 and 1479.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcatgatatattgtgttgtatgcctctagtgtc-3’ (SEQ ID NO:626) or a 5’ fragment thereof comprising 5’ -tggcatgatatattgtgttgtatgc-3 ’ (SEQ ID NO: 627), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:628 andl480.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcatgatatattgtgttgtatgtttcttgtgatgt-3’ (SEQ ID NO:629) or a 5’ fragment thereof comprising 5’-tggcatgatatattgtgttgtatgt-3’ (SEQ ID NO: 630), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:631 and 1481).

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggtggatatattgtgatgtaatccttgcggaaatat-3’ (SEQ ID NO:632) or a 5’ fragment thereof comprising 5 ’-tgggtggatatattgtgatgtaatc-3’ (SEQ ID NO:624), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NO:633, and 1479.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tgggatcatatattgtggtctaaaccctaatgacgtagacatt-3’ (SEQ ID NO:634) or a 5’ fragment thereof comprising 5’-tgggatcatatattgtggtctaaac-3’ (SEQ ID NO:635), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:636 and 1483.

In an embodiment, the plant is barley and the left border of the PT-DNA comprises 5’-tggcaagatatattgtgatgtaaatgccatcgtcact-3’ (SEQ ID NO:637) or a 5’ fragment thereof comprising 5’-tggcaagatatattgtgatgtaaat-3’ (SEQ ID NO:638), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:639 and 1484.

In an embodiment, the plant is barley and the part of the right border of the PT- DNA for integration into the genome comprises 5’-ctttgcttatcgcagaaactatcagtgtttga-3’ (SEQ ID NO:292) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO: 293) or 5’-ttga-3’ (SEQ ID NO: 62). In an embodiment, the plant is barley the part of the right border of the PT-DNA for integration into the genome comprises 5’-ctttgcttatcacagaaactatcagtgtttga-3’ (SEQ ID NO:294) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO:293) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the part of the right border of the PT- DNA for integration into the genome comprises 5’-ctttgcttatggtagaaactatcagtgtttga-3’ (SEQ ID NO:295) or a 3’ fragment thereof comprising 5’-gaaactatcagtgtttga-3’ (SEQ ID NO: 293) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the part of the right border of the PT- DNA for integration into the genome comprises 5’-gccgccatagttaccaactatcagtgtttga-3’ (SEQ ID NO:296) or a 3’ fragment thereof comprising 5’-ccaactatcagtgtttga-3’ (SEQ ID NO: 297) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the part of the right border of the PT- DNA for integration into the genome comprises 5’-gatccccctggcggagaactttcagtgtttga-3’ (SEQ ID NO:298) or a 3’ fragment thereof comprising 5’-agaactttcagtgtttga-3’ (SEQ ID NO: 299) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is barley and the right border of the PT-DNA comprises 5’-ctttgcttatcgcagaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:640) or a 3’ fragment thereof comprising 5’- gaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:641) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is barley and the right border of the PT-DNA in the barley genome comprises 5’-ctttgcttatcacagaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:642) or a 3’ fragment thereof comprising 5’- gaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:641) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is barley and the right border of the PT-DNA in the barley genome comprises 5’-ctttgcttatggtagaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:643) or a 3’ fragment thereof comprising 5’- gaaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:641) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is barley and the right border of the PT-DNA in the barley genome comprises 5’-gccgccatagttaccaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:644) or a 3’ fragment thereof comprising 5’- ccaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:645) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449). In an embodiment, the plant is barley and the right border of the PT-DNA in the barley genome comprises 5 ’ -gatccccctggcggagaactttcagtgtttgacaggatatattggcgggtaaac-3 ’

(SEQ ID NO:646) or a 3’ fragment thereof comprising 5’- agaactttcagtgtttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:647) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is barley, and the left border comprises 5’- ttggtggatatattgtgatgtaaac-3 ’ (SEQ ID NO: 1500).

In an embodiment, the plant is barley, and the left border comprises 5’- tgggtggatatattgtgatgtaaac-3 ’ (SEQ ID NO:607).

In an embodiment, the plant is barley, and the left border comprises 5’- cgggtggatatattgtgatgtaaac-3 ’ (SEQ ID NO: 1474).

In an embodiment, the plant is barley, and the left border comprises 5’- tggcatgatatattgtggtgcatgt-3 ’ (SEQ ID NO : 612) .

In an embodiment, the plant is barley, and the left border comprises 5’- cggcatgatatattgtggtgcatgt-3’ (SEQ ID NO: 1475).

In an embodiment, the plant is barley, and the left border comprises 5’- tgggtggatatattgtggcgtaaac-3 ’ (SEQ ID NO:615).

In an embodiment, the plant is barley, and the left border comprises 5’- cgggtggatatattgtggcgtaaac-3 ’ (SEQ ID NO: 1476).

In an embodiment, the plant is barley, and the left border comprises 5’- tggcaggatatattatggtgtagat-3 ’ (SEQ ID NO:618).

In an embodiment, the plant is barley, and the left border comprises 5’- cggcaggatatattatggtgtagat-3 ’ (SEQ ID NO: 1477).

In an embodiment, the plant is barley, and the left border comprises 5’- tggcatgatatattgtggtgaaaac-3 ’ (SEQ ID NO:621).

In an embodiment, the plant is barley, and the left border comprises 5’- cggcatgatatattgtggtgaaaac-3’ (SEQ ID NO: 1478).

In an embodiment, the plant is barley, and the left border comprises 5’- tgggtggatatattgtgatgtaatc-3 ’ (SEQ ID NO:624).

In an embodiment, the plant is barley, and the left border comprises 5’- cgggtggatatattgtgatgtaatc-3 ’ (SEQ ID NO: 1479).

In an embodiment, the plant is barley, and the left border comprises 5’- tggcatgatatattgtgttgtatgc-3’ (SEQ ID NO:627).

In an embodiment, the plant is barley, and the left border comprises 5’- cggcatgatatatgtgtgtatgc-3 ’ (SEQ ID NO: 1480). In an embodiment, the plant is barley, and the left border compnses 5’- tggcatgatatattgtgttgtatgt-3 ’ (SEQ ID NO:630).

In an embodiment, the plant is barley, and the left border compnses 5’- cggcatgatatattgtgttgtatgt-3 ’ (SEQ ID NO: 1481).

In an embodiment, the plant is barley, and the left border compnses 5’- tgggtggatatattgtgatgtaatc-3 ’ (SEQ ID NO:624).

In an embodiment, the plant is barley, and the left border compnses 5’- cgggtggatatattgtgatgtaatc-3 ’ (SEQ ID NO: 1482).

In an embodiment, the plant is barley, and the left border compnses 5’- tgggatcatatattgtggtctaaac-3 ’ (SEQ ID NO:635).

In an embodiment, the plant is barley, and the left border compnses 5’- cgggatcatatattgtggtctaaac-3’ (SEQ ID NO: 1483).

In an embodiment, the plant is barley, and the left border compnses 5’- tggcaagatatattgtgatgtaaat-3 ’ (SEQ ID NO:638).

In an embodiment, the plant is barley, and the left border compnses 5’- cggcaagatatattgtgatgtaaat-3 ’ (SEQ ID NO: 1484).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtgatgtaaac-3’ (SEQ ID NO:251).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-gtggatatattgtgatgtaaac-3’ (SEQ ID NO:249).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-catgatatattgtggtgcatgt-3’ (SEQ ID NO:255).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgcatgt-3’ (SEQ ID NO:257).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’ -gtggatatattgtggcgtaaac-3 ’ (SEQ ID NO:259).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtggcgtaaac-3’ (SEQ ID NO:261).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-caggatatattatggtgtagat-3’ (SEQ ID NO:263).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattatggtgtagat-3’ (SEQ ID NO:265).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-catgatatattgtggtgaaaac-3’ (SEQ ID NO:267).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgaaaac-3’ (SEQ ID NO269). In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-gtggatatattgtgatgtaatc-3’ (SEQ ID NO:271).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtgatgtaatc-3’ (SEQ ID NO:273).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-catgatatattgtgttgtatgc-3’ (SEQ ID NO:275).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtgttgtatgc-3’ (SEQ ID NO:277).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-catgatatattgtgttgtatgt-3’ (SEQ ID NO:279).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtgttgtatgt-3’ (SEQ ID NO:281).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-gatcatatattgtggtctaaac-3’ (SEQ ID NO:285).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtggtctaaac-3’ (SEQ ID NO:287).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-caagatatattgtgatgtaaat-3’ (SEQ ID NO:289).

In an embodiment, the plant is barley and PT-DNA has a processed the left border which comprises 5’-tatattgtgatgtaaat-3’ (SEQ ID NO:291).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’- cagcatatattgtggtgtaaaggatgtccacttccaa-3’ (SEQ ID NO:300) or a 5’ fragment thereof comprising 5’ -cagcatatattgtggtgtaaag-3 ’ (SEQ ID NO:301).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtgtaaaggatgtccacttccaa-3’ (SEQ ID NO:302) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaag-3’ (SEQ ID NO:303).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caccttatattgtggtgtaaatgagttgagtgtac- 3’ (SEQ ID NO:304) or a 5’ fragment thereof comprising 5’-caccttatattgtggtgtaaat-3’ (SEQ ID NO:305).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaaatgagttgagtgtac-3’ (SEQ ID NO:306) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152). In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -caggatatattgtggtttttgtaatcc-3 ’ (SEQ ID NO:307) or a 5’ fragment thereof comprising 5’-caggatatattgtggtttttgt-3’ (SEQ ID NO:308).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtttttgtaatcc-3’ (SEQ ID NO:309) or a 5’ fragment thereof comprising 5’-tatattgtggtttttgt-3’ (SEQ ID NO:310).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-ttgtatatattgtggtgtaacttcattatagaa-3’ (SEQ ID NO:311) or a 5’ fragment thereof comprising 5’-ttgtatatattgtggtgtaact-3’ (SEQ ID NO:312).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaacttcattatagaa-3’ (SEQ ID NO:313) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaact-3’ (SEQ ID NO:314).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’- caagatatattgtgatgtaaaatagactttgacttgccca-3’ (SEQ ID NO:315) or a 5’ fragment thereof comprising 5’ -caagatatattgtgatgtaaaa-3’ (SEQ ID NO:316).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtgatgtaaaatagactttgacttgccca-3’ (SEQ ID NO:317) or a 5’ fragment thereof comprising 5’-tatattgtgatgtaaaa-3’ (SEQ ID NO:318).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattctggtgatgctattatgtgctct-3’ (SEQ ID NO: 319) ora 5’ fragment thereof comprising 5’ -caggatatattctggtgatgct-3 ’ (SEQ ID NO:320).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattctggtgatgctattatgtgctct-3’ (SEQ ID NO:321) or a 5’ fragment thereof comprising 5’-tatattctggtgatgct-3’ (SEQ ID NO:322).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaatatatattgtggtataaaattggatattaa-3’ (SEQ ID NO: 323) or a 5’ fragment thereof comprising 5’ -gaatatatattgtggtataaaa-3 ’ (SEQ ID NO:324). In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtataaaattggatattaa-3’ (SEQ ID NO:325) or a 5’ fragment thereof comprising 5’-tatattgtggtataaaa-3’ (SEQ ID NO: 146).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatattttgtgttagcactgtgtata-3’ (SEQ ID NO:326) or a 5’ fragment thereof comprising 5’-caggatattttgtgttagcact-3’ (SEQ ID NO:327).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tattttgtgttagcactgtgtata-3’ (SEQ ID NO:328) or a 5’ fragment thereof comprising 5’-tattttgtgttagcact-3’ (SEQ ID NO:329).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattctggtgataacaccacaatggcat- 3’ (SEQ ID NO:330) or a 5’ fragment thereof comprising 5’-caggatatattctggtgataac-3’ (SEQ ID NO:331).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattctggtgataacaccacaatggcat-3’ (SEQ ID NO:332) or a 5’ fragment thereof comprising 5’-tatattctggtgataac-3’ (SEQ ID NO:333).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’- gaggacatattgtggtgttcatagctagattcttctaaacttattt-3’ (SEQ ID NO:334) or a 5’ fragment thereof comprising 5’ -gaggacatattgtggtgttcat-3 ’ (SEQ ID NO:335).

In an embodiment, the plant is banana and the part of the left border of the PT- DNA for integration into the genome comprises 5’- catattgtggtgttcatagctagattcttctaaacttattt-3’ (SEQ ID NO:336) or a 5’ fragment thereof comprising 5’-catattgtggtgttcat-3’ (SEQ ID NO:337).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcagcatatattgtggtgtaaaggatgtccacttccaa-3’ (SEQ ID NO: 648) or a 5’ fragment thereof comprising 5’-tggcagcatatattgtggtgtaaag-3’ (SEQ ID NO: 649), or wherein the 5’ t is replaced with a c (SEQ ID NO:650).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcaccttatattgtggtgtaaatgagttgagtgtac-3’ (SEQ ID NO:651) or a 5’ fragment thereof comprising 5’-tggcaccttatattgtggtgtaaat-3’ (SEQ ID NO:652), or wherein the 5’ t is replaced with a c (SEQ ID NO:653). In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’ -tggcaggatatattgtggtttttgtaatcc-3’ (SEQ ID NO:654) or a 5’ fragment thereof comprising 5’-tggcaggatatattgtggtttttgt-3’ (SEQ ID NO: 655), or wherein the 5’ t is replaced with a c (SEQ ID NO: 656).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggttgtatatattgtggtgtaacttcattatagaa-3’ (SEQ ID NO:657) or a 5’ fragment thereof comprising 5’-tggttgtatatattgtggtgtaact-3’ (SEQ ID NO: 658), or wherein the 5’ t is replaced with a c (SEQ ID NO:659).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcaagatatattgtgatgtaaaatagactttgacttgccca-3’ (SEQ ID NO:660) or a 5’ fragment thereof comprising 5’-tggcaagatatattgtgatgtaaaa-3’ (SEQ ID NO:661), or wherein the 5 ’ t is replaced with a c (SEQ ID NO:662).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcaggatatattctggtgatgctattatgtgctct-3’ (SEQ ID NO:663) or a 5’ fragment thereof comprising 5’ -tggcaggatatattctggtgatgct-3 ’ (SEQ ID NO:664), or wherein the 5’ t is replaced with a c (SEQ ID NO:665).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tgggaatatatattgtggtataaaattggatattaa-3’ (SEQ ID NO: 666) or a 5’ fragment thereof comprising 5’-tgggaatatatattgtggtataaaa-3’ (SEQ ID NO:667), or wherein the 5’ t is replaced with a c (SEQ ID NO:668).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcaggatattttgtgttagcactgtgtata-3’ (SEQ ID NO:669) or a 5’ fragment thereof comprising 5’ -tggcaggatattttgtgttagcact-3 ’ (SEQ ID NO:670), or wherein the 5’ t is replaced with a c (SEQ ID NO:671).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tggcaggatatattctggtgataacaccacaatggcat-3’ (SEQ ID NO:672) or a 5’ fragment thereof comprising 5’ -tggcaggatatattctggtgataac-3 ’ (SEQ ID NO:673), or wherein the 5’ t is replaced with a c (SEQ ID NO:674).

In an embodiment, the plant is banana and the left border of the PT-DNA comprises 5’-tgggaggacatattgtggtgttcatagctagattcttctaaacttattt-3’ (SEQ ID NO:675) or a 5’ fragment thereof comprising 5’ -tgggaggacatattgtggtgttcat-3 ’ (SEQ ID NO:676), or wherein the 5’ t is replaced with a c (SEQ ID NO:677).

In an embodiment, the plant is banana and the part of the right border of the PT- DNA for integration into the genome comprises 5’-aagatagatttattgataaattatcagtgtttga-3’ (SEQ ID NO:338) or a 3’ fragment thereof comprising 5’-taaattatcagtgtttga-3’ (SEQ ID NO:76) or 5’-ttga-3’ (SEQ ID NO:62). In an embodiment, the plant is banana and the part of the right border of the PT- DNA for integration into the genome comprises 5’ -tgatcctctcaagggtaaactgtcagtgtttga-3’ (SEQ ID NO:339) or a 3’ fragment thereof comprising 5’-taaactgtcagtgtttga-3’ (SEQ ID NO:340) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is banana and the part of the right border of the PT- DNA for integration into the genome comprises 5’-cttcttcagaacaaaaactttcagtgtttga-3’ (SEQ ID NO:341) or a 3’ fragment thereof comprising 5’-aaaactttcagtgtttga-3’ (SEQ ID NO: 193) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is banana and the right border of the PT-DNA comprises 5’-aagatagatttattgataaattatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:678) or a 3’ fragment thereof comprising 5’- taaattatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:463) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is banana and the right border of the PT-DNA comprises 5’-tgatcctctcaagggtaaactgtcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:679) or a 3’ fragment thereof comprising 5’- taaactgtcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:680) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is banana and the right border of the PT-DNA comprises 5’-cttcttcagaacaaaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:681) or a 3’ fragment thereof comprising 5’- aaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:559) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tctgatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:342) or a 5’ fragment thereof comprising 5’-tctgatatattgtggtgtaaaa-3’ (SEQ ID NO:343).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:344) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tctaatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:346) or a 5’ fragment thereof comprising 5’-tctaatatattgtggtgtaaaa-3’ (SEQ ID NO:347). In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-attgatatattgtggtgtaccacgcggcatgcat- 3’ (SEQ ID NO:348) or a 5’ fragment thereof comprising 5’-attgatatattgtggtgtacca-3’ (SEQ ID NO:349).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtgtaccacgcggcatgcat-3’ (SEQ ID NO:350) or a 5’ fragment thereof comprising 5’-tatattgtggtgtacca-3’ (SEQ ID NO:230).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-atggatatattgtggtataaatttcttggtca-3’ (SEQ ID NO:351) or a 5’ fragment thereof comprising 5’-atggatatattgtggtataaat-3’ (SEQ ID NO:352).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtataaatttcttggtca-3 ’ (SEQ ID NO:353) or a 5’ fragment thereof comprising 5’-tatattgtggtataaat-3’ (SEQ ID NO:354).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-ttggatttattgtggtgtaacctgtcagttcggatttt- 3’ (SEQ ID NO:355) or a 5’ fragment thereof comprising 5’-ttggatttattgtggtgtaacc-3’ (SEQ ID NO:356).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tttattgtggtgtaacctgtcagttcggatttt-3’ (SEQ ID NO:357) or a 5’ fragment thereof comprising 5’-tttattgtggtgtaacc-3’ (SEQ ID NO:358).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-agagatatattgtggtttaaaagatcagcata-3’ (SEQ ID NO:359) or a 5’ fragment thereof comprising 5’-agagatatattgtggtttaaaa-3’ (SEQ ID NO:360).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtttaaaagatcagcata-3’ (SEQ ID NO:361) or a 5’ fragment thereof comprising 5’-tatattgtggtttaaaa-3’ (SEQ ID NO:211).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgtcgtgtcttcacttcattgat-3’ (SEQ ID NO:362) or a 5’ fragment thereof comprising 5’ -caggatatattgtcgtgtcttc-3 ’ (SEQ ID NO: 1007). In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtcgtgtcttcacttcattgat-3’ (SEQ ID NO:363) or a 5’ fragment thereof comprising 5’-tatattgtcgtgtcttc-3’ (SEQ ID NO: 1008).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’- accaatatattgtggtataaactactgcaaatcttgg-3’ (SEQ ID NO:364) or a 5’ fragment thereof comprising 5’ -accaatatattgtggtataaac-3’ (SEQ ID NO:365).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtataaactactgcaaatcttgg-3’ (SEQ ID NO:366) or a 5’ fragment thereof comprising 5’-tatattgtggtataaac-3’ (SEQ ID NO:367).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-ttctatatattgtggtctaaaatatgatggaacatg- 3’ (SEQ ID NO:368) or a 5’ fragment thereof comprising 5’-ttctatatattgtggtctaaaa-3’ (SEQ ID NO:369).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtctaaaatatgatggaacatg-3’ (SEQ ID NO:370) or a 5’ fragment thereof comprising 5’-tatattgtggtctaaaa-3’ (SEQ ID NO:371).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-atgaatatattgtggtataaattacatggtcattgct- 3’ (SEQ ID NO:372) or a 5’ fragment thereof comprising 5’-atgaatatattgtggtataaat-3’ (SEQ ID NO:373).

In an embodiment, the plant is sugarcane and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtataaattacatggtcattgct-3’ (SEQ ID NO:374) or a 5’ fragment thereof comprising 5’-tatattgtggtataaat-3’ (SEQ ID NO:354).

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggtctgatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ ID NO:682) or a 5’ fragment thereof comprising 5’-tggtctgatatattgtggtgtaaaa-3’ (SEQ ID NO:683), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:684 and 1485.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5 ’-tggtctaatatattgtggtgtaaaaaacatttcatcat-3’ (SEQ IDNO:685) ora 5’ fragment thereof comprising 5’-tggtctaatatattgtggtgtaaaa-3’ (SEQ ID NO:686), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:687 and 1486.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggattgatatattgtggtgtaccacgcggcatgcat-3’ (SEQ ID NO:688) or a 5’ fragment thereof comprising 5’-tggattgatatattgtggtgtacca-3’ (SEQ ID NO:689), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:690 and 1487.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggatggatatattgtggtataaatttcttggtca-3’ (SEQ ID NO:691) or a 5’ fragment thereof comprising 5’ -tggatggatatattgtggtataaat-3 ’ (SEQ ID NO: 692), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:693 and 1488.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggttggatttattgtggtgtaacctgtcagttcggatttt-3’ (SEQ ID NO:694) or a 5’ fragment thereof comprising 5’-tggttggatttattgtggtgtaacc-3’ (SEQ ID NO:695), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:696 and 1489.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggagagatatattgtggtttaaaagatcagcata-3’ (SEQ ID NO:697) or a 5’ fragment thereof comprising 5’-tggagagatatattgtggtttaaaa-3’ (SEQ ID NO:698), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:699 and 1490.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggcaggatatattgtcgtgtcttcacttcattgat-3’ (SEQ ID NO:700) or a 5’ fragment thereof comprising 5 ’-tggcaggatatattgtcgtgtcttc-3 ’ (SEQ ID NO: 1011), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:701 and 1491.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggaccaatatattgtggtataaactactgcaaatcttgg-3’ (SEQ ID NO:702) or a 5’ fragment thereof comprising 5’-tggaccaatatattgtggtataaac-3’ (SEQ ID NO:703), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:704 and 1492.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggttctatatattgtggtctaaaatatgatggaacatg-3’ (SEQ ID NO:705) or a 5’ fragment thereof comprising 5’-tggttctatatattgtggtctaaaa-3’ (SEQ ID NO: 706), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:707 and 1493.

In an embodiment, the plant is sugarcane and the left border of the PT-DNA comprises 5’-tggatgaatatattgtggtataaattacatggtcattgct-3’ (SEQ ID NO:708) or a 5’ fragment thereof comprising 5’-tggatgaatatattgtggtataaat-3’ (SEQ ID NO:709), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs: 710 and 1493.

In an embodiment, the plant is sugarcane and the part of the right border of the PT-DNA for integration into the genome comprises 5’-ttgtgaattttaagtttaaaatatcagtgtttga- 3’ (SEQ ID NO:375) or a 3’ fragment thereof comprising 5’-taaaatatcagtgtttga-3’ (SEQ ID NO: 376) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is sugarcane and the part of the right border of the PT-DNA for integration into the genome comprises 5’-ctggtggtccaatgcaactatcagtgtttga- 3’ (SEQ ID NO:377) or a 3’ fragment thereof comprising 5’-gcaactatcagtgtttga-3’ (SEQ ID NO:378) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is sugarcane and the part of the right border of the PT-DNA for integration into the genome comprises 5’-gttcaggtgcatcaactatcagtgtttga-3’ (SEQ ID NO:379) or a 3’ fragment thereof comprising 5’-tcaactatcagtgtttga-3’ (SEQ ID NO:380) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is sugarcane and the part of the right border of the PT-DNA for integration into the genome comprises 5’-cctacctggcgcgccaactatcagtgtttga- 3’ (SEQ ID NO:381) or a 3’ fragment thereof comprising 5’-ccaactatcagtgtttga-3’ (SEQ ID NO:297) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is sugarcane and the right border of the PT-DNA comprises 5’-ttgtgaattttaagtttaaaatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:711) or a 3’ fragment thereof comprising 5’- taaaatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:712) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is sugarcane and the right border of the PT-DNA comprises 5’-ctggtggtccaatgcaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:713) or a 3’ fragment thereof comprising 5’- gcaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:714) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is sugarcane and the right border of the PT-DNA comprises 5’-gttcaggtgcatcaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:715) or a 3’ fragment thereof comprising 5’-tcaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:716) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is sugarcane and the right border of the PT-DNA comprises 5’-cctacctggcgcgccaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:717) or a 3’ fragment thereof comprising 5’- ccaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:645) or 5’- ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggtctgatatattgtggtgtaaaa-3’ (SEQ ID NO:683).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggtctgatatattgtggtgtaaaa-3’ (SEQ ID NO: 1485).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggtctaatatattgtggtgtaaaa-3’ (SEQ ID NO:686).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggtctaatatattgtggtgtaaaa-3’ (SEQ ID NO: 1486).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggattgatatattgtggtgtacca-3’ (SEQ ID NO:689).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggattgatatattgtggtgtacca-3’ (SEQ ID NO: 1487).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggatggatatattgtggtataaat-3’ (SEQ ID NO:692).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggatggatatattgtggtataaat-3’ (SEQ ID NO.1488).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggttggatttattgtggtgtaacc-3’ (SEQ ID NO:695).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggttggatttattgtggtgtaacc-3’ (SEQ ID NO: 1489),

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggagagatatattgtggtttaaaa-3’ (SEQ ID NO:698).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggagagatatattgtggtttaaaa-3 ’ (SEQ ID NO: 1490).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggcaggatatattgtcgtgtcttc-3’ (SEQ ID NO: 1011).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggcaggatatattgtcgtgtcttc-3’ (SEQ ID NO: 1491).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggaccaatatattgtggtataaac-3’ (SEQ ID NO:703).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggaccaatatattgtggtataaac-3’ (SEQ ID NO: 1492).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggttctatatattgtggtctaaaa-3’ (SEQ ID NO:706). In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggttctatatattgtggtctaaaa-3 ’ (SEQ ID NO: 1493).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- tggatgaatatattgtggtataaat-3’ (SEQ ID NO: 709).

