Barley with improved properties

Description

Barley with improved properties Technical field

[0001] The present invention relates to barley plants with improved properties. In particular, the present invention relates to barley plants with properties useful for the production of barley-based beverages, for example, beer. The present invention also relates to methods for the production of barley-based beverages, as well as to products prepared from the barley plants of the present invention. Background of the Invention

[0002] In commercial malting processes, barley grains are germinated, or malted, under controlled conditions that allow for the partial mobilization of starch and protein reserves from the starchy endosperm over a period of 4 to 6 days. The malting process is normally initiated by soaking the dried barley grain in water. This process is known as steeping, and the aim is not only to clean the grain but also to increase its moisture content to about 40% (w / w) so that the subsequent endosperm mobilization step occurs more quickly. During soaking, the water is usually drained once to allow for re-aeration of the grain. This step is known as "air resting" and is considered necessary, mainly because the submerged grain runs out of oxygen after about 16 hours.After an “air rest” of about 8 hours, the grain is typically re-immersed in water to complete the soaking treatment for another 8 hours, or in a series of re-soaking steps. The two-step soaking process... Petition 870250086789, dated 09 / 25 / 2025, page 25 / 280 2 / 224 Increasing the moisture content of dry grain to 40% or more takes about 32 hours in total.

[0003] The soaked grain is spread out for germination, during which enzymes secreted by aleuronic and scutellar epithelial cells, along with some that already exist in the starchy endosperm cells, degrade the cell walls, starch, and proteins. Under normal germination conditions, the phytohormone gibberellic acid (GA) is believed to be synthesized in the nodal region, or elsewhere in the embryo, from where it diffuses along the water gradient.

[0004] The maltster generally aims to rapidly induce the synthesis of as many starch-degrading enzymes as possible in the grains. In many commercial malting programs, GA may be added to accelerate the enzymatic secretion process from the aleurone layer. Starch-degrading enzymes, which include α and β-amylases, starch debranching enzymes (e.g., limit dextrinase), and α-glucosidases, partially depolymerize the grain's starch reserves into monosaccharides, disaccharides, oligosaccharides, and glucose. The products of starch depolymerization are subsequently used by yeast cells as a carbon source and fermented into beer ethanol.

[0005] α-Amylases play a primary role in starch degradation in the endosperm. The expression of α-amylases is tightly regulated in cereal plants. In wild barley, there is very little expression of α-amylase genes during endosperm growth and maturation, which is Petition 870250086789, dated 09 / 25 / 2025, p. 26 / 280 3 / 224 consistent with the extensive accumulation of starch during this period. During germination, α-amylase activity is increased. Tight regulation of α-amylase activity is important because abnormal α-amylase activity can have serious consequences for plant health. For example, in certain barley varieties, premature germination can occur. This may be associated with sprout and root growth, high α-amylase production, and grain wilting at maturity (Green et al., 1997). Furthermore, elevated α-amylase activity has been found to be frequently increased in wilted grains compared to normal grains (Green et al., 1997). The Himalaya barley cultivar carrying the sln1 mutation has also consistently demonstrated premature germination and elevated α-amylase production (Green et al., 1997).Overexpression of α-amylase genes in the developing endosperm of rice produced varying degrees of calcareous grains, that is, grains composed of immature and poorly compacted starch granules (Nakata et al., 2017).

[0006] Malters also aim to reduce the levels of cell wall polysaccharides in barley grain, particularly (1,3;1,4)-p-glucans and arabinoxylans. (1,3;1,4)-p-glucans can be especially problematic for brewers, as they can be extracted from malt in soluble forms that form highly viscous aqueous solutions that slow down filtration processes in the brewery and contribute to undesirable turbidity in the final beer. Thus, low levels of soluble (1,3;1,4)-p-glucan represent an important quality parameter of malting. (1,3;1,4)-p-glucan can be degraded by Petition 870250086789, dated 09 / 25 / 2025, page 27 / 280 4 / 224 glucanases and therefore high levels of (1,3;1,4)β-glucanase enzymes remain important measures of malt quality.

[0007] As noted above, the germination process typically takes about 4 to 5 days. After the controlled germination stages, the wet malt is dried from a moisture content of 40% to 4 to 5%. This drying process, called kiln drying, consumes a lot of energy and represents a significant cost for the industry. The entire process, including kiln drying, typically takes 6 to 7 days.

[0008] In the brewery, malt is milled to open the grain, and the resulting contents are extracted with hot water in a process known as mashing. The extracted material includes partially degraded starch, proteins, and cell wall molecules, as described above, and these are further degraded by endogenous enzymes from the grain that were extracted from the malt. At this stage, some brewers add additional, and usually cheaper, carbon sources (adjuncts) to support the subsequent yeast fermentation process and to offset the higher costs of malt. These adjuncts may be barley, rice, wheat, or other cereal flours from non-germinated grains, but their addition may require the concomitant addition of hydrolytic enzymes because there are not enough endogenous enzymes in the malt to degrade the adjunct components.The added enzymes are generally derived from unpurified and relatively inexpensive extracts of fungal and / or bacterial cultures. The addition of exogenous enzymes is not legal. Petition 870250086789, dated 09 / 25 / 2025, pages 28 / 280 5 / 224 in some countries, particularly where beer must be produced under strictly regulated conditions.

[0009] Further degradation of starch and other endosperm components extracted in hot water proceeds in a process known as saccharification. After mashing, the extracts are filtered, often in a clarification tank, and cooled. The extract may be boiled in the presence of hops or hop extracts and, after cooling, yeast cultures are added to ferment the released sugars into ethanol. The beer thus produced is usually matured and filtered before bottling. The beer may also be carbonated before bottling.

[0010] As described above, one of the most time-consuming and energy-intensive steps in beer production is malting. Therefore, there is a need to provide materials and methods that can reduce the time required for malting. In particular, there is a need for methods that allow for high hydrolytic enzyme activity and rapid β-glucan degradation during malting.

[0011] However, as noted above, high α-amylase activity is associated with undesirable effects on plant fitness. In particular, high α-amylase activity may be associated with reduced yield, reduced grain starch content and / or pre-harvest germination.

[0012] There is therefore a need for barley plants with a high level of hydrolytic enzymatic activity in the grains quickly after the start of germination, but which nevertheless have properties Petition 870250086789, dated 09 / 25 / 2025, page 29 / 280 6 / 224 optimal agronomic characteristics, such as high yield and, preferably, low occurrence of pre-harvest sprouting. In particular, there is a need for barley plants with a high level of α-amylase activity and / or a high level of β-glucanase activity in the grains rapidly after the start of germination and with ideal agronomic properties. Summary of the invention

[0013] The inventors have surprisingly discovered that barley plants and parts thereof comprising a mutation in the gene encoding CXE2L1, preferably a gain-of-function mutation in CXE2L1, are particularly useful for producing barley-based beverages such as beer. In particular, the inventors have found that barley plants carrying a mutation in the gene encoding CXE2L1, wherein said mutation results in the substitution of an amino acid near the putative ligand binding site in the CXE2L1 polypeptide, are advantageous. Such barley plants are particularly useful for methods of producing cereal-based beverages with reduced malting time.

[0014] Barley plants encoding a gain-of-function mutant CXE2L1 polypeptide, such as barley plants exhibiting a gain-of-function CXE2L1 mutation, can be identified by measuring α-amylase activity in the endosperm grain half without the embryo of said barley plant after being soaked in water and / or germinating, as described in the Examples presented in this descriptive report. In other words, gain of function can be indirectly determined by determining α-amylase activity in the endosperm grain half. Petition 870250086789, dated 09 / 25 / 2025, page 30 / 280 7 / 224 without the embryo. Under normal germination conditions, signaling molecules are produced in the embryo and diffuse along the water gradient. These signaling molecules induce the expression of hydrolytic enzymes in the germinating grain. Thus, normally, embryonic signaling molecules are necessary to induce the expression of hydrolytic enzymes, such as α-amylase, during germination. However, the inventors surprisingly discovered that, in a barley plant encoding a CXE2L1 polypeptide comprising a mutation, as described in this document, hydrolytic enzyme activity, in this case α-amylase activity, is also induced in germinating grains without the embryo.Thus, the inventors discovered that certain point mutations in the CXE2L1 gene result in a gain of function of the mutant CXE2L1 polypeptide, and that the grains of this barley generally have high hydrolytic activity during germination, regardless of the signaling molecules produced by the embryo.

[0015] Thus, when describing and referring in this descriptive report to a gain-of-function mutation in CXE2L1, this refers to a mutation in which barley plants carrying said mutation have α-amylase activity in the mid-grain of the endosperm without embryo of said barley plant after being soaked in water and / or germination, when determined, as described in the Examples in this descriptive report. A gain-of-function mutant CXE2L1 polypeptide is a mutant CXE2L1 polypeptide encoded by a CXE2L1 gene carrying that gain-of-function mutation. Petition 870250086789, dated 09 / 25 / 2025, page 31 / 280 8 / 224 function. Gain of function can potentially occur through different mechanisms. Without being limited by theory, it is believed that gain-of-function mutation in CXE2L1 could encode a gain-of-function mutant CXE2L1 polypeptide with increased CXE2L1 activity. However, gain-of-function mutation in CXE2L1 and a gain-of-function CXE2L1 polypeptide could also gain α-amylase activity in the endosperm grain half without the embryo of the barley plant after being soaked in water and / or germinating through other mechanisms.

[0016] As indicated above, barley plants encoding such mutant CXE2L1 polypeptide have several superior properties compared to similar barley plants that do not comprise said gain-of-function mutant. In particular, such barley plants have high hydrolase activity in germinating barley grains. For example, the plants have increased α-amylase, β-amylase, β-glucanase, and limit dextrinase activity in barley grains, particularly after germination of said grains. Interestingly, they show increased activity as early as 72 hours, preferentially after 48 hours, and most preferentially after 24 hours of germination. The grains of these barley plants also show increased β-glucan degradation during both germination and malting as well as during mashing, and a reduction in the total β-glucan content in the mash, malt, and wort.Interestingly, the decrease in β-glucan content can be observed as early as 120 hours, preferably after 96 hours. Thus, the barley plants described in this descriptive report and... Petition 870250086789, dated 09 / 25 / 2025, page 32 / 280 9 / 224 its parts that encode the mutant polypeptide CXE2L1 are particularly useful for the production of barley-based beverages, such as beer.

[0017] Even more surprising is that the inventors discovered that barley plants encoding this mutant CXE2L1 polypeptide have highly similar, in principle identical, agronomic properties compared to similar barley plants that do not express this mutant CXE2L1 polypeptide. For example, the barley plant encoding a gain-of-function mutant CXE2L1 polypeptide has a height, thousand-grain weight (TGW), and average grain size similar, if not identical, to those of a similar barley plant that does not encode a gain-of-function mutant CXE2L1 polypeptide.

[0018] Furthermore, the inventors discovered, quite surprisingly, that barley plants encoding this mutant CXE2L1 polypeptide, which also carry a mutation in a gene encoding a β-glucan synthase, exhibit a significantly greater increase in hydrolase activity in germinating barley grains, as well as significantly reduced levels of β-glucan, while maintaining normal agronomic properties.

[0019] The barley plants of the present invention have high hydrolytic enzymatic activity at the beginning of germination and, preferably, also low levels of β-glucan, making the barley plants particularly useful for reducing malting time. Thus, the barley plants of the present invention are useful for preparing malt-based beverages, in which said malt is germinated for less than Petition 870250086789, dated 09 / 25 / 2025, page 33 / 280 10 / 224 h, such as less than 72 h, for example less than 48 h, such as in the range of 24 to 96 h, such as in the range of 24 to 72 h, for example in the range of 24 to 48 h.

[0020] Thus, the present invention provides barley plants, products derived therefrom, methods for preparing these barley plants, methods for producing malt, aqueous extracts, as well as methods for producing beverages from these barley plants, according to the claims appended to this document. Brief Description of the Drawings

[0021] Figure 1. Megazyme α-amylase activity staining assay. A few individual endosperm grains (exemplified in this descriptive report by sample E11) showed high α-amylase activity, comparable in activity to the positive controls (three individual positive controls H9-H11, referred to in this descriptive report as Ref).

[0022] Figure 2. Megazyme α-amylase activity staining assay. Verification of isolated and propagated mutants. Individual endosperm grains (eight grains per mutant) from samples E11_grain1 and E11_grain2 of mutant plate 138; samples H5_grain1 and H5_grain2 from plate 139 showed high α-amylase activity, comparable in activity to the reference cultivar Quench (endosperm grains treated with GA hormone).

[0023] Figure 3. Megazyme α-amylase activity staining assay. Verification of isolated and propagated mutants. Average absorbance of eight endosperm grain halves tested shown in Figure 2. Petition 870250086789, dated 09 / 25 / 2025, page 34 / 280 11 / 224

[0024] Figure 4. α-amylase activity (U g DW-1) after 72 hours of whole grain germination, measured using the Ceralpha method (Ceralpha method according to the manufacturer's protocol, Megazyme) modified for the Gallery Plus Beermaster. The black bar indicates grain germination when submerged in a shaker flask, while the gray bar indicates grain germination in Petri dishes. Error bars indicate standard deviations (n=2).

[0025] Figure 5. α-amylase activity (U g DW-1) after 72 hours of endosperm grain germination, measured using the Ceralpha method (Ceralpha method according to the manufacturer's protocol, Megazyme) modified for the Gallery Plus Beermaster. The black bar indicates the germination of grains submerged in the shaker flask, while the gray bar indicates the germination of grains in Petri dishes. The error bars show the standard deviations (n=2).

[0026] Figure 6. Phenotype of endosperm α-amylase activity of half-grain measured with the Megazyme α-amylase activity staining assay. HENZ-a1 and HENZ-a2 endosperm half-grains, grown in New Zealand (2016 / 17) or Denmark (Fyn 2017), exhibit high α-amylase activity compared to wild-type Quench, Planet, or Paustian grains.

[0027] Figure 7 shows higher α-amylase activity in the EENZ-α1 and EENZ-α2 mutants compared with the reference cultivar Quench (wt) after incubation for 24 hours at 25°C in aerated water (90 l / h / kg). Petition 870250086789, dated 09 / 25 / 2025, page 35 / 280 12 / 224

[0028] Figure 8 shows the accumulation of hydrolytic enzyme transcripts. A. Accumulation of α-amylase (AMY1-2) transcript determined by ddPCR after 48 hours of immersion. Error bars indicate the standard deviation calculated from three technical replicates. B. Accumulation of limit dextrinase transcript determined by ddPCR after 48 hours of immersion. Error bars indicate the standard deviation calculated from three technical replicates. C. Accumulation of β-glucanase (BGL2A) transcript determined by ddPCR after 48 hours of immersion. Error bars indicate the standard deviation calculated from three technical replicates.

[0029] Figure 9 shows higher α-amylase activity in HENZ-a1 and HENZ-a2 mutants compared with the reference cultivar Quench (wt), after 24 hours of incubation at 25°C in aerated water (90 l / h / kg) (WA), followed by 24 hours of incubation at 25°C in aerated air (90 l / h / kg) (A).

[0030] Figure 10 shows the germination energy at 4 ml after 24 h, 48 h and 72 h. The data show high grain quality, with no indication of pre-harvest germination.

[0031] Figure 11 shows the germination energy at 4 ml and 8 ml after 72 hours of germination. The barley mutants HENZ-a1 and HENZ-a2 show lower sensitivity to water compared to the reference cultivar Quench (wt).

[0032] Figure 12. General description of micromalting.

[0033] Figure 13. Staining assay of the megazyme α-amylase activity of the endosperm midgrain of HENZ-a1 and F1 progenies from crosses of HENZ-a1 with Bowman, Paustian and Quench. Petition 870250086789, dated 09 / 25 / 2025, page 36 / 280 13 / 224

[0034] Figure 14. Staining assay of Megazyme α-amylase activity in the endosperm of half-grain HENZ-a1 and F2 populations of HENZ-a2 from reciprocal crosses (the top panel shows crosses between Paustian and HENZ-a1, the bottom panel shows crosses between Quench and HENZ-a2).

[0035] Figure 15. Genetic mapping of HENZ-a2 in the F2 Planet x HENZ-a2 population. On the left is the physical genomic map of chromosome 3H, the mapping interval is defined by two flanking markers and their physical positions in megabases (Mb). On the right is the genetic map of HENZ-a2 on 3HS, the marker position is shown in genetic distance (cM). The logarithm of the probabilities for each marker has been plotted alongside, indicating the confidence.

[0036] Figure 16. Candidate region for Sanger sequencing harboring the S113P (T>C) and A127V (C>T) mutations. Horvu_PLANET_3H01G160900 is the Planet reference for CXE2L1; nucleotides identical to the reference are shaded dark.

[0037] Figure 17. Candidate structural model of HENZ-α and FMT-3H. A) Surface of the protein model in dark gray with the substrate / ligand binding pocket in white. The transplanted GA4 / substrate in black spheres is located in the substrate / ligand binding pocket. B) Locations of the amino acids HENZ-α (A127V, dark gray sphere) and FMT3H (S113P, white sphere) highlighted in relation to the substrate / ligand binding pocket (on white surface) with the putative substrate GA4 as black spheres. C) As in B), with the <5 angstrom residues of the substrate-interacting residues highlighted in black rods (1 layer of Petition 870250086789, dated 09 / 25 / 2025, page 37 / 280 14 / 224 waste) and the <5 angstrom waste from the 1st layer of waste separated into white sticks (2nd layer of waste).

[0038] Figure 18. Phylogenetic tree of the CXE family of barley and rice containing the candidate gene CXE2L1 (HORVU.MOREX.r3.3HG0242030, Morex_V3 (Mascher et al. 2021)) HENZ-α and FMT-3H.

[0039] Figure 19 shows the hydrolytic enzyme activity and β-glucan content after malting of FMT-3H + Mut2. A) α-amylase activity, B) β-amylase activity and C) Limit dextrinase activity. D) β-glucan content.

[0040] Figure 20 shows the α-amylase activity measured in micromalted grains of malting barley varieties. The two bars for each variety are replicates. The results are described in more detail in Example 17.

[0041] Figure 21 shows the α-amylase activity in the endosperm of half-grain HENZ-78, HENZ-a1, HENZ-a2, Planet and Quench. Error bars indicate standard deviations. Detailed description Definitions

[0042] As used in this descriptive report, a can mean one or more, depending on the context in which it is used.

[0043] The term α-amylase refers in this descriptive report to an enzyme with α-amylase activity. In particular, an α-amylase according to the present invention is an enzyme capable of catalyzing the endohydrolysis of (1^4)-α-D-glycosidic linkages in polysaccharides containing three or more (1^4)-α-linked D-glucose units. The activity of the α-amylase can be determined by K-CERA 01 / 12 (protocol Petition 870250086789, dated 09 / 25 / 2025, p. 38 / 280 15 / 224 and kit available from Megazyme, Ireland). Barley plants contain multiple genes encoding α-amylase, which are organized into clusters. Typically, barley plants may comprise an amy1_1 cluster, an amy1_2 cluster, and an amy2 cluster. The amy1_2 cluster often comprises only one gene, but for simplicity, it is referred to in this descriptive report as the amy1_2 cluster. In addition to the clusters mentioned above, the barley plant may contain additional α-amylase genes / clusters (e.g., the amy3, amy4_1, and amy4_2 genes). Each of these clusters may comprise one or more α-amylase genes.

[0044] Examples of α-amylase gene sequences are available in the barley genome project published by Mascher et al., 2017 (Morex_V1): Mascher 2017 name gene ID amy1_1a HORVU6Hr1G078330.1 amy1_1b HORVU6Hr1G078360.1 amy1_1c HORVU6Hr1G078420.1 amy1_1d HORVU0Hr1G032700.1 amy1_1e HORVU0Hr1G032850.5 amy1_2 HORVU6Hr1G080790.1 amy2_1 HORVU7Hr1G091150.1 amy2_2 HORVU7Hr1G091240.1 amy2_3 HORVU7Hr1G091250.3 amy3 HORVU5Hr1G068350.1 amy4_1 HORVU2Hr1G071710.5 amy4 2 HORVU3Hr1G067620.1 _ Petition 870250086789, dated 09 / 25 / 2025, p. 39 / 280 16 / 224

[0045] The term adjunct, as used in this descriptive report, refers to carbon-rich raw material sources added during beer preparation, typically during mashing. The adjunct may be an ungerminated cereal grain, which may be ground together with the germinated grains prepared according to the present invention. The adjunct may also be a syrup, sugar, or similar.

[0046] The term amino acid, as used in this descriptive report, refers to a proteinogenic amino acid. Preferably, proteinogenic amino acids are one of the 20 amino acids encoded by the standard genetic code. The one- and three-letter IUPAC codes are used to name amino acids.

[0047] The term amino acid corresponding to X is used in this descriptive report to describe amino acids of a given polypeptide (e.g., a mutant CXE2L1 polypeptide) relative to the amino acids of a reference polypeptide (e.g., CXE2L1 of SEQ ID NO: 1). After alignment between said polypeptide and the reference polypeptide, an amino acid corresponds to X if it is in the same position as X in said alignment.

[0048] The term amylose refers to homopolymers of α-D-glucose. Amylose has a linear molecular structure, since its glucose units are linked almost exclusively by α-1-4-glycosidic bonds.

[0049] The term amylopectin refers to homopolymers of α-D-glucose. Amylopectin molecules contain frequent α-1-6-glycosidic linkages. These Petition 870250086789, dated 09 / 25 / 2025, p. 40 / 280 17 / 224 introduce branching points in the α-1-4 linked glucose chains, resulting in clusters of parallel chains that appear at regular intervals along the axis of the molecule.

[0050] The term approximately, when used in this descriptive report in relation to numerical values, preferably means ± 10%, more preferably ± 5%, and even more preferably ± 1%.

[0051] The term barley in reference to the process of making barley-based beverages, such as beer, particularly when used to describe the malting process, means barley grains or seeds. In all other cases, unless otherwise indicated, barley means the barley plant (Hordeum vulgare, L.), including any breeding line, cultivar or variety, while part of a barley plant may be any part of a barley plant, for example, any tissue or cells.

[0052] The term barley flour, as used in this descriptive report, refers to ground barley grains.

[0053] The term β-amylase refers to an enzyme that catalyzes the hydrolysis of (1->4)-alpha-D-glycosidic linkages in polysaccharides, in order to remove successive maltose units from the non-reducing ends of the chains. β-amylase activity can be determined by K-BETA3 (protocol and kit available from Megazyme, Ireland).

[0054] The term β-glucanase, as used in this descriptive report, refers to enzymes with potential Petition 870250086789, dated 09 / 25 / 2025, p. 41 / 280 18 / 224 to depolymerize β-glucan from cereals. Thus, unless otherwise indicated, the term “β-glucanase” refers to an endo- or exo- enzyme or a mixture thereof, characterized by (1,3;1,4)-β- and / or (1,4)-β-glucanase activity. Barley plants may comprise one or more genes encoding β-glucanase, for example, the BGL2A and / or BGL2B genes. The sequence of BGL2A is available under accession number HORVU.MOREX.r3.7 HG0750120.1, Morex_V3.

[0055] The β-glucan content used in this descriptive report can be determined by any useful method. Preferably, the content refers to the (1,3;1,4)-ε-glucan content. It is determined as the sum of the content of Glc-ε-(1->4)-Glc-ε-(1->3)-Glc (DP3) and Glc-ε-(1->4)-Glc-ε-(1->4)-Glc-ε-(1->3)-Glc (DP4) oligomers. The content of DP3 and DP4 oligomers can, for example, be determined by lichenase digestion of (1,3;1,4)-ε-glucans, followed by quantification, for example, by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0056] The term “β-glucan synthase”, as used in this descriptive report, should be considered as any protein that catalyzes the synthesis of (1,3;1,4)β-glucan and, optionally, catalyzes the polymerization of glucopyranosyl monomers. For example, (1,3;1,4)-β-glucan synthase may be a polypeptide encoded by a CsIF gene or a functional homolog thereof.

[0057] The term “charged amino acid”, as used in this descriptive report, refers to amino acids with electrically charged side chains. Petition 870250086789, dated 09 / 25 / 2025, p. 42 / 280 19 / 224 Preferably, the charged amino acid is selected from the group consisting of Arg, His, Lys, Asp, and Glu. Negatively charged amino acids are preferably selected from the group consisting of Asp and Glu.

[0058] The term chit, as used in this descriptive report, refers to the embryonic growth bud that is visible during the germination phase of a cereal grain.

[0059] The term DP, as used in this descriptive report, refers to the degree of polymerization and indicates the number of α-1,4-linked glucose units in amylopectin side chains. Thus, by way of example, DP3 refers to amylopectin side chains consisting of a sequence of 3 α-1,4-linked glucose units. Similarly, DP4 refers to amylopectin side chains consisting of a sequence of 4 α-1,4-linked glucose units. The term DP3:DP4 ratio of (1,3;1,4)-β-glucans, as used in this descriptive report, refers to the ratio of amylopectin side chains consisting of a sequence of 3 glucose units linked by α-1,4 and amylopectin side chains consisting of a sequence of 4 glucose units linked by α-1,4 within the aforementioned (1,3;1,4)-β-glucans.The DP3:DP4 ratio can be determined by digesting (1,3;1,4)-β-glucans with lichenase, followed by quantification of the released DP3 and DP4 oligomers, for example, by HPAEC-PAD.

[0060] By encoding or encoded, in the context of a specific nucleic acid, is meant the information Petition 870250086789, dated 09 / 25 / 2025, page 43 / 280 20 / 224 for translation into the specified protein. A nucleic acid or polynucleotide that codes for a protein may comprise untranslated sequences, for example, introns, within translated regions of the nucleic acid, or it may not possess such intervening untranslated sequences, for example, in cDNA. The information by which a protein is encoded is specified by the use of codons.

[0061] As used in this descriptive report, expression in the context of nucleic acids should be understood as the transcription and accumulation of mRNA. Expression used in the context of proteins refers to the translation of mRNA into a polypeptide.

[0062] The term gene means the segment of DNA involved in the production of a polypeptide chain; it includes regions that precede and follow the coding region (promoter and terminator). In addition, plant genes generally consist of exons interrupted by introns.

[0063] The term germinated grain, as used in this descriptive report, refers to a grain that has developed a visible cotyledon and a visible root and coleoptile.

[0064] The term green malt, as used in this descriptive report, refers to germinated cereal grains that have not been subjected to a drying stage in an oven. In general, these cereal grains have been germinated under controlled environmental conditions. In some embodiments, green malt is milled green malt.

[0065] The term half grain, as used in this descriptive report, refers to one of the parts of a grain, Petition 870250086789, dated 09 / 25 / 2025, page 44 / 280 21 / 224, that is, a grain or seed that has been divided in two, where one part comprises the entire embryo. Normally, these parts are of similar size. The term "endosperm half-grain without embryo," as used in this descriptive report, refers to the half of the half-grain that does not include the embryo.

[0066] The term “start of germination”, as used in this descriptive report, refers to the moment when barley grains with a water content of less than 15% come into contact with sufficient water to initiate germination. Thus, “germination”, as used in this descriptive report, also includes soaking, in addition to any germination after soaking.

[0067] The term grain is defined as comprising the caryopsis of the cereal, also called the inner seed. In addition, the grain may comprise the lemma and the palea. In most barley varieties, the lemma and palea adhere to the caryopsis and are part of the grain after threshing. However, there are also varieties of naked barley. In these, the caryopsis becomes free from the lemma and palea after threshing and threshes freely, as in wheat. The terms kernel and grain are used interchangeably in this descriptive report. [00 68] The term “kiln-dried malt”, as used in this descriptive report, refers to germinated cereal grains that have been dried in an oven. In general, said cereal grains have been germinated under controlled environmental conditions. In some embodiments, kiln-dried malt is milled kiln-dried malt. Petition 870250086789, dated 09 / 25 / 2025, p. 45 / 280 22 / 224

[0069] The term “limit dextrinase”, as used in this descriptive report, refers to an enzyme capable of catalyzing the hydrolysis of (1->6)-alpha-D-glycosidic linkages in amylopectin and pullulan and in alpha and beta limit dextrins of amylopectin and glycogen. In particular, a limit dextrinase may be an enzyme classified under EC 3.2.1.142. Limit dextrinase activity is determined by the Pullanase Limit Dextrinase Assay Kit (PullG6 method; protocol and kit available from Megazyme, Ireland).

[0070] The term “malting”, as used in this descriptive report, refers to the controlled germination of cereal grains (in particular barley grains) that occurs under controlled environmental conditions. In some embodiments, “malting” may also include a drying step of the germinated cereal grains, for example, by oven drying.

[0071] “Mashing” is the incubation of ground malt (e.g., green malt or oven-dried malt) and / or non-germinated cereal grains in water. Mashing is preferably carried out at specific temperatures and in a specific volume of water.

[0072] The term “milled” refers to material (e.g., barley grains or malt) that has been finely divided, for example, by cutting, grinding, crushing, or grinding. Barley grains can be milled while wet, using, for example, a wet grinder or mill. Milled barley grains or milled malt are sufficiently finely divided to make the material Petition 870250086789, dated 09 / 25 / 2025, page 46 / 280 23 / 224 useful for aqueous extracts. Ground barley grains or ground malt cannot be regenerated in an intact plant by essentially biological methods.

[0073] Mutations include deletions, insertions, substitutions, transversions, and point mutations in the coding and non-coding regions of a gene. Deletions can affect the entire gene or only a part of the gene. Point mutations can involve changes in a base pair and can, for example, result in premature stop codons, frameshift mutations, splice site mutations, or amino acid substitutions. Mutations according to the present invention are preferably point mutations. A gene comprising a mutation may be referred to as a mutant gene. If said mutant gene encodes a polypeptide with a sequence different from the wild type, said polypeptide may be referred to as a mutant polypeptide.

[0074] The term nonpolar amino acid, as used in this descriptive report, refers to amino acids with hydrophobic side chains. Preferably, the nonpolar amino acid is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Gly, more preferably from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Trp and Gly.

[0075] The term plant product means a product resulting from the processing of a plant or plant material. The said plant product may therefore, for example, be green malt, kiln-dried malt, wort, a beverage. Petition 870250086789, dated 09 / 25 / 2025, p. 47 / 280 24 / 224 fermented or unfermented, a food or feed product for animals.

[0076] The term “polar amino acid”, as used in this descriptive report, refers to amino acids with polar and uncharged side chains. Preferably, the polar amino acid is selected from the group consisting of Ser, Thr, Asn and Gln.

[0077] The term “offspring,” as used in this descriptive report, means a plant that is directly or indirectly descended from a particular plant. Thus, offspring are not limited to direct descendants but also include descendants after several generations. In general, the offspring of a barley plant carrying a specific mutation also carries that specific mutation. Thus, the offspring of a barley plant carrying a specific mutation in the CXE2L1 gene also carries that specific mutation.

[0078] The term “sequence identity,” as used in this descriptive report, refers to the percentage of identical amino acids or nucleotides between a candidate sequence and a reference sequence after alignment. Thus, a candidate sequence that shares 80% amino acid identity with a reference sequence requires that, after alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is determined with the aid of computational analysis, such as, without limitation, the Clustal Omega computational alignment program for alignment of Petition 870250086789, dated 09 / 25 / 2025, p. 48 / 280 25 / 224 polypeptide sequences (Sievers et al. (October 11, 2011) Molecular Systems Biology 7:539, PMID: 21988835; Li et al. (April 6, 2015) Nucleic Acids Research 43 (W1): W580-4 PMID: 25845596; McWilliam et al., (May 13, 2013) Nucleic Acids Research 41 (web server edition): W597-600 PMID: 23671338, and the standard parameters suggested therein. The Clustal Omega software is available from EMBLEBI at https: / / www.ebi.ac.uk / Tools / msa / clustalo / . Using this program with its default settings, the mature (bioactive) portion of a query and a reference polypeptide are aligned. The number of fully conserved residues is counted and divided by the length of the reference polypeptide. The MUSCLE or MAFFT algorithms can be used for nucleotide sequence alignment. Sequence identities can be calculated similarly to that indicated for amino acid sequences. The sequence identity, as provided in this descriptive report, is therefore calculated along the entire length of the reference sequence.

[0079] The term “similar genotype”, as used in this descriptive report, for example, in the context of “a barley plant carrying a mutation in the CXE2L1 gene, but with a similar genotype”, refers to said barley plants that have essentially the same genotype, except for a limited number of random and / or spontaneous mutations. For example, a parental barley plant is considered to be of similar genotype to barley plants obtained after random mutagenesis of said parental plant, except that the plants Petition 870250086789, dated 09 / 25 / 2025, p. 49 / 280 26 / 224 barley plants may carry a mutation in the CXE2L1 gene after mutagenesis.

[0080] The term starch, as used in this descriptive report, refers to a composition of one or both of the discrete macromolecules: amylose and amylopectin.

[0081] The term immersion, as used in this descriptive report, refers to the process of increasing the water content of a cereal grain.

[0082] The term stop codon, as used in this descriptive report, refers to a triplet of nucleotides in the genetic code that, within mRNA, results in the termination of translation. The term stop codon, as used in this descriptive report, also refers to a triplet of nucleotides within a gene that encodes the stop codon in mRNA. The stop codon in DNA typically has one of the following sequences: TAG, TAA, or TGA.

[0083] The term water content of a grain, as used in this descriptive report, refers to the % H2O w / w in that grain.

[0084] The term wild-type CslF6, as used in this descriptive report, refers to the polypeptide with SEQ ID NO: 7 or SEQ ID NO: 8.

[0085] The wild-type term CXE2L1, as used in this descriptive report, refers to the polypeptide with SEQ ID NO: 1.

[0086] The term wort refers to a liquid extract of malt and / or cereal grains, such as milled malt and / or milled cereal grains and, optionally, additional adjuncts. The Petition 870250086789, dated 09 / 25 / 2025, p. 50 / 280 27 / 224 Wort is generally obtained by mashing, optionally followed by spraying, which is a process of extracting residual sugars and other compounds from the grains used after mashing with hot water. Spraying is typically carried out in a lauter tun, a mash sieve, or other apparatus to allow the separation of the extracted water from the grains used, also known as mash filtration. The wort obtained after mashing is generally referred to as “first wort,” while the wort obtained after spraying is generally referred to as “second wort.” If not specified, the term wort may be first wort, second wort, or a combination of both. During conventional beer production, the wort is boiled together with the hops. Wort without hops may also be referred to as “sweet wort,” while wort boiled with hops may be referred to as “boiled wort” or simply as wort.

[0087] The enzymatic activities of cereal grains, as used in this descriptive report, refer to the activities measured in flour prepared from the specified grain type. For example, 10 U / g of alpha-amylase activity per gram of cereal grain refers to said alpha-amylase activity (10 U) measured in an aqueous extract derived from 1 g of flour (dry matter) of said cereal. α-amylase activity is determined by K-CERA. 01 / 12 (protocol and kit available from Megazyme, Ireland). β-amylase activity is determined by K-BETA3 (protocol and kit available from Megazyme, Ireland). Limit dextrinase activity is determined by the PullG6 method (protocol and kit available from Megazyme, Ireland). Petition 870250086789, dated 09 / 25 / 2025, p. 51 / 280 28 / 224

[0088] The volume of a gas, as indicated in this descriptive report, refers to the volume of said gas at 1 atm and 20°C.

[0089] The volume of O2 indicated in this descriptive report refers to the volume of O2 at 1 atm and 20°C. In embodiments of the present invention where the O2 is comprised in a gas mixture, then the total volume of the gas mixture can be determined, and the volume of O2 can be calculated as the percentage of the total volume consisting of O2. As an example, atmospheric air comprises 21% O2. Thus, the volume of O2 in atmospheric air, as used in this descriptive report, is 21% of the total volume of atmospheric air. Barley plant carrying a mutation in the CXE2L1 gene

[0090] The present invention relates to a barley plant or part thereof, wherein said barley plant has high α-amylase activity in a half endosperm grain without embryo, and wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutated CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide. The wild-type CXE2L1 is preferably the CXE2L1 of SEQ ID NO: 1, but may also be a functional variant thereof with at least 95% sequence identity with this.

[0091] In some respects, the present invention relates to a barley plant or part thereof, wherein said barley plant has high α-amylase activity in a half-grain of endosperm without embryo, wherein said barley plant carries: Petition 870250086789, dated 09 / 25 / 2025, p. 52 / 280 29 / 224 a. a point mutation in the CXE2L1 gene, in which the mutated CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, and b. a mutation in a gene that codes for a β-glucan synthase.

[0092] The wild-type CXE2L1 is preferably the CXE2L1 of SEQ ID NO: 1, but may also be a functional variant thereof with at least 95% sequence identity with it. In some embodiments, the gain-of-function CXE2L1 mutant comprises a substitution as described below.

[0093] In some aspects, the present invention relates to a barley plant or part thereof, wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity therewith, except that the mutant CXE2L1 comprises a substitution in: a. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P), provided that the plant does not contain SEQ ID NO: 5.