In an embodiment, the plant is sugarcane, and the left border comprises 5’- cggatgaatatattgtggtataaat-3 ’ (SEQ ID NO: 1494).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tctgatatattgtggtgtaaaa-3’ (SEQ ID NO:343).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tctaatatattgtggtgtaaaa-3’ (SEQ ID NO:347).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-attgatatattgtggtgtacca-3’ (SEQ ID NO:349).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtacca-3’ (SEQ ID NO:230).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-atggatatattgtggtataaat-3’ (SEQ ID NO:352).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtataaat-3’ (SEQ ID NO:354).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-ttggatttattgtggtgtaacc-3’ (SEQ ID NO:356).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tttattgtggtgtaacc-3’ (SEQ ID NO:358).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-agagatatattgtggtttaaaa-3’ (SEQ ID NO:360).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtttaaaa-3’ (SEQ ID NO:211).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’ -caggatatattgtcgtgtcttc-3 ’ (SEQ ID NO: 1007).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtcgtgtcttc-3’ (SEQ ID NO: 1008).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-accaatatattgtggtataaac-3’ (SEQ ID NO: 365).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtataaac-3’ (SEQ ID NO:367). In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-ttctatatattgtggtctaaaa-3’ (SEQ ID NO:369).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-tatattgtggtctaaaa-3’ (SEQ ID NO:371).

In an embodiment, the plant is sugarcane and PT-DNA has a processed the left border which comprises 5’-atgaatatattgtggtataaat-3’ (SEQ ID NO:373).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- ttttgaatattgtggtgtaaacctaaactccttgtgtgagtgttgaaagcttg-3’ (SEQ ID NO:382) or a 5’ fragment thereof comprising 5’-ttttgaatattgtggtgtaaac-3’ (SEQ ID NO:383).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- aatattgtggtgtaaacctaaactccttgtgtgagtgttgaaagcttg-3’ (SEQ ID NO:384) or a 5’ fragment thereof comprising 5’-aatattgtggtgtaaac-3’ (SEQ ID NO:385).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgttataacctatttgcataa-3’ (SEQ ID NO:386) or a 5’ fragment thereof comprising 5’ -caggatatattgttataacctattt-3 ’ (SEQ ID NO: 1112).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgttataacctatttgcataa-3 ’ (SEQ ID NO:387) or a 5’ fragment thereof comprising 5’-tatattgttataacctatttgcat-3’ (SEQ ID NO: 1113).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- caggatatattttggggtaggtaaagtctgcttgctg-3’ (SEQ ID NO:388) or a 5’ fragment thereof comprising 5’-caggatatattttggggtaggt-3’ (SEQ ID NO:389).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattttggggtaggtaaagtctgcttgctg-3’ (SEQ ID NO:390) or a 5’ fragment thereof comprising 5’-tatattttggggtaggt-3’ (SEQ ID NO:391).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- caggatatatttttgtgtaggggccaataagctacttttctaa-3’ (SEQ ID NO:392) or a 5’ fragment thereof comprising 5’-caggatatatttttgtgtaggg-3’ (SEQ ID NO:393).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatatttttgtgtaggggccaataagctacttttctaa- 3’ (SEQ ID NO:394) ora 5’ fragment thereof comprising 5’-tatatttttgtgtaggg-3’ (SEQ ID NO:395).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaggatatattgtggtggacaggagatg-3’ (SEQ ID NO:396) or a 5’ fragment thereof comprising 5’ -gaggatatattgtggtggacag-3 ’ (SEQ ID NO:397).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgtggtggacaggagatg-3’ (SEQ ID NO:398) or a 5’ fragment thereof comprising 5’-tatattgtggtggacag-3’ (SEQ ID NO:399).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-caggatatattgcagtttaggaactctg-3’ (SEQ ID NO:400) or a 5’ fragment thereof comprising 5’-caggatatattgcagtttagga-3’ (SEQ ID NO:401).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgcagtttaggaactctg-3’ (SEQ ID NO:402) or a 5’ fragment thereof comprising 5’-tatattgcagtttagga-3’ (SEQ ID NO:403).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaggatatattgttgtgttaggatgatatttggatc- 3’ (SEQ ID NO:404) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgttgtgttaggatgatatttggatc-3’ (SEQ ID NO:406) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5 ’-atatctatattgtggtggaaactcttttttgaaactgg- 3’ (SEQ ID NO:408) or a 5’ fragment thereof comprising 5’-atatctatattgtggtggaaac-3’ (SEQ ID NO:409).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgtggtggaaactcttttttgaaactgg-3’ (SEQ ID NO:410) or a 5’ fragment thereof comprising 5’-tatattgtggtggaaac-3’ (SEQ ID NO: 179).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’- gaggatatattgttgtgttaggctgatagttggatcatttt-3’ (SEQ ID NO:411) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405). In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgttgtgttaggctgatagttggatcatttt-3’ (SEQ ID NO:412) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaggatatattgttgtgttaggctgatatttgga-3’ (SEQ ID NO:413) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagg-3’ (SEQ ID NO:405).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’ -tatattgttgtgttaggctgatatttgga-3’ (SEQ ID NO:414) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagg-3’ (SEQ ID NO:407).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-gaggatatattgttgtgttagtctgattgtgg-3’ (SEQ ID NO:415) or a 5’ fragment thereof comprising 5’-gaggatatattgttgtgttagt-3’ (SEQ ID NO:416).

In an embodiment, the plant is potato and the part of the left border of the PT- DNA for integration into the genome comprises 5’-tatattgttgtgttagtctgattgtgg-3’ (SEQ ID NO:417) or a 5’ fragment thereof comprising 5’-tatattgttgtgttagt-3’ (SEQ ID NO:418).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggttttgaatattgtggtgtaaacctaaactccttgtgtgagtgttgaaagcttg-3’ (SEQ ID NO:718) or a 5’ fragment thereof comprising 5’ -tggttttgaatattgtggtgtaaac-3 ’ (SEQ ID NO:719), or wherein the 5’ t is replaced with a c (SEQ ID NO:720).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggcaggatatattgttataacctatttgcataa-3’ (SEQ ID NO:721) or a 5’ fragment thereof comprising 5’-tggcaggatatattgttataaccta-3’ (SEQ ID NO: 1014), or wherein the 5’ t is replaced with a c (SEQ ID NO:722).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggcaggatatattttggggtaggtaaagtctgcttgctg-3’ (SEQ ID NO:723) or a 5’ fragment thereof comprising 5’ -tggcaggatatattttggggtaggt-3’ (SEQ ID NO: 724), or wherein the 5’ t is replaced with a c (SEQ ID NO:725).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggcaggatatatttttgtgtaggggccaataagctacttttctaa-3’ (SEQ ID NO:726) or a 5’ fragment thereof comprising 5’-tggcaggatatatttttgtgtaggg-3’ (SEQ ID NO:727), or wherein the 5’ t is replaced with a c (SEQ ID NO:728). In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tgggaggatatattgtggtggacaggagatg-3’ (SEQ ID NO:729) or a 5’ fragment thereof comprising 5’ -tgggaggatatattgtggtggacag-3 ’ (SEQ ID NO:730), or wherein the 5’ t is replaced with a c (SEQ ID NO:731).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggcaggatatattgcagtttaggaactctg-3’ (SEQ ID NO:732) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgcagtttagga-3 ’ (SEQ ID NO:733), or wherein the 5’ t is replaced with a c (SEQ ID NO:734).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tgggaggatatattgttgtgttaggatgatatttggatc-3’ (SEQ ID NO:735) or a 5’ fragment thereof comprising 5’-tgggaggatatattgttgtgttagg-3’ (SEQ ID NO:736), or wherein the 5’ t is replaced with a c (SEQ ID NO:737).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tggatatctatattgtggtggaaactcttttttgaaactgg-3’ (SEQ ID NO:738) or a 5’ fragment thereof comprising 5’-tggatatctatattgtggtggaaac-3’ (SEQ ID NO:739), or wherein the 5’ t is replaced with a c (SEQ ID NO:740).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tgggaggatatattgttgtgttaggctgatagttggatcatttt-3’ (SEQ ID NO:741) or a 5’ fragment thereof comprising 5’-tgggaggatatattgttgtgttagg-3’ (SEQ ID NO:736), or wherein the 5’ t is replaced with a c (SEQ ID NO:742).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tgggaggatatattgttgtgttaggctgatatttgga-3’ (SEQ ID NO:743) ora 5’ fragment thereof comprising 5’-tgggaggatatattgttgtgttagg-3’ (SEQ ID NO:736), or wherein the 5’ t is replaced with a c (SEQ ID NO:744).

In an embodiment, the plant is potato and the left border of the PT-DNA comprises 5’-tgggaggatatattgttgtgttagtctgattgtgg-3’ (SEQ ID NO: 745) or a 5’ fragment thereof comprising 5’-tgggaggatatattgttgtgttagt-3’ (SEQ ID NO:746), or wherein the 5’ t is replaced with a c (SEQ ID NO:747).

In an embodiment, the plant is potato and the part of the right border of the PT- DNA for integration into the genome comprises 5’-agtatgatgctgtgcataaactatcagtatttga-3’ (SEQ ID NO:419) or a 3’ fragment thereof comprising 5’-taaactatcagtatttga-3’ (SEQ ID NO: 420) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the plant is potato and the part of the right border of the PT- DNA for integration into the genome comprises 5’ -gtatgtaatttcttatttgattcaaactatcagtttttga- 3’ (SEQ ID NO:748) or a 3’ fragment thereof comprising 5’-caaactatcagtttttga-3’ (SEQ ID NO: 195) or 5’-ttga-3’ (SEQ ID NO: 62). In an embodiment, the plant is potato and the part of the right border of the PT- DNA for integration into the genome comprises 5’- gaaaatgtatgaagagaggaaaaacttccagtgtttga-3’ (SEQ ID NO:421) or a 3’ fragment thereof comprising 5’-aaaacttccagtgtttga-3’ (SEQ ID NO:422) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the plant is potato and the right border of the PT-DNA comprises 5’-agtatgatgctgtgcataaactatcagtatttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:749) or a 3’ fragment thereof comprising 5’- taaactatcagtatttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:750) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is potato and the right border of the PT-DNA comprises 5’-gtatgtaatttcttatttgattcaaactatcagtttttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:751) or a 3’ fragment thereof comprising 5’- caaactatcagtttttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 561) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the plant is potato and the right border of the PT-DNA comprises 5 ’ -gaaaatgtatgaagagaggaaaaacttccagtgtttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:752) or a 3’ fragment thereof comprising 5’- aaaacttccagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:753) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

As a further example, the present invention provides a plant transfer DNA (PT- DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT- DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the PT-DNA comprises part of a right border for integration into the genome.

In an embodiment, the PT-DNA has one or more of the features defined above in relation to the Ti plasmid.

In an embodiment, the 3 ’ end of any left border provided herein is replaced with one of the following sequences, or any naturally occurring Agrobacterium left border, 5’-cggcaggatatattgaattgtaaat-3’ (SEQ ID NO: 754), 5’-cggcaggatatattcaattgtaaac-3’ (SEQ ID NO:755), 5’-cggcaggatatatttagttgtaaaa-3’ (SEQ ID NO:756), 5’- tggcaggatatattgtgatgtaaac-3 ’ (SEQ ID NO:767), 5’-tggcaggatatattgtggtgtaaac-3’ (SEQ ID NO:758), 5’-tggcaggatatatcaaagtgtaagt-3’ (SEQ ID NO:759), 5’- tggcaggatatatcgaggtgtaaaa-3 ’ (SEQ ID NO:760), 5’-tggcaggatgtattgtcatgtaaaa-3’ (SEQ ID NO:761), 5’-tggcaggatatatcgttatgtaatc-3’ (SEQ ID NO:762), 5’- tggcaggatatattggtgtgtaaac-3 ’ (SEQ ID NO:763), 5’-yggcaggatrtatyndndtgtaadh-3’ (SEQ ID NO:764), where y is t or c, r is g or a, y is t or c, n is a, c, g or t, d is a, g or t, and h is a, c or t.

In an embodiment, the 3 ’ end of any processed left border, or part of left border, herein is replaced with one of the following sequences, or any naturally occurring Agrobacterium left border, 5’-caggatatattgaattgtaaat-3’ (SEQ ID NO:765), 5’- caggatatattcaattgtaaac-3 ’ (SEQ ID NO: 766), 5’-caggatatatttagttgtaaaa-3’ (SEQ ID NO:767), 5’-caggatatattgtgatgtaaac-3’ (SEQ ID NO:768), 5’-caggatatattgtggtgtaaac-3’ (SEQ ID NO:769), 5’-caggatatatcaaagtgtaagt-3’ (SEQ ID NO:770), 5’- caggatatatcgaggtgtaaaa-3 ’ (SEQ ID NO:771), 5’-caggatgtattgtcatgtaaaa-3’ (SEQ ID NO:772), 5’-caggatatatcgttatgtaatc-3’ (SEQ ID NO:773), 5’ -caggatatattggtgtgtaaac-3 ’ (SEQ ID NO:774), 5’-caggatrtatyndndtgtaadh-3’ (SEQ ID NO:775), where y is t or c, r is g or a, y is t or c, n is a, c, g or t, d is a, g or t, and h is a, c or t.

In an embodiment, the 3 ’ end of any processed left border, or part of left border, herein is replaced with one of the following sequences, or any naturally occurring Agrobacterium left border, 5’-tatattgaattgtaaat-3’ (SEQ ID NO:776), 5’- tatattcaattgtaaac-3 ’ (SEQ ID NO:777), 5’-tatatttagttgtaaaa-3’ (SEQ ID NO:778 5’- tatattgtgatgtaaac-3’ (SEQ ID NO:251), 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779), 5’- tatatcaaagtgtaagt-3’ (SEQ ID NO:780), 5’-tatatcgaggtgtaaaa-3’ (SEQ ID NO:781), 5’- tgtattgtcatgtaaaa-3’ (SEQ ID NO:782), 5’-tatatcgttatgtaatc-3’ (SEQ ID NO:783), 5’- tatattggtgtgtaaac-3 ’ (SEQ ID NO:784), 5’-trtatyndndtgtaadh-3’ (SEQ ID NO:785), where y is t or c, r is g or a, y is t or c, n is a, c, g or t, d is a, g or t, and h is a, c or t.

In an embodiment, the sequence 5’ -tgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 786) in any right border provided herein is replaced with one of the following sequences, or any naturally occurring Agrobacterium right border, 5’-tgacaggatatataccgttgtaatt-3’ (SEQ ID NO: 787), 5’-tggcaggatatataccgttgtaatt-3’ (SEQ ID NO: 788), 5’- tggcaggatatatgccgttgtaatt-3 ’ (SEQ ID NO: 789), 5’-tgacaggatatatggtgatgtcacg-3’ (SEQ ID NO: 790), 5’ -tgacaggatatatgttcctgtcatg-3 ’ (SEQ ID NO: 791), 5’- tgacaggatatatcttgtggtcagg-3 ’ (SEQ ID NO: 792), 5’-tgacaggatttatcgttatgtcatg-3’ (SEQ ID NO: 793), 5’ -tggcaggatttattgctaagtcatc-3 ’ (SEQ ID NO: 794), 5’- tggcaggatgtatcagattgtagtg-3 ’ (SEQ ID NO: 795), or 5'-tgrcaggatdtatnnbnndgtmmb-3' (SEQ ID NO: 796), where r is g or a, d is a, g or t, n is a, c, g or t, b is c, g or t, d is a, g or t, and m is a or c.

In an embodiment, a PT-DNA of the invention, and/or or a left border thereof, does not have any additional 5’ nucleotides beyond those disclosed herein. In an embodiment, a 5 ’ fragment of a left border, or part or processed version thereof, provided herein has one, two, three, four, five or more nucleotides at the 3 ’ end corresponding to the nucleotides of a longer version of the fragment provided herein.

In an embodiment, the 5’ tgg or egg of a PT-DNA of the invention can be ttg.

In an embodiment, a T-DNA of the invention for use with Piggybac has a left border comprising TTAA.

WT-DNA

In an embodiment, the PT-DNA is wheat transfer DNA (WT-DNA). The WT- DNA may be part of a Ti plasmid, or have been processed and be in the genome of a wheat plant.

For example, the present invention provides a wheat plant having a wheat transfer DNA (WT-DNA) integrated into the genome of the plant, wherein the integrated WT- DNA only has wheat DNA, and wherein the WT-DNA comprises a polynucleotide of interest.

In an embodiment, the integrated WT-DNA has a processed WT-DNA right border, or lacks any portion of a WT-DNA right border.

In an embodiment, the integrated WT-DNA has a processed WT-DNA left border, or lacks any portion of a WT-DNA left border.

In an embodiment, the integrated WT-DNA comprises a processed WT-DNA right border, a processed WT-DNA left border and a polynucleotide of interest.

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO:797).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtggtgttggg-3’ (SEQ ID NO:798).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gtagttatattgtggtgtaaac-3’ (SEQ ID NO:799).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtagacaactc-3’ (SEQ ID NO:800).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gaggatatattgtggtgataaa-3’ (SEQ ID NO: 801).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gatcctatattgtggtgtaaaa-3’ (SEQ ID NO:802). In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -caggatatattgtgccggccta-3 ’ (SEQ ID NO:803).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-aatgatatattgtggtgtatga-3’ (SEQ ID NO:804).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -gcagatatattgtggtgtatgc-3 ’ (SEQ ID NO: 805).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -caggatatattgttggcctact-3’ (SEQ ID NO:806).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-cgggatatattgtggtgtgtgc-3’ (SEQ ID NO: 807).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-atctctatattgtggtgtaaaa-3’ (SEQ ID NO: 808).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtggttctgta-3’ (SEQ ID NO:809).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-atccagatattgtggtgtaaac-3’ (SEQ ID NO:810).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-attcagatattgtggtgtaaac-3’ (SEQ ID NO: 811).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-taggatatattgtgctgtaatt-3’ (SEQ ID NO:812).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtggtc tagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaagagaa gagtcgcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgagaattcctcatttggtttcacattcttaagcat gattactccattcttaattgggcctataactcatagcagagcatacacatttgaatttctttattatcttcttgaacaattactttttgtca attcatattccatag-3 ’ (SEQ ID NO: 1281), caggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtggtc tagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggcatttggtttcacat tcttaagcatgattactccattc-3 ’ (SEQ ID NO: 1282), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatccacc-3’ (SEQ ID NO: 1283), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatccac-3’ (SEQ ID NO: 1284), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatcca-3’ (SEQ ID NO: 1285), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatcc-3’ (SEQ ID NO: 1286), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattatc-3’ (SEQ ID NO: 1287), 5’- caggatatattgtggtgtaaactctatcaactggtactgtattat-3’ (SEQ ID NO: 1288), 5’- caggatatattgtggtgtaaactctatcaactggtactgtatta-3’ (SEQ ID NO: 1289), 5’- caggatatattgtggtgtaaactctatcaactggtactgtatt-3’ (SEQ ID NO: 1290), 5’- caggatatattgtggtgtaaactctatcaactggtactgtat-3’ (SEQ ID NO: 1291), 5’- caggatatattgtggtgtaaactctatcaactggtactgta-3’ (SEQ ID NO: 1292), 5’- caggatatattgtggtgtaaactctatcaactggtactgt-3’ (SEQ ID NO: 1293), 5’- caggatatattgtggtgtaaactctatcaactggtactg-3’ (SEQ ID NO: 1294), 5’- caggatatattgtggtgtaaactctatcaactggtact-3’ (SEQ ID NO: 1295), 5’- caggatatattgtggtgtaaactctatcaactggtac-3’ (SEQ ID NO: 1296), 5’- caggatatattgtggtgtaaactctatcaactggta-3’ (SEQ ID NO: 1297), 5’- caggatatattgtggtgtaaactctatcaactggt-3’ (SEQ ID NO: 1298), 5’- caggatatattgtggtgtaaactctatcaactgg-3’ (SEQ ID NO: 1299), 5’- caggatatattgtggtgtaaactctatcaactg-3’ (SEQ ID NO: 1300), 5’- caggatatattgtggtgtaaactctatcaact-3 ’ (SEQ ID NO: 1301), 5’- caggatatattgtggtgtaaactctatcaac-3 ’ (SEQ ID NO: 1302), 5’- caggatatattgtggtgtaaactctatcaa-3’ (SEQ ID NO: 1303), 5’-caggatatattgtggtgtaaactctatca- 3’ (SEQ ID NO: 1304), 5’-caggatatattgtggtgtaaactctatc-3’ (SEQ ID NO: 1305), 5’- tggcaggatatattgtggtgtaaactctat-3’ (SEQ ID NO: 1306), 5’-caggatatattgtggtgtaaactcta-3’ (SEQ ID NO: 1307), 5’-caggatatattgtggtgtaaactct-3’ (SEQ ID NO: 1308), 5’- caggatatattgtggtgtaaactc-3 ’ (SEQ ID NO: 1309), 5’-caggatatattgtggtgtaaact-3’ (SEQ ID NO: 1310). In an embodiment, a sequence provided above is lacking the 5’ nucleotides cagga, examples of which include, but are not limited to, 5’- tatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtggtctagggt ataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaagagaagagtc gcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgag-3’ (SEQ ID NO:813) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779) or 5’- tatattgtggtgtaaact-3’(SEQ ID NO: 1495). In an embodiment, a sequence provided above is lacking the 5’ nucleotides cagg. In an embodiment, a sequence provided above is lacking the 5’ nucleotides cag. In an embodiment, a sequence provided above is lacking the 5’ nucleotides ca. In an embodiment, a sequence provided above is lacking the 5’ nucleotide c.

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’- taggatatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacagaaa agaaaagaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgcaca aaggcccagcccacgtcggccttccact-3’ (SEQ ID NO:814) or a 5’ fragment thereof comprising 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO:797). In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’- tatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacagaaaagaaa agaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgcacaaaggc ccagcccacgtcggccttccact-3 ’ (SEQ ID NO:815) or a 5’ fragment thereof comprising 5’- tatattgtggtgtgatt-3 ’ (SEQ ID NO: 816).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtggtgttgggattctctggttct-3’ (SEQ ID NO:817) or a 5’ fragment thereof comprising 5’-caggatatattgtggtgttggg-3’ (SEQ ID NO:798).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgttgggattctctggttct-3’ (SEQ ID NO:818) or a 5’ fragment thereof comprising 5’-tatattgtggtgttggg-3’ (SEQ ID NO: 819).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gtagttatattgtggtgtaaactctatc-3’ (SEQ ID NO: 820) or a 5’ fragment thereof comprising 5’-gtagttatattgtggtgtaaac-3’ (SEQ ID NO: 799).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtaaactctatc-3’ (SEQ ID NO:821) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtagacaactc-3’ (SEQ ID NO: 822).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtagacaactct-3’ (SEQ ID NO:823).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtagacaactct-3’ (SEQ ID NO:824).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -gaggatatattgtggtgataaaagctatctc -3’ (SEQ ID NO:825) or a 5’ fragment thereof comprising 5’-gaggatatattgtggtgataaa-3’ (SEQ ID NO:801).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgataaaagctatctc -3’ (SEQ ID NO:826) or a 5’ fragment thereof comprising 5’-tatattgtggtgataaa-3’ (SEQ ID NO: 827).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gatcctatattgtggtgtaaaatatatcgagctagtagtg-3’ (SEQ ID NO: 828) or a 5’ fragment thereof comprising 5’-gatcctatattgtggtgtaaaa-3’ (SEQ ID NO: 802). In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtaaaatatatcgagctagtagtg-3’ (SEQ ID NO:829) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtgccggcctaaggaccaagagggtctac-3’ (SEQ ID NO: 830) or a 5’ fragment thereof comprising 5’ -caggatatattgtgccggccta-3 ’ (SEQ ID NO: 803).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtgccggcctaaggaccaagagggtctac-3’ (SEQ ID NO:831) or a 5’ fragment thereof comprising 5’-tatattgtgccggccta-3’ (SEQ ID NO: 832).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-aatgatatattgtggtgtatgagatcatgttcttgt-3’ (SEQ ID NO:833) or a 5’ fragment thereof comprising 5’-aatgatatattgtggtgtatga -3’ (SEQ ID NO:804).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5 ’-tatattgtggtgtatgagatcatgttcttgt-3’ (SEQ ID NO:835) ora5’ fragment thereof comprising 5’-tatattgtggtgtatga -3’ (SEQ ID NO:836).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gcagatatattgtggtgtatgcacacacccttc-3’ (SEQ ID NO:837) or a 5’ fragment thereof comprising 5’ -gcagatatattgtggtgtatgc-3 ’ (SEQ ID NO: 805).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -tatattgtggtgtatgcacacacccttc-3’ (SEQ ID NO:838) or a 5’ fragment thereof comprising 5’-tatattgtggtgtatgc-3’ (SEQ ID NO:839).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgttggcctactccagtcat-3’ (SEQ ID NO: 840) or a 5’ fragment thereof comprising 5’ -caggatatattgttggcctact-3 ’ (SEQ ID NO:806).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -tatattgttggcctactccagtcat-3 ’ (SEQ ID NO:841) or a 5’ fragment thereof comprising 5’-tatattgttggcctact-3’ (SEQ ID NO: 842).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-cgggatatattgtggtgtgtgcgctgtgtctctttc-3’ (SEQ ID NO: 843) or a 5’ fragment thereof comprising 5’-cgggatatattgtggtgtgtgc-3’ (SEQ ID NO:807).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtgtgcgctgtgtctctttc-3’ (SEQ ID NO: 844) or a 5’ fragment thereof comprising 5’-tatattgtggtgtgtgc-3’ (SEQ ID NO: 845).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’ -atctctatattgtggtgtaaaagcaag-3’ (SEQ ID NO:846) or a 5’ fragment thereof comprising 5’-atctctatattgtggtgtaaaa-3’ (SEQ ID NO:808). In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtaaaagcaag-3’ (SEQ ID NO:847) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-caggatatattgtggttctgtaatctct-3’ (SEQ ID NO:848) or a 5’ fragment thereof comprising 5’-caggatatattgtggttctgta-3’ (SEQ ID NO:809).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggttctgtaatctct-3’ (SEQ ID NO:849) or a 5’ fragment thereof comprising 5’-tatattgtggttctgta-3’ (SEQ ID NO:850).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-atccagatattgtggtgtaaacatacactgtgaat-3’ (SEQ ID NO:851) or a 5’ fragment thereof comprising 5’-atccagatattgtggtgtaaac-3’ (SEQ ID NO: 810).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gatattgtggtgtaaacatacactgtgaat-3’ (SEQ ID NO:852) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaac-3’ (SEQ ID NO: 853).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-attcagatattgtggtgtaaacatacactgtgaatgg-3’ (SEQ ID NO:854) or a 5’ fragment thereof comprising 5’-attcagatattgtggtgtaaac-3’ (SEQ ID NO:811).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-gatattgtggtgtaaacatacactgtgaatgg-3’ (SEQ ID NO: 855) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaac-3’ (SEQ ID NO: 853).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-taggatatattgtggtgtgatttgtgggccagctctcatat-3’ (SEQ ID NO:856) or a 5’ fragment thereof comprising 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO: 797).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtggtgtgatttgtgggccagctctcatat-3’ (SEQ ID NO:857) or a 5’ fragment thereof comprising 5’-tatattgtggtgtgatt-3’ (SEQ ID NO: 816).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-taggatatattgtgctgtaatttgtgggccaactctcat-3’ (SEQ ID NO:858 or a 5’ fragment thereof comprising 5’-taggatatattgtgctgtaatt-3’ (SEQ ID NO: 812).