[0094] In some respects, the present invention relates to a barley plant or part thereof, wherein said barley plant carries a point mutation in the CXE2L1 gene, Petition 870250086789, dated 09 / 25 / 2025, p. 53 / 280 30 / 224 wherein the mutated CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein the mutant CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: a. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P), provided that the plant does not contain SEQ ID NO: 5; and / or c. the amino acid corresponding to amino acid 125 of SEQ ID NO: 1, where the substitution in question is a substitution of a proline (P) for a serine (S); and / or d. the amino acid corresponding to amino acid 126 of SEQ ID NO: 1, where the substitution in question is a substitution of an alanine (A) for a threonine (T); and / or e. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a threonine (T); and / or f. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a threonine (T); and / or g. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a valine (V). Petition 870250086789, dated 09 / 25 / 2025, p. 54 / 280 31 / 224

[0095] In some respects, the present invention relates to a barley plant or part thereof, wherein said barley plant bears: a. a point mutation in the CXE2L1 gene, wherein said mutated CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: i. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, wherein said substitution is a substitution of a serine (S) for a proline (P); and also b. a mutation in a gene that codes for a β-glucan synthase.

[0096] In some respects, the present invention relates to a barley plant or part thereof, wherein said barley plant bears: a. a point mutation in the CXE2L1 gene, wherein said mutated CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: Petition 870250086789, dated 09 / 25 / 2025, p. 55 / 280 32 / 224 i. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, wherein said substitution is a substitution of a serine (S) for a proline (P); and / or iii. the amino acid corresponding to amino acid 125 of SEQ ID NO: 1, wherein said substitution is a substitution of a proline (P) for a serine (S); and / or iv. the amino acid corresponding to amino acid 126 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a threonine (T); and / or v. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a threonine (T); and / or vi. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a threonine (T); and / or vii.the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of alanine (A) for valine (V); and also b. a mutation in a gene that codes for a β-glucan synthase.

[0097] In some embodiments, the barley plant or part thereof has high α-amylase activity in half-grain endosperm without embryo. The “high α-amylase activity” is preferably an activity of Petition 870250086789, dated 09 / 25 / 2025, p. 56 / 280 33 / 224 at least 20 U / g dry weight 24 hours after soaking a half dry grain with water.

[0098] In some embodiments, the grains of the barley plant of the present invention have a reduced β-glucan content compared to barley that does not carry the mutation(s) but otherwise has a similar phenotype. In particular, the germinated grains of the barley plant of the present invention have a reduced β-glucan content 4 to 6 days after the start of germination compared to wild-type barley compared to barley that does not carry the mutation(s) but otherwise has a similar phenotype.

[0099] In some embodiments, the wild-type CXE2L1 polypeptide is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 90% sequence identity with the same, such as at least 91% sequence identity with the same, such as at least 92% sequence identity with the same, such as at least 93% sequence identity with the same, such as at least 94% sequence identity with the same, 95% sequence identity with the same, such as at least 96% sequence identity with the same, such as at least 97% sequence identity with the same, such as at least 98% sequence identity with the same, such as at least 99% sequence identity with the same, such as 100% sequence identity with the same. The CXE2L1 gene may be any gene encoding the CXE2L1 mentioned above. Petition 870250086789, dated 09 / 25 / 2025, p. 57 / 280 34 / 224

[0100] In principle, the barley plant of the present invention may comprise any type of mutation in the CXE2L1 gene that results in a gain-of-function mutant CXE2L1 polypeptide. In particular, the mutation in the CXE2L1 gene may result in the substitution of an amino acid near the ligand (i.e., substrate) binding site of the CXE2L1 polypeptide.

[0101] In some embodiments, the gain-of-function mutant CXE2L1 polypeptide comprises a substrate-interacting amino acid substitution, such as an amino acid in the active site of CXE2L1.

[0102] In some embodiments, the gain-of-function mutant CXE2L1 polypeptide comprises an amino acid substitution positioned <5 angstrom from the amino acids that interact with the CXE2L1 substrate.

[0103] In some embodiments, the gain-of-function mutant CXE2L1 polypeptide comprises an amino acid substitution positioned <5 angstrom from amino acids that are <5 angstrom from amino acids that interact with the CXE2L1 substrate.

[0104] In some embodiments, the gain-of-function mutant CXE2L1 polypeptide comprises a selected amino acid substitution from the group consisting of: a. the amino acid corresponding to S113 or A127 of SEQ ID NO: 1; or b. the amino acid corresponding to P125, A126, or A212 of SEQ ID NO: 1; or Petition 870250086789, dated 09 / 25 / 2025, p. 58 / 280 35 / 224 c. the amino acid corresponding to F13, L14, G83, G84, G85, L88, Q93, F96, H166, S167, A168, F201, S219, L220,T221, M224, L228, H300, G301, F302, I304, or R305 of SEQ ID NO: 1; or d. the amino acid corresponding to E11, D12, G15, V16, V17, Q18, R27, E30, L33, T35, Y63, Y80, F81, H82, Y86,C87, G89, S90, I91, A92, P94, N95, H97, S98, L99, C100, Y116 L118, S164, G165, G169, A170, N171, L172, A173, L198,S199, A200, F202, A203, G217, V218, T222, A223, A225, D226, Q227 W229, R230, M231, S232, L233, A244, V265, P267, S269, D270 V271, L272, F295, E298, Q299, P303, Q306, P307, S309, T311, A312, or L315 da SEQ ID NO: 1; ou e. o aminoacido correspondente to V9, V10, L19, L20, S24, V25, V26, G28, D29, A31, V32, R34, N36, G37, L39, P40 V42, V45, Q46, W47, D49, Y52, L58, S59, V60, R61, A62,R64, P65, L78, V79, L101, R102, A103, A104, A105, A109,V110, V111, L112, S113, V114, Q115, R117, A119, P120, E121,H122, R123, L124, A127, I128, D130, G131, F134, W151, F162,L163, H174, H175, V176, T177, V178, I196, L197, G204, A205, R207 T208, T210, E211, P214, P215, E216, P234, V235, A237,S238, M239, H241, P242, L243, N245, P246, L263, V264, A266,L268, R273, D274, R275, V276, Y279, V293, Q294, E296, G297,F308, E310, S313, E314, L316, R317, V318, I319, or R320 of SEQ ID NO: 1; f. the amino acid corresponding to Y86, Y116, R117, H122, R123, L124, P125, A126, I128, D129, D130, G131 or L172 of SEQ ID NO: 1; g. the amino acid corresponding to S59, V60, R61, Y63, Y80, I91, C100, V111, L112, V114 or Q115 of SEQ ID NO: 1; Petition 870250086789, dated 09 / 25 / 2025, p. 59 / 280 36 / 224 where wild CXE2L1 is CXE2L1 with SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it.

[0105] Substitutions can be a replacement of any of the amino acids mentioned above with any other amino acid.

[0106] In some embodiments, the substitution is a replacement of an alanine with a nonpolar amino acid and / or an aliphatic amino acid and / or an amino acid with a hydrophobic side chain. In some embodiments, the substitution is a replacement of an alanine with a valine.

[0107] In some embodiments, the substitution is a replacement of a serine by a small amino acid and / or an aliphatic amino acid. In some embodiments, the substitution is a replacement of a serine by a proline.

[0108] In some embodiments, the substitution in the CXE2L1 polypeptide comprises a substitution in: a. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P).

[0109] In some embodiments, the substitution in the CXE2L1 polypeptide comprises a substitution in: Petition 870250086789, dated 09 / 25 / 2025, pp. 60 / 280 37 / 224 a. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P); and / or c. amino acid 125 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of proline (P) for serine (S); and / or d. amino acid 126 of SEQ ID NO: 1, where the substitution in question is a substitution of alanine (A) for threonine (T); and / or e. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or f. amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or g. amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for valine (V).

[0110] In some embodiments, in addition to the substitution in the CXE2L1 polypeptide described above, the CXE2L1 polypeptide may comprise one or more mutations selected from the group consisting of: • the amino acid corresponding to A62 of the SEQ ID NO: 1 for V (A62V) or for T (A62T), • the amino acid corresponding to S67 of the SEQ ID NO: 1 for L (S67L), Petition 870250086789, dated 09 / 25 / 2025, p. 61 / 280 38 / 224 • the amino acid corresponding to V153 of SEQ ID NO:1 to M (V153M), • the amino acid corresponding to A212 of SEQ ID NO:1 to T (A212T) or to V (A212V), • the amino acid corresponding to E216 of SEQ ID NO:1 to K (E216K), • the amino acid corresponding to M224 of SEQ ID NO:1 to I (M224I), • the amino acid corresponding to R230 of SEQ ID NO:1 to C (R230C) or to H (R230H) or • the amino acid corresponding to S238 of SEQ ID NO:1 to N (S238N), where wild-type CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it.

[0111] In some embodiments, the substitution is selected from the substitutions listed in Table A, Table B, or Table C below. Ter denotes stop codon. Table A. High-probability ligand / substrate pocket-forming residues FIND-IT 5 angstrom of alpha-filled GA4 (representative substrate) SNP Position (CDS) SNP Position AA WT AA MUT AA 77 C > T 14 Leu Phe 247 G > A 83 Gly Ser 248 G > A 83 Gly Asp 250 G > A 84 Gly Arg 251 G > A 84 Gly Glu 253 G > A 85 Gly Ser Petition 870250086789, dated 09 / 25 / 2025, p. 62 / 280 39 / 224 254 G > A 85 Gly Asp 262 C > T 88 Leu Phe 277 C > T 93 Gln Ter 496 C > T 166 His Tyr 500 C > T 167 Ser Leu 502 G > A 168 Ala Thr 503 C > T 168 Ala Val 656 C > T 219 Ser Phe 662 C > T 221 Thr Met 672 G > A 224 Met Ile 682 C > T 228 Leu Phe 898 C > T 300 His Tyr 901 G > A 301 Gly Ser 902 G > A 301 Gly Asp 913 C > T 305 Arg Cys 914 G > A 305 Arg His Table B FIND-IT high probability 1' line 5 angstrom out of the ligand / substrate pocket forming residues SNP_Position (CDS) SNP Position AA WT AA MUT AA 31 G > A 11 Glu Lys 34 G > A 12 Asp Asn 43 G > A 15 Gly Ser 44 G > A 15 Gly Asp 46 G > A 16 Val Ile 49 G > A 17 Val Met 52 C > T 18 Gln Ter 79 C > T 27 Arg Cys 80 G > A 27 Arg His 88 G > A 30 Glu Lys Petition 870250086789, dated 09 / 25 / 2025, p. 63 / 280 40 / 224 97 C > T 33 Leu Phe 104 C > T 35 Thr Ile 244 C > T 82 His Tyr 260 G > A 87 Cys Tyr 265 G > A 89 Gly Ser 266 G > A 89 Gly Asp 269 C > T 90 Ser Phe 274 G > A 92 Ala Thr 275 C > T 92 Ala Val 280 C > T 94 Pro Ser 281 C > T 94 Pro Leu 289 C > T 97 His Tyr 293 C > T 98 Ser Leu 295 C > T 99 Leu Phe 299 G > A 100 Cys Tyr 352 C > T 118 Leu Phe 491 C > T 164 Ser Phe 493 G > A 165 Gly Ser 494 G > A 165 Gly Asp 505 G > A 169 Gly Ser 506 G > A 169 Gly Asp 508 G > A 170 Ala Thr 509 C > T 170 Ala Val 517 G > A 173 Ala Thr 518 C > T 173 Ala Val 592 C > T 198 Leu Phe 596 C > T 199 Ser Phe 598 G > A 200 Ala Thr 599 C > T 200 Ala Val 607 G > A 203 Ala Thr 608 C > T 203 Ala Val Petição 870250086789, de 25 / 09 / 2025, pág. 64 / 280 41 / 224 649 G > A 217 Gly Ser 650 G > A 217 Gly Asp 652 G > A 218 Val Met 665 C > T 222 Thr Ile 667 G > A 223 Ala Thr 668 C > T 223 Ala Val 673 G > A 225 Ala Thr 674 C > T 225 Ala Val 676 G > A 226 Asp Asn 679 C > T 227 Gln Ter 686 G > A 229 Trp Ter 687 G > A 229 Trp Ter 688 C > T 230 Arg Cys 689 G > A 230 Arg His 693 G > A 231 Met Ile 695 C > T 232 Ser Leu 730 G > A 244 Ala Thr 731 C > T 244 Ala Val 793 G > A 265 Val Ile 799 C > T 267 Pro Ser 800 C > T 267 Pro Leu 806 G > A 269 Ser Asn 808 G > A 270 Asp Asn 811 G > A 271 Val Met 814 C > T 272 Leu Phe 892 G > A 298 Glu Lys 895 C > T 299 Gln Ter 907 C > T 303 Pro Ser 908 C > T 303 Pro Leu 916 C > T 306 Gln Ter 919 C > T 307 Pro Ser Petição 870250086789, de 25 / 09 / 2025, pág. 65 / 280 42 / 224 920 C > T 307 Pro Leu 926 G > A 309 Ser Asn 932 C > T 311 Thr Met 934 G > A 312 Ala Thr 935 C > T 312 Ala Val Tabela C. FIND-IT high probability 2' row 5 angstrom outside 1st row SNP Position (CDS) SNP Position AA WT AA MUT AA 25 G > A 9 Val Ile 28 G > A 10 Val Met 55 C > T 19 Leu Phe 58 C > T He 20 Leu Phe 271 C T He 73 Ser G > A 25 Val Ile 76 G > A 26 Val Ile 82 G > A 28 Gly Ser 83 G > A 28 Gly Asp 85 G > A 29 Asp Asn 91 G > A 31 Ala Thr 92 C > T 31 Ala Val 94 G > A 32 Val Ile 100 C T 130 Cg > 14 Gys > Ar C Arg His 109 G > A 37 Glu Lys 118 C > T 40 Pro Ser 119 C > T 40 Pro Leu 124 G > A 42 Val Ile 133 G > A 45 Val Met Petition 870250086789, of 25 / 09 / 2025, p. 66 / 280 43 / 224 136 C > T 46 Gln Ter 140 G > A 47 Trp Ter 141 G > A 47 Trp Ter 145 G > A 49 Asp Asn 172 C > T 58 Leu Phe 176 G > A 59 Ser Asn 178 G > A 60 Val Ile 181 C > T 61 Arg Cys 182 G > A 61 Arg His 184 G > A 62 Ala Thr 185 C > T 62 Ala Val 191 G > A 64 Arg Lys 193 C > T 65 Pro Ser 194 C > T 65 Pro Leu 235 G > A 79 Val Met 301 C > T 101 Leu Phe 304 C > T 102 Arg Cys 305 G > A 102 Arg His 307 G > A 103 Ala Thr 308 C > T 103 Ala Val 310 G > A 104 Ala Thr 311 C > T 104 Ala Val 313 G > A 105 Ala Thr 314 C > T 105 Ala Val 325 G > A 109 Ala Thr 326 C > T 109 Ala Val 328 G > A 110 Val Ile 331 G > A 111 Val Met 338 C > T 113 Ser Phe 340 G > A 114 Val Met 343 C > T 115 Gln Ter Petição 870250086789, de 25 / 09 / 2025, pág. 67 / 280 44 / 224 349 C > T 117 Arg Cys 350 G > A 117 Arg His 355 G > A 119 Ala Thr 356 C > T 119 Ala Val 358 C > T 120 Pro Ser 359 C > T 120 Pro Leu 361 G > A 121 Glu Lys 364 C > T 122 His Tyr 367 C > T 123 Arg Cys 368 G > A 123 Arg His 370 C > T 124 Leu Phe 379 G > A 127 Ala Thr 380 C > T 127 Ala Val 388 G > A 130 Asp Asn 391 G > A 131 Gly Ser 392 G > A 131 Gly Asp 452 G > A 151 Trp Ter 453 G > A 151 Trp Ter 487 C > T 163 Leu Phe 520 C > T 174 His Tyr 523 C > T 175 His Tyr 526 G > A 176 Val Ile 530 C > T 177 Thr Met 532 G > A 178 Val Ile 589 C > T 197 Leu Phe 610 G > A 204 Gly Arg 611 G > A 204 Gly Glu 613 G > A 205 Ala Thr 614 C > T 205 Ala Val 619 C > T 207 Arg Cys 620 G > A 207 Arg His Petição 870250086789, de 25 / 09 / 2025, pág. 68 / 280 45 / 224 623 C > T 208 Thr Met 629 C > T 210 Thr Met 631 G > A 211 Glu Lys 640 C > T 214 Pro Ser 641 C > T 214 Pro Leu 643 C > T 215 Pro Ser 644 C > T 215 Pro Leu Glu A > Lys > T 234 Pro Ser 701 C > T 234 Pro Leu 703 G > A 235 Val Met 709 G > A 237 Ala Thr 710 C > T 237 Ala Val 713 G > A 238 Ser Asn 717 G > A 239 Met Ile 721 C > T 234 Pro Ser 725 C > T 242 Pro Leu 736 C > T 246 Pro Ser 737 C > T 246 Pro Leu 787 C > T 263 Leu Phe 790 G > A 264 Val Ile 796 G > A 266 Ala Thr 797 C > T 266 Ala C Valg T C > 273 Ar > 273 Ar A 273 Arg His 820 G > A 274 Asp Asn 823 C > T 275 Arg Cys 824 G > A 275 Arg His 826 G > A 276 Val Met 877 G > A 293 Val Ile Petition 870250086789, of 25 / 09 / 2025, p. 69 / 280 46 / 224 880 C > T 294 Gln Ter 886 G > A 296 Glu Lys 889 G > A 297 Gly Arg 890 G > A 297 Gly Glu 928 G > A 310 Glu Lys 938 G > A 313 Ser Asn 940 G > A 314 Glu Lys 949 C > T 317 Arg Trp 950 G > A 317 Arg Gln 952 G > A 318 Val Met 959 G > A 320 Arg Lys

[0112] In some embodiments, the CXE2L1 polypeptide comprises at least one substitution, such as at least two substitutions, such as at least three substitutions, such as at least four substitutions, such as at least five substitutions selected from any of the substitutions described in this descriptive report.

[0113] In specific embodiments, the replacement is S113P, provided that the plant does not contain SEQ ID NO: 5.

[0114] As used in this descriptive report, the terms “amino acid X substitution for amino acid Y” or “amino acid X substitution for amino acid Y” refer to amino acid X in a reference sequence (typically the wild-type sequence CXE2L1 of SEQ ID NO: 1) being substituted for amino acid Y. Amino acid X substitution for amino acid Y may also be described as XnY, where n indicates the position of the amino acid in the sequence. For example, Petition 870250086789, dated 09 / 25 / 2025, pp. 70 / 280 47 / 224 S113P refers to a replacement of Ser in position 113 with Pro. Characteristics of a barley plant carrying a mutation in CXE2L1

[0115] The hydrolytic enzymatic activity, such as α-amylase activity, of barley plants is tightly regulated. During germination, hydrolytic enzymatic activity aids in the conversion of starch into sugar. However, at other times during plant development, for example, during grain filling, hydrolytic enzymatic activity, such as α-amylase activity, is undesirable because it can result in reduced grain filling, reduced starch content, shriveled grains, and / or pre-harvest sprouting.

[0116] The present invention provides barley plants carrying a mutation in CXE2L1, wherein said mutation preferentially leads to the expression of a gain-of-function mutant CXE2L1 polypeptide. One of the main advantages of these barley plants is the increased hydrolytic enzymatic activity during germination.

[0117] The inventors discovered that, quite surprisingly, barley plants or parts thereof carrying a point mutation in CXE2L1 have highly similar, in principle identical, agronomic properties compared to barley plants or parts thereof of a similar genotype that do not carry such a point mutation in CXE2L1 and therefore do not comprise the mutant CXE2L1 polypeptide. This discovery is quite surprising because aberrant α-amylase activity can have serious consequences for the Petition 870250086789, dated 09 / 25 / 2025, page 71 / 280 48 / 224 plant health. The barley plants of the present invention are therefore preferably characterized by having highly similar and / or identical agronomic properties compared to the agronomic properties of a barley plant that does not have a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0118] In some embodiments, the aforementioned agronomic properties are selected from the group consisting of flowering time, plant height, thousand grain weight (TGW), average grain size, starch content in grains, protein content in grains, water content in grains, number of shoots, time to maturity, germination speed, time to heading and plant yield.

[0119] In some embodiments, the grains of said barley plant do not germinate on the plant before harvest. In other words, the barley plant does not have pre-harvest germination. Pre-harvest germination is highly undesirable.

[0120] Furthermore, as mentioned above, barley plants carrying a point mutation in CXE2L1 according to the present invention have high activity of one or more hydrolytic enzymes. In particular, barley plants carrying a point mutation in CXE2L1 according to the present invention have high activity of one or more hydrolytic enzymes in the grains during germination.

[0121] In some embodiments, one or more hydrolytic enzymes are selected from the group consisting Petition 870250086789, dated 09 / 25 / 2025, page 72 / 280 49 / 224 in α-amylase, β-amylase, limit dextrinase, pullulanase, β-glucanase, xylanase, glucoamylase and protease.

[0122] In some embodiments, one or more hydrolytic enzymes is selected from the group consisting of α-amylase, β-amylase and limit dextrinase.

[0123] In some embodiments, the level, as well as the amount, of mRNA transcribed from one or more genes encoding hydrolytic enzymes in the barley plant or part thereof is increased compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but which is of a similar genotype, wherein the level, as well as the amount, of mRNA is measured in whole grains, wherein said grains have been germinated for at least 12 h, as well as at least 24 h, as well as at least 36 h, as well as at least 48 h.

[0124] In some embodiments, the level, as well as the amount, of mRNA is increased by at least 5%, as well as by at least 10%, as well as by at least 15%, as well as by at least 20%, as well as by at least 30%, as well as by at least 40%, as well as by at least 50%, as well as by at least 60%, as well as by at least 70%, as well as by at least 80%, as well as by at least 90%, as well as by at least 100%, as well as by at least 200% compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but that is genotypically similar, wherein the level, as well as the amount, of mRNA is measured in whole grains, wherein said grains have been germinated for at least 12 h, as well as for at least 24 h, as well as for at least 36 h, as well as for at least 48 h. Petition 870250086789, dated 09 / 25 / 2025, p. 73 / 280 50 / 224

[0125] In some embodiments, the level, as well as the amount, of mRNA is increased between 5% and 200%, as well as between 10% and 150%, as well as between 25% and 100%, as well as between 15% and 80%, as well as between 50% and 150%, compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but that is of a similar genotype, wherein the level, as well as the amount, of mRNA is measured in whole grains, wherein said grains have been germinated for at least 12 h, as well as at least 24 h, as well as at least 36 h, as well as at least 48 h.

[0126] The level, like the quantity, of mRNA can be measured using any method known to the expert in the technical field. For example, the mRNA level can be measured using real-time reverse transcription polymerase chain reaction (RT-qPCR).

[0127] In particular, plants that have a point mutation in CXE2L1 have high α-amylase activity in the embryo-free endosperm grain half. Conversely, α-amylase activity in the embryo-free endosperm grain half of a plant that does not contain a mutant CXE2L1 polypeptide according to the present invention, but which is of a similar genotype, is very low. In principle, no α-amylase activity can be detected in the embryo-free endosperm grain half of a plant that does not express such a gain-of-function mutant CXE2L1 polypeptide, but which otherwise has a similar genotype.

[0128] In some embodiments, the high activity of α-amylase in a medium endosperm grain without Petition 870250086789, dated 09 / 25 / 2025, page 74 / 280 51 / 224 embryo is at least 5 U / g, preferably at least 10 U / g, such as at least 15 U / g, such as at least 20 U / g, such as at least 25 U / g, such as at least 30 U / g, such as at least 35 U / g, such as at least 40 U / g, such as at least 45 U / g, such as at least 50 U / g in dry weight.

[0129] In some embodiments, the high α-amylase activity in half-grain endosperm without embryo is at least 30 U / g dry weight.

[0130] In some embodiments, the high activity of α-amylase in a half endosperm grain without embryo is between 20 U / g and 60 U / g, as well as between 30 U / g and 50 U / g, as well as between 30 U / g and 45 U / g, as well as between 35 U / g and 45 U / g, as well as between 30 U / g and 40 U / g, as well as between 35 U / g and 40 U / g, as well as between 40 U / g and 50 U / g.

[0131] Preferably, the high α-amylase activity mentioned above is determined 12 to 48 hours after soaking the dried grains in water. More preferably, said high α-amylase activity is determined as described in Example 3 below.

[0132] In some embodiments, α-amylase activity in the endosperm half-grains without the embryo of said barley plant is increased at least twice compared to a barley plant that does not have a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype, such as at least 3 times, such as at least 4 times, such as at least 5 times, such as at least 6 times, such as at least 7 times, such as at least 8 times, such as at least 9 times, such as at least 10 times, such as at least 20 times, such as by Petition 870250086789, dated 09 / 25 / 2025, pp. 75 / 280 52 / 224 at least 30 times, as by at least 50 times, as by at least 100 times, as by at least 1000 times, as by at least 10,000 times compared to a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0133] In some embodiments, α-amylase activity in the endosperm half-grains without the embryo of said barley plant is increased between 2 and 10,000 times compared to a barley plant that does not have a point mutation in the CXE2L1 gene, but which is genotypically similar, such as between 5 and 100 times, such as between 20 and 100 times, such as between 3 and 50 times, such as between 5 and 25 times, such as between 10 and 75 times, such as between 30 and 60 times, such as between 300 and 500 times, such as between 100 and 10,000 times, such as between 50 and 1,000 times, such as between 500 and 5,000 times, compared to a barley plant that does not carry a point mutation in the CXE2L1 gene, but which, otherwise, It has a similar genotype.

[0134] In some embodiments, α-amylase activity in whole grains of barley plants carrying a point mutation in CXE2L1 is increased compared with α-amylase activity in whole grains of a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0135] In some embodiments, α-amylase activity in whole grains of barley plants that have a point mutation in CXE2L1 is increased by at least 5%, as well as by at least 10%, as well as by at least Petition 870250086789, dated 09 / 25 / 2025, pp. 76 / 280 53 / 224 15%, as in at least 20%, as in at least 50%, as in at least 100%, as in at least 200%, compared to a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but has a similar genotype.

[0136] In some embodiments, α-amylase activity in whole grains of barley plants carrying a point mutation in CXE2L1 is increased between 5% and 200%, as between 5% and 150%, as between 5% and 100%, as between 50% and 100%, compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0137] Preferably, the aforementioned increase in α-amylase activity is obtained 12 to 110 h, such as 12 to 96 h, for example, 24 to 48 h after the start of germination.

[0138] In some embodiments, β-amylase activity in the grains of a barley plant carrying a point mutation in CXE2L1 is increased compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but that has a similar genotype.

[0139] In some embodiments, limit dextrinase activity in the grains of a barley plant carrying a point mutation in CXE2L1 is increased compared with a similar barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0140] Preferably, the activity of the hydrolytic enzyme, such as, for example, α-amylase activity, α-amylase activity and / or limit dextrinase activity, Petition 870250086789, dated 09 / 25 / 2025, page 77 / 280 54 / 224 is measured after soaking dry half-grains in water and / or after the start of germination of the whole grain or half-grain, as applicable. Thus, preferably, hydrolytic enzymatic activity is measured after soaking in water and / or germination for at least 12 h, such as at least 24 h, such as at least 48 h, or at least 72 h.

[0141] Thus, in some embodiments, the activity of the hydrolytic enzyme is elevated and / or increased after being soaked in water and / or germinated for at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours.

[0142] In some embodiments, hydrolytic enzymatic activity is elevated and / or increased after being soaked in water and / or germination for a period between 12 hours and 72 hours, such as between 24 hours and 72 hours, such as between 24 hours and 48 hours, such as between 48 hours and 72 hours.

[0143] α-amylase activity can be measured using methods well known to the specialist in the technical area. α-amylase activity can, for example, be measured using a standardized kit, such as the Megazyme Ceralpha kit, according to the manufacturer's protocol (K-CERA 01 / 12).

[0144] β-amylase activity can be measured using methods well known to specialists in the field. β-amylase activity can, for example, be measured using a standardized kit, such as the Megazyme β-amylase assay kit (Betamyl-3), according to the manufacturer's protocol (K-BETA3). Petition 870250086789, dated 09 / 25 / 2025, pp. 78 / 280 55 / 224

[0145] β-glucanase activity can be measured using methods well known to specialists in the field. β-glucanase activity can, for example, be measured using a standardized kit, such as the LIBIOS malt β-glucanase / lichanase kit, according to the manufacturer's protocol (MBG4 Method).

[0146] Limit dextrinase activity can be measured using methods well known to the specialist in the field. Limit dextrinase activity can, for example, be measured using a standardized kit, such as the Megazyme Pullanase limit dextrinase assay kit, according to the manufacturer's protocol (PullG6 method). Barley plant

[0147] The barley plant according to the present invention may be any plant of the species Hordeum vulgare, including any breeding line, cultivar or variety.

[0148] “Wild barley”, Hordeum vulgare ssp. Spontaneum, is considered the progenitor of present-day cultivated forms of barley. Domesticated, but heterogeneous, mixtures of barley are referred to as local barley varieties. Today, most local varieties have been replaced in advanced agriculture by pure-line cultivars. Compared to local varieties, modern barley cultivars have numerous improved properties (Nevo, 1992; Pelger et al., 1992).

[0149] In this invention, the term "barley plant" includes any barley plant, such as wild barley, local varieties of barley or modern cultivars. Petition 870250086789, dated 09 / 25 / 2025, pp. 79 / 280 56 / 224 of barley. Thus, the present invention relates to any barley plant comprising a mutation in the CXE2L1 gene, as described in this descriptive report.

[0150] However, the preferred barley plants for use with the present invention are modern barley cultivars or pure lines. The barley cultivar to be used with the present invention may, for example, be selected from the group consisting of RGT Planet (Planet), Paustian, Sebastian, Quench, Celeste, Lux, Prestige, Saloon, Neruda, Harrington, Klages, Manley, Schooner, Stirling, Clipper, Franklin, Alexis, Blenheim, Ariel, Lenka, Maresi, Steffi, Gimpel, Cheri, Krona, Camargue, Chariot, Derkado, Prisma, Union, Beka, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin-choku No. 1, Kanto Late Variety Gold, Fuji Nijo, New Golden, Satukio Nijo, Seijo No.17, Akagi Nijo, Azuma Golden, Amagi Nijpo, Nishino Gold, Misato golden, Haruna Nijo, Scarlett, Rosalina and Jersey, preferably from the group consisting of Haruna Nijo, Sebastian, Quench, Celeste, Lux, Prestige, Saloon, Neruda and Power, preferably from the group consisting of Paustian, Harrington, Klages, Manley, Schooner, Stirling, Clipper, Franklin, Alexis, Blenheim, Ariel, Lenka, Maresi, Steffi, Gimpel, Cheri, Krona, Camargue, Chariot, Derkado, Prisma, Union, Beka, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin - choku No. 1, Kanto late Variety Gold, Fuji Nijo, New Golden, Satukio Nijo, Seijo No. 17, Akagi Nijo, Azuma Golden, Amagi Nijpo, Nishino Gold, Misato Golden, Haruna Nijo, Scarlett, and Jersey, preferably from the group that includes Paustian, Haruna Nijo, Sebastian, Tangent, and Lux. Petition 870250086789, dated 09 / 25 / 2025, page 80 / 280 57 / 224 Prestige, Saloon, Neruda, Power, Quench, NFC Tipple, Barke, Class, Vintage, Applaus, Bowie, Broadway, Champ, Chanson, Charles, Chimbon, Cosmopolitan, Crossway, Dragoon, Ellinor, Embrace, Etoile, Evergreen, Flair, Highway, KWS Beckie, KWS Cantton, KWS Coralie, KWS Fantex, KWS Irina, KWS Josie, KWS Kellie, LG Diablo, LG Figaro, LG Nabuco, LG Tomahawk, Laureate, Laurikka, Lauxana, Luther, Odyssey, Ovation, Prospect, RGT Elysium, RGT Observer, RGT Planet, Rotator, Sarbi, Scholar, Subway and Golden Promise.

[0151] The thermometers “RGT Planet” and “Planet” are used interchangeably in this descriptive context.

[0152] The barley plant may have any suitable form. For example, the barley plant according to the present invention may be a viable barley plant, a dried plant, a homogenized plant, a grain, or a milled barley grain. The plant may be a mature plant, an embryo, a grain, a germinated grain, a malted grain (for example, in the form of green malt or oven-dried malt), a milled malted grain, a milled grain, or the like.

[0153] The parts of barley plants may be any suitable part of the plant, such as grains, embryos, leaves, stems, roots, flowers or fractions thereof. A fraction may, for example, be a section of a grain, embryo, leaf, stem, root or flower. The parts of barley plants may also be a fraction of a homogenate or a fraction of a ground barley plant or grain.

[0154] In one embodiment of the present invention, the parts of the barley plants may be cells of said barley plant, such as viable cells that Petition 870250086789, dated 09 / 25 / 2025, page 81 / 280 58 / 224 can be propagated in vitro in tissue cultures. In other embodiments, however, the barley plant parts may be viable cells that are not capable of maturing into a whole barley plant, i.e., cells that are not reproductive material. Thus, in one embodiment, the plant part is not reproductive material.

[0155] In some embodiments, the plant or part thereof was not obtained by means of an essentially biological process. In some embodiments, the offspring of the plant or part thereof was not obtained by means of an essentially biological process. In other words, in one embodiment, the barley plant of the present invention was not obtained exclusively by means of an essentially biological process. The offspring of a barley plant obtained by a technical process is considered in this descriptive report as not having been obtained exclusively by means of an essentially biological process, because the parent plant is obtained by a technical process.

[0156] A barley plant or part thereof that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype, may, for example, be a parent plant. Thus, if the mutant is obtained by mutagenesis of a given barley cultivar, the barley plant that does not carry the point mutation in the CXE2L1 gene, but which otherwise has a similar genotype, may preferentially be a plant of that cultivar. Barley plants comprising more than one mutation Petition 870250086789, dated 09 / 25 / 2025, p. 82 / 280 59 / 224

[0157] In addition to the mutations described in this descriptive report, barley plants may also comprise one or more of the following mutations. Thus, the barley plant may comprise one or more of the following mutations.

[0158] In addition to the mutation(s) described above, the barley plant may also comprise a mutation in a gene encoding a β-glucan synthase. An important advantage of these barley plants is that the grains of said barley plant have a reduced β-glucan content. Preferably, said β-glucan is (1,3;1,4)-e-glucan.

[0159] In some embodiments, the barley plant carrying a mutation in a gene encoding a β-glucan synthase has a content of (1,3;1,4)-e-glucan: a. less than 5% of the total dry weight of the grains, for example, less than 3% of the total dry weight of the grains, preferably less than 2% of the total dry weight of the grains; and / or b. a maximum of 60% of the (1,3;1,4)-e-glucan content of a barley plant carrying a wild-type CslF6 gene, but with a similar genotype.

[0160] In some embodiments, the barley plant carrying a mutation in a gene encoding a β-glucan synthase has a content of (1,3;1,4)-e-glucan: a. in the range of 1 to 5% of the total dry weight of the grains, for example, 1 to 3% of the total dry weight of the grains, preferably 1.3 to 2% of the total dry weight of the grains; and / or b. of at least 30% and at most 60%, preferably at least 40% and at most 60% of the (1,3;1,4)-e-glucan content Petition 870250086789, dated 09 / 25 / 2025, p. 83 / 280 60 / 224 of a barley plant carrying a wild-type CslF6 gene, but with a similar genotype.

[0161] In principle, the barley plant can comprise any type of mutation in a gene encoding a β-glucan synthase that results in a loss of function of the mutant β-glucan synthase polypeptide. In some embodiments, the mutation is a deletion.

[0162] In some embodiments, the gene encoding a β-glucan synthase is CslF6. Thus, in some embodiments, the barley plant carries a mutation in the gene encoding CslF6. The said mutation may be, for example, any of the mutations described in international patent application WO 2019 / 129736.

[0163] Thus, in some embodiments, the gene encoding a β-glucan synthase is CslF6, wherein the said mutant CslF6 gene encodes a mutant CslF6 polypeptide, wherein wild-type CslF6 is CslF6 of SEQ ID NO: 7 or a functional variant thereof with at least 95% sequence identity with it.

[0164] In some embodiments, the mutant CslF6 gene encodes a loss-of-function mutant CslF6 polypeptide, where wild-type CslF6 is CslF6 of SEQ ID NO: 7 or a functional variant thereof with at least 95% sequence identity with it. In particular, the mutation may introduce a premature stop codon, a frameshift, and / or a splice site mutation.

[0165] In some embodiments, the mutant CslF6 polypeptide comprises an amino acid substitution in a membrane-bound domain of CslF6. The Petition 870250086789, dated 09 / 25 / 2025, pp. 84 / 280 The 61 / 224 amino acids located in the membrane at CslF6 are shown in Table D below. Table D. Location of amino acid sequences in the membrane of the CslF6 protein. (AA = amino acid) Position of AA in SEQ ID NO: 7 or SEQ ID NO: 8 AA Sequence Functionality 109-128 RVLIFVRLIAFTLFVIWRIS Transverse membrane (SEQ ID NO: 12) 137-158 LWVTSICGEFWFGFSWLLDQLP Transverse membrane (SEQ ID NO: 13) 700-731 LQRVAYINITTYPTFAIFLIFYTTVPAL SFVT Part of the membrane extension; Helical connection in the membrane, causing torsion; Part of the membrane extension (SEQ ID NO: 10) 741-758 TMFYVYLGIVLSTLLVIA Membrane extension (SEQ ID NO: 11) 835-857 ITPIIIIFVNIIGSAVAFAKVLD Transverse membrane (SEQ ID NO: 9) 864-882 LKVAGGVFFNFWVLFHLYPF Transverse membrane (SEQ ID NO: 14)

[0166] In some embodiments, the mutant CslF6 polypeptide comprises an amino acid substitution in a membrane-bound domain of CslF6, wherein said substitution is a substitution of a Petition 870250086789, dated 09 / 25 / 2025, page 85 / 280 62 / 224 a nonpolar amino acid is replaced by a charged amino acid or a polar amino acid is replaced by a nonpolar amino acid, wherein the membrane-bound domain is selected from the group consisting of the membrane-bound domains of CslF6 which consist of: a. amino acids 835 to 857 (SEQ ID NO: 9), or b. amino acids 700 to 731 (SEQ ID NO: 10), or w. amino acids 741 to 758 (SEQ ID NO: 11).

[0167] In some embodiments, the mutant CslF6 polypeptide comprises an amino acid substitution in a membrane-bound domain of CslF6, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 835 to 857 (SEQ ID NO: 9) of CslF6, wherein said substitution is the substitution of a nonpolar amino acid for a charged amino acid.

[0168] In some embodiments, the mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution 847, wherein said substitution is the substitution of a glycine (G) for a glutamic acid (E).

[0169] In some embodiments, the mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 741 to 758 (SEQ ID NO: 10) of CslF6, wherein said substitution is the substitution of a nonpolar amino acid for a charged amino acid. Petition 870250086789, dated 09 / 25 / 2025, p. 86 / 280 63 / 224

[0170] In some embodiments, the mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises a substitution of amino acid 748, wherein said substitution is the substitution of a glycine (G) for an aspartic acid (D).

[0171] In some embodiments, the mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 700 to 731 of CslF6 (SEQ ID NO: 11), wherein said substitution is the substitution of a polar amino acid for a nonpolar amino acid.

[0172] In some embodiments, the mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises a substitution of amino acid 709, wherein said substitution is the substitution of a threonine (T) for an isoleucine (I).

[0173] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in the gene encoding LOX-1, resulting in a complete loss of Functional LOX-1. The said mutation may be, for example, any of the mutations described in international patent application WO 2005 / 087934. For example, the barley plant may comprise a gene encoding LOX-1 comprising a premature stop codon, said codon corresponding to bases 3572-3574 of SEQ ID NO:2 of WO 2005 / 087934, or a mutation at the splice site, the mutation corresponding to base 2311 of SEQ ID NO:6 of WO 2005 / 087934. 2005 / 087934. Petition 870250086789, dated 09 / 25 / 2025, p. 87 / 280 64 / 224

[0174] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in the gene encoding LOX-2, resulting in a complete loss of functional LOX-2. The said mutation may be, for example, any of the mutations described in international patent application WO 2010 / 075860. For example, the barley plant may comprise a gene encoding LOX-2 comprising a mutation at nucleotide position 2689 of SEQ ID NO: 1 of WO 2010 / 075860, leading to the formation of a premature stop codon.

[0175] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in the gene encoding MMT, resulting in a total loss of functional MMT. The said mutation may be, for example, any of the mutations described in international patent application WO 2010 / 063288. For example, the barley plant may comprise a gene encoding MMT comprising a G^A mutation of base no. 3076 of SEQ ID NO: 3 of WO 2010 / 063288 or a gene encoding MMT comprising a G^A mutation of base no. 1462 of SEQ ID NO: 16 of WO 2010 / 063288.

[0176] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in a gene encoding a protein involved in anthocyanin and / or proanthocyanidin biosynthesis, for example, in one or more of the genes described by Jende-Strid (Genetic control of flavonoid biosynthesis in barley, Hereditas 119: 187204, 1993). The barley plant may, for example, be an anthocyanogen-free mutant, as described in US Petition 870250086789, dated 09 / 25 / 2025, pp. 88 / 280 65 / 224 4,165,387. In some embodiments, the barley plant may comprise a mutation in the gene encoding ANT-28, resulting in a complete loss of functional ANT-28. ANT-28 encodes a domain protein involved in the accumulation of proanthocyanidin in developing grains (Himi et al., The Ant28 gene for proanthocyanidin synthesis encoding the R2R3 MYB domain protein significantly affects grain dormancy in barley, Euphytica, 188:141-151, 2012). The mutation in ANT-28 can be, for example, any of the mutations described in GrainGenes (https: / / wheat.pw.usda.gov / ggpages / bgn / 42 / BGS685-757.htm; see section “BGS 608, Proanthocyanidin-free 28, ant28”), such as ant28.484. Barley plants exhibiting this / these mutation(s) are preferentially free of anthocyanogens or have a low content of them.

[0177] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in the gene encoding HRT, resulting in a complete loss of functional HRT. This mutation may be, for example, any of the mutations described in international patent application WO 2019 / 129739. The barley plant may, for example, also exhibit a mutation in the HRT gene, resulting in a mutant HRT gene encoding a protein. HRT mutant lacking one or more of the amino acids in SEQ ID NO: 2 of WO 2019 / 129739, or a mutation that results in the deletion of at least the coding region of the HRT gene, where the coding region of the HRT gene encodes a polypeptide of SEQ ID NO: 2 of WO 2019 / 129739. Preferably, the mutation is a Petition 870250086789, dated 09 / 25 / 2025, pp. 89 / 280 66 / 224 mutation resulting in a mutant HRT gene encoding a mutant HRT protein, wherein said mutant HvHRT protein carries a W170stop mutation of SEQ ID NO: 2 of WO 2019 / 129739, or the barley plant carries a mutant HvHRT gene comprising a G^A mutation of nucleotide 510 of the HvHRT coding sequence of SEQ ID NO: 1 of WO 2019 / 129739.

[0178] In addition to one or more of the mutations described above, the barley plant may also comprise a mutation in the gene encoding LDI, resulting in a complete loss of functional LDI. The mutation may be any type of mutation that results in a loss of LDI function. The said mutation may be, for example, any of the mutations described in international patent application WO 2021 / 175786. For example, the barley plant may carry a mutation in the LDI gene, resulting in a mutant LDI gene encoding a mutant LDI protein lacking one or more of the amino acids in SEQ ID NO: 1 of WO 2021 / 175786, or a mutation resulting in the deletion of at least the coding region of the LDI gene, wherein the coding region of the LDI gene encodes a polypeptide in SEQ ID NO: 1 of WO 2021 / 175786. For example, the mutation may be selected from: a. a missense mutation that results in a change from a proline to a different amino acid in one or more loop regions of the LDI, and b. a missense mutation that results in a change from a negatively charged amino acid to an uncharged amino acid in one or more regions of the alpha helix of LDI. Petition 870250086789, dated 09 / 25 / 2025, pp. 90 / 280 67 / 224

[0179] Preferably, the loop regions are selected from the group consisting of amino acids corresponding to positions 25 to 44 and amino acids corresponding to positions 56 to 62 and amino acids corresponding to positions 77 to 78 and amino acids corresponding to positions 91 to 111 and amino acids corresponding to positions 124 to 14 7 of SEQ ID NO: 1 of WO 2021 / 175786. More preferably, the alpha helix regions are selected from the group consisting of amino acids corresponding to positions 45 to 55 and amino acids corresponding to positions 63 to 76 and amino acids corresponding to positions 79 to 90 and amino acids corresponding to positions 112 to 123 of SEQ ID NO: 1 of WO 2021 / 175786.

[0180] In addition to one or more of the mutations described above, the barley plant may also have an amy1_1 haplotype characterized by said barley plants comprising an amy1_1 cluster comprising at least 5 copies of genes encoding functional α-amylases. In other words, said barley plant may have an amy1_1 haplotype characterized by said barley plants comprising an amy1_1 cluster comprising at least 5 functional genes, each gene encoding an α-amylase.

[0181] The aforementioned 5 functional genes may each encode the same α-amylase or may encode different α-amylases, such as different α-amylase variants. In some cases, two or more of the aforementioned 5 functional genes may each encode the same α-amylase, while the remaining functional genes each encode a different α-amylase. Petition 870250086789, dated 09 / 25 / 2025, pp. 91 / 280 68 / 224 different α-amylases, such as different αamylase variants.

[0182] In preferred embodiments, the amy1_1 cluster comprises at least five functional genes, each encoding an independently selected α-amylase from the group consisting of: • an α-amylase with the amino acid sequence as established in SEQ ID NO:37; • an α-amylase with the amino acid sequence as established in SEQ ID NO:38; • an α-amylase with the amino acid sequence as established in SEQ ID NO:39; • an α-amylase with the amino acid sequence as established in SEQ ID NO:40; • an α-amylase with the amino acid sequence as established in SEQ ID NO:41; • an α-amylase with the amino acid sequence as established in SEQ ID NO:42; • an α-amylase with the amino acid sequence as established in SEQ ID NO:43; • an α-amylase with the amino acid sequence as established in SEQ ID NO:44; • an α-amylase with the amino acid sequence as established in SEQ ID NO:45; • an α-amylase with the amino acid sequence as established in SEQ ID NO:46; • an α-amylase with the amino acid sequence as established in SEQ ID NO:47; and Petition 870250086789, dated 09 / 25 / 2025, page 92 / 280 69 / 224 • functional homologs of any of the above mentioned with at least 80% sequence identity with them, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with them.

[0183] In highly preferred embodiments, the barley plant comprises at least five functional genes, each encoding an independently selected α-amylase group consisting of: • an α-amylase with the amino acid sequence as set out in SEQ ID NO: 42 or a functional homolog thereof with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% sequence identity with the same; • an α-amylase with the amino acid sequence as set forth in SEQ ID NO: 43 or a functional homolog thereof with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the same; and • an α-amylase with the amino acid sequence as set forth in SEQ ID NO: 44 or a functional homolog thereof with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the same. Petition 870250086789, dated 09 / 25 / 2025, pp. 93 / 280 70 / 224

[0184] In some embodiments, the barley plant or part thereof according to this disclosure bears: a. an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said amy1_1 cluster is as described elsewhere in this document; b. a mutation in the gene encoding LOX-1, resulting in a complete loss of functional LOX-1, as described elsewhere in this document; c. optionally, a mutation in the gene encoding LOX-2, resulting in the total loss of functional LOX-2, as described elsewhere in this document; d. a mutation in the gene that codes for MMT, resulting in the total loss of functional MMT, as described elsewhere in this document; and e. a mutation in CXE2L1, as described elsewhere in this document.

[0185] In some embodiments, the barley plant or part thereof, according to the present disclosure, carries: a. an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said amy1_1 cluster is as described elsewhere in this document; b. a mutation in the gene that codes for LOX-1, resulting in the total loss of functional LOX-1; Petition 870250086789, dated 09 / 25 / 2025, pp. 94 / 280 71 / 224 c. optionally, a mutation in the gene encoding LOX-2, resulting in the total loss of functional LOX-2, as described elsewhere in this document; d. a mutation in the gene that codes for MMT, resulting in the total loss of functional MMT, as described elsewhere in this document; e. a mutation in the gene encoding ANT-28, resulting in the total loss of ANT-28 functionality, as described elsewhere in this document; and f. a mutation in CXE2L1, as described elsewhere in this document.

[0186] The aforementioned barley plant or part thereof, comprising an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase combined with one or more additional mutations, as described elsewhere in this document, such as comprising a mutation in CXE2L1, as described elsewhere in this document, may have increased α-amylase activity, increased β-amylase activity, increased free limit dextrinase activity and / or decreased β-glucan content compared to a barley plant or part thereof of similar genotype, but which does not comprise the aforementioned minimum number of copies of the functional α-amylase gene of the amy1_1 cluster and, optionally, does not comprise the aforementioned one or more additional mutations.

[0187] α-amylase activity is preferably measured as described in Example 4. Petition 870250086789, dated 09 / 25 / 2025, page 95 / 280 72 / 224

[0188] β-amylase activity is preferably measured as described in the K-BETA3 protocol (protocol and kit available from Megazyme, Ireland).

[0189] Free dextrinase activity is preferably measured as described in Example 15.

[0190] The β-glucan content is preferably measured as described in Example 10.

[0191] In some embodiments, the barley plant or part thereof has an α-amylase activity of at least 230 U / g, such as at least 240 U / g, such as at least 250 U / g, such as at least 260 U / g, such as at least 270 U / g, such as at least 280 U / g, such as at least 290 U / g, such as at least 300 U / g, such as at least 310 U / g after 6 days of germination. In some embodiments, the barley plant or part thereof has an α-amylase activity of 230 to 350 U / g, such as 240 to 330 U / g, such as 250 to 320 U / g after 6 days of germination.

[0192] In some embodiments, the barley plant or part thereof has a β-amylase activity of at least 15 U / g, such as at least 16 U / g, such as at least 17 U / g, such as at least 18 U / g after 6 days of germination. In some embodiments, the barley plant or part thereof has a β-amylase activity of 15 to 20 U / g, such as 15 to 19 U / g, such as 15 to 18 U / g after 6 days of germination.

[0193] In some embodiments, the barley plant or part thereof has a limiting dextrinase activity of at least 60 mU / g, such as at least 65 mU / g, such as at least 70 mU / g, such as at least 75 Petition 870250086789, dated 09 / 25 / 2025, pp. 96 / 280 73 / 224 mU / g, as well as at least 80 mU / g after 6 days of germination. In some embodiments, the barley plant or part thereof has a limiting free dextrinase activity of 60 to 90 mU / g, as well as 65 to 85 mU / g, as well as 70 to 80 mU / g after 6 days of germination.

[0194] In some embodiments, the barley plant or part thereof has a β-glucan content of at most 200 mg / L, such as at most 175 mg / L, such as at most 150 mg / L, such as at most 125 mg / L, such as at most 100 mg / L, such as at most 75 mg / L, such as at most 50 mg / L mg / L, such as at most 25 mg / L, such as at most 10 mg / L after 6 days of germination. In some embodiments, the barley plant or part thereof has a β-glucan content of 0 to 200 mg / L, as well as 0 to 150 mg / L, 0 to 100 mg / L, 0 to 75 mg / L, 0 to 50 mg / L, and 0 to 25 mg / L after 6 days of germination. Plant-based products

[0195] The present invention also provides plant products prepared from a barley plant or part thereof carrying a mutation in CXE2L1, wherein said CXE2L1 encodes a gain-of-function mutant CXE2L1 polypeptide, for example, any of the barley plants and parts thereof as described in this descriptive report.

[0196] The plant product can be any product prepared from a barley plant, for example, a food, an animal feed, or a beverage. Thus, the plant product can be any of the beverages described below in the section “Beverage and method of production thereof”. The plant product can also be an aqueous extract of the plant. Petition 870250086789, dated 09 / 25 / 2025, pp. 97 / 280 74 / 224 of barley and / or malt prepared from the grains of said barley plant. For example, the vegetable product may be wort. Said aqueous extract may, for example, be prepared as described below in the section “Aqueous extract and methods of production thereof”.

[0197] In one embodiment, the vegetable product is malt, for example, milled malt, green malt, milled green malt, oven-dried malt or oven-dried milled malt, such as any of the malts described below in the section “Malt and method of production thereof”. In some embodiments, the vegetable product is a malt-based product, such as malt-based beverages. Although the main use of malt is for the production of beverages, it can also be used in other industrial processes, for example, as a source of enzymes in the baking industry or in the food industry as a flavoring and coloring agent, for example, in the form of malt or malt flour or indirectly as malt syrup, etc. Thus, the vegetable product according to the present invention can be any of the products mentioned above.

[0198] In one embodiment of the present invention, the vegetable product is barley flour, that is, barley flour prepared from the grains of a barley plant according to the present invention.

[0199] In another aspect, the vegetable products according to the present invention comprise, or even consist of, syrup, such as a barley syrup, or a barley malt syrup. The vegetable product may also be Petition 870250086789, dated 09 / 25 / 2025, pp. 98 / 280 75 / 224 an extract of barley or malt. Thus, the vegetable product can be wort. Malt and its production method.

[0200] The barley plant of the present invention is particularly useful for the production of barley-based beverages, such as beer. Such barley-based beverages are normally prepared from malt.

[0201] Thus, the present invention also provides malt prepared from a barley plant carrying a mutation in the CXE2L1 gene, optionally one or more additional mutations, as described in the section “Barley plants comprising more mutations”. Said malt may be milled malt, green malt, milled green malt, kiln-dried malt or milled kiln-dried malt and prepared from barley grains of a barley plant carrying a mutation in the CXE2L1 gene, or its progeny. Said mutation may be any of the mutations in the CXE2L1 gene that results in a gain-of-function of the CXE2L1 mutant, as described in the section “Barley plants carrying a mutation in CXE2L1”.

[0202] Green malt can be prepared by malting, that is, by germinating grains under controlled environmental conditions. Typically, this germination process may involve a soaking stage of the barley grains, followed by a germination stage. Soaking and germination can also be carried out simultaneously or partially simultaneously. In some methods, malt production may include a drying stage of the germinated grains. This drying stage may be, Petition 870250086789, dated 09 / 25 / 2025, pp. 99 / 280 76 / 224 preferably, oven drying of the germinated grains at high temperatures. Thus, oven-dried malt can be prepared by subjecting green malt to an oven drying step.

[0203] Thus, in one embodiment, a malting method may comprise the following steps: a. to provide grains from a barley plant, specifically a barley plant carrying a mutation in the CXE2L1 gene; b. to grind the aforementioned barley grains; c. to germinate barley grains under predetermined conditions; and d. Dry the sprouted barley grains, preferably by oven drying.

[0204] Germinated barley grains can be prepared by a method comprising the steps of a. to provide grains from a barley plant, specifically a barley plant carrying a mutation in the CXE2L1 gene; b. soak the barley grains; c. to germinate the aforementioned barley grain.

[0205] The immersion and germination steps can be performed sequentially, simultaneously, or partially simultaneously.

[0206] The barley plants of the present invention are particularly useful for preparing malt-based beverages, wherein said malt is germinated for less than 96 hours, such as less than 72 hours, for example, less than 48 hours, such as in the range of 24 to 96 hours, as in Petition 870250086789, dated 09 / 25 / 2025, pp. 100 / 280 77 / 224 interval of 24 to 72 hours, for example in the interval of 24 to 48 hours. The germination times mentioned above refer to the total soaking and germination time.

[0207] In a preferred embodiment, soaking and germination are carried out simultaneously in a germination process, which comprises incubating barley grains in an aqueous solution, typically under aeration, for a maximum of 72 hours.

[0208] Immersion can be carried out by any conventional method known to the expert in the field. A non-limiting example involves immersion at a temperature in the range of 10 to 25°C with alternating dry and wet conditions. During immersion, for example, barley grains can be incubated wet for a period of 30 minutes to 3 hours, followed by dry incubation for a period of 30 minutes to 3 hours and, optionally, repeating the said incubation scheme in the range of 2 to 5 times. The final water content after immersion can, for example, be in the range of 40 to 50%, or in the range of 40-45%.

[0209] Germination may comprise an incubation step of grains from a barley plant carrying a mutation in the CXE2L1 gene in an aqueous solution under aeration. For example, green malt may be produced by any of the methods described in international patent applications WO 2018 / 001882, WO 2019 / 129724 or WO 2019 / 129731.

[0210] In some embodiments, the germination stage comprises Petition 870250086789, dated 09 / 25 / 2025, pp. 101 / 280 78 / 224 a. at least one incubation step of said grains in an aqueous solution, at least partially under aeration; and b. at least one stage of air incubation of said barley grains.

[0211] Germinated barley grains that have not been dried are also referred to in this descriptive report as green malt.

[0212] The water content of barley grains can be determined by determining the weight of the barley grains, followed by drying said barley grains and determining the weight of the dry barley grains. The weight difference between the wet and dry barley grains is considered water, and the water content is given as the weight of the water divided by the total weight of the barley grains (wet barley grains). The water content given in % is therefore a % w / w.

[0213] Grain germination can be carried out by any conventional method known to the expert in the field. A non-limiting example involves germination at a temperature in the range of 10 to 25°C, optionally with a temperature variation in the range of 1 to 4 days.

[0214] As mentioned above in some embodiments of the present invention, the germinated barley grains (i.e., green malt) may be oven-dried. In some embodiments, it is preferable that the green malt not be oven-dried. In particular, it is preferable that, when the green malt is prepared by a germination process comprising an incubation step of Petition 870250086789, dated 09 / 25 / 2025, page 102 / 280 79 / 224 referred to as barley grains in an aqueous solution under aeration, the green malt not be dried in an oven.

[0215] If green malt is oven-dried, this can be done at conventional temperatures, such as at least 75°C, for example, in the range of 80 to 90°C, such as in the range of 80 to 85°C. Thus, malt can, for example, be produced by any of the methods described by Hough et al. (1982). However, any other suitable method for producing malt can also be used with the present invention, such as methods for the production of specialty malts, including, but not limited to, methods of roasting malt.

[0216] Kiln-dried malt and green malt may be further processed, for example by milling. In preferred embodiments, the green malt is not kiln-dried, in which case the green malt is preferably finely divided (for example by milling or crushing) while the green malt has a water content of at least 20%.

[0217] Thus, the vegetable product according to the present invention can be any type of malt, such as unprocessed malt or ground malt, such as flour. Thus, the vegetable product can, for example, be ground malt, oven-dried malt, or ground green malt. The ground malt and the flour derived from it comprise chemical components of malt and dead cells that do not have the capacity to re-germinate.

[0218] The barley plants provided by the present invention are characterized by carrying a mutation in the CXE2L1 gene and preferably encoding a CXE2L1 polypeptide Petition 870250086789, dated 09 / 25 / 2025, page 103 / 280 80 / 224 mutant with gain of function. A major advantage of these barley plants is that the grains have higher hydrolytic enzymatic activity and greater β-glucan degradation, such as (1,3;1,4)-e-glucan degradation, during malting.

[0219] In some embodiments, the activity of one or more hydrolytic enzymes, as described in the section “Characteristics of the barley plant carrying a CXE2L1 mutation”, is increased during and / or after malting.

[0220] In some embodiments, α-amylase activity during malting of grains from a barley plant carrying a point mutation in the CXE2L1 gene is increased compared to α-amylase activity during malting of grains from a barley plant that does not carry a point mutation in the CXE2L1 gene but has a similar genotype.

[0221] In one embodiment, the malt prepared from the grains of said barley plant has a high α-amylase activity, such as, for example, an α-amylase activity of at least 200 U / g, such as at least 210 U / g, such as at least 220 U / g, such as at least 230 U / g, such as at least 240 U / g, such as at least 250 U / g.

[0222] In one embodiment, the malt prepared from the grains of said barley plant has a high α-amylase activity, such as, for example, an α-amylase activity between 200 U / g and 280 U / g, such as between 210 U / g and 260 U / g, such as between 220 U / g and 240 U / g.

[0223] In some embodiments, the activity of β-amylase during the malting of the grains of a plant Petition 870250086789, dated 09 / 25 / 2025, pp. 104 / 280 81 / 224 barley carrying a point mutation in the CXE2L1 gene shows increased β-amylase activity during malting of grains from a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0224] In one embodiment, the malt prepared from the grains of said barley plant has a high β-amylase activity, such as, for example, a β-amylase activity of at least 10 U / g, such as at least 11 U / g, such as at least 12 U / g, such as at least 13 U / g, such as at least 14 U / g, such as at least 15 U / g.

[0225] In one embodiment, the malt prepared from the grains of said barley plant has a high β-amylase activity, such as, for example, a β-amylase activity between 10 U / g and 15 U / g, such as between 10 U / g and 14 U / g, such as between 10 U / g and 13 U / g.

[0226] In some embodiments, the limited dextrinase activity during grain malting of a barley plant carrying a point mutation in the CXE2L1 gene is increased compared to the limited dextrinase activity during grain malting of a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0227] In one embodiment, the malt prepared from the grains of said barley plant has a high limiting dextrinase activity, such as, for example, a limiting dextrinase activity of at least 45 U / g, such as at least 50 U / g, such as at least 55 U / g, such as at least 60 U / g, such as at least 65 U / g. Petition 870250086789, dated 09 / 25 / 2025, pp. 105 / 280 82 / 224

[0228] In one embodiment, the malt prepared from the grains of said barley plant has a high limiting dextrinase activity, such as, for example, a limiting dextrinase activity between 45 U / g and 65 U / g, such as between 45 U / g and 55 U / g, such as between 50 U / g and 60 U / g.

[0229] In some embodiments, β-glucan degradation during malting of grains from a barley plant carrying a point mutation in the CXE2L1 gene is increased compared with β-glucan degradation during malting of grains from a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype.

[0230] In some embodiments, the wort prepared from the grains of the said barley plant has a reduced β-glucan content compared to the β-glucan content in the wort prepared from the grains of a barley plant that does not carry a point mutation in the CXE2L1 gene, but which is of a similar genotype.

[0231] In some embodiments, the β-glucan content in the wort prepared from the grains of the barley plant of the present invention is reduced by at least 10%, as well as by at least 20%, as well as by at least 30%, as well as by at least 40%, as well as by at least 50%, as well as by at least 60%, as well as by at least 70%, as well as by at least 80%, as well as by at least 90%, as well as by 100%, compared with the β-glucan content in the wort prepared from grains of a barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype. Petition 870250086789, dated 09 / 25 / 2025, pp. 106 / 280 83 / 224

[0232] In some embodiments, the β-glucan content in wort prepared from said barley plant or part thereof is a decrease of between 10% and 100%, as well as between 10% and 50%, as well as between 50% and 75%, as well as between 25% and 100%, compared with the β-glucan content in wort prepared from grains of a barley plant that does not have a point mutation in the CXE2L1 gene, but that otherwise has a similar genotype.

[0233] In one embodiment, the β-glucan content of the wort prepared from said barley plant or part thereof is at most 400 mg / L, such as at most 350 mg / L, such as at most 300 mg / L, such as at most 250 mg / L, such as at most 200 mg / L, such as at most 150 mg / L.

[0234] In one embodiment, the β-glucan content of the wort prepared from said barley plant or part thereof is between 0 and 500 mg / L, such as between 0 and 250 mg / L, such as between 50 and 250 mg / L, such as between 100 and 200 mg / L, such as between 200 and 400 mg / L, such as between 50 and 200 mg / L.

[0235] The must can, for example, be prepared as described in the section “Aqueous extract and method of production thereof”. Aqueous extract and method of production thereof.

[0236] The present invention provides barley-based beverages, as well as methods for their preparation, in which the barley plant has a mutation in the CXE2L1 gene or in its progeny. The present invention also provides aqueous extracts of grains from barley plants that have a mutation in the CXE2L1 gene. Said aqueous extract can, Petition 870250086789, dated 09 / 25 / 2025, pp. 107 / 280 84 / 224, for example, can be prepared from green malt or oven-dried malt. The aqueous extract can, in particular, be wort.

[0237] Frequently, the methods for preparing a beverage comprise a step of preparing an aqueous extract of grains from the barley plants of the present invention and / or malts prepared from barley plants of the present invention.

[0238] The aqueous extract can generally be prepared by incubating barley flour, green malt flour and / or dry malt flour in an oven in water or in an aqueous solution. This aqueous solution is also referred to as “mashing solution” in this document. In particular, the aqueous extract can be prepared by mashing.

[0239] The present invention also provides a method for producing an aqueous extract, said method comprising the steps of: a. to provide grains from a barley plant according to the present invention; b. subjecting the barley grains to a germination stage, thus obtaining germinated grains; c. finely divide the sprouted grains; d. optionally, dry the finely divided sprouted grains; e. prepare an aqueous extract of the aforementioned finely divided germinated (dried) grains, f. thus producing an aqueous extract of barley. Petition 870250086789, dated 09 / 25 / 2025, pp. 108 / 280 85 / 224

[0240] In some embodiments, step c) is carried out while the germinated grains have a water content of at least 20%, provided that the barley grains do not have a water content lower than 20% at any time between steps b) and c).

[0241] In some embodiments, step d) is not performed.

[0242] The germination stage is described in detail in the section “Malt and method of production thereof”. In some embodiments, the germination stage (a) is carried out for a maximum of 96 hours, as for a maximum of 72 hours, as for a maximum of 48 hours.

[0243] In general, the said mashing solution may be water, such as tap water, to which one or more additional agents may be added. Additional agents may be present in the aqueous solution from the outset or may be added during the process of preparing an aqueous extract. The said additional agents may be enzymes. Thus, the mashing solution may comprise one or more enzymes. The said enzymes may be added to the aqueous solution from the outset or later during the process.

[0244] The enzymes in question may, for example, be one or more hydrolytic enzymes. Suitable enzymes include lipases, starch-degrading enzymes (e.g., amylases), glucanases [preferably (1-4)- and / or (1,3;1,4)β-glucanases] and / or xylanases (such as arabinoxylanases) and / or proteases, or enzyme mixtures comprising one or more of the aforementioned enzymes, for example, Cereflo, Petition 870250086789, dated 09 / 25 / 2025, pp. 109 / 280 86 / 224 Ultraflo or Ondea Pro (Novozymes). For example, the aqueous solution may comprise one or more hydrolytic enzymes selected from the group consisting of α-amylase, β-amylase, limit dextrinase, pullulanase, β-glucanase (e.g., endo-(1,3;1,4)-β-glucanase or endo-1,4-β-glucanase), xylanase (e.g., endo- or exo-1,4-xylanase, an arabinofuranosidase or a ferulic acid esterase), glucoamylase and protease.

[0245] In one embodiment, no or only limited amounts of α-amylase are added to said mash solution.

[0246] In one embodiment, no amount or only limited amounts of dextrinase and pullulanase are added to said mash solution.

[0247] The aforementioned additional agents, preferably of food grade, may also be a salt, for example CaCl2, or an acid, for example H3PO4.

[0248] The aqueous extract is generally prepared by incubating barley flour, green malt flour and / or dry malt flour in an oven in the mash solution at one or more predetermined temperatures. The aforementioned predetermined temperature may also be referred to as the “mashing temperature” in this descriptive report. The aforementioned mashing temperatures may, for example, be conventional temperatures used for mashing. The mashing temperature is generally kept constant (isothermal mashing) or increased gradually, for example, sequentially. In both cases, grain-soluble substances Petition 870250086789, dated 09 / 25 / 2025, pp. 110 / 280 87 / 224 barley and / or malt are released into the aforementioned mash solution, thus forming an aqueous extract.

[0249] Brewing temperatures are typically in the range of 30 to 90°C, such as in the range of 40 to 85°C, for example, in the range of 50 to 85°C. Often, incubation with the mash solution includes an initial stage at lower temperatures, for example, in the range of 50 to 70°C, and a final heating stage to a higher temperature, for example, to a temperature in the range of 75 to 80°C.

[0250] After incubation in the aqueous solution, for example, in a mash vessel, the aqueous solution can be transferred to another vessel, for example, a clarification tank, and incubated for a longer time at a higher temperature.

[0251] Non-limiting examples of useful brewing protocols can be found in the brewing literature, for example, in Hough et al. (supra).

[0252] Mashing (i.e., the incubation of barley flour, green malt flour and / or kiln-dried malt flour in the mashing solution) may occur in the presence of adjuvants, which are understood to comprise any source of carbohydrates other than malt or germinated barley grains, such as, but not limited to, barley, barley or corn syrups, or rice—whether as whole grains or as processed products such as grains, syrups or starch. All adjuvants mentioned above may be used primarily as an additional source of extract (syrups are normally dosed during wort heating). The requirements for the Petition 870250086789, dated 09 / 25 / 2025, p. 111 / 280 88 / 224 processing of adjuvants in the brewery depends on the state and type of adjuvant used.

[0253] After incubation in the mashing solution, the aqueous extract can typically be separated, for example, by filtration into the aqueous extract and the undissolved residual solid particles, the latter also called residual grains. Filtration can, for example, be carried out in a filtration tank. Alternatively, filtration can be done through a mash sieve. The aqueous extract thus obtained can also be called first wort. Additional liquid, such as water, can be added to the residual grains during a process also called spraying. After spraying and filtration, a second wort can be obtained. Additional worts can be prepared by repeating the procedure. Thus, the aqueous extract can be a wort, for example, a first wort, a second wort, an additional wort, or a combination thereof.

[0254] The method of preparing an aqueous extract can, in one embodiment, be carried out using any of the apparatus described in international patent application PCT / EP2017 / 065498, for example, any of the apparatus described on pages 20-22 thereof. Additional compounds

[0255] The methods of the present invention may comprise the step of adding one or more additional compounds. Such additional compounds may be, for example, a flavoring compound, a preservative, a functional ingredient, a colorant, a sweetener, a pH regulator, or a salt. The pH regulator may Petition 870250086789, dated 09 / 25 / 2025, p. 112 / 280 89 / 224 be, for example, a buffer or an acid, such as phosphoric acid.

[0256] Functional ingredients can be any ingredient added to achieve a specific function. Preferably, a functional ingredient makes the beverage healthier. Non-limiting examples of functional ingredients include vitamins or minerals.

[0257] The preservative can be any food-grade preservative, for example, it can be benzoic acid, sorbic acid, sorbates (e.g., potassium sorbate), sulfites and / or their salts.

[0258] The additional compound can also be CO2. In particular, CO2 can be added to obtain a carbonated beverage.

[0259] The flavoring compound to be used with the present invention may be any useful flavoring compound. The flavoring compound may, for example, be selected from the group consisting of flavors, plant extracts, plant concentrates, plant parts and herbal infusions. In particular, the flavoring compounds may be hops. Beverage and its production method.

[0260] The present invention also provides barley-based beverages and methods for producing such beverages, wherein the beverages are prepared from the barley plant according to the present invention, i.e., a barley plant carrying a mutation in the CXE2L1 gene and, optionally, one or more additional mutations, as described in the section “Barley plants comprising further mutations”. Petition 870250086789, dated 09 / 25 / 2025, p. 113 / 280 90 / 224

[0261] The beverage may be an alcoholic beverage made from barley or a non-alcoholic beverage made from barley. Alcoholic beverages made from barley may be, for example, beer or distilled alcohol.

[0262] The beer in question can be any type of beer. It can be a light-colored beer, for example, selected from the group consisting of lager, pale ale, and wheat beer. Thus, the beer can, for example, be selected from the group consisting of Altbier, Amber Ale, Barley Wine, Berliner Weisse, Bière de Garde, Bitter, Blonde Ale, Bock, Brown Ale, California Common, Cream Ale, Dortmunder Export, Doppelbock, Dunkel, Dunkelweizen, Eisbock, Fruit Lambic, Golden Ale, Gose, Gueuze, Hefeweizen, Helles, India Pale Ale, Kölsch, Lambic, Light Ale, Maibock, Malt Liquor, Mild, Märzenbier, Old Ale, Oud Bruin, Pale Ale, Pilsener, Porter, Red Ale, Roggenbier, Saison, Scotch Ale, Steam Beer, Stout, Schwarzbier, Lager, Witbier, Weissbier, Weizenbier, and Weizenbock.

[0263] The distilled alcohol mentioned may be any type of distilled alcohol. In particular, distilled alcohol may be barley-based, for example, barley malt. Non-limiting examples of such distilled alcohol include whiskey and vodka.

[0264] The beverage may be a non-alcoholic beverage, such as a non-alcoholic beverage made from barley, for example, non-alcoholic beer, or non-alcoholic beverages made from malt, such as malt.

[0265] The beverage can, for example, be prepared by a method that includes the following steps: Petition 870250086789, dated 09 / 25 / 2025, pp. 114 / 280 91 / 224 a. prepare an aqueous extract using the method described in the section “Aqueous extract and method of production thereof”; b. process the aforementioned extract into a beverage.

[0266] In some embodiments, the beverage is prepared by a method comprising the following steps: a. provide grains from a barley plant according to the present invention and / or malt prepared according to the method described in the section “Malt and method of production thereof”; b. prepare an aqueous extract of said grains and / or said malt; c. process the aforementioned aqueous extract to obtain a beverage.

[0267] The aqueous extract may be boiled with or without hops, after which it may be referred to as boiled wort. The first, second and other worts may be combined and subsequently boiled. The aqueous extract may be boiled for any suitable period of time, for example, between 60 and 120 minutes.