In an embodiment, the processed left border of the WT-DNA in the wheat genome comprises 5’-tatattgtgctgtaatttgtgggccaactctcat-3’ (SEQ ID NO: 859) or a 5’ fragment thereof comprising 5’-tatattgtgctgtaatt-3’ (SEQ ID NO:860).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-aactatcagtgtgtttga-3’ (SEQ ID NO:861), or a 3’ fragment thereof comprising 5’-ttga-3’ (SEQ ID NO:62). In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-atattgctttgcttgtcccaggaactatcagtgtttga-3’ (SEQ ID NO:862), or a 3’ fragment thereof comprising 5’-ggaactatcagtgtttga-3’ (SEQ ID NO:863) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-tattgctttgcttgtcctaggaactatcagtgtttga-3’ (SEQ ID NO: 864), or a 3’ fragment thereof comprising 5’-ggaactatcagtgtttga-3’ (SEQ ID NO:863) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-atttgttgtggaaatgtctaaactttcagtgtttga-3’ (SEQ ID NO:865), or a 3’ fragment thereof comprising 5’-taaactttcagtgtttga-3’ (SEQ ID NO:866) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-tacaaaaatattcagacctaaaatatcagtgtttga-3’ (SEQ ID NO: 867), or a 3’ fragment thereof comprising 5’-taaaatatcagtgtttga-3’ (SEQ ID NO:376) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’-tgtttgaccaagttctagaataaactatcagtatttga-3’ (SEQ ID NO:868), or a 3’ fragment thereof comprising 5’-taaactatcagtatttga-3’ (SEQ ID NO:420) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’- ctggcagagattgctgtggaatgtattaaccttgacgtcgatcaaaggcccacgatgacagatgtcgaggaccgccttctcat gctgaaccgatcttgtaggtcgcaagctgtttagctatatataagttgatgcatattatggtgtggacaacatttatgtattcgaata tgataggcaactatcagtgtttga-3 ’ (SEQ ID NO: 869), or a 3’ fragment thereof comprising 5’- aactatcagtgtgtttga-3’ (SEQ ID NO:861) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the WT-DNA in the wheat genome comprises 5’ -caactatcagtgtgtttga-3 ’ (SEQ ID NO:870).

In an embodiment, the polynucleotide of interest encodes a wheat protein.

Examples of polynucleotides of interest include, but are not limited to, those which encode a wheat protein which confers enhanced resistance to disease, enhanced tolerance to salinity, increased starch production, increased yield, increased grain size, drought tolerance, or enhanced nutritional content.

In an embodiment, the disease is a fungal disease. In an embodiment, the fungal disease is a mildew, blight, rot, smut or rust. In an embodiment, the disease is a fungal disease. In an embodiment, the fungal disease is a mildew, blight, rot or rust. In an embodiment, the fungal disease is a rust.

As another example, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT), wherein the Ti plasmid comprises a wheat transfer DNA (WT-DNA) for integration into the genome of a wheat plant, wherein the WT-DNA comprises a polynucleotide of interest and only has wheat DNA, wherein the 5’ end of the WT-DNA comprises part of a left border for integration into the genome, and the 3’ end of the WT-DNA comprises part of a right border for integration into the genome.

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779).

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtg gtctagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaaga gaagagtcgcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgag-3’ (SEQ ID NO:871) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtggtgtaaac-3 ’ (SEQ ID NO:758), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs: 872 and 1482. Further embodiments of this region of the wheat genome include 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtg gtctagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggctctgcaaga gaagagtcgcggctcggggtgacttttgtttctaagctgcatgtggttctgagattgagaattcctcatttggtttcacattcttaag catgattactccattcttaattgggcctataactcatagcagagcatacacatttgaatttctttattatcttcttgaacaattactttttg tcaattcatattccatag-3’ (SEQ ID NO: 1311), tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccaccttatacgcgtcatcatttgtgccaacattggtgtg gtctagggtataaataagacagacatatgccctccatcagttaatttcagtcaaccaattcctgaatcaaccaggcatttggtttc acattcttaagcatgattactccattc-3 ’ (SEQ ID NO: 1312), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccacc-3’ (SEQ ID NO: 1313), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatccac-3’ (SEQ ID NO: 1314), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatcca-3’ (SEQ ID NO: 1315), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatcc-3’ (SEQ ID NO: 1316), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattatc-3’ (SEQ ID NO: 1317), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtattat-3’ (SEQ ID NO: 1318), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtatta-3’ (SEQ ID NO: 1319), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtatt-3’ (SEQ ID NO: 1320), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgtat-3’ (SEQ ID NO: 1321), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgta-3’ (SEQ ID NO: 1322), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactgt-3’ (SEQ ID NO: 1323), 5’- tggcaggatatattgtggtgtaaactctatcaactggtactg-3’ (SEQ ID NO: 1324), 5’- tggcaggatatattgtggtgtaaactctatcaactggtact-3’ (SEQ ID NO: 1325), 5’- tggcaggatatattgtggtgtaaactctatcaactggtac-3’ (SEQ ID NO: 1326), 5’- tggcaggatatattgtggtgtaaactctatcaactggta-3’ (SEQ ID NO: 1327), 5’- tggcaggatatattgtggtgtaaactctatcaactggt-3’ (SEQ ID NO: 1328), 5’- tggcaggatatattgtggtgtaaactctatcaactgg-3’ (SEQ ID NO: 1329), 5’- tggcaggatatattgtggtgtaaactctatcaactg-3’ (SEQ ID NO: 1330), 5’- tggcaggatatattgtggtgtaaactctatcaact-3’ (SEQ ID NO: 1331), 5’- tggcaggatatattgtggtgtaaactctatcaac-3’ (SEQ ID NO: 1332), 5’- tggcaggatatattgtggtgtaaactctatcaa-3’ (SEQ ID NO: 1333), 5’- tggcaggatatattgtggtgtaaactctatca-3’ (SEQ ID NO: 1334), 5’- tggcaggatatattgtggtgtaaactctatc-3’ (SEQ ID NO: 1335), 5’- tggcaggatatattgtggtgtaaactctat-3’ (SEQ ID NO: 1336), 5’-tggcaggatatattgtggtgtaaactcta-

3’ (SEQ ID NO: 1337), 5’-tggcaggatatattgtggtgtaaactct-3’ (SEQ ID NO: 1338), 5’- tggcaggatatattgtggtgtaaactc-3’ (SEQ ID NO: 1339), 5’-tggcaggatatattgtggtgtaaact-3’ (SEQ ID NO: 1340), or where the 5’ t is replaced with a c. In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises any of the above sequences lacking the 5’ tgg or egg.

In an embodiment, the left border comprises 5’-tggcaggatatattgtggtgtaaac-3’ (SEQ ID NO:758), or 5’-cggcaggatatattgtggtgtaaac-3’ (SEQ ID NO: 1450).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO: 797).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -caggatatattgtggtgttggg-3’ (SEQ ID NO:798) or 5’- tatattgtggtgttggg-3’ (SEQ ID NO: 819).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gtagttatattgtggtgtaaac-3’ (SEQ ID NO:799) or 5’- tatattgtggtgtaaac-3 ’ (SEQ ID NO:779).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -caggatatattgtagacaactc-3 ’ (SEQ ID NO:800) or 5’- tatattgtagacaactc-3’ (SEQ ID NO: 822).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gaggatatattgtggtgataaa-3’ (SEQ ID NO:801) or 5’- tatattgtggtgataaa-3’ (SEQ ID NO:827). In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gatcctatattgtggtgtaaaa-3’ (SEQ ID NO:802) or 5’- tatattgtggtgtaaaa-3 ’ (SEQ ID NO:345).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -caggatatattgtgccggccta-3’ (SEQ ID NO: 803) or 5’- tatattgtgccggccta-3’ (SEQ ID NO:832).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-aatgatatattgtggtgtatga-3’ (SEQ ID NO: 804) or 5’- tatattgtggtgtatga-3 ’ (SEQ ID NO: 873).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gcagatatattgtggtgtatgc-3’ (SEQ ID NO: 805) or 5’- gcagatatattgtggtgtatgc-3’ (SEQ ID NO: 805).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -caggatatattgttggcctact-3 ’ (SEQ ID NO:806) or 5’- caggatatattgttggcctact-3 ’ (SEQ ID NO:806).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -cgggatatattgtggtgtgtgc-3’ (SEQ ID NO:807) or 5’- cgggatatattgtggtgtgtgc-3’ (SEQ ID NO: 807).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-atctctatattgtggtgtaaaa-3’ (SEQ ID NO: 808) or 5’- atctctatattgtggtgtaaaa-3 ’ (SEQ ID NO: 808).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtggttctgta-3’ (SEQ ID NO: 809) or 5’- caggatatattgtggttctgta-3’ (SEQ ID NO:809).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-atccagatattgtggtgtaaac-3’ (SEQ ID NO:810) or 5’- atccagatattgtggtgtaaac-3’ (SEQ ID NO: 810).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-attcagatattgtggtgtaaac-3’ (SEQ ID NO: 811) or 5’- attcagatattgtggtgtaaac-3’ (SEQ ID NO:811).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO:797) or 5’- taggatatattgtggtgtgatt-3’ (SEQ ID NO:797).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-taggatatattgtgctgtaatt-3’ (SEQ ID NO: 812) or 5’- taggatatattgtgctgtaatt-3’ (SEQ ID NO: 812). In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’- taggatatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacagaaa agaaaagaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgcaca aaggcccagcccacgtcggccttccact-3’ (SEQ ID NO:814) or a 5’ fragment thereof comprising 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO:797).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’- tatattgtggtgtgatttgtgggccagctctcatattttacaaaactcatttggtgcccaaacaattagccaaaacagaaaagaaa agaaataaaagaaagtataaataaaaagaaaggcctggcctttactgtgccaccgggcctaagtgctacagtgcacaaaggc ccagcccacgtcggccttccact-3 ’ (SEQ ID NO:815) or a 5’ fragment thereof comprising 5’- tatattgtggtgtgatt-3 ’ (SEQ ID NO: 816).

In an embodiment, the left border of the WT-DNA comprises 5’- tggtaggatatattgtggtgtgatt-3 ’ (SEQ ID NO:874) or 5’-cggtaggatatattgtggtgtgatt-3’ (SEQ ID NO:875).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtggtgttgggattctctggttct-3’ (SEQ ID NO:817) or a 5’ fragment thereof comprising 5’ -caggatatattgtggtgttggg-3 ’ (SEQ ID NO:798).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgttgggattctctggttct-3’ (SEQ ID NO: 818) or a 5’ fragment thereof comprising 5’-tatattgtggtgttggg-3’ (SEQ ID NO:819).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gtagttatattgtggtgtaaactctatc-3’ (SEQ ID NO: 820) or a 5’ fragment thereof comprising 5’-gtagttatattgtggtgtaaac-3’ (SEQ ID NO:799).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtaaactctatc-3’ (SEQ ID NO: 821) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaac-3’ (SEQ ID NO:779).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtagacaactc-3’ (SEQ ID NO:800).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtagacaactc-3’ (SEQ ID NO:822).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtagacaactct-3’ (SEQ ID NO: 823).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtagacaactct-3’ (SEQ ID NO: 824). In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gaggatatattgtggtgataaaagctatctc -3’ (SEQ ID NO: 825) or a 5’ fragment thereof comprising 5’-gaggatatattgtggtgataaa-3’ (SEQ ID NO:801).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgataaaagctatctc -3’ (SEQ ID NO:826) or a 5’ fragment thereof comprising 5’-tatattgtggtgataaa-3’ (SEQ ID NO: 827).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gatcctatattgtggtgtaaaatatatcgagctagtagtg-3’ (SEQ ID NO: 828) or a 5’ fragment thereof comprising 5’ -gatcctatattgtggtgtaaaa-3 ’ (SEQ ID NO:802).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtaaaatatatcgagctagtagtg-3’ (SEQ ID NO: 829) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtgccggcctaaggaccaagagggtctac-3’ (SEQ ID NO: 830) or a 5’ fragment thereof comprising 5’ -caggatatattgtgccggccta-3 ’ (SEQ ID NO:803).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtgccggcctaaggaccaagagggtctac-3’ (SEQ ID NO:831) or a 5’ fragment thereof comprising 5’-tatattgtgccggccta-3’ (SEQ ID NO: 832).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-aatgatatattgtggtgtatgagatcatgttcttgt-3’ (SEQ ID NO:833) or a 5’ fragment thereof comprising 5’ -aatgatatattgtggtgtatga -3’ (SEQ ID NO:804).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtatgagatcatgttcttgt-3’ (SEQ ID NO:835) or a 5’ fragment thereof comprising 5’-tatattgtggtgtatga -3’ (SEQ ID NO: 836).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gcagatatattgtggtgtatgcacacacccttc-3’ (SEQ ID NO:837) or a 5’ fragment thereof comprising 5’ -gcagatatattgtggtgtatgc-3’ (SEQ ID NO:805).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtatgcacacacccttc-3’ (SEQ ID NO:838) or a 5’ fragment thereof comprising 5’-tatattgtggtgtatgc-3’ (SEQ ID NO: 839).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgttggcctactccagtcat-3’ (SEQ ID NO: 840) or a 5’ fragment thereof comprising 5’ -caggatatattgttggcctact-3 ’ (SEQ ID NO: 806). In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’ -tatattgttggcctactccagtcat-3 ’ (SEQ ID NO: 841) or a 5’ fragment thereof comprising 5’-tatattgttggcctact-3’ (SEQ ID NO: 842).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-cgggatatattgtggtgtgtgcgctgtgtctctttc-3’ (SEQ ID NO:843) or a 5’ fragment thereof comprising 5’ -cgggatatattgtggtgtgtgc-3 ’ (SEQ ID NO:807).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtgtgcgctgtgtctctttc-3’ (SEQ ID NO: 844) or a 5’ fragment thereof comprising 5’-tatattgtggtgtgtgc-3’ (SEQ ID NO:845).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-atctctatattgtggtgtaaaagcaag-3’ (SEQ ID NO: 846) or a 5’ fragment thereof comprising 5’-atctctatattgtggtgtaaaa-3’ (SEQ ID NO:808).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtaaaagcaag-3’ (SEQ ID NO:847) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-caggatatattgtggttctgtaatctct-3’ (SEQ ID NO:848) or a 5’ fragment thereof comprising 5’-caggatatattgtggttctgta-3’ (SEQ ID NO:809).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggttctgtaatctct-3’ (SEQ ID NO:849) or a 5’ fragment thereof comprising 5’-tatattgtggttctgta-3’ (SEQ ID NO:850).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-atccagatattgtggtgtaaacatacactgtgaat-3’ (SEQ ID NO:851) or a 5’ fragment thereof comprising 5’ -atccagatattgtggtgtaaac-3 ’ (SEQ ID NO:810).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gatattgtggtgtaaacatacactgtgaat-3’ (SEQ ID NO:852) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaac-3’ (SEQ ID NO: 853).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-attcagatattgtggtgtaaacatacactgtgaatgg-3’ (SEQ ID NO:854) or a 5 ’ fragment thereof comprising 5 ’ -attcagatattgtggtgtaaac-3 ’ (SEQ ID NO:811).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-gatattgtggtgtaaacatacactgtgaatgg-3’ (SEQ ID NO:855) or a 5’ fragment thereof comprising 5’-gatattgtggtgtaaac-3’ (SEQ ID NO: 853).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-taggatatattgtggtgtgatttgtgggccagctctcatat-3’ (SEQ ID NO:856) or a 5’ fragment thereof comprising 5’ -taggatatattgtggtgtgatt-3 ’ (SEQ ID NO:797). In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtggtgtgatttgtgggccagctctcatat-3’ (SEQ ID NO:857) or a 5’ fragment thereof comprising 5’-tatattgtggtgtgatt-3’ (SEQ ID NO:816).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-taggatatattgtgctgtaatttgtgggccaactctcat-3’ (SEQ ID NO: 858) or a 5’ fragment thereof comprising 5’-taggatatattgtgctgtaatt-3’ (SEQ ID NO: 812).

In an embodiment, the part of the left border of the WT-DNA for integration into the genome comprises 5’-tatattgtgctgtaatttgtgggccaactctcat-3’ (SEQ ID NO: 859) or a 5’ fragment thereof comprising 5’-tatattgtgctgtaatt-3’ (SEQ ID NO:860).

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtggtgttgggattctctggttct-3’ (SEQ ID NO: 876) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgtggtgttggg-3 ’ (SEQ ID NO: 877), or wherein the 5’ t is replaced with a c. see, for example but not limited to, SEQ ID NOs: 878 and 1451.

In an embodiment, the left border of the WT-DNA comprises 5’- tgggttatattgtggtgtaaactctatc-3’ (SEQ ID NO:879) or a 5’ fragment thereof comprising 5’- tgggtagttatattgtggtgtaaac-3 ’ (SEQ ID NO: 880), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NOs: 881 and 1452.

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtagacaactc-3 ’ (SEQ ID NO:882), or wherein the 5’ t is replaced with a c (SEQ ID NO:883).

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtagacaactct-3 ’ (SEQ ID NO: 884), or wherein the 5’ t is replaced with a c (SEQ ID NO:885).

In an embodiment, the left border of the WT-DNA comprises 5’- tgggaggatatattgtggtgataaaagctatctc -3’ (SEQ ID NO:886) or a 5’ fragment thereof comprising 5’-tgggaggatatattgtggtgataaa-3’ (SEQ ID NO:887), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NOs:888 and 1453.

In an embodiment, the left border of the WT-DNA comprises 5’- tgggatcctatattgtggtgtaaaatatatcgagctagtagtg-3’ (SEQ ID NO: 889) or a 5’ fragment thereof comprising 5’-tgggatcctatattgtggtgtaaaa-3’ (SEQ ID NO: 890), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO: 891 and 1454.

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtgccggcctaaggaccaagagggtctac-3’ (SEQ ID NO:892) or a 5’ fragment thereof comprising 5 ’-tggcaggatatattgtgccggccta-3 ’ (SEQ ID NO: 893), or wherein the 5 ’ t is replaced with a c, see, for example but not limited to SEQ ID NO:894 and 1455. In an embodiment, the left border of the WT-DNA comprises 5’- tggaatgatatattgtggtgtatgagatcatgttcttgt-3’ (SEQ ID NO: 895) or a 5’ fragment thereof comprising 5’-tggatgatatattgtggtgtatga -3’ (SEQ ID NO:896), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO: 897 and 1456.

In an embodiment, the left border of the WT-DNA comprises 5’- tgggcagatatattgtggtgtatgcacacacccttc-3’ (SEQ ID NO:898) or a 5’ fragment thereof comprising 5’ -tgggcagatatattgtggtgtatgc-3 ’ (SEQ ID NO: 899), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:900, and 1457.

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgttggcctactccagtcat-3’ (SEQ ID NO:901) or a 5’ fragment thereof comprising 5’ -tggcaggatatattgttggcctact-3’ (SEQ ID NO:902), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:903 and 1458.

In an embodiment, the left border of the WT-DNA comprises 5’- tggcgggatatattgtggtgtgtgcgctgtgtctctttc-3’ (SEQ ID NO:904) or a 5’ fragment thereof comprising 5’ -tggcgggatatattgtggtgtgtgc-3’ (SEQ ID NO:905), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:906 and 1459.

In an embodiment, the left border of the WT-DNA comprises 5’- tggatctctatattgtggtgtaaaagcaag-3’ (SEQ ID NO:907) or a 5’ fragment thereof comprising 5’-tggatctctatattgtggtgtaaaa-3’ (SEQ ID NO:908), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO: 909 and 1460.

In an embodiment, the left border of the WT-DNA comprises 5’- tggcaggatatattgtggttctgtaatctct-3’ (SEQ ID NO:910) or a 5’ fragment thereof comprising 5’-tggcaggatatattgtggttctgta-3’ (SEQ ID NO:911), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NON 12 and 1461).

In an embodiment, the left border of the WT-DNA comprises 5’- tggatccagatattgtggtgtaaacatacactgtgaat-3’ (SEQ ID NO:913) or a 5’ fragment thereof comprising 5’ -tggatccagatattgtggtgtaaac-3 ’ (SEQ ID NO:914), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:915 and 1462).

In an embodiment, the left border of the WT-DNA comprises 5’- tggattcagatattgtggtgtaaacatacactgtgaatgg-3’ (SEQ ID NO:916) or a 5’ fragment thereof comprising 5’-tggattcagatattgtggtgtaaac-3’ (SEQ ID NO:917), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:918 and 1463.

In an embodiment, the left border of the WT-DNA comprises 5’- tggtaggatatattgtggtgtgatttgtgggccagctctcatat-3’ (SEQ ID NO:919) or a 5’ fragment thereof comprising 5’-tggtaggatatattgtggtgtgatt-3’ (SEQ ID NO:874), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:920 and 1464. In an embodiment, the left border of the WT-DNA comprises 5’- tggtaggatatattgtgctgtaatttgtgggccaactctcat-3’ (SEQ ID NO:921 or a 5’ fragment thereof comprising 5’ -tggtaggatatattgtgctgtaatt-3 ’ (SEQ ID NO:922), or wherein the 5’ t is replaced with a c, see, for example but not limited to SEQ ID NO:923 and 1465.

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- ctggcagagattgctgtggaatgtattaaccttgacgtcgatcaaaggcccacgatgacagatgtcgaggaccgccttctcat gctgaaccgatcttgtaggtcgcaagctgtttagctatatataagttgatgcatattatggtgtggacaacatttatgtattcgaata tgataggcaactatcagtgtttga-3 ’ (SEQ ID NO: 869), or a 3’ fragment thereof comprising 5’- aactatcagtgtgtttga-3’ (SEQ ID NO:861).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-caactatcagtgtgtttga-3’ (SEQ ID NO:870), or a 3’ fragment thereof comprising 5’-aactatcagtgtgtttga-3’ (SEQ ID NO:861) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-atattgctttgcttgtcccaggaactatcagtgtttga-3’ (SEQ ID NO:862), ora 3’ fragment thereof comprising 5’ -ggaactatcagtgtttga-3’ (SEQ ID NO:863) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-tattgctttgcttgtcctaggaactatcagtgtttga-3’ (SEQ ID NO:864), or a 3’ fragment thereof comprising 5 ’-ggaactatcagtgtttga-3’ (SEQ ID NO: 863) or 5’- ttga-3’ (SEQ ID NO:62).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-atttgttgtggaaatgtctaaactttcagtgtttga-3’ (SEQ ID NO: 865), or a 3 ’ fragment thereof comprising 5 ’-taaactttcagtgtttga-3 ’ (SEQ ID NO: 866) or 5 ’-ttga- 3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5 ’-tacaaaaatattcagacctaaaatatcagtgtttga-3’ (SEQ ID NO:867), or a3’ fragment thereof comprising 5’ -taaaatatcagtgtttga-3 ’ (SEQ ID NO:376) or 5’-ttga- 3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-tgtttgaccaagttctagaataaactatcagtatttga-3’ (SEQ ID NO: 868), or a 3’ fragment thereof comprising 5’-taaactatcagtatttga-3’ (SEQ ID NO:420) or 5’-ttga-3’ (SEQ ID NO: 62).