[0268] The processing step of the aqueous extract into a beverage may include: a. heat the aforementioned aqueous extract, optionally in the presence of hops or hop extract; b. cool the aqueous extract; c. ferment the aforementioned aqueous extract with yeast, thus producing a fermented beverage.

[0269] The processing step of the aqueous extract into a beverage may include, in particular, the fermentation of Petition 870250086789, dated 09 / 25 / 2025, pp. 115 / 280 92 / 224 referred to as the aqueous extract, for example, by fermentation of the must. Thus, the beverage can be prepared by fermentation of the aqueous extract with a microorganism, such as a yeast, for example, Saccharomyces cerevisiae and / or Saccharomyces pastorianus.

[0270] Once the aqueous extract is prepared, it can be transformed into beer by any method, including conventional brewing methods. Non-limiting descriptions of examples of suitable methods for brewing beer can be found, for example, in publications by Hough et al. (1982). Numerous regularly updated methods exist for the analysis of barley and beer products, for example, but not limited to, the American Association of Cereal Chemists (1995), American Society of Brewing Chemists (1992), European Brewery Convention (1998) and Institute of Brewing (1997). It is recognized that many specific procedures are employed for a given brewery, with the most significant variations related to the preferences of local consumers. Any method of brewing beer can be used with the present invention.

[0271] The first stage of brewing beer from aqueous extract preferably involves boiling said aqueous extract as described above, followed by a subsequent cooling phase and, optionally, resting in a whirlpool. One or more additional compounds may be added to the aqueous extract, for example, one or more of the additional compounds described below in the “Additional Compounds” section. After cooling, the aqueous extract may be transferred to fermentation tanks containing Petition 870250086789, dated 09 / 25 / 2025, page 116 / 280 93 / 224 yeast, for example, brewer's yeast, such as S. pastorianus or S. cerevisiae. The aqueous extract can be fermented for any suitable period of time, generally in the range of 1 to 20 days, such as 1 to 10 days. Fermentation is carried out at any useful temperature, for example, at a temperature in the range of 10 to 20°C. The methods may also comprise the addition of one or more enzymes, for example, one or more enzymes may be added to the wort before or during fermentation. In particular, the said enzyme may be a proline-specific endoprotease. A non-limiting example of a proline-specific endoprotease is “Brewer’s Clarex”, available from DSM. In other embodiments, exogenous enzymes are not added during the methods.

[0272] During the fermentation process, which lasts several days, the sugar is converted into alcohol and CO2, along with the development of some aromatic substances. Fermentation can be stopped at any desired time, for example, when no further drop in %P is observed.

[0273] Subsequently, the beer may be further processed, for example, chilled. It may also be filtered and / or stored, a process that develops a pleasant aroma and a less yeast-like flavor. Additives may also be added. In addition, CO2 may be added. Finally, the beer may be pasteurized and / or filtered before being packaged (e.g., transferred to containers or barrels, bottled or canned). The beer may also be pasteurized by standard methods. Petition 870250086789, dated 09 / 25 / 2025, pp. 117 / 280 94 / 224 Method for preparing a barley plant comprising a CXE2L1 mutation.

[0274] Barley plants carrying a mutation in CXE2L1, for example, any of the specific mutations described in this descriptive report, can be prepared in any useful way.

[0275] For example, such barley plants can be prepared by a method comprising the steps of: a. subjecting a plurality of barley plants or barley grains to random mutagenesis, for example, by irradiation or chemical treatment, for example, treatment with sodium azide; b. identify barley plants or barley grains carrying a mutation in CXE2L1.

[0276] Such methods may also include one or more steps of breeding said barley plants / grains in order to obtain multiple barley plants / grains, each carrying random mutations.

[0277] In particular, barley plants carrying a specific mutation in the CXE2L1 gene can be prepared and identified using the FIND-IT method, which is, for example, described by Knudsen et al., 2022 and in patent application WO 2018 / 001884. The FIND-IT method allows the identification of any specific single nucleotide substitution caused by a mutagen from a library generated by random mutagenesis. Thus, even if the library is prepared by random mutagenesis, the identification of a given specific mutation is Petition 870250086789, dated 09 / 25 / 2025, pp. 118 / 280 95 / 224 reproducible, provided a sufficiently large library is created.

[0278] Thus, in some embodiments, barley plants carrying a specific mutation in the CXE2L1 gene are prepared essentially as described in international patent application WO 2018 / 001884 or as described in Knudsen et al., 2022, using primers and probes designed to identify a specific mutation in the gene. The primers are preferably designed to be able to amplify a fragment of CXE2L1 comprising the desired mutation site, and the probes are preferably designed to distinguish between the wild type and the mutant at the desired mutation site. Primers and probes suitable for identifying a CXE2L1 gene containing a C380T mutation in the CXE2L1 gene, and said gene encoding the CXE2L1 mutant comprising an A127V mutation, are described in Example 16.

[0279] Any type of mutagenic agent may be used. In some embodiments, the mutagenic agent is a chemical mutagenic agent, such as an alkylating agent, such as a nitroso compound. In some embodiments, the chemical mutagenic agent is selected from the group consisting of methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), dimethyl sulfate, N,N-diethylnitrosoamide (NDEA), and 5-bromo-2'-deoxyuridine (BUdR), or is a combination thereof. In some embodiments, the chemical mutagenic agent is selected from ethyl methanesulfonate (EMS), dimethyl sulfate, and NaN3, or is a combination thereof. In some embodiments of Petition 870250086789, dated 09 / 25 / 2025, pp. 119 / 280 In embodiment 96 / 224, the mutagenic agent is radiation. In some embodiments, the radiation is selected from X-rays, gamma rays, and ultraviolet radiation, or is a combination thereof.

[0280] Barley plants carrying a mutation in the CXE2L1 gene can also be prepared using various directed mutagenesis methods, which, for example, can be designed based on the CXE2L1 gene sequence (SEQ ID NO: 2) provided in this descriptive report. In one embodiment, the barley plant is prepared using any of the following: CRISPR, a TALEN, a zinc finger, a meganuclease, and a DNA-cutting antibiotic, as described in WO 2017 / 138986.

[0281] In one embodiment, the barley plant is prepared using the CRISPR / Cas9 technique, for example, using RNA-guided Cas9 nuclease. This can be done as described in Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186 / s13059-015-0826-7, except that the unique guide RNA sequence is designed based on the genetic sequences provided in this descriptive report. In another embodiment, the barley plant is prepared using a combination of TALEN and CRISPR / Cas9 techniques, for example, using RNA-guided Cas9 nuclease. This can be done as described in Holme et al., Plant Mol Biol (2017) 95: 111-121; DOI: 10.1007 / s11103-017-0640-6, except that TALEN and the unique guide RNA sequence are designed based on the gene sequences provided here.

[0282] In one embodiment, the barley plant is prepared using the base-based editing technique. Petition 870250086789, dated 09 / 25 / 2025, pp. 120 / 280 97 / 224 in CRISPR / Cas9. This can be done as described in Komor et al. (2016), except that the guide RNA is designed based on the genetic sequences provided in this descriptive report.

[0283] In one embodiment, the barley plant is prepared using the CRISPR / Cas9-based primary editing technique. This can be done as described in Anzalone et al. (2019), except that the primary editing guide RNA is designed based on the genetic sequences provided in this descriptive report.

[0284] In one embodiment, the cereal plant is prepared using homology-directed repair, a combination of a DNA-cutting nuclease and a donor DNA fragment. This can be done as described in Sun et al., Molecular Plant (2016) 9:628-631; DOI: https: / / doi.org / 10.1016 / j.molp.2016.01.001 except that the DNA-cutting nuclease is designed based on the gene sequences provided in this descriptive report and the donor DNA fragment is designed based on the coding sequence of the mutant cereal variant provided in this descriptive report.

[0285] The present invention also provides barley plants that carry other mutations besides the mutation in the CXE2L1 gene. Such plants can be prepared by introducing other mutations into barley plants that already carry a mutation in the CXE2L1 gene or by generating a separate barley plant that carries the other mutation, followed by crossing the barley plants to obtain a barley plant that carries both mutations. Petition 870250086789, dated 09 / 25 / 2025, pp. 121 / 280 98 / 224

[0286] In any case, the second mutation can be prepared and identified using the same methods described above for barley plants that have a mutation in the CXE2L1 gene.

[0287] In some embodiments, the method further comprises selecting barley grains, or their offspring, carrying a mutant β-glucan synthase gene. In some embodiments, the mutant β-glucan is CslF6, optionally wherein said mutant CslF6 gene encodes a loss-of-function mutant CslF6 polypeptide, wherein the wild-type CslF6 is CslF6 of SEQ ID NO: 7, optionally further wherein the mutation in the gene encoding CslF6 is a deletion.

[0288] In particular, barley plants carrying a mutation in a gene encoding a β-glucan synthase can be prepared as described above, such as using one or more of the mutagenic agents described above. Preferably, barley plants carrying a mutation in a gene encoding a β-glucan synthase are prepared and identified using the FIND-IT method. Suitable primers and probes for identifying barley plants comprising the mutations indicated in Table E below in the CslF6 gene are described in Examples 1 and 2 of international patent application WO 2019 / 129736. Table E. Mutant No. Nucleotide alteration in the coding sequence Amino acid alteration in the protein (SEQ ID NO: 1 of WO 2019 / 129736) Petition 870250086789, dated 09 / 25 / 2025, pp. 122 / 280 99 / 224 (SEQ ID NO:2 of WO 2019 / 129736) Mutant 1 G >G (2540) Gly>Glu (847) Mutant 2 G >G (2243) Gly>Asp (748) Mutant 3 G >G (2028) Trp>Stop (676) Mutant 4 G >G (2195) Gly>Asp (732) Mutant 5 C>T (2126) Thr>Ile (709)

[0289] Alternatively, barley plants carrying a mutation in the CXE2L1 gene, as well as a mutation in a gene encoding a β-glucan synthase, can be generated by crossing a barley plant carrying a mutation in the CXE2L1 gene with the barley plant designated “Mutant 2” deposited at NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland, on 12 November 2018, under NCIMB accession number 43273. The aforementioned barley plant is described in international patent application WO 2019 / 129736.

[0290] Barley plants carrying a mutation in the CXE2L1 gene and in other genes can be generated in similar ways, i.e., as described above or by crossing with barley plants comprising the said mutations.

[0291] In one embodiment of the present invention, the objective is to provide agronomically useful barley plants that exhibit a mutation in the CXE2L1 gene and, optionally, in a gene encoding a βglucan synthase. In addition to the mutation in the CXE2L1 gene and, optionally, the mutation in a gene encoding a βglucan synthase, Petition 870250086789, dated 09 / 25 / 2025, page 123 / 280 100 / 224 glucan synthase, there are additional factors that can also be considered in the art of generating a commercial variety of barley useful for malting and / or brewing and / or as a base for beverages, for example, yield and grain size, and other parameters related to malting or brewing performance. Since many (if not all) relevant characteristics have been shown to be under genetic control, the present invention also provides modern, homozygous, high-yielding malting cultivars that can be prepared from crosses with the barley plants disclosed in this publication. The skilled craftsman (experienced barley breeder) will be able to select and develop barley plants that, after crosses with other barley plants, will result in superior cultivars.Alternatively, the creator may use plants of the present invention for additional mutagenesis to generate new cultivars carrying additional mutations, in addition to the CXE2L1 gene mutation.

[0292] The present invention also comprises barley plants carrying a mutation in the CXE2L1 gene and, optionally, in a gene encoding a β-glucan synthase prepared from a plant breeding method, including self-pollination, backcrossing, crossbreeding with populations and the like. Backcrossing methods can be used with the present invention to introduce the CXE2L1 gene mutation into another cultivar.

[0293] One way to accelerate the plant breeding process involves the initial multiplication of mutants generated by applying cultivation techniques Petition 870250086789, dated 09 / 25 / 2025, pp. 124 / 280 101 / 224 tissues and regeneration. Thus, another aspect of the present invention is to provide cells that, after growth and differentiation, produce barley plants carrying the CXE2L1 gene mutation and, optionally, a gene encoding a β-glucan synthase. For example, breeding may involve traditional crosses, preparation of fertile plants derived from anthers, or use of microspore culture.

[0294] In one embodiment, the barley plant of the present invention was not obtained exclusively by means of an essentially biological process. The offspring of a barley plant obtained by a technical process is considered in this descriptive report as not being obtained exclusively by means of an essentially biological process, because the parent plant is obtained by a technical process.

[0295] In one embodiment, the barley plant has a mutation in the CXE2L1 gene, wherein said mutation was induced by chemical and / or physical agents. In another embodiment, the barley plant also has a mutation in a gene encoding a β-glucan synthase, wherein said mutation was induced by chemical and / or physical agents.

[0296] In one embodiment, the barley plant was prepared by a method involving an induced mutagenesis step or said plant is offspring of a plant prepared by a method involving an induced mutagenesis step. Thus, the barley plant may be a barley plant prepared by a method comprising the Petition 870250086789, dated 09 / 25 / 2025, pp. 125 / 280 102 / 224 following stages or progeny of a plant prepared by a method comprising the following steps: a. to supply barley grains; and b. random mutagenesis of said barley grains; and c. select barley grains or their descendants that carry a mutant CXE2L1 gene; wherein the aforementioned mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, in which the Wild-type CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it.

[0297] In one embodiment, the barley plant may be a barley plant prepared by a method comprising the following steps or offspring of a plant prepared by a method comprising the following steps: a. providing barley grains; and b. random mutagenesis of said barley grains, thereby introducing a mutation into the CXE2L1 gene; and c. select barley grains or their descendants that carry a mutant CXE2L1 gene; wherein the said mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, wherein the said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: I. amino acid 127, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or Petition 870250086789, dated 09 / 25 / 2025, pp. 126 / 280 103 / 224 ii. amino acid 113, wherein the said substitution is a substitution of a serine (S) for a proline (P), on the condition that the plant does not contain SEQ ID NO: 5.

[0298] In one embodiment, the barley plant may be a barley plant prepared by a method comprising the following steps or the offspring of a plant prepared by a method comprising the following steps: a. to supply barley grains; and b. random mutagenesis of said barley grains, thereby introducing a mutation into the CXE2L1 gene; and c. select barley grains or their descendants that carry a mutant CXE2L1 gene; wherein the said mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, wherein the said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: i. amino acid 127, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. amino acid 113, wherein the said substitution is a substitution of a serine (S) for a proline (P), provided that the plant does not contain SEQ ID NO: 5; and / or iii. amino acid 125 of SEQ ID NO: 1, wherein the said substitution is a substitution of proline (P) for serine (S); and / or Petition 870250086789, dated 09 / 25 / 2025, pp. 127 / 280 104 / 224 iv. amino acid 126 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or v. amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or vi. amino acid 212 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or vii. amino acid 212 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for valine (V).

[0299] The mutation and / or substitution in CXE2L1 may be any mutation as defined in this descriptive report. Examples Example 1 - Isolation of HENZ-α mutants from a population of CRL Quench mutants

[0300] In this descriptive report, we describe a high-throughput screening of individual barley grains derived from a Quench mutant population. The selected trait was high hydrolase activity, exemplified by α-amylase activity, in germinated barley grains. The aim of this direct genetic screening was to identify barley variants beneficial for short malting times. Results

[0301] First, a mutant population of the cultivar (cv.) Quench was engineered. The cv. Quench was mutagenized with NaN3, as described elsewhere (Knudsen et al., Petition 870250086789, dated 09 / 25 / 2025, pp. 128 / 280 105 / 224 2022), and propagated in the field in Fyn (Denmark) for consecutive years. The respective mutant population M3 cv. Quench was cultivated in Fyn in 2014, and single ears, containing mutant M4 seeds, were harvested manually.

[0302] Next, two grains from the center of an individual ear were harvested, each split into an endosperm half-grain (without embryo) and an embryo half-grain (with embryo) and stored in two individual 96-well plates in corresponding positions.

[0303] Next, the endosperm grain media were transferred to 2 ml deep 96-well plates, soaked in water. After 24 hours, the water was removed and the grain media was incubated for a further 72 hours. Subsequently, the half-grains were ground and tested for their ability to express and synthesize hydrolytic enzymes, exemplified in this descriptive report by the activity of the α-amylase enzyme, using a Megazyme α-amylase enzymatic assay (Ceralpha Method according to the manufacturer's protocol (https: / / www.megazyme.com / alpha-amylase-assaykit)). In total, 8,280 mutant grains, derived from 4,140 individual ears, were tested. Of all the endosperm half-grains tested, 19 individual endosperm half-grains showed significant α-amylase activity (Figure 1: see the representative Eli sample compared to the positive H9-11 references).It should be noted that, in several cases, two individual endosperm half-grains derived from the same ear showed the same high α-amylase activity (as described below for HENZ-11 and HENZ-2). Petition 870250086789, dated 09 / 25 / 2025, pp. 129 / 280 106 / 224

[0304] Next, the corresponding “embryo half grains” of the 19 “endosperm half grains” identified with high α-amylase were germinated, the plants were grown to maturity, and several ears per individual plant were harvested. To verify the increased hydrolytic power in these mutants, eight endosperm half grains from each propagated plant were tested again for α-amylase activity (experimental setup identical to that described above). Four mutants, originally derived from two individual mutant ears (samples E11_grain1 and E11_grain2 from plate 138; samples H5_grain1 and H5_grain2 from plate 139), showed strong alpha-amylase activity (Figure 2, 8 individual endosperm half grains; Figure 3, average of eight endosperm half grains).The grains derived from the spike samples E11_grain1 and E11_grain2 from plate 138 were renamed HENZ-a1_1 and HENZ-a1_2, respectively, and the grains derived from the spike samples H5_grain1 and H5_grain2 from plate 139 were renamed HENZ-a2_1 and HENZ-a2_2. The corresponding embryonic grain halves of the tested HENZ-a1_1 and HENZ-a1_2, and of the HENZ-a_1 and HENZ-a2_2, were germinated in a Petri dish and grown to maturity, and the harvested seeds were kept in the barley seed bank of the Carlsberg Research Laboratory. The early-harvested grains of HENZ-a1_1 and HENZ-a2_1 (greenish grains derived from the first greenhouse propagation) were sent to Norre Aaby, Fyn, Denmark, in 2016 for row propagation. The offspring of these propagated plants HENZ-a1_1 and HENZ-a2_1 were simply named “HENZ-a1” and “HENZ-a2”, respectively. Petition 870250086789, dated 09 / 25 / 2025, pp. 130 / 280 107 / 224 "HENZ-α1" and "HENZ-α2" were subsequently propagated in Fyn (DK) and Christchurch (NZ) for downstream characterization. Materials and methods

[0305] The Quench cultivar was mutagenized using NaN3, as described in Kudsen et al., 2022. The plants M1, M2, and M3 were grown in Fyn, Denmark, in 2013, in New Zealand in 2013 / 2014, and in Fyn, Denmark, in 2014, respectively. Seeds M4 from plants M3 (Fyn, Denmark, 2014) were harvested manually and were not threshed to keep the spikes intact. Two grains from individual spikes were selected for the experiment, as described in the Results.

[0306] The experimental protocol was as follows: • Transfer grain media from the original Nunc plate to the new Nunc plate (reverse orientation) o Then to the 2 ml deep well plate (forward orientation) • Use new, mechanically stable 96 deep well (2 ml) plates • Place the endosperm grain media in well plates • Add 500 µl of H2O o Store the endosperm grain media in H2O for hours in a dark, humid environment (permeable lid on the 96-well plate, damp cloth in the box, box closed) at 15°C in a cold chamber o Remove H2O after 24 h, germinate for 72 h in a dark, humid environment (permeable lid on the Petition 870250086789, dated 09 / 25 / 2025, pp. 131 / 280 108 / 224 plate of 96 wells, damp cloth in the box, box closed) at 15°C in a cold chamber. Add steel beads (5 mm diameter) • Add 400 μL of 1x extraction buffer per half grain > preheat the buffer to 40°C • Close the 96-well plate with a silicone lid • Grind the samples: GenoGinder, 6 min at 1500 rpm Centrifuge the plates for 2 min at 2000 rpm • Add 600 μL of 1x extraction buffer per grain > preheat the buffer to 40°C • Shake for 30 seconds at 1250 rpm (Mixmate) and manually agitate the samples • Incubate for 5 minutes at 40°C • Vortex for 30 seconds at 1250 rpm (Mixmate) and manually agitate the samples • Incubate for 5 minutes at 40°C • Spin the plates for 3 minutes at 3000 rpm • Remove 50 μL of the extract and dilute 1:10 with 1x extraction buffer (450 μL) • Shake for 30 seconds at 1250 rpm (mixmate) • Mix 20 μl of diluted extract with 20 μl of Ceralpha mixture in Nunc microtiter plates at room temperature • Shake for 30 seconds at 1250 rpm (mixmate) • Incubation time at room temperature for 30 minutes Add 200 μl of 1% TRISMA stop solution. Petition 870250086789, dated 09 / 25 / 2025, pp. 132 / 280 109 / 224 • Measure absorbance at 405 nm (microtiter plate reader) Example 2 - Characterization of HENZ-α mutants under different germination conditions

[0307] In this descriptive report, we describe the characterization of HENZ-α mutants, specifically α-amylase activity and sugar composition, in different germination configurations. The objective was to show that HENZ-α variants modify barley grain germination by increasing hydrolase activity, exemplified in this descriptive report by the increased α-amylase activity and the modified sugar composition in the endosperm grain. Results

[0308] After 72 hours of germination in a Petri dish, whole grains HENZ-α1 and HENZ-α2 showed 10-20% higher α-amylase activity compared to the wild type Quench (figure 4). When submerged in water, HENZ-α1 and HENZ-α2 exhibit 180% higher α-amylase activity than the wild type Quench (figure 4).

[0309] In the germination of half-grain endosperm, significant α-amylase activity was observed in HENZ-α1 and HENZ-α2, both in the Petri dish and submerged in water (figure 5). No α-amylase activity was found in wild-type Quench. Interestingly, the α-amylase activity of HENZ-α1 and HENZ-α2 was higher when the grain was submerged in water (compared to the Petri dish) (figure 5). Petition 870250086789, dated 09 / 25 / 2025, pp. 133 / 280 110 / 224

[0310] The composition of the released sugar was analyzed in germinated endosperm half-grain (sample submerged in water). As shown in Table 1, germination of HENZ-α1 and HENZ-α2 endosperm half-grain resulted in a modified sugar composition, indicating altered hydrolase activity in the HENZ-α variants. Table 1. Composition of sugar released in the germinated endosperm grain (samples submerged in water) (ppm). Sample HENZ-a1 HENZ-a2 QUENCH Glucose 93 50 1 Fruits 27 17 0 Isomaltose 2 12 0 Sucrose 178 103 3 Isomaltotriose 1 3 1 Maltose 73 45 1 Panos 6 17 0 Maltotriose 7 18 0 Maltotetraose 6 3 0 Maltopentaose 3 5 0 Maltohexaose 1 1 0 Maltoheptaose 1 2 0 Materials and methods

[0311] HENZ-a1, HENZ-a2 and their respective wild Quench types were propagated and harvested in New Zealand in 2017 / 2018.

[0312] Germination was carried out in Petri dishes or submerged in water in glass flasks, using whole grains or half endosperm grains. The half endosperm grains were dissected manually, as described elsewhere (mutant identification experiment). Petition 870250086789, dated 09 / 25 / 2025, pp. 134 / 280 111 / 224 Germination in a Petri dish

[0313] Two sheets of Whatman No. 1 filter paper were placed in a Petri dish. 100 grains per sample were germinated with the addition of 4 ml of water. The Petri dishes were stored in a dark box at 20°C for 72 hours. Germination in a jar

[0314] 100 grains per sample were submerged in 100 ml of distilled water in a 250 ml Erlenmeyer flask with airflow at 20°C for 72 hours. The flasks were shaken at 250 rpm during incubation. Two replicates were made per sample.

[0315] After 72 hours of incubation, the grains from germination in Petri dishes or flasks were collected in a 50 ml tube and lyophilized for 24 hours. The dried grains were ground using a Retsch grinder (30 Hz, 30 seconds x 2). α-amylase activity was measured using the Ceralpha method (Ceralpha method according to the manufacturer's protocol, Megazyme) modified for the Gallery Plus Beermaster. α-amylase activity was calculated in units per gram of initial dry weight (U g DW-1).

[0316] For the sugar analysis, endosperm grains were incubated and germinated in flasks as described elsewhere (submerged condition). 0.9 ml of liquid was removed and weighed from the flasks. 10% of the sample weight of 1 M KOH was added and the samples were centrifuged. The supernatant was diluted 20 times and filtered through a 0.2 μm filter. Petition 870250086789, dated 09 / 25 / 2025, pp. 135 / 280 112 / 224

[0317] The analysis was performed using the ICS-3000 Ion Chromatography System. Separation was performed on Dionex™ CarboPac™ PA100 shielding columns (4 mm x 50 mm, 8.5 µm) and analytical columns (4 mm x 250 mm, 8.5 µm). The injection volume was 10 µl. The analytes were detected by a Pulsed Electrochemical Detector with a permanent gold ED electrode for Pulsed Amperometric Detection. Example 3 - α-amylase activity in the endosperm grain half of HENZ-αI and HENZ-α2

[0318] In this descriptive report, we describe the evaluation of the phenotype of α-amylase activity in endosperm grain mid-grain after germination. α-amylase activity depends on the GA-induced de novo expression of α-amylase enzymes, initiated by the GA hormone derived from embryo and scutellum tissues (Betts et al., 2019). Thus, without an embryo, as in endosperm grain mid-grain where the embryo has been removed, no α-amylase activity is detectable in the wild-type grain. However, even without an embryo, the HENZ-α1 and HENZ-α2 endosperm grain mid-grains exhibit α-amylase activity. This characteristic is stable across years and locations. Results

[0319] Eight non-dormant grains from each accession were dissected in half, i.e., half endosperm and half embryo. The embryo half grains were placed in a 96-well Nunc plate, while the corresponding endosperm half grains were placed in a deep 96-well plate in the same order. Both the grains with half endosperm and the grains with half embryo were Petition 870250086789, dated 09 / 25 / 2025, pp. 136 / 280 113 / 224 were incubated in water for 24 hours (in the dark, at 15°C). Subsequently, the water was removed and incubation continued for a further 72 hours. α-amylase activity was measured in grains with half the endosperm using a Megazyme α-amylase activity assay. Absorbance was measured at OD 405 nm.

[0320] As shown in Figure 6 and Table 2, the HENZ-a1 and HENZ-a2 endosperm half-grains from Fyn, Denmark 2017 and New Zealand 2016 / 2017 showed high α-amylase activity, similar to the positive control (Quench endosperm half-grain with 100 nM GA). The wild types Quench, Planet, and Paustian did not show α-amylase activity. The trait remained stable across years and environments; however, the intensity of the trait showed some variation and was strongest in the grain grown in New Zealand 17. Table 2. Access Station Average DP DK17 HENZ-αI 0.90 0.63 HENZ-αZ 1.15 0.53 Quench 0.08 0.01 Planet 0.09 0.00 Paustian 0.09 0.01 NZ16 / 17 HENZ-αI 3.65 0.49 HENZ-αZ 2.36 0.98 Quench 0.09 0.00 Planet 0.10 0.02 Paustian 0.10 0.03 Extinction + 100 nM GA 4.24 0.42 Materials and methods

[0321] HENZ-α^ HENZ^2, Wild-type Quench and other wild-type accessions (Planet and Paustian) were Petition 870250086789, dated 09 / 25 / 2025, pp. 137 / 280 114 / 224 grown in New Zealand in 2016 / 2017 and in Fyn, Denmark, in 2017.

[0322] Germination and α-amylase activity assay (Megazyme Ceralpha kit, according to the manufacturer's protocol (K-CERA 01 / 12)) were performed as previously described (mutant isolation assays) with half of the Quench endosperm grain incubated at 100 nM GA as a positive control.

[0323] The experimental protocol was as follows: Each healthy grain was dissected in half. Half of the embryo grain was placed in a Nunc plate. Half of the endosperm grain was placed in the corresponding well in a 2 ml deep-well plate. Both half of the embryo grains and half of the endosperm grains were germinated in the same way, one to check seed variability and the other for an α-amylase assay. The details are described below. • Half of the grain containing the embryo was stored in a standard 96-well Nunc plate, kept in a cool, dark, and dry place, and stored for possible germination and subsequent growth. • The respective half-grain of endosperm was placed in a 2 ml deep-well plate and then treated as follows: • Add 500 µl of H2O to the deep well plate for the α-amylase assay. Store half of the endosperm grains in H2O for 24 hours in a dark, humid environment. Petition 870250086789, dated 09 / 25 / 2025, pp. 138 / 280 115 / 224 (permeable lid and damp cloth) at 15°C in a cold chamber (malt laboratory) • Remove H2O after 24 h, germinate for 72 h in a dark, humid environment (permeable lid and damp cloth) at 15°C in a cold chamber (malt laboratory) • Add steel beads (5 mm diameter) • Add 400 pl of 1x extraction buffer (preheated to 40°C) per half grain • Close the 96-well plate with a silicone lid • Grind the samples: GenoGinder,• 6 min at 1500 rpm • Spin plates for 2 minutes at 2000 rpm • Add 600 µl of 1x extraction buffer (pre-heated to 40°C) per grain • Shake for 30 seconds at 1250 rpm (Mixmate) and manually agitate the samples • Incubate for 5 minutes at 40°C • Vortex for 30 seconds at 1250 rpm (Mixmate) and manually agitate the samples • Incubate for 5 minutes at 40°C • Spin plates for 3 minutes at 3000 rpm • Remove 50 µl of the extract and dilute 1:10 with 1x extraction buffer (450 µl) • Shake for 30 seconds at 1250 rpm (Mixmate) • Add 20 µl of Ceralpha to Nunc microtiter plates and then 20 µl of the temperature-diluted extract environment. • Shake for 30 seconds at 1250 rpm (mixmate) Petition 870250086789, dated 09 / 25 / 2025, pp. 139 / 280 116 / 224 • Incubation time at room temperature for 30 minutes • Add 200 µl of 1% TRISMA stop solution • Measure absorbance at 405 nm (microtiter plate reader) Example 4 - α-amylase activity of germinated grains Materials and methods

[0324] Field-grown grain material of the wild type Quench, HENZ-αI and HENZ-α2 was germinated in a water tank, essentially as described in Example 1 of international patent application PCT / EP2017 / 065498, with the following specifications: 200 g of barley grains (dry weight) were incubated for 48 hours in a tank and covered with water containing 1 mM GA, while being subjected to an airflow of 90 L / h per kg of dry weight of barley grains. The α-amylase activity in the same material after 24 hours and after 48 hours was measured using standard Megazyme assays, as described below. Sample preparation

[0325] Prior to enzyme activity analysis, the germinated grain samples were milled using a standard Foss Cyclotech mill (Foss, Denmark), equipped with a tungsten carbide grinding ring (Foss 10004463), nickel-plated impeller (Foss 1000 2666) and a mm output screen (Foss 10001989). All enzyme activity measurements in the germinated barley grains were performed within 48 hours of sample milling. α-amylase activity Petition 870250086789, dated 09 / 25 / 2025, pages 140 / 280 117 / 224

[0326] The α-amylase activity of the germinated grains was based on the flour prepared as described above in the “Sample Preparation” section. The assays for determining α-amylase activity used a Megazyme Ceralpha kit, using standard laboratory equipment. The assays were performed according to the manufacturer's protocol (K-CERA 01 / 12), including the calculation of α-amylase activity. Results

[0327] The α-amylase activity of the germinated grains can be seen in figure 7 and in table 3. Table 3. α-amylase, [U] / [g] - 24 h α-amylase, [U] / [g] - 48 h MaltLab # time (h) Act [U] / [g] STDEVA time (h) Act [U] / [g] STDEVA weight 24 1.4 0.8 48 5.7 0.2 HENZ-αI 24 2.3 0.4 48 9.8 0.6 HENZ^2 24 2.5 0.6 48 11.1 0.6 Example 5 - Enzyme transcription levels hydrolytic processes after germination Materials and methods

[0328] 200 g of field-grown grains in Nova Zealandia in 2016 / 2017 of the wild types Quench, HENZ^1e HENZ^2 were germinated in a tank with 500 ml of water and mM GA for 48 hours with a continuous airflow (90 l / h). Subsets of sprouted grains were flash-frozen in liquid N2, freeze-dried for 48 hours, and milled (30 / s for 30 seconds) using a Retsch MM300 ball mill fitted with a 20 mm stainless steel ball per chamber (Retsch, Germany). Quantification of transcription Petition 870250086789, dated 09 / 25 / 2025, pp. 141 / 280 118 / 224

[0329] Transcript analysis of sprouted grains was based on flour prepared as described above in the “Materials and Methods” section. Sample preparation for RNA extraction was performed according to the following procedure. For each sample, approximately 100 mg of flour were weighed into 2 mL Eppendorf tubes with two 3 mm metal beads, and 1 mL of Tri-Reagent (Sigma #93289) was added. The resulting mixture was vigorously shaken. After homogenization, the sample was centrifuged at 12,000 χ² g for 10 minutes at 4°C to precipitate polysaccharides and high molecular weight DNA. The supernatant (~700 g / L) was then pipetted into a new 2 mL PhaseLock tube (Qiagen #129056), and 0.2 mL of chloroform was added. The tube was vigorously shaken by hand for 10–15 seconds to mix the chloroform and TriReagent. The sample was then incubated at room temperature for 5-15 minutes and centrifuged at 12,000 χ² g for 5 minutes at 4°C.The mixture separated into a lower red phenol-chloroform phase, an intermediate solid gel phase, and an upper colorless aqueous phase, with the RNA remaining exclusively in the aqueous phase. The aqueous phase of the sample (~400 g) was decanted into a new Eppendorf tube, and 500 g of 2-propanol were added. The sample was incubated in a freezer for 1 hour. Then, the samples were centrifuged at 12,000 χ²g for 10 minutes at 4°C, and the supernatant was removed. The RNA pellet was resuspended in 100 g of RNase-free water, and RNA cleanup was continued according to the manufacturer's instructions. Petition 870250086789, dated 09 / 25 / 2025, pp. 142 / 280 119 / 224 (Qiagen#74181). The RNA was then quantified using NanoDrop (ThermoFisher Scientific).

[0330] To synthesize complementary DNA (cDNA), the RNA sample was first normalized to a concentration of 50 ng / µl. A 200 ng (4 µl) aliquot of the RNA sample was then used for cDNA synthesis with the iScript Select cDNA Synthesis Kit, following the manufacturer's instructions (Bio-Rad #1708896). The resulting cDNA was further diluted; specifically, 180 µl of water were added to the reaction volume of 20 µl of cDNA, resulting in a 10-fold dilution. This was followed by an additional round of 10-fold dilution, resulting in a 100-fold dilution solution that was appropriate for droplet digital PCR (ddPCR). We used ddPCR for quantification of AMY1-2, LD, and BGL2A transcripts. For analytical purposes, 5 pl of cDNA were added to a 17 pl PCR mix containing 11 pl of 2x ddPCR Supermix for probes (No.dUTP; Bio-Rad), 900 nM target-specific PCR primers, 250 nM target-specific probe (6-carboxyfluorescein—FAM), 900 nM reference-specific PCR primers, and 250 nM reference-specific probes (hexachlorofluorescein—HEX). Droplet generation was performed by loading the reaction mixture into the AutoDG Droplet. Generator (Bio-Rad), according to the manufacturer's instructions. The droplet emulsion was subjected to thermal cycling using standard PCR conditions, which included denaturation at 95°C for 10 minutes, 40 PCR cycles at 94°C for 30 seconds and 55°C for 1 minute, and a final extension to 98°C for 10 minutes before... Petition 870250086789, dated 09 / 25 / 2025, pp. 143 / 280 120 / 224 microtiter plate storage at 8°C. PCR amplification was analyzed using the QX200 Droplet Reader (Bio-Rad), and the threshold was determined by comparing samples and template-free ddPCR controls. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Data were normalized to the internal actin transcription levels of each sample, and the resulting data are presented as a bar graph, representing the absolute number of transcripts detected per cDNA plate. Direct primer AMY1-2 (SEQ ID NO: 15):5'TGAAGGAGGAGATCGATC-3'; Reverse primer AMY1-2 (SEQ -ID NO: 16):5'TTGCCGTCGATCTCG-3'; Specific detection probe for AMY1-2 (SEQ ID NO: 17): 5'-AAGCTGCAGATCATGGAGGC-3'-labeled with 6-carboxyfluorescein (FAM) Direct primer LD (SEQ ID NO: 18):5'CTTCGATGGGGTTTGAAC-3'; Reverse primer LD (SEQ -ID NO: 19):5'CAGATTTCCTCACCAAAG -3'; Specific detection probe for LD (SEQ ID NO: 20): 5'-CCTGTGCAGGTGAATTCATCA-3'-labeled with 6-carboxyfluorescein (FAM) BGL2A Direct Primer (SEQ ID NO: 21):5'AACTGGGGACTCTTCTAC-3'; Reverse primer BGL2A (SEQ -ID NO: 22):5'TCAGAAGTTGATGGGGTAG-3'; Petition 870250086789, dated 09 / 25 / 2025, pp. 144 / 280 121 / 224 Specific detection probe for BGL2A (SEQ ID NO: 23): 5'-CCCAACATGCAGCACGT-3'-labeled with 6-carboxyfluorescein (FAM) Direct reference primer Actin (SEQ ID NO: 24): 5'TACAACTCCATCATGAAGTG-3'; Reference reverse primer for actin (SEQ-ID NO: 25): 5'- GATACCTGGGAACATAGTTG-3'; Specific detection probe for reference actin (SEQ ID NO: 26): 5'-AAACATCGTGCTCAGTGGTG-3'-labeled with hexachlorofluorescein (HEX). Results

[0331] HENZ-a1 and HENZ-a2 germinating seeds were tested for transcript accumulation of genes encoding α-amylase (AMY1-2; HORVU.MOREX.r3.6HG0619750.1, Morex_V3), limit dextrinase (LD; HORVU.MOREX.r3.7HG0656620.1, Morex_V3) and β-amylase (BGL2A; HORVU.MOREX.r3.7HG0750120.1, Morex_V3) in samples after 48 hours of immersion. The samples were compared with their parental control, cv. Quench. Figures 8A-C show a clear increase in the accumulated transcription of the three genes after 48 hours of continuous immersion in HENZ-a1 and HENZ-a2, compared with their control. Example 6 - α-amylase activity of germinated grains. Materials and methods.

[0332] Field-grown grain material of the wild type Quench, HENZ-a1 and HENZ-a2 was germinated in a tank with water, essentially as described in Example 1 of international patent application PCT / EP2017 / 065498, with the following specifics: 200 g of barley grains (dry weight) were incubated for 24 hours (WA) in a tank and Petition 870250086789, dated 09 / 25 / 2025, pages 145 / 280 122 / 224 covered with water, while being subjected to an airflow of 90 L / h per kg of dry weight of barley grains, followed by 24 hours of incubation at 25°C in aerated air (90 L / h / kg) (A). Sample preparation

[0333] Prior to enzyme activity analysis, the germinated grain samples were milled using a standard Foss Cyclotech mill (Foss, Denmark), equipped with a tungsten carbide grinding ring (Foss 10004463), nickel-plated impeller (Foss 1000 2666) and a mm output screen (Foss 10001989). All enzyme activity measurements in the germinated barley grains were taken within 48 hours of sample milling. α-amylase activity

[0334] The α-amylase activity of the germinated grains was based on the flour prepared as described above in the “Sample Preparation” section. The assays for determining α-amylase activity used a Megazyme Ceralpha kit, using standard laboratory equipment. The assays were performed according to the manufacturer's protocol (K-CERA 01 / 12), including the calculation of α-amylase activity. Results

[0335] The α-amylase activity of the germinated grains can be seen in figure 9 and in table 4. Table 4. α-amylase, [U] / [g] - 48 h MaltLab # (n=2) time (h) Act [U] / [g] STDEVA Petition 870250086789, dated 09 / 25 / 2025, pp. 146 / 280 123 / 224 wt 2 4 h WA + 24 h A 24 hh A WA + 24 21.3 0.2 HENZ-a1 h A _2 4 h WA + 24 24 hh A WA + 24 23.3 0.9 HENZ-a2 h A _2 4 h WA + 24 24 hh A WA + 24 25.3 0.3

[0336] Example 7 - Grain quality and germination rate Materials and methods

[0337] The protein, water and starch contents of the barley samples were determined using a Foss 1241 NIT instrument, using barley calibration (FOSS; supplied by Foss, DK). Germination test

[0338] All barley samples used in the examples were evaluated for germination index, germination energy, and water sensitivity. The data were based on a sample of 100 barley grains for a 4 mL germination test and a sample of 100 barley grains for an 8 mL germination test, according to the Analytica-EBC 3.6.2 Germination Energy of Barley Method (BRF Method).

[0339] Grain material from HENZ-a1, HENZ-a2 and the reference cultivar Quench (wt) grown in the field under similar conditions were germinated in a standard germination test: The grains were classified according to size using the Pfeuffer grain sorter; grains with sizes of 2.5 and 2.8 mm were used. Petition 870250086789, dated 09 / 25 / 2025, pp. 147 / 280 124 / 224 - 100 whole grains were placed in 2 pieces of Whatman (Grade 1, 85 mm, Cat-No. 1001-085) in 90 mm Petri dishes and 4 ml of distilled water were added. - Another 100 whole grains were placed in 2 pieces of Whatman (Grade 1.85 mm, Cat-No. 1001-085) in 90 mm Petri dishes and 8 ml of distilled water were added (high humidity conditions). The Petri dishes were sealed and stored in a dark box covered with a damp cloth at 20°C. - Petri dishes containing 4 ml were checked after 24 h, 48 h and 72 h. The germinating grains (radicle emergence) were removed from the Petri dish and the number of germinated grains was recorded. - The 8 ml Petri dishes were checked after 72 hours, the germinated grains (radicle emergence) were removed from the Petri dish and the number of germinated grains was recorded. Results

[0340] The water, protein and starch content (%) can be seen in table 5. Table 5. Water % Protein % Starch % Cooling 11.8 10.0 65.0 HENZ-a1 12.7 10.9 63.3 HENZ-a2 12.3 10.5 63.6

[0341] The germination test results can be seen in Table 6 and Figures 10 and 11. In particular, note that HENZ-a1 and HENZ-a2 have high grain quality, with no signs of pre-harvest germination. Furthermore, HENZ Petition 870250086789, dated 09 / 25 / 2025, pp. 148 / 280 125 / 224 α1 and HENZ-a2 have lower sensitivity to water compared to the control, showing better germination in excess water. Table 6. Results 4 ml 8 ml Index G Energy G 4 ml Energy G 8 ml Sensitivity 24 h (%) 48 h (%) 72 h (%) No germination 72 h (%) No germination of weight 6.4 98.0 74.0 24.0 49 43 6 2 74 26 HENZα1 7.0 96.0 91.0 5.0 57 36 3 4 91 9 HENZ-α2 6.9 96.5 92.0 4.5 56.5 36.5 3.5 3.5 92 8 Example 8 - Agronomic characteristics Materials and methods Plant material and growing conditions

[0342] Barley plants were grown in field trial plots for agronomic evaluation (7.5 m2 plots) in Fyn, Denmark, in 2017 and 2019. Grains from the field plots were harvested and threshed using a Wintersteiger Elite plot combiner (Wintersteiger AG, Germany), and grains were graded by size (2.5 mm limit) using a Pfeuffer SLN3 sample cleaner (Pfeuffer GmbH, Germany). Evaluation of yield and measurement of grain properties.

[0343] Thousand grain weight (TGW), water, starch and protein content of mature dry grains (sample size) Petition 870250086789, dated 09 / 25 / 2025, pp. 149 / 280 126 / 224 of approximately 500 grains per measurement) of field-grown plants were determined using the MARVIN digital seed analyzer (GTA Sensorik GmbH, Neubrandenburg, Germany), as described by Knudsen et al., 2022. Plant height and date of bolting.

[0344] The height of plants from two individuals per plot replicate per row was measured manually, from the beginning of the lowest internode to the end of the ear. The heading date, as defined by Alqudah and Schurbusch, 2017 (https: / / www.frontiersin.org / articles / 10.3389 / fpls.2017.00896 / full), was manually recorded in June 2017 and 2019. Results

[0345] The agronomic characteristics of HENZ-a1 and HENZ-a2 did not undergo significant changes and are similar to those of the wild type (wt) Quench. Grain parameters, such as water / starch / protein content, TGW and total yield, are wt. Plant height and heading date are wt. Barley variants with modified GA metabolism have shown, according to other authors (Stockinger, 2021; Kandemir et al., 2021), changes in TGW, yield, heading date and plant height.

[0346] The agronomic characteristics of HENZ-a1 compared to Quench can be seen in Table 7. Harvest in Fyn in 2017. Table 7. Petition 870250086789, dated 09 / 25 / 2025, pages 150 / 280 127 / 224 ID n! & 'rt! o\o 0 τ! 1 Protein (%) TGW (g) Weight (kg / plot) Plant height (cm) o\o 0 τ! 1 Protein (%) TGW (g) Weight (kg / plot) Plant height (cm) HENZ-Cyl 13.5 62.6 11.4 51.5 5.8 67 AVERAGE 62.7 11.2 51.8 5.9 66.8 HENZcil 15 63.2 10.8 51.7 5.8 66 Standard 0.3 0.3 0.7 0.1 1.3 HENZcil 14.9 62.6 11.4 51.9 6.0 69 HENZcil 13.7 62.4 11.2 53.0 6.0 66 HENZcil 14.9 62.7 11.3 51.5 6.0 66 QUENCH 14.8 63.4 11.1 51.9 5.9 64 AVERAGE 62.7 11.4 52.1 6.1 68.9 QUENCH 13.5 62.4 11.7 51.5 6.0 70 Standard 0.4 0.2 1.7 0.2 2.9 QUENCH 14.8 62.9 11.4 51.6 6.2 67 QUENCH 14.6 63 11.4 52.8 6.1 66 QUENCH 14.7 62.3 11.4 52.0 6.3 72 QUENCH 14.7 62.7 11.1 50.2 6.3 72 QUENCH 15.1 62.4 11.7 55.8 6.0 70 QUENCH 14.9 62.7 11.3 51.1 6.0 70

[0347] The agronomic characteristics of HENZ-α2 compared to Quench can be seen in Table 8. Harvest in Fyn in 2017. Table 8. Petition 870250086789, dated 09 / 25 / 2025, pp. 151 / 280 128 / 224 ID n! & 'rt! o\o 0 τ! 1 Protein (%) TGW (g) Weight (kg / plot) Plant height (cm) o\o 0 τ! 1 Protein (%) TGW (g) Weight (kg / plot) Plant height (cm) HENZa2 13.2 62.4 11.3 49.6 5.7 67 AVERAGE 62.5 11.4 51.4 5.9 68 HENZa2 14.7 62.9 11.2 52.9 6.1 71 Standard 0.2 0.2 1.5 0.2 3.9 HENZa2 14.7 62.3 11.4 52.4 6.1 73 HENZa2 14.9 62.5 11.4 52.1 6.1 65 HENZa2 15.1 62.4 11.7 50.2 5.6 64 QUENCH 14.8 63.4 11.1 51.9 5.9 64 AVERAGE 62.7 11.4 52.1 6.1 68.9 QUENCH 13.5 62.4 11.7 51.5 6.0 70 Standard 0.4 0.2 1.7 0.2 2.9 QUENCH 14.8 62.9 11.4 51.6 6.2 67 QUENCH 14.6 63 11.4 52.8 6.1 66 QUENCH 14.7 62.3 11.4 52.0 6.3 72 QUENCH 14.7 62.7 11.1 50.2 6.3 72 QUENCH 15.1 62.4 11.7 55.8 6.0 70 QUENCH 14.9 62.7 11.3 51, 1 6.0 70

[0348] The agronomic characteristics of ЗЕНЖ-αΙ compared to Quench can be seen in table 9. Harvest in Fyn in 2019. Table 9. Petition 870250086789, dated 09 / 25 / 2025, pp. 152 / 280 129 / 224 ID o\o nJ & 'rtl Protein (%) TGW (g) Weight (kg / plot) Heading (date in June) Plant height (cm) Protein (%) TGW (g) Weight (kg / plot) Heading (date in June) Plant height (cm) HENZ-Cyl 12.8 62.8 10.3 50.59 5.89 14 77 AVERAGE 60.6 11.6 51.01 6.56 14 77.5 HENZcil 13.7 58.4 12.9 51.43 7.23 14 78 Standard 3.1 1.8 0.60 0.95 0 0.7 QUENCH 13.6 63.3 10.3 52.39 5.76 14 74 AVERAGE 63.6 10.34 51.24 6.22 13.8 75.4 QUENCH 14.0 63.7 10.1 52.03 6.54 14 74 Standard 0.4 0.3 1.30 0.30 0.6 3.1 QUENCH 13.2 64.1 10.1 49.09 6.13 14 80 QUENCH 13.7 63.7 10.5 51.09 6.26 13 72 QUENCH 13.6 63.2 10.7 51.59 6.40 13 77

[0349] The agronomic characteristics of HENZ-α2 compared to Quench can be seen in Table 10. Harvest in Fyn in 2019. Table 10. ID o\o nJ & 'rtl Starch (%) Protein (%) TGW (g) Weight (kg / plot) Heading (date qtti iiinhn) Plant height (cm) Protein (%) TGW (g) Weight (kg / plot) Heading (date in June) Plant height (cm) HENZa2 13.1 62.5 10.1 50.18 6.04 14.75 / IÉDIA 62.5 10.4 50.45 5.96 14.75 HENZa2 13.0 62.4 10.7 50.72 5.87 14.75 Standard 0.1 0.4 0.39 0.12 0.0 Petition 870250086789, dated 09 / 25 / 2025, pp. 153 / 280 130 / 224 QUENCH 13.6 63.3 10.3 52.39 5.76 14 74 TED IA 63.6 10.34 51.24 6.22 13.8 75.4 QUENCH 14.0 63.7 10.1 52.03 6.54 14 74 Standard 0.4 0.3 1.30 0.30 0.6 3.1 QUENCH 13.2 64.1 10.1 49.09 6.13 14 80 QUENCH 13.7 63.7 10.5 51.09 6.26 13 72 QUENCH 13.6 63.2 10.7 51.59 6.40 13 77 Example 9 - Micromalting setup and procedure Materials and methods

[0350] The micromalting workflow can be seen in Figure 12. The micromalting procedure was carried out in a controlled environment with a temperature of 16°C and humidity around 70%. Immersion

[0351] The barley samples were placed in individual containers, each containing 100 g of seeds, and subjected to fresh water at 16°C to achieve 35% moisture content on day 1 and 41% moisture content on day 2.

[0352] The actual water absorption of the individual samples was determined as the weight difference between the initial water content, measured with the Foss 1241 NIT instrument (Foss A / S, Denmark), and the weight of the sample after removal of surface water. Germination

[0353] After the last immersion, the barley samples were kept at a degree of immersion of 41%. After each 24 Petition 870250086789, dated 09 / 25 / 2025, pp. 154 / 280 Between 131 and 224 hours, the samples were checked for moisture content and sprayed with additional water to overcome potential losses due to respiration. Oven

[0354] After the germination process, the barley samples were dried in a Curio oven using a two-stage ramp profile. The first stage of the ramp started at a set point of 27°C and ramped linearly at 2°C / h to the breakpoint at 55°C using 100% fresh air. The second linear ramp was 4°C / h, reaching a maximum of 85°C. This temperature was maintained constant for 90 minutes using 50% fresh air. Bark removal

[0355] The kiln samples were cured using a manual root removal system from the Wissenschaftliche Station für Brauerei, Munich, Germany. Example 10 - Hydrolytic enzymatic activity and β-glucan content during micromalting Materials and methods

[0356] The experiment was carried out as described in Example 9. Two independent micromalting experiments were performed per genotype. Sample preparation Enzymatic activity

[0357] Before analyzing enzymatic activity and β-glucan content, the germinated grain samples were ground using a standard Foss Cyclotech mill (Foss, Denmark), equipped with a tungsten carbide grinding ring (Foss 10004463), nickel-plated impeller (Foss 1000 Petition 870250086789, dated 09 / 25 / 2025, pp. 155 / 280 132 / 224 2666) and a 1 mm output screen (Foss 10001989). All measurements of enzymatic activity in germinated barley grains were made within 48 hours after sample milling. β-glucan content

[0358] Two methods were used to measure the β-glucan content. The data in Table 11A were produced using the standard EBC method for detecting HMW β-glucan in wort (Analytica EBC - 8.13.3 - β-Glucan in wort by automated discrete analysis - 2021). The data presented in Table 11B were produced by extracting soluble HMW β-glucan from malt flour during malting, as described in the “Sample Preparation” section.

[0359] Malt flour was extracted in distilled water at 65°C for 2 hours in a grain:water ratio of 1:4. After extraction, samples were centrifuged at 10,000 g for 10 minutes and the supernatant was analyzed for soluble HMW β-glucan using the ThermoFisher Scientific Beta-Glucan (High MW) kit method on a GalleryPlus Beer Master (ThermoFisher Scientific). Results HENZa-1 and HENZa-2 Hydrolytic enzymatic activity and β-glucan content

[0360] Hydrolytic enzymatic activity after malting was tested in HENZ«-1 and HENZ«-2, as described in “Materials and methods”. The β-glucan content after malting was measured using the standard EBC method for detecting HMW β-glucan in wort (Analytica EBC - 8.13.3 - β-Glucan in wort by automated discrete analysis - 2021). Petition 870250086789, dated 09 / 25 / 2025, pp. 156 / 280 133 / 224

[0361] The results can be seen in table 11A. Table 11A. Name Extract, fine [% dm] Extract, VZ65 [% dm] Viscosity (8.6) [mPa*s] Viscosity VZ 65 °C (8.6) [mPa*s] Protein content [% dm] Soluble nitrogen [mg / 100 g dm] Soluble nitrogen VZ65 [mg / 100 g dm] Quench NZ17 / 18 81.30125 80.65875 1.50875 1.56875 10.9375 823 786.5 HENZ-αI NZ17 / 18 82.7 81.9 1.46 1.46 11.6 938 915 HENZ-a2 NZ17 / 18 83.15 81.6 1.5 1.49 11.8 1020 958 Quench NZ 17 / 18 Lot B 28 79.26 79.35 1.57 1.68 11.6 851 739 HENZ-αΙ NZ 17 / 18 B-plot 8 80.6 80.14 1.5 1.49 12 917 798 HENZ-a2 NZ 17 / 18 Lot B 9 80.71 81 1.49 1.5 12 892 796 Quench NZ 16 / 17 Lot B 149 81.49 81.34 1.67 1.91 9.8 739 696 HENZ-αΙ NZ 16 / 17 Line 1781 82.14 81.13 1.5 1.52 10.9 880 848 HENZ-a2 NZ 16 / 17 Line 1783 82.28 81.53 1.49 1.5 11 916 794 Quench Fyn 17 Lot B #153, ​​M120 81.96 82.04 1.6 1.67 10.9 837 732 HENZ-αΙ Fyn 17 B-plot #169, M134 83.32 83.07 1.52 1.54 10.3 917 823 HENZ-a2 Fyn 17 Lot B 83.59 82.68 1.52 1.53 11.4 923 813 Petition 870250086789, dated 09 / 25 / 2025, pp. 157 / 280 134 / 224 #186, M149 Table 11A, continued. Name Kolbach Index [%] Kolbach Index VZ65 [%] FAN [mg / 100 g dm] FAN VZ65 [mg / 100 g dm] Attenuation Limit [%] Attenuation Limit VZ65 [%] Friability [%] Extinction NZ17 / 18 47.0725 45.15 168 155 81.2375 81.925 71.375 HENZ-a1 NZ17 / 18 50.74 49.5 190 163 80.4 81.7 90 HENZ-a2 NZ17 / 18 53.84 50.5 203 174 81.6 81.7 87 Quench NZ 17 / 18 B-plot 28 45.94 39.9 176 143 81.2 80.9 75 HENZ-αΙ NZ 17 / 18 Lot B 8 48 41.4 186 158 82 83.5 92 HENZ-a2 NZ 17 / 18 Lot B 9 46.47 41.5 189 152 82.2 83.5 86 Quench NZ 16 / 17 Lot B 149 47 44.2 166 141 81.5 82.7 82 HENZ-αΙ NZ 16 / 17 Row 1781 50 49 189 176 82.2 83.2 91 HENZ-a2 NZ 16 / 17 Row 1783 52.26 45.3 213 176 81.5 83.9 92 Quench Fyn 17 Lot B #153, ​​M120 48 41.9 178 159 81.9 83.9 81 Petition 870250086789, dated 09 / 25 / 2025, pp. 158 / 280 135 / 224 HENZ-a1 Fyn 17 B-plot #169, M134 55.48 49.8 194 158 81.3 84.5 94 HENZ-a2 Fyn 17 B Lot No. 186, M149 50.82 44.7 195 157 81.7 84.9 89 Table 11A, continued. Beta-glucan Name (FIA / Congress) [mg / l] Beta-glucan (FIA / VZ 65°C) [mg / l] Alpha-amylase activity [DU / g dm] Beta-amylase activity [BU / g dm] Days Temperature Target maceration degree Total loss Cooling NZ17 / 18 347.25 47 6.25 66.75 1055.625 6 14.5 45 10.3625 HENZ-α1 NZ17 / 18 <50 <50 72 1066 6 14.5 45 11.3 HENZ-α2 NZ17 / 18 <50 51 71 1216 6 14.5 45 11.8 Quench NZ 17 / 18 B-plot 28 540 414 59 1034 6 14.5 45 9.2 HENZ- α1 NZ 17 / 18 Lot B 8 0 0 69 1115 6 14.5 45 9.8 HENZ- α2 NZ 17 / 18 B-plot 9 87 83 77 1056 6 14.5 45 9.2 Quench NZ 16 / 17 Lot B 149 306 272 57 925 6 14.5 45 9.5 HENZ- α1 NZ 16 / 17 Line 1781 0 75 69 1043 6 14.5 45 10.4 HENZ- α2 NZ 16 / 17 0 0 68 1022 6 14.5 45 10.4 Petition 870250086789, dated 09 / 25 / 2025, pp. 159 / 280 136 / 224 Line 1783 Quench Fyn 17 Lot B #153, ​​M120 402 408 64 950 6 14.5 45 10 HENZ- α1 Fyn 17 B-plot #169, M134 56 99 61 948 6 14.5 45 10.7 HENZ- α2 Fyn 17 Lot B #186, M149 68 128 60 1119 6 14.5 45 10.7 β-glucan degradation

[0362] To measure β-glucan degradation over time, flour was extracted and soluble HMW β-glucan was analyzed during malting, as described in the “Sample Preparation” section.

[0363] The β-glucan content during malting can be seen in table 11B. Table 11B. Day soluble HMW β-glucan; % of weight HENZ-a1 2 99.7 HENZ-a1 3 86.7% HENZ-a1 4 80% HENZ-a1 5 44.1% HENZ-a1 6 30.9% HENZ-a1 7 40 HENZ-a2 2 104% HENZ-a2 3 96.4% HENZ-a2 4 67.9% HENZ-a2 5 42.5% Petition 870250086789, dated 09 / 25 / 2025, pp. 160 / 280 137 / 224 HENZ-a2 6 27.2% HENZ-a2 7 38.6% Example 11 - Genetic analyzes of HENZ-a1 and HENZ-a2

[0364] In this descriptive report, we describe the genetic analyses of HENZ-a1 and HENZ-a2, specifically the genetic inheritance of the HENZ-a trait and the genetic mapping of the HENZ-a trait, including the identification of closely linked markers for marker-assisted breeding. Results Genetic inheritance

[0365] Several HENZ-a1 reciprocal crosses were performed for genetic inheritance analyses (Table 12). As shown in Table 13 and Figure 13, when HENZ-a1 was used as the dam in the crosses, the F1 offspring showed increased α-amylase activity after 72 hours in endosperm half-grain germination tests. When HENZ-a1 was used as the dam in the crosses, the F1 offspring showed low α-amylase activity after 72 hours in endosperm half-grain germination tests. This was confirmed by an endosperm half-grain assay on individual F2 seeds from Paustian x HENZ-a1 reciprocal crosses, where 40 individuals with HENZ-a1 as the dam and 48 individuals with HENZ-a1 as the dam were tested. Despite the large variation observed, the average α-amylase activity was higher when HENZ-a1 was the dam in the crosses (Figure 14, Table 14).In contrast, the respective F2 of all reciprocal crosses showed half-grain endosperm amylase activity, indicating that a. Petition 870250086789, dated 09 / 25 / 2025, pp. 161 / 280 138 / 224 characteristic HENZ-a is controlled by nuclear DNA (figure 14, table 14).

[0366] For HENZ-a2, assays of endosperm mid-grain α-amylase activity were performed on individual F2 seeds from the HENZ-a2 x Quench cross (40 individuals with HENZ-a2 as mother and 48 individuals with HENZ-a2 as father). In this descriptive report, a result similar to that described for HENZ-a1 was observed, again indicating that the HENZ-a trait is controlled by nuclear DNA (figure 14, table 14).

[0367] Taken together, our results suggest that the HENZ-a1 and HENZ-a2 traits are inherited nuclearly, incompletely dominant (common for gene gain-of-function mutations), and that the trait(s) have a dose-dependent effect on barley grain germination. Table 12. Reciprocal crosses. Mother Father Mother Father Bowman HENZ-a1 HENZ-a1 Bowman PAUSTIAN HENZ-a1 HENZ-a1 PAUSTIAN PLANET HENZ-a1 HENZ-a1 PLANET QUENCH HENZ-a1 HENZ-a1 QUENCH HENZ-a2 PAUSTIAN PAUSTIAN HENZ-a2 HENZ-a2 PLANET PLANET HENZ-a2 HENZ-a2 QUENCH QUENCH HENZ-a2 Table 13. α-amylase activity in the F1 endosperm half-grain of HENZ-α1 reciprocal crosses OD 405 nm SD HENZ-a1 3.02 0.02 F1 (HENZ-a1 as mother) 0.42 0.57 Petition 870250086789, dated 09 / 25 / 2025, pp. 162 / 280 139 / 224 F1 (HENZ-a1 as sire) 0.03 0.01 Table 14. Endosperm midgrain α-amylase megazyme activity in F2 HENZ-a1 and HENZ-a2 populations from reciprocal crosses. Mean OD 405 nm SD HENZ-a1 2.22 0.20 Paustian 0.07 0.01 F2 HENZ-a1 mother 0.32 0.56 F2 HENZ-a1 father 0.19 0.27 HENZ-a2 2.43 0.47 Quench 0.11 0.06 F2 HENZ-a2 mother 0.61 0.80 F2 HENZ-a2 father 0.51 0.73 Genetic mapping

[0368] F2 individuals from two independent crosses were genotyped using a 50,000 SNP array. Among the 50,000 detectable SNPs in this array, 6,722 markers were polymorphic between Paustian and HENZ-a1 and therefore useful for HENZ-a1 mapping studies. After quality filtering and redundancy checking, 930 polymorphic markers were used for genetic mapping and trait association analyses. In this descriptive report, HENZ-a1 was mapped to the short arm of chromosome 3H (markers JHI-Hv50k-2016-160542 and BOPA2_12_30571), however, due to the general lack of sufficient polymorphic markers on the short arm of 3H, it was not possible to define an exact range (with flanking markers). We therefore conclude that HENZ-a1 is located on the short arm of chromosome 3H (Figure 15). Petition 870250086789, dated 09 / 25 / 2025, pp. 163 / 280 140 / 224

[0369] In the second F2 population, derived from the Planet x HENZ-a2 cross, 4734 polymorphic markers were identified. After quality filtering and redundancy verification, 738 polymorphic markers were used for genetic mapping and trait association analyses. In this descriptive report, HENZ-a2, like HENZ-a1, was mapped to the short arm of chromosome 3H, but in a more defined interval between the flanking markers JHI-Hv50k-2016-164728 and SCRI_RS_209249 (Figure 15). Materials and methods: Genetic inheritance

[0370] To study genetic inheritance, we designed several reciprocal crosses, as shown in Table 12. Paustian, Planet, and Quench were used as cross parents because they do not exhibit the half-grain endosperm α-amylase phenotype. HENZ-a1, HENZ-a2, Quench, Paustian, and Planet were sown in the greenhouse in 2018. For each cross, two ears were made. The crossed ears (F1 seeds – developing into F1 plants) were harvested. Some seeds were saved for genetic inheritance analyses, and the remaining seeds were sown again in the greenhouse. F2 reciprocal crosses of Paustian and HENZa1, Quench, and HENZ-a2 were also used for genetic inheritance analyses. Several ears per cross were harvested before emergence for the production of double haploids (DH), as described elsewhere (Olsen 1987). The remaining ears of corn (F2 seeds - which develop into F2 plants) were grown to maturity and harvested. Petition 870250086789, dated 09 / 25 / 2025, pp. 164 / 280 141 / 224 Genetic mapping

[0371] After genetic inheritance analyses, two F2 populations of Paustian x HENZ-a1 and Planet x HENZ-a2 were selected for genetic mapping. For each population, 94 single grains, along with two parents, were sown in a 96-compartment tray. Leaf samples were harvested at the seedling stage and genotyped using a 50K SNP array (Knudsen et al., 2022). The two populations were grown to maturity in the greenhouse, and F3 seeds were harvested from individual F2 ears (referred to in this descriptive report as F2:3 grains).

[0372] Next, the endosperm half-grain phenotype was evaluated in the F2:3 grain. As shown previously, the HENZ-α grain phenotype is allelic dose dependent in crossbreeding experiments, and therefore the trait may be segregating in individual F2:3 grains derived from an F2 ear. Therefore, F2:3 grains derived from an F2 ear were tested as a set: 40 individual F2:3 grains from a single F2 plant were tested in partial endosperm grain germination assays and pooled before the α-amylase activity assay.

[0373] For each individual in the population, 40 healthy grains were desiccated and germinated in Petri dishes in 2.5 ml of milliQ water for 96 hours at 15°C. Subsequently, all samples were lyophilized and ground. From each ground sample, three technical replicates of 100 mg each were used for the α-amylase activity assay. The assay protocol is as follows: Petition 870250086789, dated 09 / 25 / 2025, pp. 165 / 280 142 / 224 • Add 1000 μL of 1x extraction buffer to 100 mg of flour in a 2 ml tube. • Pre-warm the chilled buffer to room temperature • Shake vigorously for 20 seconds • Incubate for 5 minutes at room temperature • Shake vigorously for 20 seconds • Incubate for 5 minutes at room temperature • Shake vigorously for 20 seconds • Incubate for 5 minutes at room temperature • Centrifuge the tube at 1000g for 10 minutes • Remove 50 μL of the extract and dilute 1:10 with 1x extraction buffer (450 μL) • Shake • Run the assay in 2 hours • The diluted extract and the fresh Ceralpha solution should be at room temperature before the assay. • Add 20 μl of diluted extract to the plate and add 20 μl of Ceralpha. • Mix • Incubation time at 40°C 20 min • Add 200 μl of 1% TRISMA stop solution • Mix • Measure absorbance at 400 nm (microtiter plate reader) Example 12 - Identification of HENZ-α through a genomic and transcriptomic approach Petition 870250086789, dated 09 / 25 / 2025, pp. 166 / 280 143 / 224

[0374] In this descriptive report, we describe the identification of candidate mutations in HENZ-a1 and HENZ-a2 through RNA-seq of germinal endosperm from HENZ-a1, HENZ-a2 and Quench half-grain, and by whole genome sequencing (WGS) of HENZ-a1, HENZ-a2 and Quench mutant siblings. Table 15. Material used in whole genome sequencing. Material Description HENZ-a1 A set of individuals from a single plant, which was genotyped at 50K and whose α-amylase activity in the mid-grain endosperm was confirmed DH of HENZ-a1 18-0364-05, a DH line from the cross HENZα1 x Planet HENZ-a2 A set of individuals from a single plant, which was genotyped at 50K and whose α-amylase activity in the mid-grain endosperm was confirmed DH of HENZ-a2 18-0382-06, a DH line from the cross HENZα2 x Planet Wild type Quench 12 mutant siblings from the original Quench M4 library, all tested to have wt mid-grain endosperm germination phenotype (no α-amylase activity) Table 16. List of candidate mutations identified by WGS, possibly causing the HENZ-α trait. Chromosomal position Mutation Impact Annotation Planet_V1 Planet_V1 ID chr3H: 78372266 C->T synonym, p.G54 Plant / MSJ11-3 protein, putative Horvu_PLANET_3H01G154400 Petition 870250086789, dated 09 / 25 / 2025, pp. 167 / 280 144 / 224 chr3H: 85403135 C->T missense, p.A127V Alpha / beta hydrolase superfamily protein Horvu_PLANET_ 3H01G160900 Candidate gene, CXE2L1 chr3H: 150437562 G->A 500 bp downstream ABIL1 protein Horvu_PLANET_ 3H01G207400 chr3H: 310461429 G->A Intron Mitochondrial processing peptidase beta subunit, mitochondrial, putative Horvu_PLANET_ 3H01G285600 chr3H: 385389840 G->A missense, p.A560T ATP- and zinc-dependent FtsH1 metalloprotease Horvu_PLANET_ 3H01G324300 Results Whole genome sequencing (WGS)

[0375] After initial filtering of bi-allelic SNPs in extended gene coding regions (genes including 500 bp regions upstream and downstream), WGS identified 56,469 SNPs on chromosome 3H. In addition, we only considered SNPs that were different between all wt samples and the HENZ-a1 or HENZ-a2 mutant samples. SNPs unique to either mutant line were retained for further analysis.

[0376] For all remaining SNPs, RNA-seq (TPM) quantification of the germinated endosperm grain middle was used as an additional filtering criterion. In this descriptive report, only genes that showed expression in the germinated endosperm grain middle were considered. Petition 870250086789, dated 09 / 25 / 2025, pp. 168 / 280 145 / 224

[0377] Finally, all SNPs outside the individual mapping ranges for HENZ-a1 and HENZ-a2 were excluded. Subsequently, we reduced the number of candidate SNPs, as shown in Table 16. One of the candidate SNPs is in the gene Horvu_PLANET_3H01G160900 (Planet_V1) or ID HORVU.MOREX.r3.3HG0242030 (Morex_V3), annotated as encoding a protein from the Alpha / beta-Hydrolases superfamily, or more specifically a Carboxylesterase (CXE) CXE2L1. Both HENZ-a1 and HENZ-a2 mutants (in both cases, original mutant and DH) carry the same C->T nucleotide transition in the CXE2L1 gene, likely induced by NaN3 (Knudsen et al., 2022). The mutant CXE2L1 gene encodes a mutant CXE2L1 protein comprising an amino acid exchange from alanine 127 to valine 127 (A127>V). From our results, HENZα1 and HENZ-a2 are very likely derived from the same mutation event induced after mutagenic treatment with NaN3.

[0378] Next, we tested barley cultivars that reportedly exhibit rapid beta-glucan degradation during malting using Sanger sequencing for the A127V (C>T) mutation in the CXE2L1 gene. As shown in Figure 16, none of these lines exhibited the A127V (C>T) mutation. However, surprisingly, several FMT-3H type lines exhibited another CXE2L1 mutation in this Sanger sequencing fragment, namely S113P (T>C). Materials and methods Whole genome sequencing (WGS) Petition 870250086789, dated 09 / 25 / 2025, pp. 169 / 280 146 / 224

[0379] HENZ-αI and HENZ-α2 seeds harvested from the F2 mapping populations were selected for WGS. Their identities were verified by 50K genotyping and endosperm half-grain phenotyping. In addition to the original mutant plants, two DH lines from Planet crosses were included. For wild-type controls, 12 ears were chosen based on the original endosperm half-grain screening data (original experiment for HENZ-α isolation; the chosen lines did not show α-amylase activity in the endosperm half-grain germination tests). Four grains were collected from each of these 12 ears, and the endosperm half-grain germination tests were repeated as previously described. All four grains from each of the 12 mutant siblings were negative for the half-grain endosperm phenotype, and therefore, the 12 mutant siblings were chosen as wild-type controls for WGS.In total, 16 accessions were grown in the greenhouse, and leaf samples were collected at the seedling stage. Subsequently, the leaves were freeze-dried and sent to LGC for Illumina PE250 sequencing.

[0380] Sequencing reads were trimmed to remove low-quality bases and adapters using cutadapt (v4.2; python3.9). The remaining high-quality reads were mapped to the RGT Planet_V1 barley reference genome assembly using minimap (v2.24), sorted using novosort, and converted to cram format using samtools (v1.15). Variant identification was performed using the mpileup and call functions of bcftools. Petition 870250086789, dated 09 / 25 / 2025, pages 170 / 280 147 / 224 Variants were annotated with SnpEff (v5.1). Gene names were extracted using SnpSift (v5.1) and genotypes were extracted with bcftools (v1.15). Only biallelic SNPs in the 500 bp upstream and 500 bp downstream regions of the genes were considered. SNPs, associated genes, and their quantification by RNA-seq from endosperm half-grain germination were all analyzed in R (v4.0.2). RNA-seq

[0381] HENZ-a1, HENZ-a2, and Quench were propagated in New Zealand in 2016 / 2017. We dissected 200 grains from each sample, and 100 endosperm grain mediums were germinated as described elsewhere (REF for other experiments). As a positive control, Quench endosperm grain medium was germinated with 4 ml of water containing 100 nM GA3. Grain mediums were sampled 24 h, 48 h, and 72 h after soaking (three biological replicates each) and lyophilized. Grain mediums (30-200 mg) were milled in liquid nitrogen before RNA isolation using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer's instructions. Total RNA from the samples was sequenced at La Trobe University, Australia. Transcript abundance was estimated as transcripts per million (TPM) using kallisto (v0.48.0) with a standard 31-mer and 100 bootstraps. Sanger Sequencing

[0382] Several commercial barley varieties (Table 17 - referred to in this descriptive report collectively as “FMT-3H type lines”) known for the rapid degradation of beta-glucan during malting were selected together Petition 870250086789, dated 09 / 25 / 2025, pp. 171 / 280 148 / 224 with HENZ-αI, HENZ-α2, Quench and Planet for Sanger sequencing. Leaf material was harvested at the seedling stage and DNA was extracted using the EchoLUTION Plant DNA Kit (BioEcho Life Sciences GmbH). Based on Horvu_PLANET_3H01G160900 (Planet_V1), two primers were designed to amplify a 429 bp genetic region (LU88F CTTAGTGTCCGCGCATACAG, LU88R CGCGGAGAGGAGGATGTAC).