In an embodiment, the right border comprises 5’ -tgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:786). In an embodiment, the right border of the WT-DNA comprises 5’- aactatcagtgtgtttgatgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 924) or 5’- tgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:786).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- ctggcagagattgctgtggaatgtattaaccttgacgtcgatcaaaggcccacgatgacagatgtcgaggaccgccttctcat gctgaaccgatcttgtaggtcgcaagctgtttagctatatataagttgatgcatattatggtgtggacaacatttatgtattcgaata tgataggcaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:925), or a 3’ fragment thereof comprising 5’-aactatcagtgtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:926).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’-caactatcagtgtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:927), or a 3’ fragment thereof comprising 5’- aactatcagtgtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 926) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- atattgctttgcttgtcccaggaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:928), or a 3’ fragment thereof comprising 5’-ggaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:929) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- tattgctttgcttgtcctaggaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:930), or a 3’ fragment thereof comprising 5’-ggaactatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:929) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- atttgttgtggaaatgtctaaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:931), or a 3’ fragment thereof comprising 5’-taaactttcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:932) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- tacaaaaatattcagacctaaaatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:933), ora3’ fragment thereof comprising 5’-taaaatatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:712) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the part of the right border of the WT-DNA for integration into the genome comprises 5’- tgtttgaccaagttctagaataaactatcagtatttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:934), or a 3’ fragment thereof comprising 5’-taaactatcagtatttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:750) or 5’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the PT-DNA is a wheat transfer DNA (WT-DNA) and the left border comprises 5’-tggcaggatatattgtggtgtaaact-3’ (SEQ ID NO: 1340).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtggtgtaaac-3’ (SEQ ID NO: 1450).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcaggatatattgtggtgttggg-3 ’ (SEQ ID NO: 877).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtggtgttggg-3’ (SEQ ID NO: 1451).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tgggtagttatattgtggtgtaaac-3 ’ (SEQ ID NO: 880).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cgggtagttatattgtggtgtaaac-3 ’ (SEQ ID NO: 1452).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcaggatatattgtagacaactc-3 ’ (SEQ ID NO:882).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtagacaactc-3’ (SEQ ID NO:883).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcaggatatattgtagacaactct-3 ’ (SEQ ID NO:884).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtagacaactct-3 ’ (SEQ ID NO: 885).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tgggaggatatattgtggtgataaa-3 ’ (SEQ ID NO:887).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cgggaggatatattgtggtgataaa-3 ’ (SEQ ID NO: 1453).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tgggatcctatattgtggtgtaaaa-3’ (SEQ ID NO: 890).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cgggatcctatattgtggtgtaaaa-3 ’ (SEQ ID NO: 1454).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcaggatatattgtgccggccta-3’ (SEQ ID NO:893).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtgccggccta-3 ’ (SEQ ID NO: 1455). In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggatgatatattgtggtgtatga -3’ (SEQ ID NO:896).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggatgatatattgtggtgtatga -3’ (SEQ ID NO: 1456).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tgggcagatatattgtggtgtatgc-3’ (SEQ ID NO:899).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cgggcagatatattgtggtgtatgc-3 ’ (SEQ ID NO: 1457).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcaggatatattgttggcctact-3 ’ (SEQ ID NO:902).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgttggcctact-3’ (SEQ ID NO: 1458).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggcgggatatattgtggtgtgtgc-3 ’ (SEQ ID NO:905).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcgggatatattgtggtgtgtgc-3 ’ (SEQ ID NO: 1459).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggatctctatattgtggtgtaaaa-3’ (SEQ ID NO:908).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggatctctatattgtggtgtaaaa-3 ’ (SEQ ID NO: 1460).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5 ’- tggcaggatatattgtggttctgta-3 ’ (SEQ ID NO:911).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggcaggatatattgtggttctgta-3 ’ (SEQ ID NO: 1461).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggatccagatattgtggtgtaaac-3 ’ (SEQ ID NO:914).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggatccagatattgtggtgtaaac-3 ’ (SEQ ID NO: 1462).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggattcagatattgtggtgtaaac-3 ’ (SEQ ID NO: 917).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggattcagatattgtggtgtaaac-3 ’ (SEQ ID NO: 1463).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggtaggatatattgtggtgtgatt-3 ’ (SEQ ID NO: 874).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggtaggatatattgtggtgtgatt-3 ’ (SEQ ID NO: 1464). In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- tggtaggatatattgtgctgtaatt-3 ’ (SEQ ID NO: 922).

In an embodiment, the PT-DNA is a WT-DNA and the left border comprises 5’- cggtaggatatattgtgctgtaatt-3 ’ (SEQ ID NO: 1465).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-caggatatattgtggtgtaaact-3’ (SEQ ID NO: 1310).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-taggatatattgtggtgtgatt-3’ (SEQ ID NO:797).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-caggatatattgtggtgttggg-3’ (SEQ ID NO:798).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-gtagttatattgtggtgtaaac-3’ (SEQ ID NO:799).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-caggatatattgtagacaactc-3’ (SEQ ID NO:800).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’ -gaggatatattgtggtgataaa-3’ (SEQ ID NO:801).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-gatcctatattgtggtgtaaaa-3’ (SEQ ID NO:802).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’ -caggatatattgtgccggccta-3 ’ (SEQ ID NO:803).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-aatgatatattgtggtgtatga-3’ (SEQ ID NO:804).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’ -gcagatatattgtggtgtatgc-3’ (SEQ ID NO:805).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’ -caggatatattgttggcctact-3 ’ (SEQ ID NO:806).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-cgggatatattgtggtgtgtgc-3’ (SEQ ID NO:807).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-atctctatattgtggtgtaaaa-3’ (SEQ ID NO:808).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-caggatatattgtggttctgta-3’ (SEQ ID NO:809).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’ -atccagatattgtggtgtaaac-3’ (SEQ ID NO:810).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-attcagatattgtggtgtaaac-3’ (SEQ ID NO:811). In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaact-3’ (SEQ ID NO: 1495).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-taggatatattgtgctgtaatt-3’ (SEQ ID NO:812).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtgatt-3’ (SEQ ID NO:816).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgttggg-3’ (SEQ ID NO:819).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtagacaactc-3’ (SEQ ID NO:822).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-caggatatattgtagacaactct-3’ (SEQ ID NO:823).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtagacaactct-3’ (SEQ ID NO:824).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgataaa-3’ (SEQ ID NO:827).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtgccggccta-3’ (SEQ ID NO:832).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtatga -3’ (SEQ ID NO:836).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtatgc-3’ (SEQ ID NO:839).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgttggcctact-3’ (SEQ ID NO:842).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtgtgc-3’ (SEQ ID NO:845).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtggttctgta-3’ (SEQ ID NO:850).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-gatattgtggtgtaaac-3’ (SEQ ID NO:853).

In an embodiment, the plant is wheat and PT-DNA has a processed the left border which comprises 5’-tatattgtgctgtaatt-3’ (SEQ ID NO:860).

As another example, the present invention provides a wheat transfer DNA (WT- DNA) for integration into the genome of a wheat plant, wherein the WT-DNA comprises a polynucleotide of interest and only has wheat DNA, wherein the 5’ end of the WT- DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the WT-DNA comprises part of a right border for integration into the genome.

In an embodiment, the WT-DNA has one or more of the features defined above in relation to the Ti plasmid.

ST-DNA

In an embodiment, the PT-DNA is sorghum transfer DNA (ST-DNA). The ST- DNA may be part of a Ti plasmid, or have been processed and be in the genome of a sorghum plant.

For example, the present invention provides a soybean plant having a soybean transfer DNA (ST-DNA) integrated into the genome of the plant, wherein the integrated ST-DNA only has soybean DNA, and wherein the ST-DNA comprises a polynucleotide of interest.

In an embodiment, the integrated ST-DNA has a processed ST-DNA right border, or lacks any portion of a ST-DNA right border.

In an embodiment, the integrated ST-DNA has a processed ST-DNA left border, or lacks any portion of a ST-DNA left border.

In an embodiment, the integrated ST-DNA comprises a processed ST-DNA right border, a processed ST-DNA left border and a polynucleotide of interest.

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’- cattatatattgtggtgtaaatataattttttttatgaataacaatgcacaatattggatgtgtgttttagccttggatgacaagtagtg agaggagagggaagagagtgagtattgatatatgtgcaattatgtttaaagtagatgttgagagaacttggatttcgtgaataat atttataggttgagaattatgagttaaggaggtgagagtacgtctta-3’ (SEQ ID NO:935) or a 5’ fragment thereof comprising 5’-cattatatattgtggtgtaaat-3’ (SEQ ID NO: 936).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’- tatattgtggtgtaaatataattttttttatgaataacaatgcacaatattggatgtgtgttttagccttggatgacaagtagtgagag gagagggaagagagtgagtattgatatatgtgcaattatgtttaaagtagatgttgagagaacttggatttcgtgaataatatttat aggttgagaattatgagttaaggaggtgagagtacgtctta-3’ (SEQ ID NO:937) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’- cagcatatattcttgtgtaaattaggactgcatcatcgaccaatgtttaaccaacatctttataatgacataatatttcacttgacatt gtaatatccaagacataggaattgttactagtaaaaggacatgaaaattgtacctgtgtgtagatataaacttggagaatatttag gacaatcagttatgtcatgtcgcataacatcatcttttaacttgaatccatggtgaatctctttgaagtttggcaaacttctgagtgtt atacatagcaggttatgtagtagtactttttctccatccttaacaatcccttcttga-3’ (SEQ ID NO:938) or a 5’ fragment thereof comprising 5’-cagcatatattcttgtgtaaat-3’ (SEQ ID NO:939).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’- tatattcttgtgtaaattaggactgcatcatcgaccaatgtttaaccaacatctttataatgacataatatttcacttgacattgtaatat ccaagacataggaattgttactagtaaaaggacatgaaaattgtacctgtgtgtagatataaacttggagaatatttaggacaatc agttatgtcatgtcgcataacatcatcttttaacttgaatccatggtgaatctctttgaagtttggcaaacttctgagtgttatacata gcaggttatgtagtagtactttttctccatccttaacaatcccttcttga-3’ (SEQ ID NO:940) or a 5’ fragment thereof comprising 5’-tatattcttgtgtaaat-3’ (SEQ ID NO: 941).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-cattatatattgtggtgtaaatataattctttttatga-3’ (SEQ ID NO: 942) or a 5’ fragment thereof comprising 5’-cattatatattgtggtgtaaat-3’ (SEQ ID NO:936).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tatattgtggtgtaaatataattctttttatga-3’ (SEQ ID NO:944) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-aattatatattgtggtgtaaaaaaattaaat-3’ (SEQ ID NO:945) or a 5’ fragment thereof comprising 5’-aattatatattgtggtgtaaaa-3’ (SEQ ID NO: 946).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tatattgtggtgtaaaaaaattaaat-3’ (SEQ ID NO:947) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-atgataatattgtggtgtaaactttgaccaaaattt-3’ (SEQ ID NO:948) or a 5’ fragment thereof comprising 5’-atgataatattgtggtgtaaac-3’ (SEQ ID NO:949).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-aatattgtggtgtaaactttgaccaaaattt-3’ (SEQ ID NO:950) or a 5’ fragment thereof comprising 5’-aatattgtggtgtaaac-3’ (SEQ ID NO:385).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-caggatatattgtgtttgataag-3’ (SEQ ID NO:951) or a 5’ fragment thereof comprising 5’-caggatatattgtgtttgataa-3’ (SEQ ID NO:952).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tatattgtgtttgataag-3’ (SEQ ID NO:953) or a 5’ fragment thereof comprising 5’-tatattgtgtttgataa-3’ (SEQ ID NO:954). In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tattatatattgtggtgtaacttattattta-3’ (SEQ ID NO:955) or a 5’ fragment thereof comprising 5’-tattatatattgtggtgtaact-3’ (SEQ ID NO: 956).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tatattgtggtgtaacttattattta-3’ (SEQ ID NO:957) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaact-3’ (SEQ ID NO:314).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-gaatttatattgtggtgtaaatttttgattaa-3’ (SEQ ID NO: 958) or a 5’ fragment thereof comprising 5’-gaatttatattgtggtgtaaat-3’ (SEQ ID NO:959).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-tatattgtggtgtaaatttttgattaa-3’ (SEQ ID NO:960) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’-aagcttatattgtggtgtaaagtcttacacatg-3’ (SEQ ID NO:961) or a 5’ fragment thereof comprising 5’-aagcttatattgtggtgtaaag-3’ (SEQ ID NO:962).

In an embodiment, the processed left border of the ST-DNA in the soybean genome comprises 5’ -tatattgtggtgtaaagtcttacacatg-3’ (SEQ ID NO:963) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaag-3’ (SEQ ID NO:303).

In an embodiment, the processed right border of the ST-DNA in the soybean genome comprises 5’- ctacacatcttcgagaagatacggtgctagcctaaggtttgctggtttgcgctttctatgtactgatgcatgaatgcatgggtcac agctaaccatattgggctgaggttgttgccttccgttttggccttatacttcttgcttcttgtagatcctttgcttcctgcagtactatc cctatcagtgtttga-3 ’ (SEQ ID NO:964), or a 3’ fragment thereof comprising 5- atccctatcagtgtttga-3 ’ (SEQ ID NO:965) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the ST-DNA in the soybean genome comprises 5’-ggatacaattaatttttttttgaaactatcagtctttga-3’ (SEQ ID NO:966), or a 3’ fragment thereof comprising 5’-gaaactatcagtctttga-3’ (SEQ ID NO:967) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the ST-DNA in the soybean genome comprises 5’-tggtcatccagaattagagaaacaatcagtgtttga-3’ (SEQ ID NO:968), or a 3’ fragment thereof comprising 5’-gaaacaatcagtgtttga-3’ (SEQ ID NO: 969) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the processed right border of the ST-DNA in the soybean genome comprises 5’-tgagacatgtatggtgaaactctcagtgtttga-3’ (SEQ ID NO:970), or a 3’ fragment thereof comprising 5’-gaaactctcagtgtttga-3’ (SEQ ID NO:971) or 5’-ttga-3’ (SEQ ID NO:62). In an embodiment, the processed right border of the ST-DNA in the soybean genome comprises 5’-cctatcagtgtttga-3’ (SEQ ID NO:972).

In an embodiment, the polynucleotide of interest encodes a soybean protein.

Examples of polynucleotides of interest include, but are not limited to, those which encode a soybean protein which confers enhanced resistance to disease, enhanced tolerance to salinity, increased yield, increased grain size, drought tolerance, or enhanced nutritional content.

As another example, the present invention provides a tumor-inducing (Ti) plasmid for Agrobacterium mediated gene transfer (AMGT), wherein the Ti plasmid comprises a soybean transfer DNA (ST-DNA) for integration into the genome of a soybean plant, wherein the ST-DNA comprises a polynucleotide of interest and only has soybean DNA, wherein the 5 ’ end of the ST-DNA comprises part of a left border for integration into the genome, and the 3’ end of the ST-DNA comprises part of a right border for integration into the genome.

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’- cattatatattgtggtgtaaatataattttttttatgaataacaatgcacaatattggatgtgtgttttagccttggatgacaagtagtg agaggagagggaagagagtgagtattgatatatgtgcaattatgtttaaagtagatgttgagagaacttggatttcgtgaataat atttataggttgagaattatgagttaaggaggtgagagtacgtctta-3’ (SEQ ID NO:935) or a 5’ fragment thereof comprising 5’-cattatatattgtggtgtaaat-3’ (SEQ ID NO: 936).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’- tatattgtggtgtaaatataattttttttatgaataacaatgcacaatattggatgtgtgttttagccttggatgacaagtagtgagag gagagggaagagagtgagtattgatatatgtgcaattatgtttaaagtagatgttgagagaacttggatttcgtgaataatatttat aggttgagaattatgagttaaggaggtgagagtacgtctta-3’ (SEQ ID NO:937) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the left border comprises 5’- tggcattatatattgtggtgtaaatataattttttttatgaataacaatgcacaatattggatgtgtgttttagccttggatgacaagta gtgagaggagagggaagagagtgagtattgatatatgtgcaattatgtttaaagtagatgttgagagaacttggatttcgtgaat aatatttataggttgagaattatgagttaaggaggtgagagtacgtctta-3’ (SEQ ID NO:973) or a 5’ fragment thereof comprising 5’-tggcattatatattgtggtgtaaat-3’ (SEQ ID NO:974), or wherein the 5’ t is replaced with a c (SEQ ID NO:975). In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’- cagcatatattcttgtgtaaattaggactgcatcatcgaccaatgtttaaccaacatctttataatgacataatatttcacttgacatt gtaatatccaagacataggaattgttactagtaaaaggacatgaaaattgtacctgtgtgtagatataaacttggagaatatttag gacaatcagttatgtcatgtcgcataacatcatcttttaacttgaatccatggtgaatctctttgaagtttggcaaacttctgagtgtt atacatagcaggttatgtagtagtactttttctccatccttaacaatcccttcttga-3’ (SEQ ID NO:938) or a 5’ fragment thereof comprising 5’-cagcatatattcttgtgtaaat-3’ (SEQ ID NO:939).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’- tatattcttgtgtaaattaggactgcatcatcgaccaatgtttaaccaacatctttataatgacataatatttcacttgacattgtaatat ccaagacataggaattgttactagtaaaaggacatgaaaattgtacctgtgtgtagatataaacttggagaatatttaggacaatc agttatgtcatgtcgcataacatcatcttttaacttgaatccatggtgaatctctttgaagtttggcaaacttctgagtgttatacata gcaggttatgtagtagtactttttctccatccttaacaatcccttcttga-3’ (SEQ ID NO:940) or a 5’ fragment thereof comprising 5’-tatattcttgtgtaaat-3’ (SEQ ID NO: 941).

In an embodiment, the left border comprises 5’- tggcagcatatattcttgtgtaaattaggactgcatcatcgaccaatgtttaaccaacatctttataatgacataatatttcacttgac attgtaatatccaagacataggaattgttactagtaaaaggacatgaaaattgtacctgtgtgtagatataaacttggagaatattt aggacaatcagttatgtcatgtcgcataacatcatcttttaacttgaatccatggtgaatctctttgaagtttggcaaacttctgagt gttatacatagcaggttatgtagtagtactttttctccatccttaacaatcccttcttga-3’ (SEQ ID NO:976) or a 5’ fragment thereof comprising 5’-tggcagcatatattcttgtgtaaat-3’ (SEQ ID NO:977), or wherein the 5’ t is replaced with a c (SEQ ID NO:978).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-cattatatattgtggtgtaaatataattctttttatga-3’ (SEQ ID NO:942) or a 5’ fragment thereof comprising 5’ -cattatatattgtggtgtaaat-3 ’ (SEQ ID NO: 936).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tatattgtggtgtaaatataattctttttatga-3’ (SEQ ID NO:944) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 142).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-aattatatattgtggtgtaaaaaaattaaat-3’ (SEQ ID NO:945) or a 5’ fragment thereof comprising 5’-aattatatattgtggtgtaaaa-3’ (SEQ ID NO: 946).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tatattgtggtgtaaaaaaattaaat-3’ (SEQ ID NO:947) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-atgataatattgtggtgtaaactttgaccaaaattt-3’ (SEQ ID NO:948) or a 5’ fragment thereof comprising 5’ -atgataatattgtggtgtaaac-3 ’ (SEQ ID NO:949). In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-aatattgtggtgtaaactttgaccaaaattt-3’ (SEQ ID NO:950) or a 5’ fragment thereof comprising 5’-aatattgtggtgtaaac-3’ (SEQ ID NO:385).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’ -caggatatattgtgtttgataag-3’ (SEQ ID NO:951) or a 5’ fragment thereof comprising 5’-caggatatattgtgtttgataa-3’ (SEQ ID NO:952).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tatattgtgtttgataag-3’ (SEQ ID NO:953) or a 5’ fragment thereof comprising 5’-tatattgtgtttgataa-3’ (SEQ ID NO:954).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tattatatattgtggtgtaacttattattta-3’ (SEQ ID NO:955) or a 5’ fragment thereof comprising 5’-tattatatattgtggtgtaact-3’ (SEQ ID NO:956).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tatattgtggtgtaacttattattta-3’ (SEQ ID NO:957) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaact-3’ (SEQ ID NO:314).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-gaatttatattgtggtgtaaatttttgattaa-3’ (SEQ ID NO:958) or a 5’ fragment thereof comprising 5’-gaatttatattgtggtgtaaat-3’ (SEQ ID NO:959).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’ -tatattgtggtgtaaatttttgattaa-3 ’ (SEQ ID NO:960) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-aagcttatattgtggtgtaaagtcttacacatg-3’ (SEQ ID NO: 961) or a 5’ fragment thereof comprising 5’-aagcttatattgtggtgtaaag-3’ (SEQ ID NO:962).

In an embodiment, the part of the left border of the ST-DNA for integration into the genome comprises 5’-tatattgtggtgtaaagtcttacacatg-3’ (SEQ ID NO:963) or a 5’ fragment thereof comprising 5’-tatattgtggtgtaaag-3’ (SEQ ID NO:303).

In an embodiment, the left border of the ST-DNA comprises 5’- tggcattatatattgtggtgtaaatataattctttttatga-3’ (SEQ ID NO:979) or a 5’ fragment thereof comprising 5’ -tggcattatatattgtggtgtaaat-3 ’ (SEQ ID NO:974), or wherein the 5’ t is replaced with a c (SEQ ID NO: 980).

In an embodiment, the left border of the ST-DNA comprises 5’- tggaattatatattgtggtgtaaaaaaattaaat-3’ (SEQ ID NO:981) or a 5’ fragment thereof comprising 5’-tggaattatatattgtggtgtaaaa-3’ (SEQ ID NO:982), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:983 and 1468. In an embodiment, the left border of the ST-DNA comprises 5’- tggatgataatattgtggtgtaaactttgaccaaaattt-3’ (SEQ ID NO:984) or a 5’ fragment thereof comprising 5’-tggatgataatattgtggtgtaaac-3’ (SEQ ID NO:985), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:986 and 1469.

In an embodiment, the left border of the ST-DNA comprises 5’- tggcaggatatattgtgtttgataag-3 ’ (SEQ ID NO:987) or a 5’ fragment thereof comprising 5’- tggcaggatatattgtgtttgataa-3 ’ (SEQ ID NO:988), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:989 and 1470.

In an embodiment, the left border of the ST-DNA comprises 5’- tggtattatatattgtggtgtaacttattattta-3’ (SEQ ID NO: 990) ora 5’ fragment thereof comprising 5’-tggtattatatattgtggtgtaact-3’ (SEQ ID NO:991), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:992 and 1471.

In an embodiment, the left border of the ST-DNA comprises 5’- tgggaatttatattgtggtgtaaatttttgattaa-3’ (SEQ ID NO:993) or a 5’ fragment thereof comprising 5’ -tgggaatttatattgtggtgtaaat-3 ’ (SEQ ID NO:994), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:995 and 1472.

In an embodiment, the left border of the ST-DNA comprises 5’- tggaagcttatattgtggtgtaaagtcttacacatg-3’ (SEQ ID NO:996) or a 5’ fragment thereof comprising 5’-tggaagcttatattgtggtgtaaag-3’ (SEQ ID NO:997), or wherein the 5’ t is replaced with a c, see, for example but not limited to, SEQ ID NOs:998 and 1473.

In an embodiment, the part of the right border of the ST-DNA for integration into the genome comprises 5’- ctacacatcttcgagaagatacggtgctagcctaaggtttgctggtttgcgctttctatgtactgatgcatgaatgcatgggtcac agctaaccatattgggctgaggttgttgccttccgttttggccttatacttcttgcttcttgtagatcctttgcttcctgcagtactatc cctatcagtgtttga-3 ’ (SEQ ID NO:964), or a 3’ fragment thereof comprising 5- atccctatcagtgtttga-3 ’ (SEQ ID NO:965) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the part of the right border of the ST-DNA for integration into the genome comprises 5’ -ggatacaattaatttttttttgaaactatcagtctttga-3’ (SEQ ID NO:966), or a 3’ fragment thereof comprising 5’-gaaactatcagtctttga-3’ (SEQ ID NO:967) or 5’-ttga- 3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the ST-DNA for integration into the genome comprises 5’-tggtcatccagaattagagaaacaatcagtgtttga-3’ (SEQ ID NO:968), or a 3’ fragment thereof comprising 5’-gaaacaatcagtgtttga-3’ (SEQ ID NO:969) or 5’-ttga- 3’ (SEQ ID NO: 62).

In an embodiment, the part of the right border of the ST-DNA for integration into the genome comprises 5’-tgagacatgtatggtgaaactctcagtgtttga-3’ (SEQ ID NO:970), ora 3’ fragment thereof comprising 5’-gaaactctcagtgtttga-3’ (SEQ ID NO:971) or 5’-ttga-3’ (SEQ ID NO:62).

In an embodiment, the right border of the ST-DNA comprises 5’- ctacacatcttcgagaagatacggtgctagcctaaggtttgctggtttgcgctttctatgtactgatgcatgaatgcatgggtcac agctaaccatattgggctgaggttgttgccttccgttttggccttatacttcttgcttcttgtagatcctttgcttcctgcagtactatc cctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:999), or a 3’ fragment thereof comprising 5-atccctatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1000) or 5’- ttgacaggatatattggcgggtaaac-3 ’ (SEQ ID NO:449).

In an embodiment, the right border of the ST-DNA comprises 5’- ggatacaattaatttttttttgaaactatcagtctttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1001), or a 3’ fragment thereof comprising 5’-gaaactatcagtctttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1002) or 5 ’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the right border of the ST-DNA comprises 5’- tggtcatccagaattagagaaacaatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1003), or a 3’ fragment thereof comprising 5’-gaaacaatcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1004) or 5’ -ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 449).

In an embodiment, the right border of the ST-DNA comprises 5’- tgagacatgtatggtgaaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1005), or a 3’ fragment thereof comprising 5’-gaaactctcagtgtttgacaggatatattggcgggtaaac-3’ (SEQ ID NO: 1006) or 5 ’-ttgacaggatatattggcgggtaaac-3’ (SEQ ID NO:449).

In an embodiment, the PT-DNA is a soybean transfer DNA (ST-DNA) and the left border comprises 5’-tggcagcatatattcttgtgtaaat-3’ (SEQ ID NO:977).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggcagcatatattcttgtgtaaat-3 ’ (SEQ ID NO: 1466).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggcattatatattgtggtgtaaat-3 ’ (SEQ ID NO:974).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggcattatatattgtggtgtaaat-3’ (SEQ ID NO: 1467).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggaattatatattgtggtgtaaaa-3’ (SEQ ID NO:982).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggaattatatattgtggtgtaaaa-3 ’ (SEQ ID NO: 1468).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggatgataatattgtggtgtaaac-3’ (SEQ ID NO:985).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggatgataatattgtggtgtaaac-3 ’ (SEQ ID NO: 1469). In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggcaggatatattgtgtttgataa-3 ’ (SEQ ID NO:988).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggcaggatatattgtgtttgataa-3 ’ (SEQ ID NO: 1470).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggtattatatattgtggtgtaact-3 ’ (SEQ ID NO: 991).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggtattatatattgtggtgtaact-3 ’ (SEQ ID NO: 1471).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tgggaatttatattgtggtgtaaat-3 ’ (SEQ ID NO:994).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cgggaatttatattgtggtgtaaat-3 ’ (SEQ ID NO: 1472).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- tggaagcttatattgtggtgtaaag-3 ’ (SEQ ID NO:997).

In an embodiment, the PT-DNA is a ST-DNA and the left border comprises 5’- cggaagcttatattgtggtgtaaag -3’ (SEQ ID NO: 1473).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-cagcatatattcttgtgtaaat-3’ (SEQ ID NO: 939).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-cattatatattgtggtgtaaat-3’ (SEQ ID NO:936).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-aattatatattgtggtgtaaaa-3’ (SEQ ID NO:946).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-atgataatattgtggtgtaaac-3’ (SEQ ID NO:949).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-caggatatattgtgtttgataa-3’ (SEQ ID NO:952).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tattatatattgtggtgtaact-3’ (SEQ ID NO:956).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-gaatttatattgtggtgtaaat-3’ (SEQ ID NO:959).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-aagcttatattgtggtgtaaag-3’ (SEQ ID NO: 962).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaat-3’ (SEQ ID NO: 152).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattcttgtgtaaat-3’ (SEQ ID NO:941). In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaaa-3’ (SEQ ID NO:345).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-aatattgtggtgtaaac-3’ (SEQ ID NO:385).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattgtgtttgataa-3’ (SEQ ID NO:954).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaact-3’ (SEQ ID NO: 314).