[0383] The PCR mixture was prepared in a 25 µl volume of 12.5 µl red extraction PCR mixture, 1.25 µl primer 1, 1.25 µl primer 2, 5 µl water, and 5 µl template DNA. An adapted PCR program was used: 3 min denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 62°C for 1 min and extension at 72°C for 1 min, finally extension for 10 min, and then stored at 8°C. The PCR products were sent to Eurofins Genomics for purification and Sanger sequencing. The sequencing results were analyzed on CLC Workbench 21 (QIAGEN). The programming was shaded at https: / / www.bioinformatics.org / sms2 / color_align_cons.html Table 17. Material sequenced by Sanger. Access Reproduction Company Accordine Ackermann Saatzucht LG Tosca Limagrain Fandaga Nordsaat Focus Secobra Lexy Breun Avalon Breun Explorer Secobra HENZ-αΙ HENZ-a2 Planeta RAGT Quench Syngenta Petition 870250086789, dated 09 / 25 / 2025, pp. 172 / 280 149 / 224 Example 13 - Visualization of HENZ-α and FMT-3H mutations identified in a structural model of the CXE2L1 protein. Results

[0384] A structural model of the HENZ-α and FMT-3H mutant CXE2L1 proteins was made. In Figure 17A, the surface of the protein model is shown in dark gray, with the substrate / ligand binding pocket in white. The transplanted substrate / GA4 (representative substrate) in black spheres is located in the substrate / ligand binding pocket. From the structural model, we predict that alanine at position 127 adopts an alpha-helical conformation away from the substrate / ligand binding pocket marked in black (Figure 17B). The branched Cbeta VAL residue generally shows a reduced propensity to form alpha-helical conformations compared to alanine. Thus, it is likely that the HENZ-α A127V mutation causes structural rearrangements in the protein core that alter the binding properties of the substrate / ligand binding cavity.

[0385] Based on the structural model, it is still possible to identify additional residues that may affect the interaction with the ligand. Mutation of these residues can potentially result in similar effects on the protein core and on the binding properties of the substrate / ligand binding cavity, such as S113P (FMT-3H) and A127V (HENZ-α). The potentially relevant residues are grouped according to: i) distance to the binder / substrate pocket and ii) distance to A127 and S113, respectively.

[0386] The relevant residues are listed below. Distance to the binder / substrate pocket Petition 870250086789, dated 09 / 25 / 2025, pp. 173 / 280 150 / 224

[0387] Using the model, the following functional or ligand-interacting residues (i.e., substrate-interacting residues) were identified: (“amino acid number”, “amino acid”) ('13', 'PHE'), ('14', 'LEU'), ('83', 'GLY'), ('84', 'GLY'), ('85', 'GLY'), ('88', 'LEU'), ('93' 'GLN'), ('96', 'PHE'), ('166', 'HIS'), ('167', 'SER'), ('168', 'ALA'), ('201', 'PHE'), ('219', 'SER'), ('220', 'LEU'), ('221', 'THR'), ('224', 'MET'), ('228', 'LEU'), ('300', 'HIS'), ('301', 'GLY'), ('302', 'PHE'), ('304', 'ILE'), ('305', 'ARG')

[0388] In addition, <5 angstrom residues of the residues that interact with the ligand / substrate were identified (highlighted in black in Figure 17C; 1 layer of residues): (“amino acid number”, “amino acid”) ('11', 'GLU'), ('12', 'ASP'), ('15', 'GLY'), ('16', VAL'), ('17', GLU'), ('33', TYR'), ('81', CYS'), ('89', ALA'), ('94', 'VAL'), ('18', 'LEU'), ('35', 'PHE'), ('82', 'GLY'), ('90', 'PRO'), ('95', 'GLN'), ('27', 'THR'), ('63', 'HIS'), ('86', 'SER'), ('91', 'ASN'), ('97', 'ARG') 'WING'), ('202', 'VAL'), ('226', 'ARG'), 'LEU'), ('164', 'ALA'), ('198', 'PHE'), ('222', 'ASP'), ('231', ('100', 'SER'), ('171', 'LEU'), ('203', 'THR'), ('227', 'MET'), 'CYS'), ('165', 'ASN'), ('199', 'ALA'), ('223', 'GLN'), ('232', ('116', 'GLY'), ('172', 'SER'), ('217', 'ALA'), ('229', 'SER'), 'TYR'), ('169', 'LEU'), ('200', 'GLY'), ('225', 'TRP'), ('233', Petition 870250086789, dated 09 / 25 / 2025, pp. 174 / 280 151 / 224 'LEU'), ('244', 'ALA'), ('265', 'VAL'), ('267', 'PRO'), ('269', 'SER'), ('270', 'ASP'), ('271', 'VAL'), ('272', 'LEU'), ('295', 'PHE'), ('298', 'GLU'), ('299', 'GLN'), ('303', 'PRO'), ('306', 'GLN'), ('307', 'PRO'), ('309', 'SER'), ('311', 'THR'), ('312', 'ALA'), ('315', 'LEU')

[0389] In addition, ^5 angstrom residues were identified from the first residue layer (highlighted in white sticks in figure 17C; second residue layer);

[0390] Note that the 2' layer of residue includes A127 and S113. (“amino acid number”, amino acid) ('9', 'VAL'), ('10', 'VAL'), ('19', 'LEU'), ('20', 'LEU'), ('24', 'SER'), ('25', 'VAL'), ('26', 'VAL') ('28', 'GLY'), ('29', 'ASP'), ('31', 'ALA'), ('32', 'VAL'), ('34', 'ARG'), ('36', 'ASN'), ('37', 'GLU'), ('39', 'LEU'), ('40', 'PRO'), ('42', 'VAL'), ('45', 'VAL'), ('46', 'GLN'), ('47', 'TRP'), ('49', 'ASP'), ('52', 'TYR'), ('58', 'LEU'), ('59', 'SER'), ('60', 'VAL'), ('61', 'ARG'), ('62', 'ALA'), ('64', 'ARG'), ('65', 'PRO'), ('78', 'LEU'), ('79', 'VAL'), ('101', 'LEU'), ('102', 'ARG') ('103', 'ALA'), ('104', 'ALA'), ('105', 'ALA'), ('109', 'ALA'), ('110', 'VAL'), ('111', 'VAL'), ('112', 'LEU'), ('113', 'SER'), ('114', 'VAL'), ('115', 'GLN'), ('117', 'ARG'), ('119', 'ALA') ('120', 'PRO'), ('121', 'GLU'), ('122', 'HIS') ('131', 'GLY'), ('134', 'PHE'), ('151', 'TRP'), ('162', 'PHE') Petition 870250086789, dated 09 / 25 / 2025, pages 175 / 280 152 / 224 ('163', 'LEU'), ('174', 'HIS'), ('175', 'HIS'), ('176', 'VAL'), ('177', 'THR ('196', 'ILE'), 'ALA'), ('207', 'ARG ('210', 'THR'), 'PRO'), ('216', 'GLU ('235', 'VAL'), 'MET'), ('241','HIS ('243', 'LEU'), 'LEU'), ('264','VAL ('268', 'LEU'), 'ARG'), ('276','VAL ('293', 'VAL'), ), ('178', 'VAL') ('197', 'READ'),('204), ('208', 'THR') ('211', 'GLU') ('279', 'TYR') ('294', 'GLN'),('296', 'GLY'), ('205', 'PRO'), ('215', 'SER'), ('239', 'PRO'), ('263', 'ASP'), ('275', 'GLU'), ('297', 'GLY'), ('308', 'PHE'), ('310', 'GLU'), ('313', 'SER'), ('314', 'GLU'), ('316', 'LEU'), ('317', 'ARG'), ('318', 'VAL'), ('319', 'ILE'), ('320', 'ARG') Distance to A127 and S113

[0391] Using the model, the following residues were identified as being close, i.e., <5 angstrom within A127: (“amino acid number”, “amino acid”) TYR, 116 TYR, 117 ARG, 122 HIS, 123 ARG, 124 LEU, 125 PRO, 126 ALA, 128 ILE, 129 ASP, 130 ASP, 131 GLY, 172 LEU

[0392] Using the model, the following residuals were identified as being close, i.e., <5 angstrom within, S113: (“amino acid number”, “amino acid”) Petition 870250086789, dated 09 / 25 / 2025, pp. 176 / 280 153 / 224 SER, 60 VAL, 61 ARG, 63 TYR, 80 TYR, 91 ILE, 100 CYS, 111 VAL, 112 LEU, 114 VAL, 115 GLN Materials and methods Structural modeling and visualization

[0393] Alphafold2 (Jumper et al., 2021), implemented in Colabfold v. 1.3.0 (Mirdita et al., 2022), with msa_mode MMseqs2 (UniRef+Environmental) and use_amber=false, was used to generate a structural prediction of residue 1-327 of CXE2L1 (SEQ ID NO: 1, HORVU.MOREX.r3.3HG0242030, Morex_V3). AlphaFill (Hekkelman et al., 2023) was used to transplant the representative GA4 ligand of PDB:3ebl.A (~30% identity) into the CXE2L1 alphafold model.

[0394] Pymol (V. 1.5.0.3) was used for visualization of the protein model. Example 14 - Phylogeny of the Carboxylesterase (CXE) family in barley and rice, which includes HENZ-α from barley and the candidate gene FMT-3H CXE2L1 (HORVU.MOREX.r3.3HG0242030, Morex_V3) Results

[0395] The candidate gene HENZ-Alpha CXE2L1 (HORVU.MOREX.r3.3HG0242030, Morex_V3) belongs to the Carboxylesterase (CXE) family, in which some members hydrolyze esters of short-chain fatty acids and others are involved in the perception of the hormone GA. To place the candidate gene in the phylogeny of the CXE family, we identified CXE genes in barley and rice, aligned them, and constructed a maximum likelihood phylogenetic tree. Petition 870250086789, dated 09 / 25 / 2025, pp. 177 / 280 154 / 224

[0396] The phylogenetic tree shows 5 subclades, where subclade 5 contains the known GID1 GA receptor from barley and rice and our HENZ-α CXE2L1 candidate gene (figure 18). Example 15 - Generation and testing of double mutants

[0397] The CXE2L1 gene of FMT-3H type lines was sequenced as described in Example 12, and was found to comprise a mutant CXE2L1 gene encoding a mutant CXE2L1 polypeptide comprising an S113P substitution.

[0398] An FMT-3H type line carrying the S113P substitution (referred to in this descriptive report as FMT-3H) was crossed with a barley strain comprising a G^A mutation of nucleotide 2243 of the coding sequence of the HvCslF6 gene (SEQ ID NO: 2 of WO 2019 / 129736), resulting in said HvCslF6 gene encoding a mutant HvCslF6 protein comprising a Gly^Asp mutation of amino acid 748 of SEQ ID NO: 1 of WO 2019 / 129736. The aforementioned barley strain was generated as described in Examples 1 and 2 of WO 2019 / 129736, and was also deposited with NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, on November 12, 2018, receiving the NCIMB accession number 43273.

[0399] From these crosses, offspring were selected that contained both the S113P substitution in the CXE2L1 polypeptide and the G^A mutation of nucleotide 2243 of the coding sequence of the HvCslF6 gene. The resulting barley plant comprised both mutations and is designated FMT-3H + Mut2. Petition 870250086789, dated 09 / 25 / 2025, pages 178 / 280 155 / 224

[0400] The hydrolytic enzyme activity and β-glucan degradation after malting were tested as described in “Materials and methods” above in the aforementioned barley plant.

[0401] The properties of barley plants are presented in tables 18 and 19 below. Table 18. Name ID Color, EBC H2O Water content, % Fine milled extract (as is), mass % standard deviation average standard deviation average standard deviation average FMT-3H + Mut2 FMT-3H + Mut2 0.12 4.9 0.1 4.43 0.43 81.04 Control RGT Planet 0.36 3.98 0.07 4.74 0.35 80.64 Table 19. Name ID Fine extract (dry), mass % Soluble nitrogen (dry), % standard deviation mean standard deviation mean FMT-3H + Mut2 Control FMT-3H + Mut2 RGT Planet 0.43 84.8 0.39 84.65 0.01 0.67 0.03 0.59 Water % Protein % Starch % FMT-3H + Mut2 FMT-3H + Mut2 12.4 9.6 64.1 RGT Planet Control 12.6 8.3 α-amylase activity

[0402] When measuring α-amylase activity after malting, the flour was prepared as described above in the “Sample Preparation” section. The α-amylase activity assays Petition 870250086789, dated 09 / 25 / 2025, pp. 179 / 280 156 / 224 amylase followed the recommendations provided with the Megazyme Ceralpha kit.

[0403] α-amylase activity can be seen in figure 19A and table 20A. Table 20A. Alpha-amylase Activity [U] / [g] STDEVA mut2+FMT-3H 233.0 6.4 RGT Planet 177.5 6.3 β-amylase activity

[0404] When measuring β-amylase activity after malting, the flour was prepared as described above in the “Sample Preparation” section. The β-amylase activity assays followed the recommendations provided with the Megazyme Betamyl kit (K-BETA3).

[0405] β-amylase activity can be seen in figure 19B and table 20B. Table 20B. β-amylase Activity [U] / [g] STDEVA mut2+FMT-3H 11.5 0.8 RGT Planet 9.4 0.3 Dextrinase threshold activity

[0406] To measure the limiting activity of free dextrinase after malting, the flour was prepared as described above in the “Sample Preparation” section. The limiting activity of dextrinase was determined using the Megazyme Limit Dextrizyme PullG6 method. The assays, Petition 870250086789, dated 09 / 25 / 2025, pages 180 / 280 157 / 224 including activity measurements, were performed according to the manufacturer's protocol (PullG6 method).

[0407] Free dextrinase activity can be seen in figure 19C and table 20C. Table 20C. Free dextrinase — KB Action [U] / [g] STDEVA mut2+FMT-3H 46.8 5.8 RGT Planet 41.0 2.4 β-glucan content

[0408] The measurement of β-glucan content after malting was performed using the standard EBC method for detection of HMW β-glucan in wort (Analytica EBC - 8.13.3 - β-Glucan in wort by automated discrete analysis - 2021).

[0409] The β-glucan content can be seen in figure 19D and in table 20D. Table 20D. B-glucan HMW soluble mg / l STDEVA mut2+FMT-3H 167.8 13.2 RGT Planet 531.7 34.4 Example 16 - Generation and testing of the FIND-IT HENZ78 mutant Materials and methods

[0410] A unique ddPCR assay was designed specifically to distinguish between the mutant barley allele and the wild-type allele of CXE2L1 at nucleotide position 380 in the wild-type coding sequence (HORVU.MOREX.r3.3HG0242030, Morex_V3). The detection probe Petition 870250086789, dated 09 / 25 / 2025, pages 181 / 280 The 158 / 224 mutant was complementary to the coding sequence, containing a T base at nucleotide position 380. The reference detection probe was complementary to the coding sequence, containing a C base at nucleotide position 380. Two flanking primers were designed to amplify the genomic sequence around nucleotide 380 in the coding sequence.

[0411] The following primers and probes were specifically designed for the CXE2L1 gene and used to screen a NaN3-mutaged RGT Planet library, as described elsewhere (Knudsen et al., 2022): Target-specific direct primer (SEQ ID NO: 27): 5'- AACTTCCACTCGCTCTG -3'; Target-specific reverse primer (SEQ ID NO: 28): 5'- CAGGAAAGGAAAGAGCC -3'; Specific detection probe for mutants (SEQ ID NO: 29): 5'-CGATGACCGGCGGG-3'-labeled with 6-carboxyfluorescein (FAM); Specific detection probe for reference (SEQ ID NO: 30): 5'-CGATGGCCGCGG-3'-labeled with hexachlorofluorescein (HEX).

[0412] The HENZ-78 mutant in the table below was identified in the dd-PCR assay. Name Nucleotide exchange in the coding sequence (SEQ ID NO: 2) Amino acid alteration in the protein (SEQ ID NO: 1) HENZ-78 C380T A^ a V (127)

[0413] An assay of the half-grain α-amylase activity of the HENZ-78 mutant was performed as described. Petition 870250086789, dated 09 / 25 / 2025, pp. 182 / 280 159 / 224 in Example 3. The half-grain α-amylase activities of Planet, HENZ-a1, HENZ-a2, and Quench were tested in the same assay (Planet as a reference for HENZ-78; Quench as a reference for HENZ-a1, HENZ-a2). Results

[0414] The endosperm half-grains of HENZ-78, HENZa1 and HENZ-a2 all showed high α-amylase activity. The endosperm half-grains of Quench and Planet did not, in principle, show any α-amylase activity. The results can be seen in Figure 21 and in the table below.

[0415] Table showing the activity of α-amylase in the endosperm grain half of the barley varieties indicated below. Average Sample (U g 1 ) STANDARD DEVIATION HENZ-78 15.5 4.3 PLANET 0.6 0.4 HENZ-a1 13.1 2.2 HENZ-a2 8.6 1.5 QUENCH 0.2 0.2 Example 17 - α-amylase activity of micromalted grains in malting barley varieties with distinct amy1 1 haplotypes and determination of the number of causative copies. Materials and methods

[0416] Based on the Morex_V1 genome assembly, a PCR assay targeting a polymorphic region upstream of the amy1_1 copies was developed and used to select a diverse panel of malting barley and breeding lines. A total of 10 diverse haplotypes were found, indicating potential copy number variation (CNV) in modern varieties. Two of these Petition 870250086789, dated 09 / 25 / 2025, pp. 183 / 280 160 / 224 haplotypes were designated haplotypes B (amy1_1-B) and Q (amy1_1-Q).

[0417] Samples of non-dormant grains from a selected group of malting barley varieties were micromalted as described in Example 9. α-amylase activity was measured using the Ceralpha method (Ceralpha Method _R-CAAR4, Megazyme) modified for Gallery Plus Beermaster (Thermo Fisher Scientific, USA).

[0418] To quantify the number of copies of the amy1_1 gene, digital droplet PCR (ddPCR) was performed on barley genomic DNA. Two ddPCR assays were designed: one for the amy1_1 gene and another for a reference gene with a known copy number (HvGW2).

[0419] The primers and probes for the amy1_1 assay were as follows: - Direct primer: [5'-CGACCACCTCAACCTG-3'] - Reverse primer: [5'-GTAAATCTTGGCGACGT-3'] - Probe: [5'- FAM - ATGTCGGCCTTGAGC-3']

[0420] The primers and probes for the HvGW2 assay were as follows: - Direct primer: [5'-AATGGTACGAGAGGAAGG-3'] - Reverse primer: [5'-GGCATAGCTTGTCCCA-3'] - Probe: [5'-HEX-ATCACTGGTCAGAGGGT-3']

[0421] Different fluorescent markers were used for the probes in order to allow the simultaneous performance of two assays in the same reaction. For each ddPCR reaction, the following components were prepared: 11 μl of 2x ddPCR Supermix for Probes (Bio-Rad), 900 nM of each primer, 250 nM of probe, 5 μl of genomic DNA template and Petition 870250086789, dated 09 / 25 / 2025, pp. 184 / 280 161 / 224 nuclease-free water to a final volume of 22 μL. Droplets were generated using a QX200 Droplet Generator (Bio-Rad). The PCR plate containing the droplets was heat-sealed at 180°C for 5 seconds with a pierceable foil, using a PX1 PCR plate sealer (Bio-Rad, Hercules, CA, USA), followed by PCR amplification (Uno96, VWR, Radnor, PA, USA) as follows: enzyme activation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, followed by annealing / extension at 55°C for 1 minute, ending with enzyme deactivation at 98°C for 10 minutes. All steps had a ramp of 2°C / s. The fluorescence of each drop was then measured using a QX200 drop reader (Bio-Rad), and the data were analyzed for copy number variation using QuantaSoft software (Bio-Rad). Results

[0422] Micromaltings focused on barley varieties with haplotypes amy1_1-B or amy1_1Q. Fig. 20 indicates a trend for barley cultivars with the amy1_1-B haplotype to exhibit globally higher α-amylase activity than barley cultivars with the amy1_1-Q haplotype.

[0423] Barley cultivars in the amy1_1-Q haplotype group were found by ddPCR sequencing to have a lower number of copies of the amy1_1 gene (4 copies) than barley cultivars in the amy1_1-B haplotype group (5 copies) - see Table 21 below. Table 21. Analysis of the number of copies per ddPCR Petition 870250086789, dated 09 / 25 / 2025, pp. 185 / 280 162 / 224 Haplotype amy1_1 Number of copies amy1_1 Barley cultivar 4 Quench amy1 1-Q 4 RGT Planet 4 Admiral 4 Navigator 5 Barke amy1 1-B 5 Paustian 5 Rubinesse

[0424] This indicates that a copy number of 5 for amy1_1 increases alpha-amylase activity. Example 18 - Playback marker Amy1_1-B

[0425] In order to develop a suitable reproduction marker for high-throughput genotyping, primers specific for amy1_1 copies were designed based on the Morex_V1 genome assembly, later improved when the Morex_V2 genome assembly was published.

[0426] A specific SNP locus for amy1_1-B (GGCGCCAGGCATGATCGGGTGGTGGCCAGCCAAGGCGGTGACCTTCGTGGACA ACCACGACACCGGCTCCACGCAGCACATGTGGCCCTTCCCTTCTGACA[A / G]GGTCAT GCAGGGATATGCGTACATACTCACGCACCCAGGGACGCCATGCATCGTGAGTTCGTCGT ACCAATACATCACATCTCAATTTTCTTTTCTTGTTTCGTTCATAA) was identified and used for the development of the KASP marker (LGC Biosearch Technologies, Hoddesdon, United Kingdom). This unique SNP resides near the end of the second exon compared to all sequenced copies in modern cultivars. At this position, the B copy has an A nucleotide, while all other B copies, as well as copies of other exons, have an A nucleotide. Petition 870250086789, dated 09 / 25 / 2025, pp. 186 / 280 163 / 224 haplotypes have the nucleotide G, resulting in an amino acid exchange B-amy1_1-R327K.

[0427] Amy1_1 copies were amplified into representative lines of 10 haplotypes, barcoded, and pooled into a sample, as instructed in the PacBio amplicon sequencing (Pacific Biosciences, CA, USA). After purification, the sample was sent to BGI Tech Solutions Co. Ltd. (Hong Kong, China) for library construction and sequencing. Amy1_1 sequences were analyzed for Morex, Barke, Quench, and other haplotypes. Example 19 - Development of new high-yielding barley varieties with high / early enzymatic activities Materials and methods

[0428] Combinations of various characteristics, as described elsewhere in this document, were combined in the same barley variety using common breeding techniques for barley crossing. The resulting barley variety was then evaluated for enzymatic activity (α-amylase, β-amylase, limit dextrinase) and β-glucan content.

[0429] α-amylase activity was measured essentially as described in Example 4.

[0430] β-amylase activity was determined essentially as described in K-BETA3 (protocol and kit available from Megazyme, Ireland).

[0431] Free dextrinase activity was determined essentially as described in Example 15. Petition 870250086789, dated 09 / 25 / 2025, pages 187 / 280 164 / 224

[0432] The β-glucan content was determined essentially as described in Example 10. Results

[0433] The following varieties of barley were generated: Variety 1 - Genotype: • LOX-1 null • MMT null • CXE2L1 gene encoding a mutant CXE2L1 polypeptide comprising an S113P substitution; • amy1_1-B Variety 2 - Genotype: • Null LOX-1 • Null MMT • Null HRT • Null ANT-28 • HvCslF6 gene encoding a mutant HvCslF6 protein comprising a Gly^Asp mutation of amino acid 748 of SEQ ID NO: 1 of WO 2019 / 129736 • CXE2L1 gene encoding a mutant CXE2L1 polypeptide comprising an S113P substitution; • amy1_1-B

[0434] As shown in Table 22 below, Variety 1 exhibits improved enzymatic activities for α-amylase, β-amylase, and limited free dextrinase compared to the reference barley variety RGT Planet. Variety 2 exhibits improved enzymatic activities for α-amylase and limited dextrinase in Petition 870250086789, dated 09 / 25 / 2025, pages 188 / 280 165 / 224 compared to the reference barley variety RGT Planet. Furthermore, Variety 1 and Variety 2 show a significant reduction in β-glucan content. Table 22. Enzymatic activities and β-glucan content after 6 days of malting. Barley Variety α-amylase activity (U / g) β-amylase activity (U / g) Limited free dextrinase activity (mU / g) β-glucan content (mg / L) Variety 1 319 18 78 0 Variety 2 295 13 80 0 RGT PLANET 228 14 58 103 Example 20 - Micromalting Materials and methods

[0435] The micromalting procedure was carried out in a controlled environment with a temperature of 16°C and humidity around 70%. Micromalting was conducted as shown in Fig. 12, but with a water target of 43%. Drying was carried out at 27-55°C (increasing by 2°C per hour), followed by 55-85°C (increasing by 4°C per hour) and finally, 1 hour at 85°C. Varieties of barley CW3a + HENZ-a1

[0436] This line is descended (double haploid) from a cross between HENZ-a1 (mother) carrying the A127V substitution and a breeding line (father; CW3a) carrying a G^A mutation at nucleotide 2243 of the coding sequence of the HvCslF6 gene (SEQ ID NO: 2 of WO 2019 / 129736), resulting in the aforementioned HvCslF6 gene encoding an HvCslF6 protein. Petition 870250086789, dated 09 / 25 / 2025, pp. 189 / 280 166 / 224 mutant comprising a Gly^Asp mutation of amino acid 748 of SEQ ID NO: 1 of WO 2019 / 129736. The CW3a strain was generated as described in Examples 1 and 2 of WO 2019 / 129736, and was also deposited at NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland, on November 12, 2018, receiving the NCIMB accession number 43273.

[0437] From these crosses, offspring were selected that contained both the A127V substitution in the CXE2L1 polypeptide and the G^A mutation of nucleotide 2243 of the coding sequence of the HvCslF6 gene. The resulting barley plant comprised both mutations and is designated CW3a + HENZ-a1. HENZ-a1 and HENZ-a2

[0438] The HENZ-a1 and HENZ-a2 lines all exhibit the A127V mutation and are all individual propagations (four replicates each) from the same original HENZα1 and HENZ-a2 mutant seed lots. The plants were grown and the respective seeds harvested in individual plots (8 m2) within the same field, but in different locations. As described in Example 1, HENZ-a1 and HENZ-a2 are derived from a Quench mutant population. HENZ-«1_DH

[0439] Lines HENZ-«1_DH1 to HENZ-«1_DH6 are all sister plants (double haploids derived from the same cross) carrying the A127V substitution. The plants were grown and the respective seeds harvested in individual plots (8 m2) in the field. FMT-3HDH Petition 870250086789, dated 09 / 25 / 2025, pages 190 / 280 167 / 224

[0440] Lines FMT-3H_DH1 to FMT-3H_DH9 are all sister plants (double haploids derived from the same cross) carrying the S113P substitution. The plants were grown and the respective seeds harvested in individual plots (8 m2). Controls

[0441] Planet-1, Planet-127, Quench-131, Quench-141, Quench-148 and Quench-158 were grown in field plots (8 m2). Planet-1 and Planet-127 are references for DH lines, Quench-131, Quench-141, Quench-148 and Quench-158 are references for HENZ-a1 and HENZ-a2. Immersion

[0442] The barley samples were placed in individual containers, each containing 100 g of seeds, and subjected to fresh water at 16°C to achieve 33% moisture content on day 1 and 43% moisture content on day 2.

[0443] The actual water absorption of the individual samples was determined as the weight difference between the initial water content, measured with the Foss 1241 NIT instrument (Foss A / S, Denmark), and the weight of the sample after removal of surface water. Germination

[0444] After the last immersion, the barley samples were kept at a degree of immersion of 43%. After each 24 hours, the samples were checked for moisture content and sprayed with additional water to overcome possible losses due to respiration. Oven Petition 870250086789, dated 09 / 25 / 2025, pp. 191 / 280 168 / 224

[0445] After the germination process, the barley samples were dried in a Curio oven using a two-stage heating profile. The first heating stage began at a defined temperature of 27°C and a linear heating at 2°C / h until the breaking point at 55°C, using 100% fresh air. The second linear ramp was 4°C / h, reaching a maximum of 85°C. This temperature was maintained constant for 90 minutes using 50% fresh air. Must

[0446] The wort analysis was performed in accordance with the EBC protocol on Malt Extract: Congress Mash (4.5.1). Enzymatic activity of malt

[0447] Enzyme activity was measured using the Megazyme methods modified for Gallery Plus Enzyme Master (Thermo Fisher Scientific, USA).

[0448] α-amylase: α-amylase assay kit (Ceralpha method)

[0449] β-Amylase: β-Amylase Assay Kit (Betamyl-3)

[0450] Limit-free dextrinase: Pullulanase / limit-dextrinase assay kit (PullG6 method)

[0451] β-glucanase: Malt β-glucanase / lichanase (MBG4 method) β-glucan content of the wort

[0452] β-glucan was measured by EBC 8.13.3 using Thermo Fisher reagents from kit D14622_H P_ BetaGlucan (High MW). Must viscosity Petition 870250086789, dated 09 / 25 / 2025, pp. 192 / 280 169 / 224

[0453] The viscosity of the wort was measured by the EBC 4.8 with the Rheotest LK 2.2 viscometer. Results

[0454] The tables below show the protein content of the barley grain, the activity of the hydrolytic enzymes in the malt, and the values ​​of the wort analysis measured according to the EBC congress mashing procedure for the various barley lines tested. The standard deviation for each value is shown when more than two samples were tested.

[0455] The table below shows the enzymatic activity in malt after micromalting. In previous examples, it was shown that the activity of α-amylase and β-glucanase was higher during germination in plants carrying the mutations described in this application. Since enzymatic activity is higher during germination, the β-glucan content of the wort from these plants is lower, as can be seen in the tables below. Sample Name Protein Alphaamylase (U / g) (+ / -) Betaamylase (U / g) (+ / -) CW3a + HENZ-a1 11.8 268 6 21 1 FMT-3H_DH1 11.9 248 14 20 1 FMT-3H_DH2 10.2 255 6 16 1 FMT-3H_DH3 10.8 219 3 16 0 FMT-3H_DH4 11.0 192 4 14 1 FMT-3H_DH5 11.4 218 8 20 1 FMT-3H_DH6 11.6 234 12 17 1 FMT-3H_DH7 11.4 202 0 16 1 FMT-3H_DH8 11.9 278 5 18 1 Petition 870250086789, dated 09 / 25 / 2025, pp. 193 / 280 170 / 224 FMT-3H_DH9 11.2 232 15 17 1 HENZ-«1_DH1 12.1 232 11 18 1 HENZ-«1_DH2 10.9 269 1 18 1 HENZ-«1_DH3 11.8 242 1 17 1 HENZ-«1_DH4 11.5 210 5 18 1 HENZ-1 9 18 1 HENZ-a1 12.4 213 10 19 0 HENZ-a2 12.4 217 8 19 0 HENZ-a2 13.4 218 3 21 0 HENZ-a2 11.5 213 1 17 1 HENZ-a2 11.6 219 8 18 1 Planet-1 12.1 213 2 20 0 Planet-127 11.2 229 9 16 0 QUENCH-131 12.2 179 4 17 0 QUENCH-141 12.1 188 6 19 0 QUENCH-148 11.5 182 2 17 1 QUENCH-158 12.3 185 6 17 1 Sample Name Dextrinase-Free Limit (mU / g) (+ / -) Beta-glucanase (mU / g) (+ / -) Beta-glucan (mg / l) (+ / -) CW3a + HENZ-a1 88 3 261 2 48 2 FMT-3H_DH1 45 1 271 12 82 7 FMT-3H_DH2 57 4 287 19 55 0 FMT-3H_DH3 44 4 245 12 53 4 FMT-3H_DH4 37 6 211 15 98 11 FMT-3H_DH5 43 4 240 11 51 2 Petition 870250086789, dated 09 / 25 / 2025, pp. 194 / 280 171 / 224 FMT-3H_DH6 30 8 247 27 58 6 FMT-3H_DH7 36 4 239 6 65 5 FMT-3H_DH8 65 9 288 12 56 4 FMT-3H_DH9 37 7 230 7 105 15 HENZ-«1_DH1 37 4 221 5 100 15 HENZ-«1_DH2 61 2 298 7 45 1 HENZ-a1_DH3 47 3 235 10 57 7 HENZ-a1_DH4 38 1 205 13 70 7 HENZ-a1_DH5 42 6 245 12 49 0 HENZ-a1_DH6 58 4 288 7 52 6 HENZ-a1 35 1 243 25 69 HENZ-a1 33 1 252 6 87 HENZ-a1 39 1 249 14 60 HENZ-a1 33 0 242 8 83 HENZ-a2 42 12 248 11 62 HENZ-a2 36 4 239 2 82 HENZ-a2 42 3 244 7 57 HENZ-a2 43 4 260 13 69 Planet-1 28 4 229 27 440 Planet-127 39 3 243 21 311 QUENCH-131 22 5 195 23 512 QUENCH-141 21 2 188 6 544 QUENCH-148 24 7 190 29 411 QUENCH-158 22 6 195 22 534 Sample Name Viscosity, mPas (+ / -) Soluble Proteins (dry), % (+ / -) Soluble Nitrogen (dry), % (+ / -) CW3a + HENZ-a1 1.50 0.01 4.60 0.00 0.73 0.01 FMT-3H_DH1 1.52 0.00 4.45 0.07 0.72 0.01 FMT-3H_DH2 1.51 0.00 4.15 0.07 0.67 0.01 FMT-3H_DH3 1.51 0.00 4.30 0.00 0.69 0.01 FMT-3H_DH4 1.56 0.01 4.30 0.00 0.69 0.00 FMT-3H_DH5 1.51 0.00 4.30 0.00 0.69 0.01 FMT-3H_DH6 1.53 0.01 4.25 0.07 0.68 0.01 FMT-3H_DH7 1.53 0.01 4.35 0.07 0.70 0.02 Petition 870250086789, dated 09 / 25 / 2025, pages 195 / 280 172 / 224 FMT-3H_DH8 1,51 0,01 4,65 0,07 0,75 0,01 FMT-3H_DH9 1,52 0,01 4,30 0,00 0,69 0,01 HENZ-«1_DH1 1,53 0,01 4,70 0,00 0,75 0,00 HENZ-«1_DH2 1,49 0,00 4,60 0,00 0,73 0,00 HENZ-«1_DH3 1,51 0,00 4,60 0,00 0,73 0,00 HENZ-«1_DH4 1,52 0,01 4,25 0,07 0,68 0,01 HENZ-«1_DH5 1,51 0,00 4,25 0,07 0,68 0,01 HENZ-«1_DH6 1,49 0,01 4,70 0,14 0,75 0,01 HENZ-a1 1,53 4,30 0,69 HENZ-a1 1,53 4,40 0,70 HENZ-a1 1,52 4,20 0,67 HENZ-a1 1,53 4,40 0,70 HENZ-a2 1,52 4,30 0,68 HENZ-a2 1,51 4,40 0,71 HENZ-a2 1,52 4,20 0,67 HENZ-a2 1,52 4,50 0,72 Planet-1 1,63 4,30 0,68 Planet-127 1,58 4,00 0,64 QUENCH-131 1,66 4,00 0,64 QUENCH-141 1,65 3,90 0,62 QUENCH-148 1,62 3,80 0,61 QUENCH-158 1,67 3,90 0,62 Sample Name Protein (dry), % by mass (+ / -) Total Nitrogen / Dry Matter, % by mass (+ / -) FAN (free alpha-amino nitrogen), mg / l (+ / -) CW3a + HENZα1 11.33 0.06 1.81 0.01 168.07 3.65 FMT-3H_DH1 11.20 0.14 1.79 0.02 161.95 3.32 FMT-3H_DH2 9.85 0.07 1.58 0.01 149.55 5.16 FMT-3H_DH3 10.20 0.14 1.63 0.01 154.00 0.57 FMT-3H_DH4 10.50 0.00 1.68 0.00 160.70 0.85 FMT-3H_DH5 10.70 0.14 1.71 0.02 151.00 1.70 FMT-3H_DH6 11.20 0.28 1.79 0.04 134.70 1.41 Petition 870250086789, dated 09 / 25 / 2025, pp. 196 / 280 173 / 224 FMT-3H_DH7 10,75 0,21 1,73 0,04 147,75 1,48 FMT-3H_DH8 11,10 0,00 1,78 0,01 164,65 6,58 FMT-3H_DH9 10,80 0,00 1,72 0,00 157,65 2,33 HENZ-«1_DH1 12,10 1,94 155,75 0,92 HENZ-«1_DH2 10,60 0,00 1,70 0,01 169,00 2,40 HENZ-«1_DH3 11,30 0,00 1,80 0,00 156,85 3,46 HENZ-«1_DH4 11,15 0,07 1,78 0,01 139,90 1,27 HENZ-«1_DH5 10,90 0,00 1,74 0,00 141,90 0,71 HENZ-«1_DH6 5,65 7,99 0,91 1,28 167,15 1,20 HENZ-a1 11,30 1,80 149,60 HENZ-a1 12,50 2,00 153,00 HENZ-a1 10,70 1,71 151,90 HENZ-a1 12,20 1,95 153,80 HENZ-a2 11,90 1,90 151,50 HENZ-a2 13,00 2,08 150,10 HENZ-a2 10,90 1,75 155,20 HENZ-a2 11,80 1,89 157,00 Planet-1 12,40 1,99 141,10 Planet-127 10,40 1,67 138,00 QUENCH-131 12,00 1,92 123,50 QUENCH-141 12,10 1,93 120,40 QUENCH-148 10,80 1,72 123,10 QUENCH-158 11,40 1,82 125,60 Sample Name Fine Extract (dry), % by mass (+ / -) Ground Fine Extract (as is), but % (+ / -) pH (+ / -) CW3a + HENZ-a1 80.60 0.34 77.44 0.31 6.26 0.01 FMT-3H_DH1 82.30 0.06 78.99 0.06 6.29 0.00 FMT-3H_DH2 83.40 0.68 79.97 0.62 6.26 0.01 FMT-3H_DH3 82.44 0.47 79.00 0.48 6.24 0.04 FMT-3H_DH4 83.82 0.25 80.49 0.28 6.23 0.01 FMT-3H_DH5 82.22 0.71 78.76 0.57 6.24 0.00 Petition 870250086789, dated 09 / 25 / 2025, pp. 197 / 280 174 / 224 FMT-3H_DH6 81,40 0,17 78,12 0,08 6,32 0,00 FMT-3H_DH7 81,96 0,30 78,57 0,28 6,24 0,02 FMT-3H_DH8 82,76 0,42 79,25 0,28 6,22 0,05 FMT-3H_DH9 82,40 0,04 78,91 0,06 6,20 0,04 HENZ-«1_DH1 81,98 0,46 78,48 0,42 6,17 0,01 HENZ-«1_DH2 82,64 0,00 79,20 0,06 6,21 0,01 HENZ-a1_DH3 82,03 0,40 78,61 0,23 6,21 0,01 HENZ-a1_DH4 82,12 0,01 78,75 0,00 6,29 0,01 HENZ-a1_DH5 81,32 0,83 78,02 0,88 6,23 0,01 HENZ-a1_DH6 82,27 0,37 78,81 0,47 6,15 0,03 HENZ-a1 80,97 77,42 6,36 HENZ-a1 79,49 75,98 6,35 HENZ-a1 81,85 78,38 6,31 HENZ-a1 81,29 77,80 6,32 HENZ-a2 80,97 77,42 6,32 HENZ-a2 78,93 75,49 6,30 HENZ-a2 81,33 77,89 6,31 HENZ-a2 81,10 77,78 6,33 Planet-1 81,00 77,34 6,27 Planet-127 81,42 77,81 6,30 QUENCH-131 79,64 76,25 6,33 QUENCH-141 79,00 75,58 6,35 QUENCH-148 80,84 77,41 6,32 QUENCH-158 79,67 76,34 6,34 Sample Name Color (+ / -) H2O Water Content, % (+ / -) CW3a + HENZ-a1 3.83 0.12 3.92 0.02 FMT-3H_DH1 4.90 0.14 4.03 0.01 FMT-3H_DH2 3.85 0.07 4.12 0.04 FMT-3H_DH3 3.95 0.07 4.17 0.04 FMT-3H_DH4 4.55 0.92 3.98 0.04 FMT-3H_DH5 3.90 0.57 4.20 0.14 FMT-3H_DH6 3.60 0.14 4.04 0.11 Petition 870250086789, dated 09 / 25 / 2025, pp. 198 / 280 175 / 224 FMT-3H_DH7 4.15 0.21 4.15 0.01 FMT-3H_DH8 4.85 0.21 4.24 0.14 FMT-3H_DH9 4.40 0.28 4.24 0.12 HENZ-a1_DH1 3.80 0.00 4.27 0.03 HENZ-a1_DH2 3.85 0.07 4.17 0.08 HENZ-a1_DH3 4.60 0.00 4.17 0.18 HENZ-a1_DH4 3.50 0.14 4.10 0.01 HENZ-a1_DH5 3.90 0.14 4.06 0.09 HENZ-a1_DH6 4.10 0.00 4.21 0.14 HENZ-a1 3.40 4.38 HENZ-a1 3.10 4.41 HENZ-a1 3.40 4.24 HENZ-a1 3.50 4.29 HENZ-a2 3.40 4.38 HENZ-a2 3.30 4.36 HENZ-a2 3.50 4.23 HENZ-a2 3.90 4.09 Planet-1 3.50 4.52 Planet-127 3.70 4.43 QUENCH-131 3.10 4.26 QUENCH-141 3.20 4.33 QUENCH-148 3.10 4.24 QUENCH-158 3.30 4.18 Example 21 - Half-grain assay of additional mutants Material and methods Generation of mutants FIND-IT