In an embodiment, the plant is soybean and PT-DNA has a processed the left border which comprises 5’-tatattgtggtgtaaag-3’ (SEQ ID NO:303).

As a further example, the present invention provides a soybean transfer DNA (ST- DNA) for integration into the genome of a soybean plant, wherein the ST-DNA comprises a polynucleotide of interest and only has soybean DNA, wherein the 5 ’ end of the ST-DNA comprises part of, or has, a left border for integration into the genome, and the 3’ end of the ST-DNA comprises part of a right border for integration into the genome.

In an embodiment, the ST-DNA has one or more of the features defined above in relation to the Ti plasmid.

Transposable Elements and Transposases

In an aspect, the present invention provides transfer DNA (T-DNA), the T-DNA comprising a first component and a second component, wherein i) the first component is a plant transfer DNA (PT-DNA) for integration into the genome of a plant, wherein the PT-DNA comprises a polynucleotide of interest and only has DNA from a plant, wherein the 5’ end of the PT-DNA comprises part of, or has, a left border for integration into the genome, and the 3 ’ end of the PT-DNA comprises part of a right border for integration into the genome, and ii) the second component is within the first component, wherein the second component is a transposable element comprising a polynucleotide encoding a selectable marker.

As used herein, a “transposable element” is a defined polynucleotide that can be inserted and/or excised from a larger polynucleotide and the genome. A PT-DNA of the invention is an example of a transposable element. However, in some examples of the invention, provided is a T-DNA, more specifically a PT-DNA of the invention, which comprises a transposable element within it that is not a T-DNA useful for Agrobacterium mediated gene transfer. Examples of such transposable elements useful for the genetic manipulation of plants are well known in the art and include Piggybac, Ac/Ds, Gypsy, Copia, LINES and SINES (see, for example, Quesneville, 2020; Yusa, 2015; Nishizawa- Yokoi and Toki, 2023).

At the ends of a transposable element an inverted terminal repeat is usually present, although the 5’ repeat does not have to be identical the 3’ repeat. Examples of terminal ends for a Piggybac transposable element are provided as SEQ ID NO’s 1388, 1391, 1397, and 1398.

As used herein, a transposase is an enzyme that facilitates the insertion and/or excision of the transposable element. Examples include the Piggybac transposase family including mutants and variants such as the Hyperactive piggyBac transposase (hyPBase) (Eckermann et al 2018) and those described in US9783790 or US 10,415,022, Ac/Ds and DAYSLEEPER.

In an embodiment, the transposase is a Piggybac transposase. This includes the various variants of Piggybac such as HyperPBase (see, for example, Yusa, 2015).

The Piggybac transposon system employs a genetically engineered transposase enzyme to insert a gene into a cell's genome. During transposition, the Piggybac transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites (Yusa, 2011 and 2015; Nishizawa-Yokoi and Toki, 2023; WO 2010/099301; WO 2024/211509).

Examples of selectable markers useful for the invention include, but are not limited to, herbicide resistance (tolerance) or antibiotic resistance.

The herbicide may be, for example, glufosinate, glufosinate -ammonium, bromoxynil, glyphosate, sulfonylureas and/or chlorsulfuron.

In another embodiment, the herbicide resistance gene is the Bar or the Pat gene.

Antibiotics, and resistance genes thereto, are well described in the art, and include those belonging to the penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfoamides, glycopeptides, aminoglycosides, carbapenems classes and the like. The antibiotic may be selected from hygromycin, bleomycin, streptomycin, spectinomycin, G418, kanamycin, ampicillin, amoxicillin, doxycycline, ciprofloxacin and/or clindamycin.

In another embodiment, the antibiotic resistance gene is neomycin phosphotransferase II (nptll) gene.

In an embodiment, the second component (transposable element) is within or directly adjacent (i.e. no nucleotides between the 3’ end of the left border and the 5’ end of the transposable element) to the left border of the PT-DNA. In an embodiment, the second component (transposable element) is within 1,000 bp of the 5’ end of the first component. In an embodiment, the second component (transposable element) is within 500 bp of the 5’ end of the first component. In an embodiment, the second component (transposable element) is within 250 bp of the 5’ end of the first component. In an embodiment, the second component (transposable element) is within 200 bp of the 5 ’ end of the first component. In an embodiment, the second component (transposable element) is within 150 bp of the 5’ end of the first component. In an embodiment, the second component (transposable element) is within 100 bp of the 5’ end of the first component. In an embodiment, the second component (transposable element) is within 50 bp of the 5’ end of the first component.

In an embodiment, the second component (transposable element) is within a region which is not part of the polynucleotide of interest, such as a stuffer region (which is also plant DNA). In an embodiment, the second component (transposable element) is within a region which is part of the polynucleotide of interest adjacent to the left border, such as an untranslated region whereby the selectable marker is present if the full construct DNA or PT-DNA is integrated into the plant genome.

In an embodiment, when present in a plant cell the second component can be excised from the T-DNA by a transposase leaving only the PT-DNA. More specifically, a PT-DNA of the invention remains. It will be understood the PT-DNA in the plant has processed left and right borders.

In an alternate embodiment, when present in a plant the second component can be excised from the T-DNA by a transposase leaving a small region of the second component such as lObp or less, 9bp or less, 8bp or less, 7bp or less, 6bp or less, preferably 5bp or less, 4bp or less, 3bp or less, 2bp or less or a single bp. Similarly, the terms “absence” and “lacking” when used in relation to the second component can refer to the entire second component having been removed or a small region as defined above remaining.

Also provided is a plant having a T-DNA of the invention comprising a transposable element. Consistent with the present disclosure, the T-DNA will typically have processed 5’ and 3’ ends as defined herein.

The present invention provides a method of producing a genetically modified plant, the method comprising crossing a plant having a T-DNA of the invention comprising a transposable element with a transgenic plant expressing a transposase which is capable of excising the transposable element. The plants produced from the cross, or progeny thereof, can be used to identify plants where the transposase has removed, completely or partially (such as having a “small region” remaining as described above), the transposable element. Such plants can be further crossed and screened to produce a plant having a PT-DNA of the invention but lacking the transgene expressing the transposase. In an embodiment, the method produces a plant which only has DNA from that Genus, that species or a sexually compatible species.

The present invention also provides a method of identifying a plant having a T- DNA of the invention comprising a transposable element. Standard methods can be used to analyse a sample from the plant for the T-DNA or a transcription product thereof such as an mRNA or protein expressed from the polynucleotide of interest.

The present invention also provides a method of identifying a plant having a T- DNA or PT-DNA of the invention lacking the transposable element. Standard methods can be used to analyse a sample from the plant for the T-DNA or a transcription product thereof such as an mRNA or protein expressed from the polynucleotide of interest. Similarly, a sample can be screened using routine procedures to determine if a plant also lacks the selectable marker, or the polynucleotide encoding therefor.

Polynucleotides and Genes

The present invention refers to various polynucleotides. As used herein, a "polynucleotide" or "nucleic acid" or "nucleic acid molecule" means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. Less preferred polynucleotides include tRNA, siRNA, shRNA and hpRNA. It may be DNA or RNA of cellular, genomic or synthetic origin, for example made on an automated synthesizer, and may be combined with carbohydrate, lipids, protein or other materials, labelled with fluorescent or other groups, or attached to a solid support to perform a particular activity defined herein, or comprise one or more modified nucleotides not found in nature, well known to those skilled in the art. The polymer may be single -stranded, essentially double -stranded or partly double -stranded. Basepairing as used herein refers to standard basepairing between nucleotides, including G:U basepairs. "Complementary" means two polynucleotides are capable of basepairing (hybridizing) along part of their lengths, or along the full length of one or both. A "hybridized polynucleotide" means the polynucleotide is actually basepaired to its complement. The term "polynucleotide" is used interchangeably herein with the term "nucleic acid".

As used herein, the term "gene" includes any deoxyribonucleotide sequence which includes a protein coding region or which is transcribed in a cell but not translated, as well as associated non-coding and regulatory regions. Such associated regions are typically located adjacent to the coding region or the transcribed region on both the 5’ and 3’ ends for a distance of about 2 kb on either side. In this regard, the gene may include control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals in which case the gene is referred to as a "chimeric gene". The sequences which are located 5 ’ of the coding region and which are present on the mRNA are referred to as 5 ’ nontranslated sequences. The sequences which are located 3’ or downstream of the coding region and which are present on the mRNA are referred to as 3 ’ non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene.

A genomic form or clone of a gene containing the transcribed region may be interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences", which may be either homologous or heterologous with respect to the “exons” of the gene. An "intron" as used herein is a segment of a gene which is transcribed as part of a primary RNA transcript but is not present in the mature mRNA molecule. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. "Exons" as used herein refer to the DNA regions corresponding to the RNA sequences which are present in the mature mRNA or the mature RNA molecule in cases where the RNA molecule is not translated. An mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above. A gene may be introduced into an appropriate vector for extrachromosomal maintenance in a cell or, preferably, for integration into the host genome.

As used herein, a "chimeric gene" refers to any gene that comprises covalently joined sequences that are not found joined in nature. Typically, a chimeric gene comprises regulatory and transcribed or protein coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. In an embodiment, the protein coding region of a polynucleotide of the invention is operably linked to a promoter or polyadenylation/terminator region which is heterologous to the native gene, thereby forming a chimeric gene. The term "endogenous" is used herein to refer to a substance that is normally present or produced in an unmodified plant at the same developmental stage as the plant under investigation. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, "recombinant nucleic acid molecule", "recombinant polynucleotide" or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA/RNA technology. The terms "foreign polynucleotide" or "exogenous polynucleotide" or "heterologous polynucleotide" and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulation.

Foreign or exogenous genes may be genes that are inserted into a non-native organism or cell, native genes introduced into a new location within the native host, or chimeric genes. Alternatively, foreign or exogenous polynucleotides may be the result of editing the genome of the organism or cell, or progeny derived therefrom. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The term "genetically modified" includes introducing genes into cells by transformation or transduction, gene editing, mutating genes in cells and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny.

Furthermore, the term "exogenous" in the context of a polynucleotide (nucleic acid) refers to a polynucleotide whose presence in a cell has come from external means, it can include introducing a polynucleotide in a cell that does not naturally comprise the polynucleotide or increasing the copy number of a polynucleotide within a cell by introducing one or more copies of the polynucleotide into a cell comprising an endogenous polynucleotide. The cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide, for example an exogenous polynucleotide which increases the expression of an endogenous polypeptide, or a cell which in its native state does not produce the polypeptide. Increased production of a polypeptide of the invention is also referred to herein as “over-expression”. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically, such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest. The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 2,100 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 2,100 nucleotides. Preferably, the query sequence is at least 2,600 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 2,600 nucleotides. Even more preferably, the query sequence is at least 2,800 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 2,800 nucleotides. Even more preferably, the GAP analysis aligns two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99. 1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In a further embodiment, the present invention relates to polynucleotides which are substantially identical to those specifically described herein. As used herein, with reference to a polynucleotide the term "substantially identical" means the substitution of one or a few (for example 2, 3, or 4) nucleotides whilst maintaining at least one activity of the native protein encoded by the polynucleotide. In addition, this term includes the addition or deletion of nucleotides which results in the increase or decrease in size of the encoded native protein by one or a few (for example 2, 3, or 4) amino acids whilst maintaining at least one activity of the native protein encoded by the polynucleotide.

The present invention also relates to the use of oligonucleotides, for instance in methods of screening for a polynucleotide of, or encoding a polypeptide of, the invention. As used herein, "oligonucleotides" are polynucleotides up to 50 nucleotides in length. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. They can be RNA, DNA, or combinations or derivatives of either. Oligonucleotides are typically relatively short single stranded molecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length. When used as a guide for a genome editing enzyme (e.g. an endonuclease such as CRISPR Cas9), probe or as a primer in an amplification reaction, the minimum size of such an oligonucleotide is the size required for the formation of a stable hybrid between the oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 22 nucleotides, even more preferably at least 25 nucleotides in length. Oligonucleotides of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule. Examples of oligonucleotides of the invention include those with a nucleotide sequence provided as any one of SEQ ID NOs: I to 12 and 1031 to 1044.

As those skilled in the art would be aware, the sequence of the oligonucleotide primers described herein can be varied to some degree without effecting their usefulness for the methods of the invention. A "variant" of an oligonucleotide disclosed herein (also referred to herein as a "primer" or "probe" depending on its use) useful for the methods of the invention includes molecules of varying sizes of, and/or are capable of hybridising to the genome close to that of, the specific oligonucleotide molecules defined herein. For example, variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region. Furthermore, a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise the target region. In addition, variants may readily be designed which hybridise close (for example, but not limited to, within 50 nucleotides or within 100 nucleotides) to the region of the genome where the specific oligonucleotides defined herein hybridise.

The present invention includes oligonucleotides that can be used as, for example, guides for RNA-guided endonucleases, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Probes and/or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.

Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or genome editing on the nucleic acid). A variant of a polynucleotide or an \T1 oligonucleotide of the invention includes molecules of varying sizes of, and/or are capable of hybridising to, the wheat genome close to that of the reference polynucleotide or oligonucleotide molecules defined herein. For example, variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region. Furthermore, a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise to the target region. In addition, variants may readily be designed which hybridise close to, for example to within 50 nucleotides, the region of the plant genome where the specific oligonucleotides defined herein hybridise. In particular, this includes polynucleotides which encode the same polypeptide or amino acid sequence but which vary in nucleotide sequence by redundancy of the genetic code. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants.

Nucleic Acid Constructs

The present invention includes nucleic acid constructs comprising polynucleotides discussed herein, and vectors and host cells containing these, methods of their production and use, and uses thereof. The present invention refers to elements which are operably connected or linked. "Operably connected" or "operably linked" and the like refer to a linkage of polynucleotide elements in a functional relationship. Typically, operably connected nucleic acid sequences are contiguously linked and, where necessary to join two protein coding regions, contiguous and in reading frame. A coding sequence is "operably connected to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single RNA, which if translated is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.

As used herein, the term "cis-acting sequence", "cis-acting element" or "cis- regulatory region" or "regulatory region" or similar term shall be taken to mean any sequence of nucleotides, which when positioned appropriately and connected relative to an expressible genetic sequence, is capable of regulating, at least in part, the expression of the genetic sequence. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In preferred embodiments of the present invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence. "Operably connecting" a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein-encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription of that polynucleotide. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide which is approximately the same as the distance between that promoter and the protein coding region it controls in its natural setting; i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.

"Promoter" or "promoter sequence" as used herein refers to a region of a gene, generally upstream (5') of the RNA encoding region, which controls the initiation and level of transcription in the cell of interest. A "promoter" includes the transcriptional regulatory sequences of a classical genomic gene, such as a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily (for example, some PolIII promoters), positioned upstream of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected.

"Constitutive promoter" refers to apromoter that directs expression of an operably linked transcribed sequence in many or all tissues of an organism such as a plant. The term constitutive as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in level is often detectable. "Selective expression" as used herein refers to expression almost exclusively in specific organs of, for example, the plant, such as, for example, endosperm, embryo, leaves, fruit, tubers or root. In a preferred embodiment, a promoter is expressed selectively or preferentially in leaves and/or stems of a plant, preferably a cereal plant. Selective expression may therefore be contrasted with constitutive expression, which refers to expression in many or all tissues of a plant under most or all of the conditions experienced by the plant.

Selective expression may also result in compartmentation of the products of gene expression in specific plant tissues, organs or developmental stages such as adults or seedlings. Compartmentation in specific subcellular locations such as the plastid, cytosol, vacuole, or apoplastic space may be achieved by the inclusion in the structure of the gene product of appropriate signals, eg. a signal peptide, for transport to the required cellular compartment, or in the case of the semi-autonomous organelles (plastids and mitochondria) by integration of the transgene with appropriate regulatory sequences directly into the organelle genome.

A "tissue-specific promoter" or "organ-specific promoter" is a promoter that is preferentially expressed in one tissue or organ relative to many other tissues or organs, preferably most if not all other tissues or organs in, for example, a plant. Typically, the promoter is expressed at a level 10-fold higher in the specific tissue or organ than in other tissues or organs.

In an embodiment, the promoter is a stem-specific promoter, a leaf-specific promoter or a promoter which directs gene expression in an aerial part of the plant (at least stems and leaves) (green tissue specific promoter) such as a ribulose- 1,5- bisphosphate carboxylase oxygenase (RUBISCO) promoter.

Examples of stem-specific promoters include, but are not limited to those described in US 5,625,136, and Bam et al. (2008).

The promoters contemplated by the present invention may be native to gene of interest, native to the host plant to be transformed or may be derived from an alternative plant source for example a sexually compatible relative, where the region is functional in the host plant. In an embodiment, a polynucleotide(s) of interest in a PT-DNA of the invention will have a promoter from a sexually compatible plant to which the PT-DNA is to be inserted.

Where relevant, other sources include the Agrobacterium T-DNA genes, such as the promoters of genes for the biosynthesis of nopaline, octapine, mannopine, or other opine promoters, tissue specific promoters (see, e.g., US 5,459,252 and WO 91/13992); promoters from viruses (including host specific viruses), or partially or wholly synthetic promoters. Numerous promoters that are functional in mono- and dicotyledonous plants are well known in the art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkel et al., 1983; Barker et al., 1983); including various promoters isolated from plants and viruses such as the cauliflower mosaic virus promoter (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989, supra) and US 5,164,316.

Alternatively, or additionally, the promoter may be an inducible promoter or a developmentally regulated promoter which is capable of driving expression of the introduced polynucleotide at an appropriate developmental stage of the, for example, plant. Other c/.s-acting sequences which may be employed include transcriptional and/or translational enhancers. Enhancer regions are well known to persons skilled in the art, and can include an ATG translational initiation codon and adjacent sequences. When included, the initiation codon should be in phase with the reading frame of the coding sequence relating to the foreign or exogenous polynucleotide to ensure translation of the entire sequence if it is to be translated. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from a foreign or exogenous polynucleotide. The sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.

The nucleic acid construct of the present invention may comprise a 3' nontranslated sequence from about 50 to 1,000 nucleotide base pairs which may include a transcription termination sequence. A 3' non-translated sequence may contain a transcription termination signal which may or may not include a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing. A polyadenylation signal functions for addition of polyadenylic acid tracts to the 3' end of a mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. Transcription termination sequences which do not include a polyadenylation signal include terminators for Poll or PolIII RNA polymerase which comprise a run of four or more thymidines. Examples of suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from an octopine synthase (ocs) gene or nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983). Suitable 3' non-translated sequences may also be derived from plant genes such as the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed.

As the DNA sequence inserted between the transcription initiation site and the start of the coding sequence, i.e., the untranslated 5’ leader sequence (5’UTR), can influence gene expression if it is translated as well as transcribed, one can also employ a particular leader sequence. Suitable leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence. For example, such leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as for example described by Joshi (1987).

Vectors

The present invention includes use of vectors for manipulation or transfer of genetic constructs. By "chimeric vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably is doublestranded DNA and contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or capable of integration into the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a cell, is integrated into the genome of the recipient cell and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene, a herbicide resistance gene or other gene that can be used for selection of suitable transformants. Examples of such genes are well known to those of skill in the art.

The nucleic acid construct of the invention can be introduced into a vector, such as a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells.

By "marker gene" is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can "select" based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, cold, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by "screening" (e.g., P-glucuronidase, luciferase, GFP, RUBY or other enzyme activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not have to be linked.

To facilitate identification of transformants, the nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the plant cells of choice. The marker gene and the foreign or exogenous polynucleotide of interest do not have to be linked, since co-transformation of unlinked genes as, for example, described in US 4,399,216 is also an efficient process in plant transformation.

Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptll) gene conferring resistance to kanamycin, strepinomycin resistance gene, paromomycin, G418; a glutathione-S- transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al. (1988), a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5 -methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidA gene encoding a P-glucuronidase (GUS) enzyme for which various chromogenic substrates are known, a P-galactosidase gene encoding an enzyme for which chromogenic substrates are known, an aequorin gene (Prasher et al., 1985), which may be employed in calciumsensitive bioluminescence detection; a green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al., 1986), which allows for bioluminescence detection, and others known in the art. By "reporter molecule" as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into the genome of, for example, the plant. Accordingly, the nucleic acid comprises appropriate elements which allow the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector which can be incorporated into the genome of a plant cell.

One embodiment of the present invention includes a recombinant vector, which includes at least one polynucleotide molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Ge Ivin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5’ and 3’ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

The level of a protein of the invention may be modulated by increasing the level of expression of a nucleotide sequence that codes for the protein in a plant cell, or decreasing the level of expression of a gene encoding the protein in the plant, leading to modified pathogen resistance. The level of expression of a gene may be modulated by altering the copy number per cell, for example by introducing a synthetic genetic construct comprising the coding sequence and a transcriptional control element that is operably connected thereto and that is functional in the cell. A plurality of transformants may be selected and screened for those with a favourable level and/or specificity of transgene expression arising from influences of endogenous sequences in the vicinity of the transgene integration site. A favourable level and pattern of transgene expression is one which results in a substantial modification of pathogen resistance or other phenotype. Alternatively, a population of mutagenized seed or a population of plants from a breeding program may be screened for individual lines with altered pathogen resistance or other phenotype associated with pathogen resistance.

Polypeptides

Genetically modified plants and host cells, such as transgenic plants or cells, of the invention may comprise an exogenous polynucleotide encoding a polypeptide of the invention. In these instances, the plants and cells produce a recombinant polypeptide. The term "recombinant" in the context of a polypeptide refers to the polypeptide encoded by an exogenous polynucleotide when produced by a cell, which polynucleotide has been introduced into the cell or a progenitor cell by recombinant DNA or RNA techniques such as, for example, transformation and/or gene editing. The cell may comprises a non- endogenous gene that causes an altered amount of the polypeptide to be produced. In an embodiment, the endogenous gene has been modified to increase its expression, such as by replacing the native promoter with one that results in increased gene expression. In an embodiment, the number of copies of a polynucleotide endogenous to the plant is increased.

The terms "polypeptide" and "protein" are generally used interchangeably.

In an aspect, a DNA construct of the invention encodes Sr33, Sr26, Sr22, Sr62 and Sr 13.

Examples of other plant pathogen resistance polypeptides that can be encoded by a DNA construct, T-DNA of the invention or PT-DNA of the invention include, Lr34, Lrl, Lr3, Lr2a, Lr3ka, Lrl l, Lrl3, Lrl6, Lrl7, Lrl8, Lr21, LrB, Sr61, Lr67, Sr50, Sr2 and Sr35, as well as those described in Mapurangaet al. (2022) orHossenini et al. (2023).

As used herein, the term “Sr33” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 13. Some variants of the Sr33 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia graminis such as the Ug99 group of races of Puccinia graminis f. sp. tritici (WO 2014/194371; Periyannan et al., 2013).

As used herein, the term “Sr26” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 14. Some variants of the Sr26 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia graminis (WO 2020/107057; Zhang et al., 2019).

As used herein, the term “Sr22” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 15. Some variants of the Sr22 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia graminis (Bukhari et al., 2020).

As used herein, the term “Sr62” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 16. Some variants of the Sr62 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia graminis (WO 2023/056269; Yu et al., 2022).

As used herein, the term “Sr 13” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 17. Some variants of the Sr 13 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia graminis (Gill et al., 2021).

In an aspect, a DNA construct of the invention encodes YrUl, Yr36, Yr46, Yr5 and Yrl5.

Examples of other plant pathogen resistance polypeptides that can be encoded by a DNA construct, T-DNA of the invention or PT-DNA of the invention include, Lr34, Lrl, Lr3, Lr2a, Lr3ka, Lrl l, Lrl 3. Lrl6, Lrl7, Lrl8, Lr21, LrB, Sr61, Sr50, Sr2 and Sr35, as well as those described in Mapuranga et al. (2022) or Hossenini et al. (2023).

As used herein, the term “YrUl” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 1271. Some variants of the YrUl protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia striiformis such as Puccinia striiformis f. sp. Tritici (Wang et al., 2020).

As used herein, the term “Yr36” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 1272. Some variants of the Yr36 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia striiformis such as Puccinia striiformis f. sp. Tritici (Fu et al., 2009).

As used herein, the term “Yr46”, also known as Lr67, relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 1273. Some variants of the Yr46 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia striiformis such as Puccinia striiformis f. sp. Tritici (WO 2015/024066; US20220127635A1).

As used herein, the term ‘Yr5” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 1274. Some variants of the Yr5 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia striiformis such as Puccinia striiformis f. sp. Tritici (Marchal et al., 2018).

As used herein, the term ‘Yr 15” relates to a protein family which share high primary amino acid sequence identity, for example at least 85%, at least 90% or at least 95% identity with the amino acid sequences provided as SEQ ID NO: 1275. Some variants of the Yr 15 protein family, when expressed in a plant, confer upon the plant resistance to at least one strain of Puccinia striiformis such as Puccinia striiformis f. sp. Tritici (Klymiuk et al., 2018).

The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 700 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 700 amino acids. More preferably, the query sequence is at least 800 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 800 amino acids. Even more preferably, the query sequence is at least 900 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 900 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length.

As used herein a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide such as when expressed in a plant, such as wheat, confers (enhanced) resistance to one or more fungal pathogen(s) when compared to a corresponding wild-type plant not expressing the polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity but are preferably at least 350 or at least 374 amino acid residues long. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.

With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99. 1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides discussed herein can be prepared by introducing appropriate nucleotide changes into a polynucleotide encoding the polypeptide, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired characteristics. Preferred amino acid sequence mutants have one, two, three, four or less than 10 amino acid changes relative to the reference wildtype polypeptide.

Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using CRISPR Cas 9 or alternative endonucleases, directed evolution, rational design strategies or mutagenesis (see below). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if, when expressed in a plant, such as wheat, confer (enhanced) resistance to one or more fungal pathogen(s). For instance, the method may comprise producing a transgenic plant expressing the mutated/altered DNA and determining the effect of the pathogen on the growth of the plant.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Where it is desirable to maintain a certain activity it is preferable to make no, or only conservative substitutions, at amino acid positions which are highly conserved in the relevant protein family. Examples of conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

In a preferred embodiment a mutant/variant polypeptide has one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. In a preferred embodiment, the changes are not in one or more of the motifs which are highly conserved between the different polypeptides provided herewith. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.

Directed Evolution

In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out variants that have the desired qualities, for example, increased activity. Further rounds of mutation and selection are then applied. A typical directed evolution strategy involves three steps:

1) Diversification: The gene encoding the protein of interest is mutated and/or recombined at random to create a large library of gene variants. Variant gene libraries can be constructed through error prone PCR (see, for example, Leung, 1989; Cadwell and Joyce, 1992 ), from pools of DNase I digested fragments prepared from parental templates (Slemmer, 1994a; Stemrner, 1994b; Crameri et al., 1998, Coco et al., 2001) from degenerate oligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures of both, or even from undigested parental templates (Zhao et al., 1998; Eggert et al., 2005; Jezequek et ah, 2008) and are usually assembled through PCR. Libraries can also be made from parental sequences recombined ?/? vivo or in vitro by either homologous or non-homologous recombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Variant gene libraries can also be constructed by sub-cloning a gene of interest into a suitable vector, transforming the vector into a "mutator" strain such as the E. colt

XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. Variant gene libraries can also be constructed by subjecting the gene of interest to DNA shuffling (i.e., in vitro homologous recombination of pools of selected mutant genes by random fragmentation and reassembly) as broadly described by Harayama (1998).

Table 1. Exemplary substitutions

2) Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. Screens enable the identification and isolation of high-performing mutants by hand, while selections automatically eliminate all nonfunctional mutants. A screen may involve screening for the presence of known conserved amino acid motifs. Alternatively, or in addition, a screen may involve expressing the mutated polynucleotide in a host organsim or part thereof and assaying the level of activity.

3) Amplification: The variants identified in the selection or screen are replicated many fold, enabling researchers to sequence their DNA in order to understand what mutations have occurred.

Together, these three steps are termed a "round" of directed evolution. Most experiments will entail more than one round. In these experiments, the "winners" of the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or polynucleotide mutants are characterized using biochemical methods.

Rational Design

A protein can be designed rationally, on the basis of known information about protein structure and folding. This can be accomplished by design from scratch (de novo design) or by redesign based on native scaffolds (see, for example, Hellinga, 1997; and Lu and Berry, Protein Structure Design and Engineering, Handbook of Proteins 2, 1153- 1157 (2007)). Protein design typically involves identifying sequences that fold into a given or target structure and can be accomplished using computer models. Computational protein design algorithms search the sequence-conformation space for sequences that are low in energy when folded to the target structure. Computational protein design algorithms use models of protein energetics to evaluate how mutations would affect a protein's structure and function. These energy functions typically include a combination of molecular mechanics, statistical (i.e. knowledge-based), and other empirical terms. Suitable available software includes IPRO (Interative Protein Redesign and Optimization), EGAD (A Genetic Algorithm for Protein Design), Rosetta Design, Sharpen and Abalone.

Recombinant Cells

Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention, or progeny cells thereof. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, particle bombardment/biolistics, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. In an embodiment, a T-DNA of the invention is used in Agrobacterium mediated gene transfer (AMGT) to produce a recombinant cell.

A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred host cells are plant cells, more preferably cells of a cereal plant, more preferably barley or wheat cells, and even more preferably a wheat cell.

Plant cells may be from a monocot such as a maize, wheat, oat, barley or banana, or from a dicot such as a soybean, grape vine, potato or tomato.

Genome Editing

Endonucleases can be used to generate single strand or double strand breaks in genomic DNA. The genomic DNA breaks in eukaryotic cells are repaired using non- homologous end joining (NHEJ) or homology directed repair (HDR) pathways. NHEJ may result in imperfect repair resulting in unwanted mutations and HDR can enable precise gene insertion by using an exogenous supplied repair DNA template. CRISPR- associated (Cas) proteins have received significant interest although transcription activator-like effector nucleases (TALENs) and zinc -finger nucleases are still useful, the CRISPR-Cas system offers a simpler, versatile and cheaper tool for genome modification (Doudna and Charpentier, 2014).

The CRISPR-Cas systems are classed into three major groups using various nucleases or combinations on nuclease. In class 1 CRISPR-Cas systems (types I, III and IV), the effector module consists of a multi-protein complex whereas class 2 systems (types II, V and VI) use only one effector protein (Makarova et al., 2015). Cas includes a gene that is coupled or close to or localised near the flanking CRISPR loci. Haft et al. (2005) provides a review of the Cas protein family.

The nuclease is guided by the synthetic small guide RNA (sgRNAs or gRNAs) that may or may not include the tracRNA resulting in a simplification of the CRISPR- Cas system to two genes; the endonuclease and the sgRNA (Jinek et al. 2012). The sgRNA is typically under the regulatory control of a U3 or U6 small nuclear RNA promoter. The sgRNA recognises the specific gene and part of the gene for targeting. The protospacer adjacent motif (PAM) is adjacent to the target site constraining the number of potential CRISPR-Cas targets in a genome although the expansion of nucleases also increases the number of PAM’s available. There are numerous web tools available for designing gRNAs including CHOPCHOP (http://chopchop.cbu.uib.no), CRISPR design https://omictools.com/crispr-design-tool, E-CRISP http://www.e- crisp.org/E-CRISP/, Geneious or Benchling https://benchling.com/crispr.

CRISPR-Cas systems are the most frequently adopted in eukaryotic work to date using a Cas9 effector protein typically using the RNA-guided Streptococcus pyogenes Cas9 or an optimised sequence variant in multiple plant species (Luo et al., 2016). Luo et al. (2016) summarises numerous studies where genes have been successfully targeted in various plant species to give rise to indels and loss of function mutant phenotypes in the endogenous gene open reading frame and/or promoter. Due to the cell wall on plant cells the delivery of the CRISPR-Cas machinery into the cell and successful transgenic regenerations have used Agrobacterium tumefaciens infection (Luo et al., 2016) or plasmid DNA particle bombardment or biolistic delivery. Vectors suitable for cereal transformation include pCXUNcas9 (Sun et al, 2016) or pYLCRISPR/Cas9Pubi-H available from Addgene (Ma et al., 2015, accession number KR029109.1).

Alternative CRISPR-Cas systems refer to effector enzymes that contain the nuclease RuvC domain but do not contain the HNH domain including Casl2 enzymes including Casl2a, Casl2b, Casl2f, Cpfl, C2cl, C2c3, and engineered derivatives. Cpfl creates double-stranded breaks in a staggered manner at the PAM-distal position and being a smaller endonuclease may provide advantages for certain species (Begemann et al., 2017). Other CRISPR-Cas systems include RNA-guided RNAses including Casl3, Casl3a (C2c2), Casl3b, Casl3c.

Sequence Insertion or Integration

The CRISPR-Cas system can be combined with the provision of a nucleic acid sequence to direct homologous repair for the insertion of a sequence into a genome. Targeted genome integration of plant transgenes enables the sequential addition of transgenes at the same locus. This “cis gene stacking” would greatly simplify subsequent breeding efforts with all transgenes inherited as a single locus. When coupled with CRISPR/Cas9 cleavage of the target site the transgene can be incorporated into this locus by homology-directed repair that is facilitated by flanking sequence homology. This approach can be used to rapidly introduce new alleles without linkage drag or to introduce allelic variants that do not exist naturally. Nickases

The CRISPR-Cas II systems use a Cas9 nuclease with two enzymatic cleavage domains a RuvC and HNH domain. Mutations have been shown to alter the double strand cutting to single strand cutting and resulting in a technology variant referred to as a nickase or a nuclease-inactivated Cas9. The RuvC subdomain cleaves the non- complementary DNA strand and the HNH subdomain cleaves that DNA strand complementary to the gRNA. The nickase or nuclease-inactivated Cas9 retains DNA binding ability directed by the gRNA. Mutations in the subdomains are known in the art for example S.pyogenes Cas9 nuclease with a D10A mutation or H840A mutation.

Genome Base Editing or Modification

Base editors have been created by fusing a deaminase with a Cas9 domain (W O 2018/086623). By fusing the deaminase can take advantage of the sequence targeting directed by the gRNA to make targeted cytidine (C) to uracil (U) conversion by deamination of the cytidine in the DNA. The mismatch repair mechanisms of the cell then replace the U with a T. Suitable cytidine deaminases may include APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G and CDA1. Further, the Cas9-deaminase fusion may be a mutated Cas9 with nickase activity to generate a single strand break. It has been suggested that the nickase protein was potentially more efficient in promoting homology-directed repair (Luo et al., 2016).

Vector Free Genome Editing or Genome Modification

More recently methods to use vector free approaches using Cas9/sgRNA ribonucleoproteins have been described with successful reduction of off-target events. The method requires in vitro expression of Cas9 ribonucleoproteins (RNPs) which are transformed into the cell or protoplast and does not rely on the Cas9 being integrated into the host genome, thereby reducing the undesirable side cuts that has been linked with the random integration of the Cas9 gene. Only short flanking sequences are required to form a stable Cas9 and sgRNA stable ribonucleoprotein in vitro. Woo et al. (2015) produced pre-assembled Cas9/sgRNA protein/RNA complexes and introduced them into protoplasts of Arabidopsis. rice, lettuce and tobacco and targeted mutagenesis frequencies of up to 45% observed in regenerated plants. RNP and in vitro demonstrated in several species including dicot plants (Woo et al., 2015), and monocots maize (Svitashev et al., 2016) and wheat (Liang et al., 2017). Genome editing of plants using CRISPR-Cas 9 in vitro transcripts or ribonucleoproteins are fully described in Liang et al. (2018) and Liang et al. (2019). Method for Gene Insertion

The method comprises introducing into a plant cell the DNA sequence of interest referred to as the donor DNA, the endonuclease and sgRNA gene targeting the site of integration. The endonuclease generates a break in the target site allowing the first and second regions of homology of the donor DNA to undergo homologous recombination with their corresponding genomic regions of homology. The cut genomic DNA acts as an acceptor of the DNA sequence. The resulting exchange of DNA between the donor and the genome results in the integration of the polynucleotide of interest of the donor DNA into the strand break in the target site in the plant genome, thereby altering the original target site and producing an altered genomic sequence.

The donor DNA may be introduced by any means known in the art. For example, a plant having a target site is provided. The donor DNA may be provided to the plant by known transformation methods including, Agrobacterium-mediated transformation or biolistic particle bombardment. The RNA guided Cas or other endonuclease cleaves at the target site, the donor DNA is inserted into the transformed plant's genome.

Genetically Modified Plants

The term "plant" as used herein as a noun refers to whole plants and refers to any member of the Kingdom Plantae, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plantlets and germinated seeds from which roots and shoots have emerged are also included within the meaning of "plant". The term "plant parts" as used herein refers to one or more plant tissues or organs which are obtained from a plant and which comprises genomic DNA of the plant. Plant parts include vegetative structures (for example, leaves, stems), roots, floral organs/structures, seed (including embryo, cotyledons, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same. The term "plant cell" as used herein refers to a cell obtained from a plant or in a plant and includes protoplasts or other cells derived from plants, gamete-producing cells, and cells which regenerate into whole plants. Plant cells may be cells in culture. By "plant tissue" is meant differentiated tissue in a plant or obtained from a plant ("explant") or undifferentiated tissue derived from immature or mature embryos, seeds, roots, shoots, fruits, tubers, pollen, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as calli. Exemplary plant tissues in or from seeds are cotyledon, embryo and embryo axis. The invention accordingly includes plants and plant parts and products comprising these.

As used herein, the term "seed" refers to "mature seed" of a plant, which is either ready for harvesting or has been harvested from the plant, such as is typically harvested commercially in the field, or as "developing seed" which occurs in a plant after fertilisation and prior to seed dormancy being established and before harvest.

As noted above, a genetically modified plant of the invention may be a transgenic plant. A "transgenic plant" as used herein refers to a plant that contains an introduced nucleic acid construct. That is, transgenic plants (transformed plants) contain genetic material (a transgene) that they did not contain prior to the transformation. The transgene may include genetic sequences obtained from or derived from a plant cell, or another plant cell, or a synthetic sequence which may be synthesised in the plant cell or external to the plant cell. Typically, the transgene of the invention has been introduced into the plant by human manipulation such as, for example, by Agrobacterium mediated transformation. The genetic material is preferably stably integrated into the genome of the plant. The introduced genetic material may comprise sequences that naturally occur in the same species but in a different genomic location or rearranged order or in a different arrangement of elements, for example an antisense sequence. Plants containing such sequences are included herein in "transgenic plants".

As used herein, the term "compared to a corresponding wild-type plant", or similar phrases, refers to a plant or grain which comprises at least 85%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99%, of the genotype of a plant or grain of the invention but does not have the genetic modification(s) of interest which for example reduces the pathogenicity of the fungal pathogen on the plant. Preferably, the corresponding wildtype plant is of the same cultivar or variety as the progenitor of the genetically modified plant of interest, or a sibling plant line which lacks the construct, often termed a "segregant", or a plant of the same cultivar or variety transformed with an "empty vector" construct, and may be a non-genetically modified plant. "Wild type", as used herein, refers to a cell, tissue, polypeptide or plant that has not been modified according to the invention. Wild-type cells, tissue, polypeptide or plants may be used as controls to compare levels of expression of an exogenous nucleic acid or the extent and nature of trait modification with cells, tissue or plants modified as described herein. In an embodiment, the wild-type plant is an isogenic plant lacking the genetic modification (s).

Genetically modified plants, as defined in the context of the present invention include progeny of the plants which have been genetically modified, wherein the progeny comprise the genetic modification of interest. Such progeny may be obtained by selffertilisation of the primary genetically modified plant or by crossing such plants with another plant of the same species. This would generally be to modulate the production of at least one protein defined herein in the desired plant or plant organ. Genetically modified plant parts include all parts and cells of said plants comprising the genetic modification such as, for example, cultured tissues, callus and protoplasts.

Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Target plants include, but are not limited to, the following: cereals (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); grapes; beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black-berries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape or other Brassicas, mustard, poppy, olives, sunflowers, safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers).

As used herein, the term "wheat" refers to any species of the Genus Triticum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. Wheat includes "hexapioid wheat" which has genome organization of AABBDD, comprised of 42 chromosomes, and "tetrapioid wheat" which has genome organization of AABB, comprised of 28 chromosomes. Hexapioid wheat includes T. aestivum. T. spelta, T. macha. T. compactum, T. sphaerococcum. T. vavilovii, and interspecies cross thereof. A preferred species of hexapioid wheat is T. aestivum ssp aestivum (also termed "breadwheat"). Tetrapioid wheat includes T. durum (also referred to herein as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross thereof. In addition, the term "wheat" includes potential progenitors of hexapioid or tetrapioid Triticum sp. such as T. uartu, T. monococcum or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T. tauschii (also known as Aegilops squarrosa or Aegilops iauschii) for the D genome. Particularly preferred progenitors are those of the A genome, even more preferably the A genome progenitor is T. monococcum. A wheat cultivar for use in the present invention may belong to, but is not limited to, any of the above-listed species. Also encompassed are plants that are produced by conventional techniques using Triticum sp. as a parent in a sexual cross with a non-Triticum species (such as rye [Secale cereale \). including but not limited to Triticale.

As used herein, the term "barley" refers to any species of the Genus Hordeum, including progenitors thereof, as well as progeny thereof produced by crosses with other species. It is preferred that the plant is of a Hordeum species which is commercially cultivated such as, for example, a strain or cultivar or variety of Hordeum vulgar e or suitable for commercial production of grain.

Genetically modified plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide of the present invention in the desired plant or plant organ. Genetically modified plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

In a preferred embodiment, the genetically modified plants are homozygous for each and every genetic modification that has been introduced (such as a transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced genetic modifications(s), such as, for example, in Fl progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.

In an embodiment, PT-DNA, WT-DNA, ST-DNA of the invention is delivered by Agrobacterium mediated gene transfer (AMGT).

Agrobacterium-mcAxaicA transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium -mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.

Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (editors), Springer-Verlag, New York, (1985): 179-203). Moreover, technological advances in vectors for Agrobacterium -mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi -linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where Agrobacierium-mcA\ c( transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.

Other methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, US 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, US 4,945,050 and US 5,141,131); (3) viral vectors (Clapp, 1993; Uu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).

It is also to be understood that two different genetically modified plants can also be mated/crossed to produce offspring that contain two independently segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out- crossing with a non- genetically modified plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, Breeding Methods for Cultivar Development, J. Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, San Diego, (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Genetically modified embryos and seeds are similarly regenerated. The resulting genetically modified rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the genetic modification(s) is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous genetically modified plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A genetically modified plant of the present invention containing a desired genetic modification is cultivated using methods well known to one skilled in the art.

Methods for transforming dicots, primarily by use of Agrobacterium iumefaciens. and obtaining transgenic plants have been published for cotton (US 5,004,863, US 5,159,135, US 5,518,908); soybean (US 5,569,834, US 5,416,011); Brassica (US 5,463,174); peanut (Cheng et al., 1996); grape (Nakajima et al., 2020) and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat, maize and barley for introducing genetic modification(s) into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, CA 2,092,588, AU 61781/94, AU 667939, US 6,100,447, WO 97/048814, US 5,589,617, US 6,541,257, Ishida et al. (2014) and other methods are set out in WO 99/14314. In an embodiment, transgenic cereal plants such as wheat or barley plants are produced by Agrobacterium tumefaciens mediated transformation procedures. Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts. The regenerable wheat cells are preferably from the scutellum of immature embryos, mature embryos, callus derived from these, or the meristematic tissue.

To confirm the presence of the genetic modification in cells and plants, a polymerase chain reaction (PCR) amplification, sequencing, or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once genetically modified plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

Marker Assisted Selection

Marker assisted selection is a well-recognised method of selecting for heterozygous plants required when backcrossing with a recurrent parent in a classical breeding program. The population of plants in each backcross generation will be heterozygous for the gene of interest normally present in a 1: 1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles of the gene. By extracting DNA from, for example, young shoots and testing with a specific marker for the introgressed desirable trait, early selection of plants for further backcrossing is made whilst energy and resources are concentrated on fewer plants. To further speed up the backcrossing program, the embryo from immature seeds (25 days post anthesis) may be excised and grown up on nutrient media under sterile conditions, rather than allowing full seed maturity. This process, termed "embryo rescue", used in combination with DNA extraction at the three leaf stage and analysis of at least one polynucleotide/polypeptide of the invention that confers upon the plant resistance to one or more fungal pathogen(s), allows rapid selection of plants carrying the desired trait, which may be nurtured to maturity in the greenhouse or field for subsequent further backcrossing to the recurrent parent.

Any molecular biological technique known in the art can be used in the methods of the present invention. Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with suitably labelled probes, single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage or a combination thereof (see, for example, Lemieux, 2000; Langridge et al. , 2001). The invention also includes the use of molecular marker techniques to detect polymorphisms linked to alleles of the (for example) polynucleotide and/or genetic modification of the invention which confers upon the plant resistance to one or more fungal pathogen(s). Such methods include the detection or analysis of restriction fragment length polymorphisms (RFLP), RAPD, amplified fragment length polymorphisms (AFLP) and microsatellite (simple sequence repeat, SSR) polymorphisms. The closely linked markers can be obtained readily by methods well known in the art, such as Bulked Segregant Analysis, as reviewed by Langridge et al., (2001).

In an embodiment, a linked loci for marker assisted selection is at least within IcM, or 0.5cM, or O.lcM, or 0.0 IcM from a gene encoding a polypeptide of the invention.

The "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or "set of primers" consisting of "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are known in the art, and are taught, for example, in "PCR" (M.J. McPherson and S.G Moller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCR can be performed on cDNA obtained from reverse transcribing mRNA isolated from plant cells comprising a polynucleotide and/or genetic modification of the invention which confers upon the plant resistance to one or more fungal pathogen(s). However, it will generally be easier if PCR is performed on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR. Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences. Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon. Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can result in the introduction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences. Primers may also contain additional sequences and/or contain modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence. The terms target or target sequence or template refer to nucleic acid sequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and can be found for example in Ausubel et al., (supra) and Sambrook et al., (supra). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence. Fungal Resistance

A DNA construct of the invention can be used to enhance resistance one or more fungal pathogen(s) in a plant such as a wheat plant.

As used herein, “resistance” is a relative term in that the presence of a polypeptide of the invention (i) reduces the disease symptoms of a plant comprising the polypeptide that confers resistance, relative to a plant lacking the gene, and/or (ii) reduces pathogen reproduction or spread on a plant or within a population of plants comprising the gene. Resistance as used herein is relative to the “susceptible” response of a plant to the same pathogen. Typically, the presence of the resistance gene improves at least one production trait of a plant comprising the gene when infected with the pathogen, such as grain yield, when compared to a corresponding wild-type plant infected with the pathogen but lacking the gene. The corresponding wild-type plant may have some level of resistance to the pathogen, or may be classified as susceptible. Thus, the terms “resistance” and “enhanced resistance” are generally used herein interchangeably. Furthermore, a polypeptide of the invention does not necessarily confer complete pathogen resistance, for example when some symptoms still occur or there is some pathogen reproduction on infection but at a reduced amount within a plant or a population of plants. Resistance may occur at only some stages of growth of the plant, for example in adult plants (fully grown in size) and less so, or not at all, in seedlings, or at all stages of plant growth. In an embodiment, resistance occurs at adult and seedling stage. By using a genetic modification strategy to express a polypeptide of the invention in a plant, and/or to increase the level of the polypeptide in a plant, the plant of the invention can be provided with resistance throughout its growth and development. Enhanced resistance can be determined by a number of methods known in the art such as analysing the plants for the amount of pathogen and/or analysing plant growth or the amount of damage or disease symptoms to a plant in the presence of the pathogen, and comparing one or more of these parameters to a corresponding wild-type plant lacking a genetic modification s) of the invention.

In a particularly preferred embodiment the one or more fungal pathogen(s) causes a disease in the plant such as, but not limited to, stem rust, leaf rust, stripe rust, downy or powdery mildew, smut, blight such as head blight, pod and stem blight, rots such as crown rot, common root rot, foot rot, stem rot, mold (e.g. white mold or pink snow mold), spot blotch, blast, leaf blight, anthracnose and southern com blight.

In an embodiment, the one or more fungal pathogen(s) at least infect one or more of the followings plants; wheat, soybean, barley, oats, rye, rice, maize or sorghum. In an embodiment, the one or more fungal pathogen(s) is a Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolaris sp., Oidium sp., Gibberella sp., Cochliobolus sp., Exserohilum sp., Uredo sp. Microdochium sp., Helminthosporium sp., Monographella sp., Colletotrichum sp., Uromyces sp., Phakopsora sp..or Erysiphe sp..

In an embodiment, the one or more fungal pathogen(s) is a Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolaris sp., Cochliobolus sp., Exserohilum sp. or Erysiphe sp..

In an embodiment, the fungal pathogen is a Puccinia sp.. In an embodiment, the Puccinia sp. is Puccinia graminis, Puccinia triticina, Puccinia tritici-duri, Puccinia recondita or Puccinia striiformis.

In an embodiment, the Puccinia graminis is Puccinia graminis f. sp. tritici (Ug99).

In an embodiment, the Puccinia recondita is Puccinia recondita f. sp. tritici.

In an embodiment, the Fusarium sp. is Fusarium pseudograminearum. Fusarium graminearum Group II, Fusarium avenaceum. Fusarium culmorum and Fusarium nivale.

In an embodiment, the Blumeria sp. is Blumeria graminis. In an embodiment, the Blumeria sp. is Blumeria graminis f. sp. tritici.

In an embodiment, the Bipolaris sp. is Bipolaris sorokiniana.

In an embodiment, the Gibberella sp. is Gibberella avenacea or Gibberella zeae.

In an embodiment, the Erysiphe sp. is Erysiphe graminis. In an embodiment, the Erysiphe graminis is Erysiphe graminis f. sp. tritici.

In an embodiment, the Exserohilum sp. is Exserohilum turcicum.

In an embodiment, the Magnoporthe sp. is Magnaporthe grisea.

In an embodiment, the Uredo sp. is Uredo glumarum.

In an embodiment, the Microdochium sp. or Microdochium nivale.

In an embodiment, the Monographella sp. is Monographella nivalis.

In an embodiment, the Cochliobolus sp. is Cochliobolus sativus.

In an embodiment, the Helminthosporium sp. is Helminthosporium sativum.

In an embodiment, the Oidium sp. is Oidium monilioides .

In an embodiment, the Colletotrichum sp. is Colletotrichum sublineolum.

In an embodiment, the Uromyces sp. is Uromyces eragrostidis .

In an embodiment, the Phakopsora sp. is P. pachyrhizi and P. meibomiae. Plant/Grain Processing

Grain/seed of the invention, preferably cereal grain and more preferably wheat grain, or other plant parts of the invention, can be processed to produce a food ingredient, food or non-food product using any technique known in the art.

In one embodiment, the product is whole grain flour such as, for example, an ultrafme-milled whole grain flour, or a flour made from about 100% of the grain. The whole grain flour includes a refined flour constituent (refined flour or refined flour) and a coarse fraction (an ultrafme-milled coarse fraction).

Refined flour may be flour which is prepared, for example, by grinding and bolting cleaned grain such as wheat or barley grain. The particle size of refined flour is described as flour in which not less than 98% passes through a cloth having openings not larger than those of woven wire cloth designated "212 micrometres (U.S. Wire 70)" . The coarse fraction includes at least one of: bran and germ. For instance, the germ is an embryonic plant found within the grain kernel. The germ includes lipids, fiber, vitamins, protein, minerals and phytonutrients, such as flavonoids. The bran includes several cell layers and has a significant amount of lipids, fibre, vitamins, protein, minerals and phytonutrients, such as flavonoids. Further, the coarse fraction may include an aleurone layer which also includes lipids, fibre, vitamins, protein, minerals and phytonutrients, such as flavonoids. The aleurone layer, while technically considered part of the endosperm, exhibits many of the same characteristics as the bran and therefore is typically removed with the bran and germ during the milling process. The aleurone layer contains proteins, vitamins and phytonutrients, such as ferulic acid.

Further, the coarse fraction may be blended with the refined flour constituent. The coarse fraction may be mixed with the refined flour constituent to form the whole grain flour, thus providing a whole grain flour with increased nutritional value, fiber content, and antioxidant capacity as compared to refined flour. For example, the coarse fraction or whole grain flour may be used in various amounts to replace refined or whole grain flour in baked goods, snack products, and food products. The whole grain flour of the present invention (i.e. -ultrafme-milled whole grain flour) may also be marketed directly to consumers for use in their homemade baked products. In an exemplary embodiment, a granulation profile of the whole grain flour is such that 98% of particles by weight of the whole grain flour are less than 212 micrometres.

In further embodiments, enzymes found within the bran and germ of the whole grain flour and/or coarse fraction are inactivated in order to stabilize the whole grain flour and/or coarse fraction. Stabilization is a process that uses steam, heat, radiation, or other treatments to inactivate the enzymes found in the bran and germ layer. Flour that has been stabilized retains its cooking characteristics and has a longer shelf life.

In additional embodiments, the whole grain flour, the coarse fraction, or the refined flour may be a component (ingredient) of a food product and may be used to product a food product. For example, the food product may be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, an English muffin, a muffin, a pita bread, a quickbread, a refrigerated/frozen dough product, dough, baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a ready to eat meal, stuffing, a microwaveable meal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling, baby food, a baking mix, a batter, a breading, a gravy mix, a meat extender, a meat substitute, a seasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo mein noodles, an ice cream inclusion, an ice cream bar, an ice cream cone, an ice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, an extruded snack, a fruit and grain bar, a microwaveable snack product, a nutritional bar, a pancake, a par- baked bakery product, a pretzel, a pudding, a granola-based product, a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animal food or pet food.

In alternative embodiments, the whole grain flour, refined flour, or coarse fraction may be a component of a nutritional supplement. For instance, the nutritional supplement may be a product that is added to the diet containing one or more additional ingredients, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics and fibre. The whole grain flour, refined flour or coarse fraction of the present invention includes vitamins, minerals, amino acids, enzymes, and fibre. For instance, the coarse fraction contains a concentrated amount of dietary fibre as well as other essential nutrients, such as B- vitamins, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. For example 22 grams of the coarse fraction of the present invention delivers 33% of an individual's daily recommend consumption of fiber. The nutritional supplement may include any known nutritional ingredients that will aid in the overall health of an individual, examples include but are not limited to vitamins, minerals, other fibre components, fatty acids, antioxidants, amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/or other nutritional ingredients. The supplement may be delivered in, but is not limited to the following forms: instant beverage mixes, ready-to-drink beverages, nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews, chewable tablets, and pills. One embodiment delivers the fiber supplement in the form of a flavoured shake or malt type beverage, this embodiment may be particularly attractive as a fibre supplement for children.

In an additional embodiment, a milling process may be used to make a multi -grain flour or a multi-grain coarse fraction. For example, bran and germ from one type of grain may be ground and blended with ground endosperm or whole grain cereal flour of another type of cereal. Alternatively, bran and germ of one type of grain may be ground and blended with ground endosperm or whole grain flour of another type of grain. It is contemplated that the present invention encompasses mixing any combination of one or more of bran, germ, endosperm, and whole grain flour of one or more grains. This multigrain approach may be used to make custom flour and capitalize on the qualities and nutritional contents of multiple types of cereal grains to make one flour.

It is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be produced by any milling process known in the art. An exemplary embodiment involves grinding grain in a single stream without separating endosperm, bran, and germ of the grain into separate streams. Clean and tempered grain is conveyed to a first passage grinder, such as a hammermill, roller mill, pin mill, impact mill, disc mill, air attrition mill, gap mill, or the like. After grinding, the grain is discharged and conveyed to a sifter. Further, it is contemplated that the whole grain flour, coarse fraction and/or grain products of the present invention may be modified or enhanced by way of numerous other processes such as: fermentation, instantizing, extrusion, encapsulation, toasting, roasting, or the like.

Malting

A malt-based beverage provided by the present invention involves alcohol beverages (including distilled beverages) and non-alcohol beverages that are produced by using malt as a part or whole of their starting material. Examples include beer, happoshu (low-malt beer beverage), whisky, low-alcohol malt-based beverages (e.g., malt-based beverages containing less than 1% of alcohols), and non-alcohol beverages.

Malting is a process of controlled steeping and germination followed by drying of the grain such as barley and wheat grain. This sequence of events is important for the synthesis of numerous enzymes that cause grain modification, a process that principally depolymerizes the dead endosperm cell walls and mobilizes the grain nutrients. In the subsequent drying process, flavour and colour are produced due to chemical browning reactions. Although the primary use of malt is for beverage production, it can also be utilized in other industrial processes, for example as an enzyme source in the baking industry, or as a flavouring and colouring agent in the food industry, for example as malt or as a malt flour, or indirectly as a malt syrup, etc.

In one embodiment, the present invention relates to methods of producing a malt composition. The method preferably comprises the steps of:

(i) providing grain, such as barley or wheat grain, of the invention,

(ii) steeping said grain,

(iii) germinating the steeped grains under predetermined conditions and

(iv) drying said germinated grains.

For example, the malt may be produced by any of the methods described in Hoseney (Principles of Cereal Science and Technology, Second Edition, 1994: American Association of Cereal Chemists, St. Paul, Minn.). However, any other suitable method for producing malt may also be used with the present invention, such as methods for production of speciality malts, including, but limited to, methods of roasting the malt.

Malt is mainly used for brewing beer, but also for the production of distilled spirits. Brewing comprises wort production, main and secondary fermentations and posttreatment. First the malt is milled, stirred into water and heated. During this "mashing", the enzymes activated in the malting degrade the starch of the kernel into fermentable sugars. The produced wort is clarified, yeast is added, the mixture is fermented and a post-treatment is performed.

EXAMPLES

EXAMPLE 1 - Materials and Methods

DNA extraction and PCR amplification

Leaf tissue or callus tissue was freeze dried and macerated with a ball bearing using a Nimbus liquid handling robot (Hamilton, USA), followed by the addition of extraction buffer (0.1 M Tris pH 8.0, 0.05 M EDTA, 1.25% SDS) and heating for 1 h at 65 °C. Samples were then treated with 6M NH3Ac and DNA precipitated from the resultant supernatant with isopropanol. The DNA was then washed in 70% ethanol and resuspended in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). lOOng samples of DNA were then PCR amplified using GoTaq DNA polymerase (Promega) and a C100 Touch Thermo Cycler (Bio-Rad), 0.5 pmols of each primer. Products were resolved by gel electrophoresis using 1.0% agarose gels.

Production of constructs containing plant border sequence substitutes

Vector pCSIRO was used as a backbone cloning vector to produce binary vectors containing plant substitute border sequences. Gibson assembly was undertaken by Epoch Life Science (Missouri City, Texas, USA) to replace the entire coding region of the Agrobacterium left border sequence and Bar selection sequence with wheat LB 1 or LB2, while the original Agrobacterium right border was also substituted by the wheat RB 1. The attRl and attR2 sequences for gateway cloning were not affected, which enabled the sequential addition of RUBY reporter gene sequences and BAR selectable marker genes by Gateway recombination (see below). All binary vectors were introduced into Agrobacterium strain AGL1 by electroporation and selected for resistance to Kanamycin.

Production of wheat 5 Sr cisgenic construct

The resulting vector with wheat LB1/RB1 was used to stack 5 (five) wheat Stem rust resistance (Sr) genes to produce the cisgenic construct using protocols described previously (Luo et al., 2021) with the following modifications. The Sr 13 gene sequence, cloned into vector pDONORl (Luo et al, 2021), was then introduced into the wheat LB1/RB1 vector (pCSIRO pMl) using LR Gateway recombination (Gateway LR Clonase® II enzyme mix, Thermo Fisher, Carlsbad, CA, USA). Following this reaction, the sample was restricted with I-Scel which cleaves only pDONORl and the original wheat LB 1 /RBI vector, but not the recombinant vector. The restricted sample was then transformed into E. coli strain H10 and positive colonies identified as expressing lacZ (i.e. blue). Plasmid DNA was then isolated from a positive colony and the wheat LB1/RB1/Srl3 binary vector subjected to BP recombination (Gateway BP Clonase® II enzyme mix, Thermo Fisher) which removed lacZ and regenerated an attRl site. The Sr62 gene in donor vector pDONOR2 was then incorporated into wheat LB 1/RB 1 /Sr 13- \LacZ. again by LR Gateway recombination followed by Pl-Scel restriction and positive lacZ selection. Plasmid DNA was isolated from a lacZ positive E. coli colony and lacZ removed and attRl regenerated again by BP recombination. Sequential reiteration of this cloning strategy was used to insert the Sr22. Sr26 and Sr33 gene sequence from pDONORl, pDONOR2 and pDONORl, respectively.

Prior to the addition of the next Sr gene in this cloning strategy Gateway cloning scars were removed using CRISPR/Cas9 based targeted deletion. The last remaining lacZ and donor vector sequence was also removed using CRISPR/Cas9 based targeted deletion. In detail, gRNAs were designed and synthesized in vitro to target both 5’ and 3 ’ ends of Sr genes. For wheat RB sequence, a gRNA was designed to target 5 ’ end and for wheat LB 1 sequence, a gRNA targeting 3 ‘ end was designed (Liu et al., 2015) (Table 2). The non-wheat sequences between Sr genes, or between wheat border sequences (RB or LB1) and Sr genes were removed by sequential in vitro digest using CRISPR Cas 9 and the relevant two gRNAs (with New England Biolabs (NEB) Notting Hill, Vic, Cas9 nuclease cat# M0386S) flanking the unwanted sequences followed by self-ligation (with NEB T4 DNA ligase Cat #: M0202S) and transformation into E.coli cells.

To design oligos for PCR to obtain DNA which was used as a template for gRNA synthesis in vitro the following primers were used.

(i) Primer 1 was used with specific sequence for each guide to the position intended to cut: GATCACTAATACGACTCACTATA-gg (SEQ ID NO: 1042) (20 bp guide sequence specific for the cutting site)-GTTTTAGAGCTAGAAA (SEQ ID NO: 1043). The two g’s are included in the transcript and required for transcription. If the guide-specific sequence starts with one or more g’s, the same number of g’s can be deleted from the primer.

(ii) Primer 2 (always the same):

AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTA TTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQ ID NO: 1044).

PCR was performed using primers (i) and (ii) to make DNA template for T7 RNA polymerase using following steps. After the PCR reaction, the DNA was purified using Bioline PCR clean up kit Bio-52060. 150ng of DNA was used for gRNA synthesis (T7 RNA polymerase, NEB E2050) in 20ul. To remove the unwanted cloning sequences in the plasmid, lul of each of the two gRNAs flanking the unwanted sequence was used to digest plasmid DNA (~200ng) with Cas9 (NEB M0386) in 20ul. After the digest, 5ul was used to perform self-ligation using T4 DNA ligase (NEB M0202). The ligation product was transformed into NEB 10-beta cell (NEB C3020) for deletion screening.

The target sequences for gRNAs synthesis are in the table below.

The T-DNA of the final 5 Sr gene stack (wheat LB 1/Sr33/Sr26/Sr22/Sr62/Sr 13/wheat RBI) did not contain any sequences that were not derived from the wheat genome which includes sexually compatible species, except for the 8 bases 5’ of the wheat LB1. The final stack does not contain an Agrobacterium border sequence, selectable marker genes, Gateway recombination sites or cloning scars.

Table 2. Target sequences for gRNAs synthesis.

Target names Target sequence Targeting region

RB-M1- gggTGGTCtggcagagattgctg crRNA_wheat wheat RB to Srl3 3'end

Srl3_3crRNA_R

wheat RB to Srl3 3'end gggACATCTTTAATTGTAGCAAT

Srl3_5crRNA (SEQ ID NO:1017) Srl3 5' end to Sr62 5' end gggCGCAGGTGGTGATTAGATAA

Sr62-5crRNA-R (SEQ ID NQ:1018) Srl3 5' end to Sr62 5' end gggACGTACTCAAAGAAAAACTC

Sr62-3-crRNA (SEQ ID NQ:1019) Sr62 3' end to Sr22 5' end gggATTGCGAACTTTACTGTCAA

Sr22-5crRNA_R (SEQ ID NQ:1020) Sr62 3' end to Sr22 5' end

Sr22- gggACGAATCGTATATGTAAAAA

3crRNAnew (SEQ ID NQ:1021) Sr22 3' end to Sr26 5' end

Sr26- ggGTTGGCGGCCGCcatgggcg

5crRNA_Rm (SEQ ID NQ:1022) Sr22 3' end to Sr26 5' end

Sr26- ggGacagacatccgatgatatg

3crRNAnew (SEQ ID NQ:1023) Sr26 3' end to Sr33 5' end

Sr33-5- gggCAAGATGTCAAA l 1 1 I AAAA crRNA_Rm (SEQ ID NQ:1024) Sr26 3' end to Sr33 5' end gggCTACTCATTAGGAACTCGAG

Sr33-3-crRNA (SEQ ID NQ:1025) wheat LB1 to Sr33 3'end wheat_LBl- Gggactcttctcttgcagagcc crRNA-R (SEQ ID NQ:1026) wheat LB1 to Sr33 3'end

The T-DNA of the final 5 Sr gene stack (wheat LB 1 / Sr33/Sr26/Sr22/Sr62/Sr 13/wheat RB 1 ) did not contain any sequences that were not derived from the wheat genome which includes sexually compatible species, except for the 8 bases 5’ of the wheat LB1. The final stack does not contain an Agrobacterium border sequence, selectable marker genes, Gateway recombination sites or cloning scars. Production of wheat 5Yr cisgenic construct with synthetic transposable PiggyBac 35s Bar selection marker

Five wheat yellow rust resistance (Yr5/Yrl5/Yr36/Yr46(Lr67)/YrUl) genes were sequentially introduced into the wheat LB 1 /RBI vector to produce the pMl_5Yr_PiggyBac_35SBar_wheat_T_DNA insert in binary vector pCSIRO (Figure 15) using previously described protocols (Luo et al., 2021) with the following modifications. The Yrl5 gene sequence, in vector pDONORl (Luo et al, 2021), was introduced into the wheat LB1/RB1 vector (pCSIRO pMl) using LR Gateway recombination (Gateway LR Clonase® II enzyme mix, Thermo Fisher, Carlsbad, CA, USA). Following this reaction, the sample was restricted with I-Scel which cleaves only pDONORl and the original wheat LB 1/RB 1 vector, but not the recombinant vector. The restricted sample was then transformed into E. coli strain H10p and colonies selected using positive lacZ selection (i.e. blue colonies selected). Plasmid DNA was then isolated from a positive colony and the wheat LB1/RB1/Yrl5 binary vector subjected to BP recombination (Gateway BP Clonase® II enzyme mix, Thermo Fisher) to remove lacZ and regenerated an attRl site. The Yr5 gene in donor vector pDONOR2 was then incorporated into wheat LB 1 /RBI /Yr 15- \ acZ. again by LR Gateway recombination followed by Pl-Scel restriction and positive IctcZ selection. Plasmid DNA was isolated from a lacZ positive E. coli colony and IctcZ removed and attRl regenerated again by BP recombination. Sequential reiteration of this cloning strategy was used to insert the Yr5. Yr46(Lr67), Yr36 and YrUl gene sequences from pDONORl, pDONOR2 and pDONORl, respectively. However, prior to the addition of the next Yr gene, Gateway att sites were first removed using CRISPR/Cas-mediated in vitro gene editing as described further below.

A PiggyBac transposon carrying a 35S-BAR selectable marker sequence in pDONOR2 was then inserted in between YrU 1 and the wheat LB sequence by Gateway recombination. The original wheat LB1 sequence corresponds to nucleotides 631979550 to 631979754 of chr 7D of the Chinese Spring v.2 genome sequence. This sequence was replaced with nucleotides 631979550 to 631979912 of chr 7D with the PiggyBAC/35SBAR transposon inserted between nucleotides 631979821 and 631979822 of this wheat sequence. Subsequent CRIPSR/Cas removal of lacZ and donor vector sequence (described below) also resulted in a 76 bp deletion of the newly added wheat genomic DNA sequence corresponding to nucleotides 631979618 to 631,979,694. The excision of Piggy BAC/35 SB AR therefore recreates the 631979550 to 631979912 chr 7D sequence albeit with a 76 bp deletion of endogenous wheat DNA (Figure 18). During the sequential Gateway cloning process, prior to the insertion of the next sequence, cloning scars (i.e remnant att sites) were removed using CRISPR/Cas9 based in vitro targeted deletion. This process was also used to remove lacZ and donor vector sequence afterthe addition of PiggyBac/35SBAR sequence described above. In each case two gRNAs were designed (see Table 3) that flanked the unwanted cloning sequence to be removed. Plasmids were incubated with both guide RNAS and purified Cas9 protein (NEB, USA) for 4 hrs at 37°C followed by vector self-ligation using T4 DNA ligase (NEB, USA) and subsequent transformation into E. coli. Plasmids in E. coli that contained the appropriate deletion were then identified by PCR analysis using primers that flanked the deletion site.

Table 3. gRNA guide sequences for removing cloning scars.

SEQ

Target name ID NO Target sequence Targeting region

RB-M1- 1183 crRNA_wheat gggTGGTCtggcagagattgctg wheat RB to Yr51 3' end

Yrl5_3crRNA_R 1184 gggTGCATGTGATCAATAAT

TTG wheat RB to Yr513' end

1185 gggCCAG IAG I 1 1 1 1 IAG I IA

Yrl5_5crRNA AA Yrl5 5' end to Yr5 5' end

1186 ggGTCGTCGTCGCTGCGCT

Yr5-5crRNA-R CGC Yrl55' end to Yr5 5' end

1187 gggAGGCGCGCCGATTCCA

Yr5-3-crRNA GACG Yr5 3' end to Lr675' end

1188 gggTCACTAGTGATTTCTTAT

Lr67-5crRNA_R GA Yr5 3' end to Lr67 5' end

Lr67-3-crRNA ¹¹⁸⁹ gggTTTGCAAGCTTTCGTATT

CG Lr673 end to Yr365 end

Yr36-5-CrRNA- 1190 gggTTGGCGGCCGCAATAG

R AGAG Lr673' end to Yr365' end

1191 ggGCGGCCGCTCAGTTTAC

Yr36-3-CrRNA AAC Yr363' end to YrU1 5' end

YrUl-5-CrRNA- 1192 R gGGTGCGGCCGCtcatgattt Yr363’ end to YrUl 5’ end

„ , _M , A U93 YrUl 3' end to wheat LB1 extended 3'

YrUl-3-CrRNA gggcatgcggccgccaggccct end

Lb_Ml_extra_cr 1194 YrUl 3' end to wheat LB1 extended 3'

RNA_R ggaggatacactagatattcta end wheat_LBl- 1195 wheat LB1 extended 5’ end to wheat crRNA-R gggactcttctcttgcagagcc LB1 (original unextended)

Lb_Ml_crRNA_ 1196 wheat LB1 extended 5’ end to wheat

F ggagattgagaattcctcatt LB1 (original unextended) EXAMPLE 2 - Identification of wheat polynucleotides producing genetically modified wheat

Efficiency of transformation is frequently reported and monocots remain somewhat recalcitrant to Agrobacterium transformation (de Saeger et al., 2021). Transformation efficiency is typically examined by comparing the number of independent transformed events or lines obtained relative to the number of explants transformed. The quality of the events is reviewed by looking at the presence of undesirable vector sequence that may have integrated with the T-DNA. A desirable transformant contains the complete sequence between LB and RB absent of any rearrangements or truncations and is introduced as a single copy into the plant cell genome without any vector backbone sequence.

Experiments to substitute the bacterial border sequences with plant genomic sequences to develop transgenic plants that contain no non-endogenous plant DNA sequences were undertaken. The goal of the experiments is to effectively replace the 21 bases of LB sequence and 3 base of RB sequence that are transferred to the plant during Agrobacterium transformation with endogenous plant sequences. Jen and Chilton (1987) had previously investigated the structural properties of the border sequences and noted that deletions of parts or whole of the 25 bp repeats resulted in loss of activity. The identification of a functional plant derived LB sequence was clearly going to be more challenging given the requirement for up to 21 identical nucleotides. Other Agrobacterium sequences that are not transferred as part of the T-DNA sequences were maintained in binary vectors used in these studies since they do not readily integrate into the plant genome.

Previous studies have suggested that the terminal 25 bp repeat sequences present in Agrobacterium tumefacians Ti plasmids and synthetic binary plasmids are sufficient to mediate DNA transfer from bacterium to plant cells (Shaw et al, 1984; Srinivasan et al, 1989). Sequence degeneracy exists between and amongst some functional border repeat sequences of octopine type and nopaline type Ti plasmids suggesting there is some degree of permissive flexibility in border sequences that still enables T-DNA transfer (figure 2). However, adjacent sequences not included in the transferred T-DNA region improve transformation efficiency, such as so-called overdrive sequence that is in juxtaposition to the RB (Peralta et al., 1986).

BLAST search analysis was undertaken on publicly available plant genome sequences of Chinese Spring wheat (IWGS Chinese Spring v. 2.1), maize, soybean, indica rice, japonica rice, sorghum, banana, potato and tomato using the 25 bp nopaline Ti plasmid LB sequence (TGGCAGGATATATTGTGGTGTAAAC) (SEQ ID NO: 758). No plant sequences with 100% identity to this LB sequence were identified in any genome with the best match being identity for 21 of the 25 LB nucleotides in the wheat genome (examples shown in Table 4). Plant sequences with at least 16 nucleotides identical to the LB were identified in all of plant the genomes analysed (Table 4).

The part of the RB from the Agrobacterium that transfers to the plant encodes nucleotides TGA which are 5’ of the enzymatic cleavage site. Whilst others have focussed on replacing the RB (e.g. the retracted paper by Rommens et al., 2005) since only a portion of the RB 5’ of the cleavage site transfers as T-DNA, the approach taken here was to determine if the RB and flanking sequence derived from a plant genomic sequence that replaces part of the native RB 5’ of the enzymatic cleavage site effects the Agrobacterium endonuclease cleavage and/or DNA strand transfer efficiency. Given the abundance of plant sequences encoding TGA a search for a substitute RB (TGACAGGATATATTGGCGGGTAAAC) (SEQ ID NO:786) was expanded to identify plant sequences with similarity to native Agrobacterium sequences immediately 5’ of the nopaline RB. A comparison of several published binary vector sequences showed that the endogenous Agrobacterium sequence TTAAACTATCAGTGTT (SEQ ID NO: 1027) was often (but not always) present in juxtaposition to the RB in these vectors (Figure 3). The sequence TTAAACTATCAGTGTTTGA (SEQ ID NO: 1028) was therefore searched for as a RB alternative in the plant genomes described above. No identical sequences were detected but similar sequences encoding mismatches were observed (examples shown in Table 5).

EXAMPLE 3 - Binary plasmid construct design with WT-DNA borders

To determine if the proposed wheat derived (WT-DNA) replacement LB and RB sequences can completely or partly substitute for bona fide bacterial border T-DNA sequences and still facilitate T-DNA transfer a binary construct, plasmid pMO, was produced using standard transformation vector pCSIRO as a backbone. All T-DNA border sequences and intervening T-DNA was removed from the pCSIRO backbone.

The inventors identified that the design of the WT-DNA LB sequence can be semi-wheat/ Agrobacterium to obtain the desired wheat plant sequence only product from the method described. As observed for bona fide T-DNA border sequences the frequency of integration of the 5 nt CAGGA next to the cleavage site is low and does not routinely integrate (Kim et al., 2003; Kleinboelting et al., 2015; Gong et al., 2021). As such the sequence selection for WT-DNA LB design may or may not need to replace these nucleotides, since following a simple analysis step the integration of these nucleotides can be identified and plants discarded. Table 4. Plant LB-like sequences.

Wheat genomic sequences were incorporated and used to substitute the bona fide Agrobacterium LB and RB sequences. Nucleotides 631979550 -631979773 of wheat chromosome 7D (IWGSC Chinese Spring v. 2.1) were used as a WT-DNA LB sequence as it encodes 16 bases (nucleotides 631979550-631979566) identical to the 3’ end ofthe 25 bp Agrobacterium LB repeat (tatattgtggtgtaaac) (SEQ ID NO:779) (Figure 4). The resulting WT-DNA LB sequence in pMO encodes 8 nucleotides of the Agrobacterium LB sequence (Figure 4, boxed in yellow) combined with the 16 bases from the wheat genome. This WT-DNA LB sequence is therefore semi-wheat. The WT-DNA LB is cleaved such that 5 nucleotides (CAGGA) may be transferred as part of the T-DNA that are not derived from the wheat genome. The WT-DNA LB used for the pMO design was TATATTGTGGTGTAAAC (SEQ ID NO: 1029).

In addition, the WT-DNA RB was comprised of nucleotides 553784436 - 553784630 of wheat chromosome 5D (IWGSC Chinese Spring genome v2.1) which replace part (3 bp) of the native Agrobacterium RB sequence and include a 13 bp (aactatcagtgtt) (SEQ ID NO: 1030) region of wheat sequence (Figure 4) with identity to sequence immediately 5 ’ of typical Agrobacterium RB sequences and used in a number of standard binary vector constructs (Figure 3). The remaining 22 bp ofthe RB sequence (boxed in Figure 4) are not derived from the wheat genome and although synthetic, are effectively Agrobacterium in origin. These latter Agrobacterium sequences are not transferred as part of the T-DNA strand.

Included in the T-DNA sequence, between these two potential WT-DNA border replacement sequences, was a RUBY reporter construct under the regulatory control of a maize polyubiquitin promoter sequence, to create plasmid pMO (Figure 5). Upon expression the RUBY reporter gene converts tyrosine into a bright red, visible pigment, betalain (as described by He et al., 2020), making the RUBY reporter an efficient an easy tool for visually detecting transformation success.