[0456] A NaN3-mutaged RGT Planet library was selected for specific mutations in the gene encoding CXE2L1. The mutants in the table below were identified. Petition 870250086789, dated 09 / 25 / 2025, pp. 199 / 280 176 / 224 Name Nucleotide exchange in the coding sequence (SEQ ID NO: 2) Amino acid alteration in the protein (SEQ ID NO: 1) HENZ-79 G141A W ^ STOP (47) HENZ-80 C373T P ^ S (125) HENZ-81 C380T A ^ T (127) HENZ-82 C635T A ^ V (212) HENZ-83 G364A A ^ T (212) HENZ-84 G37 6A A ^ T (126)

[0457] The mutants were identified using FIND-IT, as already described for HENZ-78 in Example 16. In short, a ddPCR screening method essentially as described in international patent application PCT / EP2017 / 065516. More specifically, a set of randomly mutagenized barley grains was prepared, followed by the preparation of an ordered library, as described in international patent application PCT / EP2017 / 065516 in WS1 and WS2 on pages 66-69, as well as in Examples 1 to 2. The mutants in the table above were identified and selected as described in international patent application PCT / EP2017 / 065516 in WS3 and WS4 on pages 67-72, as well as in Examples 3 to 15, using the primers and probes specified in the table below. In particular, the screening was carried out essentially as described in international patent application PCT / EP2017 / 065516 in WS3 and Examples 3 to 7, using the primers and probes specified in the table below.The individual barley grains carrying the genetic mutation were identified essentially as described in the application. Petition 870250086789, dated 09 / 25 / 2025, pages 200 / 280 177 / 224 international patent PCT / EP2017 / 065516 in WS4 (pp. 69-72) and in Examples 8 to 15, using the primers and probes specified in the table below. The primers and probes were specifically designed for the identification of each of the specific mutants. Table. Primers and probes for specific mutants. Name Target-specific direct primer Target-specific reverse primer Reference-specific detection probe labeled with hexachlorofluorescein (HEX) Mutant-specific detection probe labeled with 6-carboxyfluorescein (FAM) HENZ-79 CAAACGAGCCGTTGC GTATGCGCGGACACT A TGCAGTGGAAGGACG TGCAGTGAAAGGACGT HENZ-80 AACTTCCACTCGCTC TG CAGGAAAGGAAAGAC GC CCGCGGGGAGGC CCGCGGAGAGGCG HENZ-81 CTGTCCGTGCAGTAC C GCAGCCAGGAAAGGA A CCCCGCGGCCAT CCCGCGACCATCG HENZ-82 GTACATCCTCCTCTC CG ATCGCGGTCGTCAAG AGGGTCGGCCTCC AGGGTCGACCTCCG HENZ-83 GTACATCCTCCTCTC CG ATCGCGGTCGTCAAG ACGGAGGCCGACC ACGGAGACCGACCC HENZ- 84 CTGTCCGTGCAGTAC C GCAGCCAGGAAAGGA A CTCCCCGCGGCC CTCCCCACGGCCA Plant propagation

[0458] Barley plants with specific mutations in the CXE2L1 gene are grown in 2-liter pots in a greenhouse with 16 hours of light and 8 hours of darkness until they reach maturity. Half-grain assay Petition 870250086789, dated 09 / 25 / 2025, pages 201 / 280 178 / 224

[0459] A half-grain assay of the aforementioned FIND-IT mutants is performed as described in Example 3. Planet and Quench are used as controls.

[0460] HENZ-79 is expected to produce less favorable results. Petition 870250086789, dated 09 / 25 / 2025, pages 202 / 280 179 / 224 Items 1. A barley plant or part thereof, wherein said barley plant has high α-amylase activity in a half endosperm grain without embryo and wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant gain-of-function CXE2L1 polypeptide. 2. The barley plant or part thereof, in which wild-type CXE2L1 is a polypeptide with SEQ ID NO: 1 or a functional homolog thereof that shares at least 95% sequence identity with it, preferably wild-type CXE2L1 is a polypeptide with SEQ ID NO: 1. 3. A barley plant or part thereof, wherein said barley plant has high α-amylase activity in a half-grain endosperm without embryo, and wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein wild-type CXE2L1 is a polypeptide of SEQ ID NO: 1 or a functional homolog thereof that shares at least 95% sequence identity with it, preferably wild-type CXE2L1 is a polypeptide of SEQ ID NO: 1. 4. The barley plant or part thereof in accordance with any of the above items, wherein the mutant CXE2L1 polypeptide comprises a substitution of an amino acid selected from the group consisting of: a. the amino acid corresponding to S113 or A127 of SEQ ID NO: 1; or Petition 870250086789, dated 09 / 25 / 2025, pp. 203 / 280 180 / 224 b. the amino acid corresponding to P125, A126, or A212 of SEQ ID NO: 1; or c. the amino acid corresponding to F13, L14, G83, G84, G85, L88, Q93, F96, H166, S167, A168, F201, S219, L220, T221, M224, L228, H300, G301, F302, I304 or R305 of SEQ ID NO: 1; or d. the amino acid corresponding to E11, D12, G15, V16, V17, Q18, R27, E30, L33, T35, Y63, Y80, F81, H82, Y86, C87, G89, S90, I91, A92, P94, N95, H97, S98, L99, C100, Y116, L118, S164, G165, G169, A170, N171, L172, A173, L198, S199, A200, F202, A203, G217, V218, T222, A223, A225, D226, Q227, W229, R230, M231, S232, L233, A244, V265, P267, S269, D270, V271, L272, F295, E298, Q299, P303, Q306, P307, S309, T311 A312 or L315 da SEQ ID NO: 1;ou e. o aminoacido correspondente to V9, V10, L19, L20, S24, V25, V26, G28, D29, A31, V32, R34, N36, G37, L39, P40 V42, V45, Q46, W47, D49, Y52, L58, S59, V60, R61, A62,R64, P65, L78, V79, L101, R102, A103, A104, A105, A109,V110, V111, L112, S113, V114, Q115, R117, A119, P120, E121,H122, R123, L124, A127, I128, D130, G131, F134, W151, F162,L163, H174, H175, V176, T177, V178, I196, L197, G204, A205, R207 T208, T210, E211, P214, P215, E216, P234, V235, A237, S238 M239, H241, P242, L243, N245, P246, L263, V264', A266, L268, R273, D274, R275, V276, Y279, V293, Q294, E296, G297, F308 E310, S313, E314, L316, R317, V318, I319 or R320 of SEQ ID NO: 1; f. the amino acid corresponding to Y86, Y116, R117, H122, R123, L124, P125, A126, I128, D129, D130, G131 or L172 of SEQ ID NO: 1; Petition 870250086789, dated 09 / 25 / 2025, pp. 204 / 280 181 / 224 g. the amino acid corresponding to S59, V60, R61, Y63, Y80, I91, C100, V111, L112, V114 or Q115 of SEQ ID NO: 1; where wild CXE2L1 is CXE2L1 with SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it. 5. A barley plant or part thereof, wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: a. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113, in which the aforementioned substitution is a substitution of a serine (S) for a proline (P); provided that the plant does not contain SEQ ID NO: 5. 6. A barley plant or part thereof, wherein said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: Petition 870250086789, dated 09 / 25 / 2025, pp. 205 / 280 182 / 224 a. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113, in which the aforementioned substitution is a substitution of a serine (S) for a proline (P); and / or c. the amino acid corresponding to amino acid 125 of SEQ ID NO: 1, where the substitution in question is a substitution of a proline (P) for a serine (S); and / or d. the amino acid corresponding to amino acid 126 of SEQ ID NO: 1, where the substitution in question is a substitution of an alanine (A) for a threonine (T); and / or e. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a threonine (T); and / or f. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of an alanine (A) for a threonine (T); and / or g. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, wherein the said substitution is a substitution of alanine (A) for valine (V); provided that the plant does not contain SEQ ID NO: 5. 7. A barley plant or part thereof, wherein said barley plant has high α-amylase activity in a half-grain of endosperm without an embryo, wherein said barley plant bears: a. a point mutation in the CXE2L1 gene, wherein the mutated CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, wherein the wild-type CXE2L1 Petition 870250086789, dated 09 / 25 / 2025, pp. 206 / 280 183 / 224 is CXE2L1 with SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, and b. a mutation in a gene that codes for a β-glucan synthase. 8. A barley plant or part thereof, in which the said barley plant bears: a. a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution in: i. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, wherein said substitution is a substitution of a serine (S) for a proline (P); and also b. a mutation in a gene that codes for a β-glucan synthase. 9. A barley plant or part thereof, in which the said barley plant bears: a. a point mutation in the CXE2L1 gene, wherein said mutated CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least Petition 870250086789, dated 09 / 25 / 2025, pp. 207 / 280 184 / 224 95% sequence identity with it, except that the CXE2L1 mutant comprises a substitution in: I. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of an alanine (A) for a valine (V); and / or ii. the amino acid corresponding to amino acid 113 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of a serine (S) for a proline (P); and / or iii. the amino acid corresponding to amino acid 125 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of a proline (P) for a serine (S); and / or iv. the amino acid corresponding to amino acid 126 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of an alanine (A) for a threonine (T); and / or v. the amino acid corresponding to amino acid 127 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of an alanine (A) for a threonine (T); and / or vi. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of an alanine (A) for a threonine (T); and / or vii. the amino acid corresponding to amino acid 212 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of an alanine (A) for a valine (V); and also b. a mutation in a gene that codes for a β-glucan synthase. 10. The barley plant or part thereof in accordance with any of the above items, where the barley plant Petition 870250086789, dated 09 / 25 / 2025, pp. 208 / 280 185 / 224 has high α-amylase activity in a medium endosperm grain without the embryo. 11. The barley plant or part thereof in accordance with any of the above items, in which the grains of said barley plant have a reduced content of β-glucan. 12. A barley plant or part thereof in accordance with any of the above items, wherein the gain-of-function mutant CXE2L1 polypeptide comprises an amino acid substitution selected from the group consisting of: a. the amino acid corresponding to S113 or A127 of SEQ ID NO: 1; or b. the amino acid corresponding to P125, A126, or A212 of SEQ ID NO: 1; or c. the amino acid corresponding to F13, L14, G83, G84, G85, L88, Q93, F96, H166, S167, A168, F201, S219, L220, T221, M224, L228, H300, G301, F302, I304 or R305 of SEQ ID NO: 1; or d. the amino acid corresponding to E11, D12, G15, V16, V17, Q18, R27, E30, L33, T35, Y63, Y80, F81, H82, Y86, C87, G89, S90, I91, A92, P94, N95, H97, S98, L99, C100, Y116, L118, S164, G165, G169, A170, N171, L172, A173, L198, S199, A200, F202, A203, G217, V218, T222, A223, A225, D226, Q227, W229, R230, M231, S232, L233, A244, V265, P267, S269, D270, V271, L272, F295, E298, Q299, P303, Q306, P307, S309, T311, A312 or L315 of SEQ ID NO: 1; or e. the amino acid corresponding to V9, V10, L19, L20, S24, V25, V26, G28, D29, A31, V32, R34, N36, G37, L39,P40, V42, V45, Q46, W47, D49, Y52, L58, S59, V60, R61, A62,R64, Petition 870250086789, dated 09 / 25 / 2025, pages 209 / 280 186 / 224 P65, L78, V79, L101, R102, A103, A104, A105, A109, V110, V111, L112, S113, V114, Q115, R117, A119, P120, E121, H122, R123, L124, A127, I128, D130, G131, F134, W151, F162, L163, H174, H175, V176, T177, V178, I196, L197, G204, A205, R207, T208, T210, E211, P214, P215, E216, P234, V235, A237, S238, M239, H241, P242, L243, N245, P246, L263, V264, A266, L268, R273, D274, R275, V276, Y279, V293, Q294, E296, G297, F308, E310, S313, E314, L316, R317, V318, I319 or R320 of SEQ ID NO: 1; f. the amino acid corresponding to Y86, Y116, R117, H122, R123, L124, P125, A126, I128, D129, D130, G131 or L172 from SEQ ID NO: 1; g. the amino acid corresponding to S59, V60, R61, Y63, Y80, I91, C100, V111, L112, V114 or Q115 of SEQ ID NO: 1; wherein the wild-type CXE2L1 is the CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with the same. 13. The barley plant or part thereof according to any of the above items, wherein the substitution at CXE2L1 is a substitution of an alanine for a valine or a substitution of a serine for a proline, for example, wherein said substitution comprises a substitution at: a. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P). Petition 870250086789, dated 09 / 25 / 2025, pp. 210 / 280 187 / 224 14. The barley plant or part thereof according to any of the above items, wherein the substitution at CXE2L1 is a substitution of an alanine for a valine, or a substitution of a proline for a serine, or a substitution of an alanine for a threonine, or a substitution of a serine for a proline, for example, wherein said substitution comprises a substitution at: a. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein the said substitution is a substitution of a serine (S) for a proline (P); and / or c. amino acid 125 of SEQ ID NO: 1, where the aforementioned substitution is a substitution of proline (P) for serine (S); and / or d. amino acid 126 of SEQ ID NO: 1, where the substitution in question is a substitution of alanine (A) for threonine (T); and / or e. amino acid 127 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or f. amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or g. amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for valine (V). Petition 870250086789, dated 09 / 25 / 2025, pp. 211 / 280 188 / 224 15. The barley plant according to any of the above items, wherein said barley plant, in addition to the substitution in the CXE2L1 polypeptide, comprises one or more mutations selected from the group consisting of: a. the amino acid corresponding to A62 of SEQ ID NO:1 for V (A62V) or for T (A62T), b. the amino acid corresponding to S67 of SEQ ID NO:1 for L (S67L), c. the amino acid corresponding to V153 of SEQ ID NO:1 to M (V153M), d. the amino acid corresponding to A212 of SEQ ID NO:1 for T (A212T) or for V (A212V), e. the amino acid corresponding to E216 of SEQ ID NO:1 for K (E216K), f. the amino acid corresponding to M224 of SEQ ID NO:1 to I (M224I), g. the amino acid corresponding to R230 of SEQ ID NO:1 for C (R230C) or for H (R230H) or h. the amino acid corresponding to S238 of SEQ ID NO:1 to N (S238N), where wild-type CXE2L1 is the CXE2L1 of SEQID NO: 1 or a functional variant thereof with at least 95% sequence identity with it. 16. The barley plant according to any of the above items, where the substitution in the CXE2L1 polypeptide is selected from one or more of the mutations listed in Table A, Table B, and Table C. Petition 870250086789, dated 09 / 25 / 2025, pp. 212 / 280 189 / 224 17. The barley plant or part thereof, according to any of the preceding items, provided that the plant does not contain SEQ ID NO: 5. 18. A barley plant or part thereof meeting any of the preceding items, wherein one or more agronomic properties of the barley plant are similar to or superior to the agronomic properties of a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but is of a similar genotype, wherein said agronomic properties are, for example, flowering time, plant height, thousand grain weight (TGW), average grain size, starch content in grains, protein content in grains, water content in grains and yield of the barley plant. 19. The barley plant or part thereof according to any of the preceding items, in which the high α-amylase activity in a half-grain of endosperm without the embryo is at least 5 U / g, preferably at least 10 U / g, such as at least 15 U / g, such as at least 20 U / g, such as at least 25 U / g, such as at least 30 U / g, such as at least U / g, such as at least 40 U / g, at least 45 U / g, or at least 50 U / g, on a dry weight basis. 20. The barley plant or part thereof in accordance with any of the preceding items, in which the high α-amylase activity in a half grain of endosperm without embryo is at least 30 U / g on a dry weight basis. 21. The barley plant or part thereof in accordance with any of the above items, in which the α-amylase activity in the endosperm half-grains without embryo of said plant is present. Petition 870250086789, dated 09 / 25 / 2025, pp. 213 / 280 190 / 224 barley plant is increased by at least 2 times, as for example by at least 3 times, as for example by at least 4 times, as for example by at least 5 times, as for example by at least 6 times, as at least 7 times, as at least 8 times, as at least 9 times, as at least times, as at least 20 times, as at least 30 times, as at least 50 times, as at least 100 times, as at least 1000 times, as at least 10,000 times compared to a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but which is of similar genotype. 22. A barley plant or part thereof according to any of the preceding items, in which the activity of «α-amylase activity in the endosperm grains without the embryo of said barley plant is increased between 2 and 10,000 times, as between 5 and 100 times, as between 20 and 100 times, as between 3 and 50 times, as between 5 and 25 times, as between 10 and 75 times, as between 30 and 60 times, as between 300 and 500 times, compared with a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but is of similar genotype. 23. A barley plant or part thereof meeting any of the preceding items, in which the α-amylase activity in the whole grain is increased by at least 5%, as by at least 10%, as by at least 15%, as by at least 20%, as by at least 50%, as by at least 100%, as by at least 200%, compared with a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype. Petition 870250086789, dated 09 / 25 / 2025, pp. 214 / 280 191 / 224 24. A barley plant or part thereof meeting any of the preceding criteria, in which the α-amylase activity in the whole grain is increased between 5% and 200%, or between 5% and 150%, or between 5% and 100%, or between 50% and 100%, compared with a reference barley plant that does not have a point mutation in the CXE2L1 gene, but is of a similar genotype. 25. The barley plant or part thereof, in which α-amylase activity is measured after the half grain and whole grain have been soaked in water for at least 24 hours, such as at least 48 hours, such as at least 72 hours. 26. A barley plant or part thereof that meets any of the above criteria, in which the level, such as the quantity, of mRNA transcribed from one or more genes encoding hydrolytic enzymes in the barley plant or part thereof is increased compared to a reference barley plant that does not carry a point mutation in the gene. CXE2L1, but which is of a similar genotype, where the level, as the quantity, of mRNA is measured in whole grains, where said grains have been germinated for at least 12 h, as at least 24 h, as at least 36 h, as at least 48 h. 27. The barley plant or part thereof according to any of the preceding items, wherein one or more hydrolytic enzymes is selected from the group consisting of α-amylase, β-amylase, limit dextrinase, pullulanase, β-glucanase, xylanase, glucoamylase and protease, preferably wherein one or more hydrolytic enzymes is selected from Petition 870250086789, dated 09 / 25 / 2025, pp. 215 / 280 192 / 224 group consisting of α-amylase, β-glucanase and limit dextrinase. 28. A barley plant or part thereof in accordance with any of the preceding items, in which the level, such as the quantity, of mRNA is increased by at least 5%, such as by at least 10%, such as by at least 15%, such as by at least 20%, such as by at least 30%, such as by at least 40%, such as by at least 50%, such as by at least 60%, such as by at least 70%, such as by at least 80%, such as by at least 90%, such as by at least 100%, such as by at least 200% compared with a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but which is of a similar genotype, in which the level, such as the quantity, of mRNA is measured in whole grains of barley, in which said grains have been germinated for at least 12 h, such as at least 24 h, such as at least 36 h, such as at least 200% 48 hours. 29. A barley plant or part thereof meeting any of the above criteria, wherein the level, as the quantity, of mRNA is increased between 5% and 200%, between 10% and 150%, between 25% and 100%, between 15% and 80%, or between 50% and 150% compared with a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but which is of a similar genotype, wherein the level, as the quantity, of mRNA is measured in whole grains, wherein said grains have been germinated for at least 12 h, at least 24 h, at least 36 h, or at least 48 h. 30. The barley plant or part thereof in accordance with any of the above items, wherein a malt is prepared. Petition 870250086789, dated 09 / 25 / 2025, pp. 216 / 280 193 / 224 from the grains of said barley plant has a high α-amylase activity, such as, for example, an α-amylase activity of at least 200 U / g, such as at least 210 U / g, such as at least 220 U / g, such as at least 230 U / g, such as at least 240 U / g, such as at least 250 U / g. 31. The barley plant or part thereof according to any of the preceding items, wherein a malt prepared from the grains of said barley plant has a high α-amylase activity, such as, for example, an α-amylase activity between 200 U / g and 280 U / g, such as between 210 U / g and 260 U / g, such as between 220 U / g and 240 U / g. 32. The barley plant or part thereof according to any of the preceding items, wherein a malt prepared from grains and seeds of said barley plant has high β-amylase activity, such as, for example, a β-amylase activity of at least 10 U / g, such as at least 11 U / g, such as at least 12 U / g, such as at least 13 U / g, such as at least 14 U / g, such as at least 15 U / g. 33. The barley plant or part thereof according to any of the preceding items, wherein a malt prepared from the grains of said barley plant has a high β-amylase activity, such as, for example, a β-amylase activity between 10 U / g and 15 U / g, or between 10 U / g and 14 U / g, or between 10 U / g and 13 U / g. 34. The barley plant or part thereof according to any of the above items, in which a malt prepared from the grains of said barley plant has a Petition 870250086789, dated 09 / 25 / 2025, pp. 217 / 280 194 / 224 high limiting dextrinase activity, such as, for example, a limiting dextrinase activity of at least 45 U / g, such as at least 50 U / g, such as at least 55 U / g, such as at least 60 U / g, such as at least 65 U / g. 35. The barley plant or part thereof according to any of the above items, wherein a malt prepared from the grains of said barley plant has a high threshold dextrinase activity, such as, for example, a threshold dextrinase activity between 45 U / g and 65 U / g, or between 45 U / g and 55 U / g, or between 50 U / g and 60 U / g. 36. A barley plant or part thereof in accordance with any of the above items, wherein the wort prepared from the grains of said barley plant has a reduced β-glucan content compared to the β-glucan content in wort prepared from the grains of a reference barley plant that does not carry a point mutation in the CXE2L1 gene, but is of a similar genotype. 37. A barley plant or part thereof according to any of the preceding items, in which the β-glucan content in the wort prepared from the grains of said barley plant is reduced by at least 10%, as well as by at least 20%, as well as by at least 30%, as well as by at least 40%, as well as by at least 50%, as well as by at least 60%, as well as by at least 70%, as well as by at least 80%, as well as by at least 90%, as well as by 100%, compared with the β-glucan content in the wort prepared from grains of a reference barley plant that does not have a point mutation in the CXE2L1 gene, but that otherwise has a similar genotype. Petition 870250086789, dated 09 / 25 / 2025, pp. 218 / 280 195 / 224 38. A barley plant or part thereof in accordance with any of the preceding items, in which the β-glucan content in the wort prepared from said barley plant or part thereof is reduced by between 10% and 100%, or between 10% and 50%, or between 50% and 75%, or between 25% and 100%, compared with the β-glucan content in the wort prepared from grains of a reference barley plant that does not have a point mutation in the CXE2L1 gene, but which otherwise has a similar genotype. 39. The barley plant or part thereof in accordance with any of the preceding items, wherein the β-glucan content of the wort prepared from said barley plant or part thereof is a maximum of 400 mg / L, such as a maximum of 350 mg / L, such as a maximum of 300 mg / L, such as a maximum of 250 mg / L, such as a maximum of 200 mg / L, such as a maximum of 150 mg / L mg / L. 40. The barley plant or part thereof in accordance with any of the preceding items, wherein the β-glucan content of the wort prepared from said barley plant or part thereof is between 0 and 500 mg / L, as well as between 0 and 250 mg / L, as well as between 50 and 250 mg / L, as well as between 100 and 200 mg / L, as well as between 200 and 400 mg / L, as well as between 50 and 200 mg / L. 41. The barley plant or part thereof in accordance with any of the above items, where the reference barley plant of similar genotype is the barley cultivar Quench or Planet. Petition 870250086789, dated 09 / 25 / 2025, pp. 219 / 280 196 / 224 42. The barley plant or part thereof according to any of the above items, wherein the β-glucan is (1,3;1,4)-β-glucan. 43. The barley plant or part thereof in accordance with any of the above items, where the barley plant carries a mutation in the gene that codes for CslF6. 44. The barley plant or part thereof according to any of the above items, wherein said barley plant has a content of (1,3;1,4)-ε-glucan: a. in the range of 1 to 5% of the total dry weight of the grains, for example, 1 to 3% of the total dry weight of the grains, preferably 1.3 to 2% of the total dry weight of the grains; and / or b. of at least 30% and at most 60%, preferably at least 40% and at most 60% of the (1,3;1,4)-e-glucan content of a barley plant carrying a wild-type CslF6 gene, but otherwise of the same genotype. 45. A barley plant or part thereof in accordance with any of the preceding items, wherein the gene encoding a β-glucan synthase is CslF6, wherein said mutant CslF6 gene encodes a mutant CslF6 polypeptide, wherein wild-type CslF6 is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, or a functional variant thereof with at least 95% sequence identity therewith. 46. ​​A barley plant or part thereof that meets any of the above criteria, where the mutation in the gene encoding CslF6 is a deletion. Petition 870250086789, dated 09 / 25 / 2025, pp. 220 / 280 197 / 224 47. A barley plant or part thereof in accordance with any of the preceding items, wherein the said mutant CslF6 gene encodes a loss-of-function mutant CslF6 polypeptide, wherein the wild-type CslF6 is the CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, or a functional variant thereof with at least 95% sequence identity with it. 48. A barley plant or part thereof conforming to any of the above, wherein the mutant CslF6 polypeptide comprises an amino acid substitution in a membrane-located domain of CslF6, wherein said substitution is a substitution of a nonpolar amino acid for a charged amino acid or a substitution of a polar amino acid for a nonpolar amino acid, wherein the membrane-located domain is selected from the group consisting of membrane-located domains of CslF6 consisting of: a. amino acids 835 to 857 (SEQ ID NO: 9), or b. amino acids 700 to 731 (SEQ ID NO: 10), or w. amino acids 741 to 758 (SEQ ID NO: 11). 49. The barley plant or part thereof according to any of the preceding items, wherein the mutant CslF6 polypeptide comprises an amino acid substitution in a membrane-bound domain of CslF6, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 835 to 857 (SEQ ID NO: 9) of CslF6, wherein the Petition 870250086789, dated 09 / 25 / 2025, pp. 221 / 280 198 / 224 The aforementioned substitution is the replacement of a nonpolar amino acid with a charged amino acid. 50. The barley plant or part thereof in accordance with any of the above items, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution 847, wherein said substitution is the substitution of a glycine (G) for a glutamic acid (E). 51. The barley plant or part thereof according to any of the preceding items, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 741 to 758 (SEQ ID NO: 10) of CslF6, wherein said substitution is the substitution of a nonpolar amino acid for a charged amino acid. 52. The barley plant or part thereof according to any of the above items, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises a substitution of amino acid 748, wherein said substitution is the substitution of a glycine (G) for an aspartic acid (D). 53. The barley plant or part thereof according to any of the preceding items, wherein the said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises an amino acid substitution in the transmembrane domain consisting of amino acids 700 to 731 of CslF6 (SEQ ID NO: 11), wherein the Petition 870250086789, dated 09 / 25 / 2025, pp. 222 / 280 199 / 224 The aforementioned substitution is the replacement of a polar amino acid with a non-polar amino acid. 54. The barley plant or part thereof in accordance with any of the above items, wherein said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises a substitution of amino acid 709, wherein said substitution is the substitution of a threonine (T) for an isoleucine (I). 55. A barley plant or part thereof conforming to any of the above, wherein the barley plant comprises a mutation in one or more additional genes, for example, one or more of the following mutations: a. a mutation in the gene that codes for LOX-1, resulting in the complete loss of functional LOX-1; b. a mutation in the gene that codes for LOX-2, resulting in the total loss of functional LOX-2; c. a mutation in the gene that codes for MMT, resulting in the total loss of MMT functionality; d. a mutation in the gene that codes for ANT-28, resulting in the total loss of ANT-28 functionality; e. a mutation in the gene that codes for HRT, resulting in the total loss of HRT function; f. a mutation in the gene that codes for LDI, resulting in the total loss of LDI function. 56. A barley plant or part thereof, in which the said barley plant bears: a. an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said α-amylase has an amino acid sequence Petition 870250086789, dated 09 / 25 / 2025, pp. 223 / 280 200 / 224 selected independently from the group consisting of SEQ ID Nos: 31-41 and their respective functional counterparts with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with them; and b. a mutation in one or more selected genes from the group consisting of CXE2L1, LOX-1, LOX-2, MMT, ANT-28, HRT, LDI and a gene encoding a β-glucan synthase, such as CslF6. 57. The barley plant or part thereof as defined in item 56, wherein the said mutation in CXE2L1 is defined in accordance with any of items 1 to 42. 58. The barley plant or part thereof in accordance with any of items 56 to 57, wherein the said mutation in one or more genes selected from the group consisting of LOX1, LOX-2, MMT, ANT-28, HRT and LDI is defined in accordance with item 55. 59. The barley plant or part thereof in accordance with item 56, in which the said mutation in the said gene encoding the said β-glucan synthase is defined in accordance with any of items 43 to 52. 60. The barley plant or part thereof in accordance with item 58, in which the gene encoding the β-glucan synthase is CslF6. 61. The barley plant or part thereof in accordance with any of items 56 to 59, in which the said barley plants bear Petition 870250086789, dated 09 / 25 / 2025, pp. 224 / 280 201 / 224 a. an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said α-amylase has an independently selected amino acid sequence from the group consisting of SEQ IDs 37-47 and their respective functional homologs with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with them; b. a mutation in the gene that codes for LOX-1, resulting in the total loss of functional LOX-1; c. optionally, a mutation in the gene that codes for LOX-2, resulting in total loss of functional LOX-2; d. a mutation in the gene that codes for MMT, resulting in total loss of MMT functionality; and e. a mutation in CXE2L1, as defined in any of items 1 to 42. 62. The barley plant or part thereof in accordance with any of items 56 to 61, wherein said barley plants bear a a. an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said α-amylase has an independently selected amino acid sequence from the group consisting of SEQ IDs 37-47 and their respective functional homologs with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with them; Petition 870250086789, dated 09 / 25 / 2025, pp. 225 / 280 202 / 224 b. a mutation in the gene that codes for LOX-1, resulting in the complete loss of functional LOX-1; c. optionally, a mutation in the gene that codes for LOX-2, resulting in the total loss of functional LOX-2; d. a mutation in the gene that codes for MMT, resulting in the total loss of MMT functionality; e. a mutation in the gene that codes for ANT-28, resulting in the total loss of ANT-28 function; and f. a mutation in CXE2L1, as defined in any of items 1 to 42. 63. A barley plant or part thereof in accordance with any of items 56 to 62, wherein the amy1_1 cluster comprises at least 5 functional genes, each encoding an α-amylase, wherein said α-amylase is independently selected from the group consisting of: • an α-amylase with the amino acid sequence as set out in SEQ ID NO: 42 or a functional homolog thereof with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the same; • an α-amylase with the amino acid sequence as set forth in SEQ ID NO: 43 or a functional homolog thereof with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the same; and • an α-amylase with the amino acid sequence as set forth in SEQ ID NO: 44 or a functional homolog thereof Petition 870250086789, dated 09 / 25 / 2025, pp. 226 / 280 203 / 224 same with at least 80% sequence identity with the same, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the same. 64. The barley plant or part thereof in accordance with any of items 56 to 63, wherein the α-amylase activity of said barley plant or part thereof is at least 230 U / g, such as at least 240 U / g, such as at least 250 U / g, such as at least 260 U / g, such as at least 270 U / g, such as at least 280 U / g, such as at least 290 U / g, such as at least 300 U / g, such as at least 310 U / g, such as from 230 to 350 U / g, such as from 250 to 320 U / g after 6 days of germination. 65. The barley plant or part thereof in accordance with any of items 56 to 64, wherein the β-amylase activity of said barley plant or part thereof is at least 15 U / g, such as at least 16 U / g, such as at least 17 U / g, such as at least 18 U / g, such as 15 to 20 U / g, such as 15 to 18 U / g after 6 days of germination. 66. The barley plant or part thereof, in accordance with any of items 56 to 64, wherein the free dextrinase activity of said barley plant or part thereof is at least 60 mU / g, such as at least 65 mU / g, such as at least 70 mU / g, such as at least 75 mU / g, such as at least 80 mU / g, such as from 60 to 90 mU / g, such as from 65 to 85 mU / g after 6 days of germination. 67. The barley plant or part thereof in accordance with any of items 56 to 66, wherein the β-glucan content of said barley plant or part thereof is not more than 200 mg / L, Petition 870250086789, dated 09 / 25 / 2025, pp. 227 / 280 204 / 224 such as a maximum of 150 mg / L, such as a maximum of 100 mg / L, such as a maximum of 75 mg / L, such as a maximum of 50 mg / L, such as a maximum of 25 mg / L, such as a maximum of 10 mg / L, such as from 0 to 200 mg / L, such as from 0 to 100 mg / L, such as from 0 to 50 mg / L after 6 days of germination. 68. The barley plant or part thereof according to any of the preceding items, wherein the part of the plant is a grain, a seed, a cell and / or a part of the plant that is not reproductive material. 69. The barley plant or part thereof in accordance with any of the preceding items, wherein the plant or part thereof was not obtained by means of an essentially biological process and / or wherein the progeny of the plant or part thereof was not obtained by means of an essentially biological process. 70. A vegetable product comprising or prepared from the barley plant or parts thereof, in accordance with any of the preceding items. 71. The vegetable product according to any of the preceding items, wherein the vegetable product is a malt, such as, for example, ground malt, green malt and / or ground green malt. 72. The vegetable product according to any of the preceding items, wherein the vegetable product is a beverage prepared from said barley plant or part thereof. 73. The plant product according to any of the preceding items, wherein the plant product is an aqueous extract or a beverage prepared from the grains of said plant. Petition 870250086789, dated 09 / 25 / 2025, pages 228 / 280 205 / 224 barley plant and / or from malt comprising processed grains from said barley plant. 74. The plant-based product according to any of the preceding items, wherein the said beverage is beer. 75. A method for preparing a barley plant or part thereof with α-amylase activity in the endosperm grain half without the embryo, the method comprising the steps of: a. to supply barley grains; and b. random mutagenesis of barley grains; and c. select barley grains or their offspring that carry a mutant CXE2L1 gene; wherein the aforementioned mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, in which the Wild-type CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with the same, provided that the plant does not contain SEQ ID NO: 5. 76. A method of preparing a barley plant or part thereof, the method comprising the steps of: a. to supply barley grains; and b. random mutagenesis of said barley grains, thereby introducing a mutation into the CXE2L1 gene; and c. select barley grains or their offspring that carry a mutant CXE2L1 gene; wherein the said mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, wherein the said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: X or a functional variant thereof with at least 95% identity. Petition 870250086789, dated 09 / 25 / 2025, pp. 229 / 280 Sequence 206 / 224 is the same, except that the CXE2L1 mutant comprises a substitution in: i. amino acid 127, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. amino acid 113, wherein the said substitution is a substitution of a serine (S) for a proline (P); provided that the plant does not contain SEQ ID NO: 5. 77. A method of preparing a barley plant or part thereof, the method comprising the steps of: a. to supply barley grains; and b. random mutagenesis of said barley grains, thereby introducing a mutation into the CXE2L1 gene; and c. select barley grains or their progeny that carry a mutant CXE2L1 gene; wherein the said mutant CXE2L1 gene encodes a gain-of-function mutant CXE2L1 polypeptide, wherein the said mutant CXE2L1 is the CXE2L1 of SEQ ID NO: X or a functional variant thereof with at least 95% sequence identity with it, except that the mutant CXE2L1 comprises a substitution at: i. amino acid 127, wherein the said substitution is a substitution of an alanine (A) for a valine (V); and / or ii. amino acid 113, wherein the said substitution is a substitution of a serine (S) for a proline (P); and / or iii. amino acid 125 of SEQ ID NO: 1, wherein the said substitution is a substitution of proline (P) for serine (S); and / or Petition 870250086789, dated 09 / 25 / 2025, pp. 230 / 280 207 / 224 iv. amino acid 126 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); and / or v. amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or vi. amino acid 212 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or vii. amino acid 212 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for valine (V); provided that the plant does not contain SEQ ID NO: 5. 78. The method according to any of the above items, where the mutation and / or substitution in CXE2L1 is as defined in any of the above items. 79. The method according to any of the preceding items, wherein the method further comprises the selection of barley grains or their offspring that carry a mutant βglucan synthase gene. 80. The method according to any of the preceding items, wherein the said mutant β-glucan is CslF6, optionally wherein the said mutant CslF6 gene encodes a loss-of-function mutant CslF6 polypeptide, wherein wild-type CslF6 is CslF6 of SEQ ID NO: 7, optionally further wherein the mutation in the gene encoding CslF6 is a deletion. Petition 870250086789, dated 09 / 25 / 2025, pp. 231 / 280 208 / 224 81. A method for preparing dry malt in an oven, said method comprising the steps of a. to provide grains from a barley plant in accordance with any of the above items; b. to soak the aforementioned grains; c. to germinate soaked grains under predetermined conditions; d. Dry the sprouted grains. 82. The method according to any of the above items, wherein the barley plant or part thereof is defined in any of the above items. 83. A method for producing an aqueous extract, said method comprising the following steps: a. to provide grains from a barley plant in accordance with any of the preceding items; b. subjecting the barley grains to a germination stage, thus obtaining germinated grains; c. finely divide the sprouted grains; d. optionally, dry the finely divided sprouted grains; e. prepare an aqueous extract of the aforementioned finely divided germinated (dried) grains, f. thus producing an aqueous extract of barley. 84. The method according to any of the preceding items, wherein step c) is carried out while the germinated grains have a water content of at least 20%, provided that the barley grains do not have a water content lower than 20% at any time between steps b) and c). Petition 870250086789, dated 09 / 25 / 2025, pp. 232 / 280 209 / 224 85. The method according to any of the items above, where step d) is not performed. 86. The method according to any of the previous items, in which the germination stage is carried out for a maximum of 96 hours, or for a maximum of 72 hours, or for a maximum of 48 hours. 87. A method for producing a beverage, said method comprising the following steps: a. to supply grains from a barley plant in accordance with any of the above items and / or malt in accordance with any of the above items; b. prepare an aqueous extract of said grains and / or said malt; c. process the aforementioned aqueous extract into a beverage. 88. A method for producing a beverage, said method comprising the steps of: a. prepare an aqueous extract using the method described in any of the above items; b. process the aforementioned extract to obtain a beverage. 89. The method according to any of the preceding items, in which the processing step comprises a fermentation step with a microorganism, such as yeast. 90. The method according to any of the preceding items, wherein the barley plant is defined in any of the preceding items. 91. The method according to any of the preceding items, wherein the grains of the barley plant are as defined in any of the preceding items. Petition 870250086789, dated 09 / 25 / 2025, pages 233 / 280 210 / 224 92. The method according to any of the preceding items, wherein the malt is as defined in any of the preceding items. 93. The method according to any of the previous items, where the beverage is beer. 94. The method according to any of the above items, where the beverage is a light-colored beer, for example, selected from the group consisting of lager, pale ale, and wheat beer. 95. The method according to any of the above items, where the beverage is a lager beer. 96. The method according to any of the above items, where the beer is a non-alcoholic beer. Sequence overview SEQ ID NO. Name Description or sequence 1 CXE2L1 Amino acid sequence of CXE2L1 (ID HORVU.MOREX.r3.3HG0242030.1, Morex_V3) MSGGTAPRVVEDFLGVVQLLSDGSVVRGDEAVLRTNEPLPD VTGVQWKDVLYHPAHGLSVRAYRPASSVAGGSKLPVLVYFH GGGYCLGSIAQPNFHSLCLRAAAEIPAVVLSVQYRLAPEHR LPAAIDDGASFLSWLRGQAGLGAGADPWLVESADFAQIFLS GHSAGANLAHHVTVRVASGQIAVTPVRVVGYILLSAFFAGA ERTATEADPPEGVSLTTAMADQLWRMSLPVGASMDHPLANP FGPESPSLAPVELPPALVVAPLSDVLRDRVLGYGARLKDMG KAVEVVQFEGEQHGFPIRQPFSETASELLRVIRRFVYSGN 2 CXE2L1 DNA sequence of CXE2L1 (gene ID HORVU.MOREX.r3.3HG0242030, Morex_V3) ATGTCCGGCGGCACGGCACCGCGCGTTGTGGAGGACTTCCT CGGCGTCGTGCAGCTCCTCAGCGACGGCTCCGTCGTTCGCG GTGACGAAGCCGTCCTCCGCACAAACGAGCCGTTGCCGGAC Petition 870250086789, dated 09 / 25 / 2025, pp. 234 / 280 211 / 224 GTCACCGGTGTGCAGTGGAAGGACGTCCTGTACCACCCCGC GCACGGCCTTAGTGTCCGCGCATACAGGCCGGCGTCGTCCG TGGCCGGCGGCAGCAAGCTCCCTGTGCTGGTGTACTTCCAC GGCGGGGGCTACTGCCTCGGCTCCATCGCGCAGCCCAACTT CCACTCGCTCTGCCTCCGCGCCGCAGCCGAGATACCCGCCG TCGTGCTGTCCGTGCAGTACCGCCTCGCCCCCGAGCACCGC CTCCCCGGCCATCGACGACGGCCGCGTCTTTCCTTTCCTG GCTGCGCGGCCAGGCCGGGCTTGGCGCTGGCGCCGACCCGT GGCTCGTGGAGTCGGCCGATTTCGCCCAGATCTTCCTCTCC GGCCACTCGGCGGGTGCCAACCTGGCCCACCACGTCACGGT CCGGGTCGCGTCGGGGCAGATCGCGGTCACCCCGGTGCGCG TCGTCGGGTACATCCTCCTCTCCGCGTTCTTCGCTGGGGCC GAGCGCACGGCGACGGAGGCCGACCCTCCGGAGGGGGTGTC CTTGACGACCGCGATGGCCGACCAGCTCTGGCGCATGTCGC TGCCGGTGGGGGCGAGCATGGACCACCCGCTGGCCAACCCG TTCGGCCCGGAGAGCCCCAGCCTCGCGCCGGTGGAGCTCCC GCCGGCGCTCGTCGTCGCGCCGTTGAGCGACGTGCTCCGCG ACCGCGTGCTAGGGTACGGGGCGAGGCTGAAGGACATGGGG AAGGCCGTCGAGGTTGTCCAGTTCGAGGGGGAGCAGCATGG TTTCCCAATCCGCCAGCCGTTCAGCGAGACGGCCAGTGAGT TGCTGCGGGTGATCAGGCGGTTCGTCTACAGCGGCAACTGA 3 CXE2L1-A127V Amino acid sequence of CXE2L1 with A127V mutation MSGGTAPRVVEDFLGVVQLLSDGSVVRGDEAVLRTNEPPLPDVTGVQWKDVLYHPAHGLSVRAYRPASSVAGGSKLPVLVYFH GGGYCLGSIAQPNFHSLCLRAAAEIPAVVLSVQYRLAPEHR LPAVIDDGASFLSWLRGQAGLGAGADPWLVESADFAQIFLS GHSAGANLAHHVTVRVASGQIAVTPVRVVGYILLSAFFAGA ERTATEADPPEGVSLTTAMADQLWRMSLPVGASMDHPLANP FGPESPSLAPVELPPALVVAPLSDVLRDRVLGYGARLKDMG KAVEVVQFEGEQHGFPIRQPFSETASELLRVIRRFVYSGN 4 CXE2L1-S113P Amino acid sequence of CXE2L1 with S113P mutation Petition 870250086789, dated 09 / 25 / 2025, pages 235 / 280 212 / 224 MSGGTAPRVVEDFLGVVQLLSDGSVVRGDEAVLRTNEPLPD VTGVQWKDVLYHPAHGLSVRAYRPASSVAGGSKLPVLVYFH GGGYCLGSIAQPNFHSLCLRAAEIPAVVLPVQYRLAPEHR LPAAIDDGASFLSWLRGQAGLGALSVGALDWLDFAQ GHSAGANLAHHVTVRVASGQIAAVTPVRVVGYILLSAFFAGA ERTATEADPPEGVSLTTAMADQLWRMSLPVGASMDHPLANP FGPESPSLAPVELPPALVVAPLSDVLRDRVLGYGARLKDMG KAVEVVQFEGEQHGFPIRQPFSETASELLRVIRVYRFVN-HSGMT Marcador CCGCTGCTATTCTGTGAGCATAGGTTTACTTCATGCTACCT CTGTCCTGTAAATATATGATGTTTTAGCAGCTCAAATGAAC TGCTAAAACGTTCATACTATGATACGGAGAGTACATTT TACTAACCTGCCGAATGTGGGGATTTTTTCTCTATGCATA GGCTTAGTTTCTGCTTAGCTTAGCTTAGCTTAGCTTAGCTTAGTCTAGTCATTT CATACTCCCTCCG 6 The HENZ-α locus is the FMT-3H marker CCGCTGCTATTCTGTGAGCATAGGTTTACTTCATGCTACCT CTGTCCTGTAAATATATGATGTTTTAGCAGCTCAAATGAAC TGCTAAAACGTTCATACTATGATACGGAGGGAGTACATTT TACTAACCTGCCTTGTTGATTGGTTGATT GGCTTAGTTTCTGCTGCTATTCTGTGGGCATAAGTTTACTT CATACTCCCTCCG 7 CslF6 Amino acid sequence of cellulose synthase-like CslF6 (CslF6) (based on NCBI GenBank number: EU267181.1) 8 CslF6 Amino acid sequence of CslF6 containing A590T polymorphism 9 Amino acid sequence located in the CslF6 membrane (amino acids 835 to 857) ITPIIIIFVNIIGSAVAFAKVLD. Petition 870250086789, dated 09 / 25 / 2025, pages 236 / 280 213 / 224 10 Amino acid sequence located in the CslF6 membrane (amino acids 700 to 731) LQRVAYINITTYPTFAIFLIFYTTVPALSFVT 11 Amino acid sequence located in the CslF6 membrane (amino acid 741 to 758) TMFYVYLGIVLSTLLVIA 12 Amino acid sequence located in the CslF6 membrane (amino acids 109-128) RVLIFVRLIAFTLFVIWRIS 13 Amino acid sequence located in the CslF6 membrane (amino acids 137-158) LWVTSICGEFWFGFSWLLDQLP 14 Amino acid sequence located in the CslF6 membrane LKVAGGVFFNFWVLFHLYPF Petition 870250086789, dated 09 / 25 / 2025, pages 237 / 280 214 / 224 (amino acids 864-882) 15 Direct primer AMY1-2 5'- TGAAGGAGGAGATCGATC-3' 16 Reverse primer AMY1-2 5'- TTGCCGTCGATCTCG-3' 17 Specific detection probe for AMY1-2 5'- AAGCTGCAGATCATGGAGGC-3'-labeled with 6-carboxyfluorescein (FAM) 18 Direct primer LD 5'- CTTCGATGGGGTTTGAAC-3' 19 Reverse primer LD 5'- CAGATTTCCTCACCAAAG-3' 20 Specific detection probe for LD 5'- CCTGTGCAGGTGAATTCATCA-3'-labeled with 6-carboxyfluorescein (FAM) 21 Direct primer BGL2A 5'- AACTGGGGACTCTTCTAC-3' 22 Reverse primer BGL2A 5'-TCAGAGTTGATGGGGTAG-3' 23 Specific detection probe for BGL2A 5'-CCCAACATGCAGCACGT-3'-labeled with 6-carboxyfluorescein (FAM) 24 Direct reference primer for actin 5'-TACAACTCCATCATGAAGTG-3' 25 Reverse reference primer for actin 5'-GATACCTGGGAACATAGTTG-3' Petition 870250086789, dated 09 / 25 / 2025, pp. 238 / 280 215 / 224 26 Specific detection probe for reference actin 5'- AAACATCGTGCTCAGTGGTG -3'-labeled with hexachlorofluorescein (HEX) 27 Specific direct primer for target 5'- AACTTCCACTCGCCTTG -3' 28 Specific reverse primer for target 5'- CAGGAAAGGAAAGACGC -3' 29 Specific detection probe for mutants 5'- CGATGACCGCGGG -3'-labeled with 6-carboxyfluorescein (FAM) 30 Specific detection probe for reference 5'- CGATGGCCGCGG -3'-labeled with hexachlorofluorescein (HEX) 31 CTTAGTGTCCGCGCATACAG 32 CGCGGAGAGGAGGATGTAC 33 GCGCCGCAGCCGAGATACCCGCCGTCGTGCTGTCCGTGCAG TACCGCCTCGCCCCCGAGC 34 GCGCCGCAGCCGAGATACCCGCCGTCGTGCTGCCCGTGCAG TACCGCCTCGCCCCCGAGC 35 ACCGCCTCCCCGCGGCCATCGACGACGGCGCGTCTTTCCTT TCCTGGCTGCGCGGCCAGG 36 ACCGCCTCCCCGCGGTCATCGACGACGGCGCGTCTTTCCTT TCCTGGCTGCGCGGCCAGG 37 HORVU.MOREX.r 3.6HG0617430. 1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITHVWLPPASQSVAEQGYMPGRLYD LDASKYGNKAQLKSLIGALHGKGVKAIADIVINHRTAEHKD Petition 870250086789, dated 09 / 25 / 2025, pp. 239 / 280 216 / 224 GRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNPDTG ADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFA KGYSADVAKIYIDRSEPSFAVAEIWTSLAYGGDGKPNLNQD QHRQELVNWVDKVGGKGPTATTDFTTKGILNVAVEGELWRL RGTDGKAPGMIGWWPAKAVTFVDNHDTGSTQMHWPFPSDRV MQGYAYILTHPGTPCIFYDHFFDWGLKEEIDRLVSRTRHG IHNESKLQIIEADADLYLAEIDGKVIVKLGPRYDVGNLIPG GFKVAAHGNDYAVWEKI 38 HORVU.MOREX.r 3.6HG0617450. 1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITHVWLPPASQSVAEQGYMPGRLYD LDASKYGNKAQLKSLIGALHGKGVKAIADIVINHRTAEHKD GRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNPDTG ADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFA KGYSADVAKIYIDRSEPSFAVAEIWTSLAYGGDGKPNLNQD QHRQELVNWVDKVGGKGPTATTDFTTKGILNVAVEGELWRL RGTDGKAPGMIGWWPAKAVTFVDNHDTGSTQHMWPPFSDRV MQGYAYILTHPGTPCIFYDHFFDWGLKEEIDRLVSVRTRHG IHNESKLQIIEADADLYLAEIDGKVIVKLGPRYDVGNLIPG GFKVAAHGNDYAVWEKI 39 HORVU.MOREX.r 3.6HG0617470.1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITHVWLPPASQSVAEQGYMPGRLYD LDASKYGNKAQLKSLIGALHGKGVKAIADIVINHRTAEHKD GRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNPDTG ADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFA KGYSADVAKIYIDRSEPSFAVAEIWTSLAYGGDGKPNLNQD QHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVEGELWRL RGTDGKAPGMIGWWPAKAVTFVDNHDTGSTQHMWPFPSDRV MQGYAYILTHPGTPCIFYDHFFDWGLKEEIDRLVSVRTRHG IHNESKLQIIEADADLYLAEIDGKVIVKLGPRYDVGNLIPG GFKVAAHGNDYAVWEKI 40 HORVU.MOREX.r 3.6HG0617480. 1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITHVWLPPASQSVAEQGYMPGRLYD LDASKYGNKAQLKSLIGALHGKGVKAIADIVINHRTAEHKD GRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNPDTG. Petition 870250086789, dated 09 / 25 / 2025, pages 240 / 280 217 / 224 ADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFA KGYSADVAKIYIDRSEPSFAVAEIWTSLAYGGDGKPNLNQD QHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVEGELWRL RGTDGKAPGMIGWWPAKAVTFVDNHDTGSTQHMWPFPSDRV MQGYAYILTHPGTPCIFYDHFFDGLKEEIDRLVSVRTRHG IHNESKLQIIEDADLYLAEIDGKVIVKLGPRYDVGNLIPG GFKVAAHGNDYAVWEKI 41 HORVU.MOREX.r 3.6HG0617510.1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITHVWLPPASQSVAEQGYMPGRLYD LDASKYGNKAQLKSLIGALHGKGVKAIADIVINHRTAEHKD GRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNPDTG ADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFA KGYSADVAKIYIDRSEPSFAVAEIWTSLAYGGDGKPNLNQD QHRQELVNWVDKVGSKGPATTFDFTTKGILNVAVEGELWRL RGTDGKAPGMIGWWPAKAVTFVDNHDTGSTQHMWPFPSDRV MQGYAYILTHPGTPCIFYDHFFDWGLKEEIDRLVSVRTRHG IHSESKLQIIEADADLYLAEIDGKVIVKLGPRYDVGNLIPG GFKVAAHGNDYAVWEKI 42 Amyl 1- B cópia1 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRV QKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK PNLNQDQHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDKVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHSESKLQIIEADADLYLAEIDGKIIVKLGPR YDVGNLIPAGFKVAAHGND YAVWEKI. Petição 870250086789, de 25 / 09 / 2025, pág. 241 / 280 218 / 224 43 Amyl 1- B cópia2 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRV QKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK PNLNQDQHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDRVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHSESKLQIIEADADLYLAEIDGKVIVKLGPR YDVGNLIPGGFKVAAHGND YAVWEKI 44 Amyl 1- B cópia3 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRV QKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK PNLNQDQHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDRVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHSESKLQIIDADADLYLAEIDGKVIVKLGPR YDVGNLIPGGFKVAAHGND YAVWEKI 45 Amyl 1- Q cópial MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGGWYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE Petition 870250086789, dated 09 / 25 / 2025, pages 242 / 280 219 / 224 HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRV QKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK PNLNQDQHRQELVNWVDKVGSKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDRVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHSESKLQIIEADADLYLAEIDGKVIVKLGPR YDVGNLIPGGFKVAAHGND YAVWEKI 46 Amyl 1- Q cópia2 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRV QKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK PNLNQDQHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDRVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHNESKLQIIEADADLYLAEIDGKVIVKLGPR YDVGNLIPAGFKVAAHGND YAVWEKI 47 Amyl 1- Q cópia3 MANKHLSLSLFLVLLGLSASLASGQVLFQGFNWESWKHNGG WYNFLMGKVDDIAAAGITH VWLPPASQSVAEQGYMPGRLYDLDASKYGNKAQLKSLIGAL HGKGVKAIADIVINHRTAE HKDGRGIYCIFEGGTPDARLDWGPHMICRDDRPYADGTGNP DTGADFGAAPDIDHLNLRVQKELVEWLNWLKADIGFDGWRFDFAKGYSADVAKIYIDRSE PSFAVAEIWTSLAYGGDGK Petition 870250086789, dated 09 / 25 / 2025, pages 243 / 280 220 / 224 PNLNQDQHRQELVNWVDKVGGKGPATTFDFTTKGILNVAVE GELWRLRGTDGKAPGMIGW WPAKAVTFVDNHDTGSTQHMWPFPSDRVMQGYAYILTHPGT PCIFYDHFFDWGLKEEIDR LVSVRTRHGIHSESKLQIIEADADLYLAEIDGKVIVKLGSR YDVGNLIPAGFKVAAHGND YAVWEKI 48 HENZ-78 Target-specific direct primer AACTTCCACTCGCCTTG 49 HENZ-79 Target-specific direct primer CAAACGAGCCGTTGC 50 HENZ-80 Target-specific direct primer AACTTCCACTCGCCTTG 51 HENZ-81 Target-specific direct primer CTGTCCGTGCAGTACC 52 HENZ-82 Target-specific direct primer GTACATCCTCCTCTCCG 53 HENZ-83 Direct primer specifically for target GTACATCCTCCTCTCCG 54 HENZ-84 Direct primer CTGTCCGTGCAGTACC Petition 870250086789, dated 09 / 25 / 2025, pages 244 / 280 221 / 224 Target-specific reverse primer 55 HENZ-78 Target-specific reverse primer CAGGAAAGGAAAGAACGC 56 HENZ-79 Target-specific reverse primer GTATGCGCGGACACTA 57 HENZ-80 Target-specific reverse primer CAGGAAAGGAAAGACGC 58 HENZ-81 Target-specific reverse primer GCAGCCAGGAAAGGAA 59 HENZ-82 Target-specific reverse primer ATCGCGGTCGTCAAG 60 HENZ-83 Target-specific reverse primer ATCGCGGTCGTCAAG 61 HENZ-84 Primer GCAGCCAGGAAAGGAA Petition 870250086789, dated 09 / 25 / 2025, pp. 245 / 280 222 / 224 Target-specific reverse 62 HENZ-78 Reference probe CGATGGCCGCGG 63 HENZ-79 Reference probe TGCAGTGGAAGGACG 64 HENZ-80 Reference probe CCGCGGGGAGGC 65 HENZ-81 Reference probe CCCCGCGGCCAT 66 HENZ-82 Reference probe AGGGTCGGCCTCC 67 ​​HENZ-83 Reference probe ACGGAGGCCGACC 68 HENZ-84 Reference probe CTCCCCGCGGCC 69 HENZ-78 Mutant probe CGATGACCGCGGG 70 HENZ-79 Mutant probe TGCAGTGAAAGGACGT 71 HENZ-80 Mutant probe CCGCGGAGAGGCG 72 HENZ-81 Mutant probe CCCGCGACCATCG 73 HENZ-82 Mutant probe AGGGTCGACCTCCG 74 HENZ-83 Mutant probe ACGGAGACCGACCC 75 HENZ-84 Mutant Probe CTCCCCACGGCCA Petition 870250086789, dated 09 / 25 / 2025, pp. 246 / 280 223 / 224 References