EXAMPLE 4 - Functional characterisation WT-DNA LB sequence analysis

Plasmid pMO (Figure 5) was introduced by electroporation into Agrobacterium strain AGL1 carrying a disarmed Ti plasmid pMP90. Stock cultures were grown overnight at 28°C in liquid LB media containing the appropriate antibiotics with vigorous shaking. Bacteria were resuspended in infiltration buffer (10 mm MES pH 5.6, 10 mm MgCL and 150 pm acetosyringone) at ODgoo nm of 0.8 and then syringe infiltrated into 4- week-old Nicotiana benthamiana plants grown under controlled environment conditions. Three control AGL1 Agrobacterium strains were also infiltrated into N. benthamiana.. The first encoded the same Ubi-RUBY transgene as pMO except this reporter gene was cloned into a standard binary vector (pCSIRO-RUBY) that contained conventional Agrobacterium border sequences and a 35S-BAR selectable marker gene (Figure 5). The second strain carried a conventional binary vector (pHDE) that encoded the RUBY reporter gene under the regulatory control of the cauliflower mosaic 35S promoter (Figure 5). The third Agrobacterium strain carried a 35S-YFP gene on a standard binary vector, pAM (Figure 5).

Two days post-infiltration strong betalain accumulation was observed at 35S- RUBY infiltration sites while only faint or no pigment accumulation was seen at the remaining infiltration sites (Figure 5). However, 5 days post-infiltration strong betalain accumulation was observed at both pMO pMO and pCSIRO/RUBY infiltration sites. No betalain accumulation occurred at control sites infiltrated with Agrobacterium carrying a YFP reporter gene (Figure 5). The faster betalain accumulation observed at 35S-RUBY infiltration sites compared with the Ubi-RUBY encoding vectors is likely due to the 35 S promoter showing higher expression in N. benthamiana compared with the monocot derived maize ubiquitin promoter. These data demonstrate that the wheat nucleotides 549874103-549874297 of chromosome 5D comprising WT-DNA borders in the pMO vector effectively substituted for part of the bona fide Agrobacterium RB sequences present in pCSIRO/RUBY in promoting T-DNA transfer. These data do not demonstrate that the WT-DNA UB sequence is functional as previous studies have demonstrated successful T-DNA transfer by Agrobacterium in the absence of a UB sequence, and that such transfer was associated with transfer of the entire binary plasmid.

EXAMPLE 5 - Functional characterisation WT-DNA LB sequence analysis

To test for functional cleavage of the WT-DNA LB sequence a second set of constructs were produced. The Agrobacterium LB and RB sequences were replaced in binary plasmid pCSIRO with the same WT-DNA sequences described above and used in pMO. A Ubi-RUBY and 35S-BAR gene were then inserted to produce pM/WT-DNA LB/RUBY/BAR/WT-DNA RB (Figure 6). Plasmid pM/RUBY/BAR/WT-DNA RB was then produced by removing the WT-DNA LB sequence, while pM/RUBY/WT-DNA LB/BAR/WT-DNA RB was created by inserting the WT-DNA LB sequence between the Ubi-RUBY and 35S-BAR genes (Figure 6). If functional the WT-DNA LB sequence of this latter plasmid would be cleaved during Agrobacterium T-DNA transfer, meaning the Ubi-RUBY gene would not be transferred as part of the T-DNA and no betalain accumulation would occur. These constructs, in addition to the control pCSIRO/RUBY, were each introduced into Agrobacterium strain AGL1 and these cultures then infiltrated into N. benthamiana leaves as described and results are shown in Figure 6.

Both plasmids pM/WT-DNA LB/RUBY/BAR/WT-DNA RB and pCSIRO/RUBY generated strong betalain accumulation at the infiltration sites 5 days post-infiltration (Figure 6). Significantly less betalain accumulation occurred at infiltration sites where pM/RUBY/WT-DNA LB/BAR/WT-DNA RB was introduced suggesting the WT-DNA LB sequence is subject to cleavage in most, but not all, DNA transfers. For comparison, plasmid pM/RUBY/BAR/WT-DNA RB infiltration, which contains no LB (i.e. no Agrobacterium native LB sequence or WT-DNA LB sequence), produced stronger betalain accumulation than the WT-DNA containing pM/RUBY/WT- DNA LB/BAR/WT-DNA RB, demonstrating that the WT-DNA LB was functional and the data is consistent with the conclusion that some cleavage of the WT-DNA LB sequence occurred during T-DNA transfer for this plasmid.

EXAMPLE 6 - Wheat plant transformation using WT-DNA borders

Wheat transformation using Agrobacterium tumefaciens strain AGL1 carrying the plasmid of interest was undertaken using Japan Tobacco transformation protocol as described by Ishida et al. (2015) using wheat cultivar Fielder embryo explants, as modified by Richardson et al. (2014). Briefly, seeds from cv Fielder were harvested 12- 14 days post-anthesis were surface sterilized in a 0.8 % sodium hypochlorite solution for 10 minutes. Embryos were removed from the seed under aseptic conditions and cocultivated with Agrobacterium strains containing binary constructs of interest for 2 days on WLS-AS medium (Ishida et al., 2015) in the dark. After co-cultivation embryonic axes were excised with a scalpel and explants were then transferred to WLS-Res medium and placed in the dark at 24°C. After 5 days explants were transferred to WLS-P5 callus induction media containing 5 mg/ml of phosphinothricin (PPT). Two weeks later callus was bisected and placed on WLS-P10 (10 mg/L of PPT) for 3 weeks in the dark. Callus was then regenerated on LSZP5 (5 mg/L PPT) medium in 200 pmols m-2 s-1 light at 24°C. Shoots were transferred to LSF-P5 (5 mg/L PPT) medium to allow root formation and once robust root systems were developed plants were transferred to the glasshouse.

Of the 83 calli infected with Agrobacterium strain AGL1 carrying pMO, 25 (30%) calli showed evidence of betalain accumulation at cell foci that ranged from small to more substantial (Figure 7a). Control explants were transformed with the conventional binary plasmid pCSIRO/RUBY in the same experiment. Of the 25 explants infected with Agrobacterium strain AGL1 carrying pCSIRO/RUBY 15 (60%) showed betalain accumulation (Figure 7b). Statistical analyses by pairwise T-test show no significant difference (P=0.09) between these two treatments, implying that the WT-DNA RB sequence was approximately equal in efficacy to the bona fide Agrobacterium RB sequence. Calli transformed with pMO were then regenerated in the absence of selection and one RUBY expressing plant grown out to maturity (Figure 7c and 7d).

EXAMPLE 7 - Functional analysis of WT-DNA sequences in transformed wheat by PCR analysis

To show evidence of WT-DNA LB cleavage in wheat cells transformed with pMO, DNA was extracted from 5 calli that showed betalain accumulation in the above experiment and three calli from the same experiment that did not show betalain accumulation. PCR amplification was undertaken using primer sets that surrounded the WT-DNA LB sequence (Figure 8) using the following primers P-LB1: ggagctgttggctggctgg (SEQ ID NO: 1031), P-LB2: ggcaggatatattgtggtgtaaactc (SEQ ID NO: 1032), P-LB3: gtggtgtaaactctatcaactggtactg (SEQ ID NO: 1033), P-LB4: ggtactgtattatccaccttatacgcg (SEQ ID NO: 1034), P-LB5: ccttatacgcgtcatcatttgtgc (SEQ ID NO: 1035), P-LBR: caacgaacaggtcactatcag (SEQ ID NO: 1036), P-RB1 attctaataaacgctcttttctcttag (SEQ ID NO: 1037), P-RB2: gtttacccgccaatatatcctgtcaaac (SEQ ID NO: 1038), P-RB3: tcaaacactgatagttgcctatcatattcg (SEQ ID NO: 1039), P-RB4: cagcttgcgacctacaagatcg(SEQ ID NO: 10), P-RB5:gattgagtgtgtcttgactttg (SEQ ID NO: 1040) and P-RBR: catcgatgataattcgagctcgg (SEQ ID NO: 1041).

The vector pMO was used as a positive control for PCR amplification with amplification products produced for all five primer sets (Figure 8, lane +). No amplification products from PCR1 were obtained from any callus DNA for binary vector sequence in juxtaposition to the WT-DNA LB indicating that the vector backbone was not transferred in the predicted T-DNA sequence. In contrast amplification products were obtained from DNA of betalain accumulating calli shown in lane 4 and for all other primer pairs, while faint amplification products were obtained in some instances for callus DNA in lane 8. These data provide evidence of cleavage of the WT-DNA LB. Although, this experiment is not well controlled in that each betalain positive callus piece transformed with pMO will vary greatly in the number of transformed cells, particularly as regeneration occurred in the absence of selection.

A similar PCR amplification experiment was used to investigate cleavage of the WT-DNA RB sequence using the same callus tissue DNA extracts. Again, no evidence of binary vector sequence indicating the backbone sequence was not transferred (Figure 9, PCR 1 and part of PCR 2) was detected in these callus DNAs. In contrast, PCR amplification products PCR 3, 4 and 5 were detected in callus DNA (Figure 9a, lane 4) while all DNA samples from callus with betalain accumulation showed amplification of the PCR 5 product. It is well established that T-DNA integration into plant genomes is often imprecise which can lead to exclusion of terminal transferred DNA sequences (Kim et al., 2003; Kleinboelting et al., 2015; Gong et al., 2021) which may explain the absence of amplification of PCR 3 and 4 products in three of the five betalin positive callus DNAs.

EXAMPLE 8 - Transformation of wheat with stem rust resistant multigene stack

A larger wheat transformation experiment using phosphinothricin (PPT) selection was then undertaken using the binary vector constructs shown in Figure 10. As described above pCSIRO/RUBY is a standard binary vector containing Agrobacterium LB and RB sequences. In vector pMl the border sequences of pCSIRO/RUBY have been replaced with wheat nucleotides 553784436 - 553784630 of chromosome 5D as comprising WT- DNA RB substitute sequence and nucleotides 631979550 - 631979773 of wheat chromosome 7D (IWGSC v. 2) as a WT-DNA LB substitute sequence. These are the same WT-DNA sequences used in binary plasmid pMO (Figure 5), however, pMO does not contain a BAR selectable marker gene that confers resistance to phosphinothricin. Binary plasmid pML2 differed to pMLl in that the WT-DNA LB sequence was substituted with wheat chromosome 5A nucleotides 669141979 - 669142155 (IWGSC Chinese Spring v.2.1). This sequence encodes a 25 bp WT-DNA LB repeat sequence that is entirely derived from the wheat genome, although it has 4 nucleotide mismatches when compared with the bona fide Agrobacterium 25 bp RB repeat sequence (Figure 11).

These three constructs were separately introduced into Agrobacterium strain AGL1 by electroporation and used to transform wheat cultivar Fielder, again using the Japan Tobacco wheat transformation protocol (Ishida et al., 2014) as described. However, in these experiments each construct was also co-transformed with a second Agrobacterium strain carrying a large 40 kb construct that encodes five cloned wheat stem rust resistance genes flanked by the same wheat WT-DNA LB and WT-DNA RB sequences used in pMl (Figure 10). Approximately 170 Fielder wheat embryos were Agrobacterium infected for each co-transformation and PPT resistant transgenic plants produced in each case with transformation efficiencies shown in Table 6. PPT resistant wheat transgenics were produced using binary constructs pMl and pM2 with transformation efficiencies not dissimilar to the standard Agrobacterium transformation vector pCSIRO/RUBY. Table 6. Generation of transgenic Fielder plants using constructs shown in Figure 9.

PCR analyses were then undertaken on the 110 transgenic plants produced by cotransformation with these RUBY + Sr vectors to investigate the presence of the wheat Sr gene stack construct. Primers that spanned Sr resistance gene junctions and border sequence junctions were used for PCR amplification as shown in Figure 12, with corresponding primer sequences shown in above for the LB and RB analysis of the pMO transgenics in Example 7. In total 34% of the 110 transgenic plants produced a junction sequence amplification product that spanned the Sri 3 gene and WT-DNA RB sequence. This frequency reduced for sequence further 5’ of the wheat T-DNA RB sequence, which is not unexpected given the RB end of the WT-DNA is the first to be transferred into the plant cells. These data show that WT-DNA border sequences can successfully promote the T-DNA transfer of both very large and smaller sized T-DNA sequences, with efficiencies not dissimilar to the native Agrobacterium LB and RB sequences present in the pCSIRO/RUBY plasmid.

PCR analysis indicate that of the 37 plants that were PCR positive for the Srl3 gene/WT-DNA RB junction, all junction sequences could be amplified from only two lines (Figure 12). T1 progeny from these two lines were then analysed for co-segregation of the wheat Sr gene stack construct and the 35S-BAR encoding binary vector they were co-transformed which was pMl and pM2, respectively (Figure 10). PCR was used to identify pMl and pM2, while primers specific for the Srl3 gene and WT-DNA RB sequence were used to identify the wheat Sr gene stack construct. Plant 1 (line 82) was self-fertilised to produce a T1 family of 31 individuals. From Plant 1 (line 82), cosegregation of pMl and the Sr gene stack was observed amongst 27 of the 31 T1 progeny. The expected ratio is 23.25 : 7.75. Chi square P = 0.11. ie. consistent with a single locus. The Ruby/Bar segregated 30 red: 1 green. These numbers are consistent with two independent Ruby/Bar loci being present in this family (Chi P= 0.48).

All 27 plants that had the Sr stack were red ie. inherited at least one copy of Ruby/Bar. 3 plants were red but did not have the Sr stack. 1 plant was green and did not have the Sr stack. These numbers fit one locus having the Sr stack+ linked Ruby/Bar and a second Ruby/Bar locus being present that is unlinked (P=0.3).

Plant 2 (line 107) was backcrossed to Fielder. 32 Fl progeny plants were produced. Of the progeny the following were obtained 12 Red and SR positive plants, 9 Red and Sr negative plants, 9 Green and SR positive plants and 2 Green and Sr negative plants. The resulting progeny fit into a two unlinked loci model (P=0.08). Importantly, the backcrossed material 9 plants were recovered that had the Sr stack but did not have Ruby/Bar meaning in these recovered plants the Sr stack had segregated away from the selectable marker plasmid resulting in entirely cisgenic wheat plants comprising the Sr stack.

T1 progeny from the second plant were then reanalysed for all wheat Sr gene stack junction sequences shown in Figure 12. Cosegregation between all junction sequence amplification products was observed suggesting this transgenic event incorporated all genes present in the Sr gene stack.

Expression of genes encoded in the wheat Sr gene stack was than analysed by quantitative RT-PCR of RNA extracted from progeny of this second plant. RNA was extracted and pooled from T1 plants carrying the gene stack and a second RNA pool made from null sibs. Quantitative RT-PCR was undertaken using gene specific primers and Sr amplification products quantified relative to transcripts from the wheat GAPDH gene. Gene expression products were obtained for all 5 Sr genes present in the Sr gene stack at levels comparable to control lines that each contained equivalent single endogenous genes. These data are consistent with all 5 Sr genes at the locus being expressed.

EXAMPLE 9 - Identification of ST-DNA border sequences

To determine if plant sequences from other species can also substitute for Agrobacterium LB and RB sequences, three sequences from the soybean genome (Figure 13) were used as ST-DNA border replacements in binary vector pCSIRO/RUBY . These sequences were used to produce constructs pSl,pS2 and pS3 which each encoded a Ubi- RUBY reporter gene in addition to a 35S-BAR selectable marker gene (Figure 14). In addition, construct pSl contained the WT-DNA LB substitute sequence, wheat LB1, and a soybean ST-DNA RB substitute sequence (soy RB2) whereas ST-DNA border sequences in pS2 and pS3 were derived from soybean sequences only, as shown in Figure 13. These three binary vectors were separately introduced into an Agrobacterium strain AGL1 by electroporation in addition to control plasmid 35-RUBY/pHDE which is a standard Agrobacterium binary vector encoding a 35S-RUBY reporter gene (Figure 14). Agrobacterium strains containing these constructs were then infdtrated into N. benthamina leaves and RUBY reporter expression observed at all infection sites 5 dpi. These data demonstrate that the soybean ST-DNA border sequences encoded in pS 1 , pS2 and pS3 are sufficient to mediate T-DNA transfer and produce transient gene expression in Agrobacterium infected plant cells.

EXAMPLE 10 - Transformation of wheat with a yellow rust resistance multigene stack

A wheat transformation experiment was undertaken using the binary vector pMl_5Yr_PiggyBac_35SBar_wheat_T_DNA shown in Figure 15 and constructed as described below. This plant T-DNA sequence encodes yellow rust resistance genes Yr5, Yrl5, Yr36, Yr46(Lr67) and YrUl, with a PiggyBac transposon sequence carrying a 35 SB AR selectable marker gene located within a modified wheat LB sequence. During the cloning process a 76 bp deletion was introduced into the wheat LB sequence that was only due to the cloning process and not important for function. Next generation constructs will not contain these types of deletions and consist only of contiguous wheat DNA with a PiggyBac transposon inserted at a TTAA sequence of choice.

In the presence of the corresponding transposase protein, which is encoded a Ubi- hyPBase transposase gene, PiggyBac can excise thereby removing both itself and its selectable marker cargo sequence leaving a final insertion in the wheat genome as shown in Figure 16. For this to occur, plants containing the 5Yr gene/PiggyBac construct are crossed to a second Fielder transgenic wheat line expressing the corresponding Ubi- hyPBase transposase gene, with a map of this gene shown in Figure 17. Progeny from these hybrid plants are then screened for individuals that contain the 5 Yr genes and no longer have the PiggyBac/35SBAR transposon due to its excision, and also do not contain the Ubi-hyPBase transposase transgene due to segregation. Unlike many transposons, when PiggyBac excises it leaves no footprint scars and thereby returns the wheat border sequence back to its original native wild-type sequence.

245 embryos were infected with Agrobacterium containing the 5 Yr gene/PiggyBac construct and 8 PPT resistant TO plants were obtained. Seven primer pairs (Table 7) were used to amplify junction sequences between Yr genes and Yr gene/wheat border sequences. DNAs from two TO plants, 5YrPiggyB#16 and 5YrPiggyB#18 showed amplification of all junction sequences.

T able 7. Primers to amplify junction sequences between Y r genes and Y r gene/wheat border sequences.

In parallel, independent transformation experiments were undertaken to introduce the Ubi-hyPBase gene into Fielder wheat. The Ubi-HyPBase transgene in pDONORl (Figure 17) was introduced into the binary vector pCSIRO-STACK and used for wheat transformation .

122 embryos were transformed with this construct and transgenic wheat plants selected using Bar selection. 50 TO transgenic plants were produced. RT-QPCR was then undertaken on 8 Ubi-hyPBase TO plants to identify lines that showed higher levels of expression. Five higher expressing lines were identified and then crossed to either 5YrPiggyB#16 or 5YrPiggyB#18.

The resultant hybrid plants were then analysed by PCR junction sequence analysis described above to determine if all junction sequences had been coinherited. Approximately half (21/44) of the progeny from plant 5YrPiggyB#18 inherited all junction sequences consistent with a single locus encoding all 5 genes being present. Much fewer (10/79) progeny of plant 5YrPiggyB#16 showed Yr gene coinheritance and hence this line was discarded as unlikely to carry a full-length sequence.

PiggyBac transposition was then investigated in hybrid plants derived from crossing 5YrPiggyB#18 with either Ubi-hyPBase lines #19, #21 or #24. PCR primers (LB3 and YrUl_3 out) were used that flank the PiggyBac transposon and which amplify an 745 bp product after transposon excision (Figure 18). 14 of the 21 hybrid plants that putatively contain a full length 5 Yr sequence also showed evidence of PiggyBac transposition. Sanger sequencing of these transposon excision amplification products from three plants showed that in each case the PiggyBac transposon-had precisely excised to leave no footprint and therefore restore the wheat genomic sequence. However, a second set of PCR primers that amplified part of PiggyBac and an immediately adjacent wheat sequence showed that each of these plants were chimeric and also had cells in which the transposon had not exercised, which is typical of first- generation, transposon x transposase hybrid plants. Progeny from the hybrid plants showing active transposition will be PCR screened for the presence of all 5 Yr genes, the absence of the PiggyBac transposon and the absence of the Ubi-hyPBase transgene locus.

EXAMPLE 11 - Barlev PT-DNA borders

All Agrobacterium T-DNA border sequences and intervening T-DNA was removed from the backbone of binary vector pCSIRO and barley genomic sequences were used to substitute the bona fide Agrobacterium LB and RB sequences, while a Ubi- RUBY reporter gene was placed inside the barely border sequences. Nucleotides 500797397 - 500797357 of barley chromosome 6H (Ensembl Plants Hordeum vulgare MorexV3_pseudomolecules_assembly -Genomic sequence) was used as a substitute LB sequence (Figure 19) in plasmid pMBl while nucleotides 151772483 - 151772512 of barley chromosome 1H were as a LB in plasmid pMB2 (Figures 19 and 21). For both plasmids pMBl and pMB2 adjacent RB sequence was replaced with nucleotides 50835791 - 508357886 of barley chromosome 5H (Figure 20). Those nucleotides boxed in in Figure 20 is sequence not derived from the wheat genome and are effectively Agrobacterium in origin. These Agrobacterium sequences are not transferred as part of the T-DNA strand. Inserted in the intervening sequence, between these two potential “plant border” sequences, was a. RUBY reporter construct under the regulatory control of a maize polyubiquitin promoter sequence, to create plasmids pMBl and pMB2 (Figure 20). Upon expression, the RUBY reporter gene converts tyrosine into the bright red, visible pigment, betalain (He et al., 2020).

Plasmids pMBl and pMB2 were introduced into Agrobacterium strain AGL1 carrying a disarmed Ti plasmid pMP90, by electroporation. Stock cultures were grown overnight at 28°C in liquid LB media containing the appropriate antibiotics with vigorous shaking. Bacteria were resuspended in infiltration buffer (10 mm MES pH 5.6, 10 mm MgCh and 150 pm acetosyringone) at ODgoo nm of 0.8 and then syringe infiltrated into 4- week-old Nicotiana benthamiana plants grown under controlled environment conditions. An AGL1 Agrobacterium strain that carried a 35S-YFP gene on a standard binary vector, pAM, was also infiltrated as a negative control (Figure 21).

Five days post-infiltration betalain accumulation was observed at both pMB 1 and pMB2 infiltration sites while no betalain accumulation occurred at control sites infiltrated with the Agrobacterium carrying a YFP reporter gene (Figure 22). These data demonstrate that this PT-DNA can be used to insert a polynucleotide of interest into the barley genome.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

This application claims priority from AU 2023903851 filed 29 November 2023, the entire contents of which is incorporated herein by reference.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. REFERENCES

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Claims

1. A DNA construct comprising genes encoding i) a first polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 13, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 13, ii) a second polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 14, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 14, iii) a third polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 15, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 15, iv) a fourth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 16, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 16, v) a fifth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 17, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 17, wherein each gene comprises a promoter that is capable of directing expression of the genes in a cell of a plant, and wherein each polypeptide confers enhanced resistance to one or more fungal pathog en(s).

2. The DNA construct of claim 1, wherein one or more or all of the following apply i) the first polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 13, ii) the second polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 14, iii) the third polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 15, iv) the fourth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 16, or v) the fifth polypeptide comprises a sequence of amino acids provided as SEQ ID NO: 17.

3. The DNA construct of claim 1 or claim 2 which comprises a sequence of nucleotides provided as SEQ ID NO:28, or a nucleotide sequence which is at least 80% identical, at least 90% identical or at least 95% identical to SEQ ID NO:28.

4. The DNA construct according to any one of claims 1 to 3, wherein each polypeptide confers resistance to Puccinia sp. such as Puccinia graminis.

5. A DNA construct comprising genes encoding: i) a first polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1271, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1271, ii) a second polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1272, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1272, iii) a third polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1273, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1273, iv) a fourth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1274, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1274, v) a fifth polypeptide comprising a sequence of amino acids provided as SEQ ID NO: 1275, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 1275, wherein each gene comprises a promoter that is capable of directing expression of the genes in a cell of a plant, and wherein each polypeptide confers enhanced resistance to one or more fungal pathog en(s).

6. A T-DNA or chimeric vector comprising the DNA construct according to any one of claims 1 to 5.

7. A recombinant cell comprising the DNA construct according to any one of claims 1 to 5, or the T-DNA or vector of claim 6.

8. A method of producing the cell of claim 7, the method comprising the step of introducing the DNA construct according to any one of claims 1 to 5, or the T-DNA or vector of claim 6, into a cell.

7. A genetically modified plant, or part thereof, comprising the DNA construct according to any one of claims 1 to 5.

8. The plant of claim 7 which has enhanced resistance to one or more fungal pathogen(s) when compared to a corresponding wild-type plant lacking the DNA construct.

9. The plant of claim 7 or claim 8 which is a wheat plant.

10. The plant according to any one of claims 7 to 9 which only comprises DNA found in a Triticum sp..

11. A method of producing a plant with a DNA construct according to any one of claims 1 to 5, the method comprising the steps of i) introducing a DNA construct according to any one of claims 1 to 5 to a plant cell, ii) regenerating a plant with the DNA construct from the cell, and iii) optionally harvesting seed from the plant, and/or iv) optionally producing one or more progeny plants, thereby producing the plant.

12. A method of producing a plant with a DNA construct according to any one of claims 1 to 5, the method comprising the steps of i) crossing two parental plants, wherein at least one plant comprises a DNA construct according to any one of claims 1 to 5, ii) screening one or more progeny plants from the cross in i) for the presence or absence of the DNA construct, and iii) selecting a progeny plant comprising the DNA construct, thereby producing the plant.

13. A method for identifying a plant which has enhanced resistance to one or more fungal pathogen(s), the method comprising the steps of i) obtaining a sample from a plant, and ii) screening the sample for the presence or absence of a DNA construct according to any one of claims 1 to 5.

14. A method of producing a plant part, the method comprising, a) growing a plant according to any one of claims 7 to 10, and b) harvesting the plant part.

15. A method of producing flour, wholemeal, starch, seedmeal, oil or other product obtained from seed, the method comprising; a) obtaining seed of a plant according to any one of claims 7 to 10, and b) extracting the flour, wholemeal, starch, seedmeal, oil or other product.

16. A product produced from a plant according to any one of claims 7 to 10, and/or a part thereof, wherein the product comprises the DNA construct.

17. A method of preparing a food product, the method comprising mixing seed of a plant according to any one of claims 7 to 10, or flour, wholemeal, starch, seedmeal or oil from the seed, with another food ingredient.

18. Use of a plant according to any one of claims 7 to 10 for controlling or limiting one or more fungal pathog en(s) in crop production.