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[0467] Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Zídek, A., Potapenko, A. and Bridgland, A., 2021. Forecast Petition 870250086789, dated 09 / 25 / 2025, pp. 247 / 280 224 / 224 highly accurate protein structure with AlphaFold. Nature, 596(7873), pp. 583-589.

[0468] Mascher, M., Gundlach, H., Himmelbach, A., Beier, S., Twardziok, SO, Wicker, T., ... & Bayer, M. (2017). An ordered sequence capture of the chromosomal conformation of the barley genome. Nature, 544(7651), 427433.

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[0471] Nakata M, Fukamatsu Y, Miyashita T, Hakata M, Kimura R, Nakata Y, Kuroda M, Yamaguchi T and Yamakawa H (2017) High temperature-induced expression of rice alpha-amylases in the developing endosperm produces calcareous grains. Front. Plant Sci. 8:2089. doi: 10.3389 / fpls.2017.02089

[0472] Olsen, FL 1987. Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare. The effect of ammonium nitrate, glutamine and asparagine as nitrogen sources - Carlsberg Research Communications 52: 393-404

[0473] Stockinger, EJ. The cultivation of winter-hardy malted barley. Plants 2021, 10, 1415. https: / / doi.org / 10.3390 / plants10071415. Petition 870250086789, dated 09 / 25 / 2025, pages 248 / 280

Claims

1. A barley plant or part thereof, characterized in that said barley plant has high α-amylase activity in a half-grain endosperm without an embryo, and in that said barley plant carries a point mutation in the CXE2L1 gene, wherein said mutant CXE2L1 gene encodes a mutant CXE2L1 polypeptide, wherein the wild-type CXE2L1 is a polypeptide of SEQ ID NO: 1 or a functional homolog thereof that shares at least 95% sequence identity with it, preferably the wild-type CXE2L1 is a polypeptide of SEQ ID NO:

1.

2. Barley plant or part thereof, according to claim 1, characterized in that the mutant CXE2L1 polypeptide comprises an amino acid substitution selected from the group consisting of: a. the amino acid corresponding to S113 or A127 of SEQ ID NO: 1; or b. the amino acid corresponding to P125, A126 or A212 of SEQ ID NO: 1; or c. the amino acid corresponding to F13, L14, G83, G84, G85, L88, Q93, F96, H166, S167, A168, F201, S219, L220, T221, M224, L228, H300, G301, F302, I304 or R305 of SEQ ID NO: 1; or d. the amino acid corresponding to E11, D12, G15, V16, V17, Q18, R27, E30, L33, T35, Y63, Y80, F81, H82, Y86, C87, G89, S90, I91, A92, P94, N95, H97, S98, L99, C100, Y116, L118, S164, G165, G169, A170, N171, L172, A173, L198, S199, A200, F202, A203, G217, V218, T222, A223, A225, D226, Q227, W229, R230, M231, S232, L233, A244, V265, P267, S269, D270, Petition 870250086789, dated 09 / 25 / 2025, page.249 / 280 2 / 8 V271, L272, F295, E298, Q299, P303, Q306, P307, S309, T311, A312 ou L315 da SEQ ID NO: 1; ou e. o aminoácido correspondente to V9, V10, L19, L20, S24, V25, V26, G28, D29, A31, V32, R34, N36, G37, L39, P40, V42, V45, Q46, W47, D49, Y52, L58, S59, V60, R61, A62, R64, P65, L78, V79, L101, R102, A103, A104, A105, A109, V110, V111, L112, S113, V114, Q115, R117, A119, P120, E121, H122, R123, L124, A127, I128, D130, G131, F134, W151, F162, L163, H174, H175, V176, T177, V178, I196, L197, G204, A205, R207, T208, T210, E211, P214, P215, E216, P234, V235, A237, S238, M239, H241, P242, L243, N245, P246, L263, V264, A266, L268, R273, D274, R275, V276, Y279, V293, Q294, E296, G297, F308, E310, S313, E314, L316, R317 V318, I319 or R320 da SEQ ID NO: 1; f. o aminoacido correspondente to Y86, Y116, R117, H122, R123, L124, P125, A126, I128, D129, D130, G131 or L172 da SEQ ID NO: 1; g.the amino acid corresponding to S59, V60, R61, Y63, Y80, I91, C100, V111, L112, V114, or Q115 of SEQ ID NO: 1; where wild-type CXE2L1 is CXE2L1 of SEQ ID NO: 1 or a functional variant thereof with at least 95% sequence identity with it.

3. Barley plant or part thereof, according to any one of claims 1 to 2, characterized in that the substitution in CXE2L1 is a substitution of an alanine for a valine, a substitution of a proline for a serine, a substitution of an alanine for a threonine, or a substitution of a serine for a proline. Petition 870250086789, dated 09 / 25 / 2025, pp. 250 / 280 3 / 8 4. Barley plant or part thereof, according to any one of claims 1 to 3, characterized in that the substitution in CXE2L1 is a substitution in: a. amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of an alanine (A) for a valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein said substitution is a substitution of a serine (S) for a proline (P); and / or c. amino acid 125 of SEQ ID NO: 1, wherein said substitution is a substitution of proline (P) for serine (S); and / or d. amino acid 126 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or e. amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for threonine (T); and / or f. amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for threonine (T); g.amino acid 212 of SEQ ID NO: 1, where the said substitution is a substitution of alanine (A) for valine (V).

5. Barley plant or part thereof, according to any one of claims 1 to 4, characterized in that the substitution at CXE2L1 is a substitution of an alanine for a valine or a substitution of a serine for a proline, for example, wherein said substitution comprises a substitution at: a. amino acid 127 of SEQ ID NO: 1, wherein said substitution is a substitution of alanine (A) for valine (V); and / or b. amino acid 113 of SEQ ID NO: 1, wherein said substitution is a substitution of a serine (S) for a proline (P).

6. A barley plant or part thereof, according to any one of claims 1 to 5, characterized in that said mutant CXE2L1 gene encodes the mutant CXE2L1 polypeptide with gain of function.

7. Barley plant or part thereof, according to any one of claims 1 to 6, characterized in that the grains of said barley plant have a reduced β-glucan content.

8. Barley plant or part thereof according to any one of 1 to 7, characterized in that the β-glucan content of the wort prepared from said barley plant or part thereof is at most 300 mg / L, such as at most 250 mg / L, such as at most 200 mg / L, such as at most 150 mg / L.

9. A barley plant or part thereof according to any one of 1 to 8, characterized in that said barley plant carries a mutation in a gene encoding a β-glucan synthase.

10. Barley plant or part thereof, according to claim 8, characterized in that the gene encoding a β-glucan synthase is CslF6, wherein the said mutant CslF6 gene encodes a mutant CslF6 polypeptide, wherein the wild-type CslF6 is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, or a functional variant thereof with at least 95% sequence identity therewith, optionally wherein the said mutant CslF6 polypeptide is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, except that the mutant CslF6 comprises a substitution of: a. amino acid 847, wherein the said substitution is the substitution of a glycine (G) by a glutamic acid (E); and / or b. amino acid 748, wherein the said substitution is the substitution of a glycine (G) by an aspartic acid (D); and / or c. a substitution of amino acid 709, wherein the said substitution is the substitution of a threonine (T) by an isoleucine (I).

11. A barley plant or part thereof, according to claim 8, characterized in that the gene encoding a β-glucan synthase is CslF6, wherein said mutated CslF6 gene encodes a loss-of-function mutant CslF6 polypeptide, wherein the wild-type CslF6 is CslF6 of SEQ ID NO: 7 or SEQ ID NO: 8, or a functional variant thereof with at least 95% sequence identity therewith.

12. Barley plant or part thereof, according to any of claims 1 to 11, characterized in that it has the condition that the plant does not contain SEQ ID NO:

5. Petition 870250086789, dated 09 / 25 / 2025, p. 253 / 280 6 / 8 13. Barley plant or part thereof, according to any one of claims 1 to 12, characterized in that the high α-amylase activity in a half grain of endosperm without the embryo is at least 5 U / g, preferably at least 10 U / g on a dry weight basis.

14. Barley plant or part thereof, according to any one of claims 1 to 13, characterized in that the α-amylase activity is measured after soaking half the grain and the whole grain in water for at least 24 h, such as at least 48 h, such as at least 72 h.

15. Barley plant or part thereof, according to any one of claims 1 to 14, characterized in that said barley plant comprises an amy1_1 cluster comprising at least 5 functional genes, each encoding an α-amylase, wherein said α-amylase has an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 37-47, or their respective functional homologs with at least 80%, as at least 85%, as at least 90%, as at least 95%, as at least 98%, as at least 99% sequence identity with them.

16. A barley plant or part thereof, according to any one of claims 1 to 15, characterized in that the barley plant comprises a mutation in one or more additional genes, for example, one or more of the following mutations: a. a mutation in the gene encoding LOX-1, resulting in the total loss of functional LOX-1; Petition 870250086789, dated 09 / 25 / 2025, p. 254 / 280 7 / 8 b. a mutation in the gene encoding LOX-2, resulting in the total loss of functional LOX-2; c. a mutation in the gene encoding MMT, resulting in the total loss of MMT functionality; d. a mutation in the gene encoding ANT-28, resulting in the total loss of ANT-28 functionality; e. a mutation in the gene encoding HRT, resulting in the total loss of HRT function; f. a mutation in the gene encoding LDI, resulting in the total loss of LDI function.

17. Vegetable product characterized in that it comprises the barley plant or part thereof as defined in any of claims 1 to 16.

18. Vegetable product, according to claim 17, characterized in that the vegetable product is a malt, such as, for example, ground malt, green malt and / or ground green malt.

19. Method for producing an aqueous extract, said method being characterized in that it comprises the steps of: a. providing grains of a barley plant as defined in any one of claims 1 to 16; b. subjecting the barley grains to a germination step, thereby obtaining germinated grains; c. finely dividing said germinated grains; d. optionally, drying the finely divided germinated grains; e. preparing an aqueous extract of said finely divided (dried) germinated grains, Petition 870250086789, dated 09 / 25 / 2025, pp. 255 / 280 8 / 8 f. thus producing an aqueous extract of barley.

20. A method for producing a beverage, said method being characterized in that it comprises the steps of: a. providing grains from a barley plant as defined in any one of claims 1 to 16, and / or malt as defined in claim 18; b. preparing an aqueous extract of said grains and / or said malt; c. processing said aqueous extract into a beverage, such as beer.

21. Method according to claim 20, characterized in that the processing step comprises a fermentation step with a microorganism, such as yeast.

22. Method according to any one of claims 20 to 21, characterized in that the aqueous extract is prepared as defined in claim 19.

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