Plant cells and plants having reduced content of steroidal glycoalkaloids and methods of obtaining the same

Abstract

A method for obtaining genetically modified plant cells includes the steps of: i. providing plant cells, and ii. modifying the genome of said plant cells to reduce or suppress the expression of at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM ISO 2 The genes described herein each have a sequence having at least 90%, 90%, and 92% sequence identity with SEQ ID NO:41, 43, or 45, and 47, respectively, or encode amino acid sequences having at least 90% sequence identity with SEQ ID NO:42, 44, or 46, and 48, respectively. A gene-modified plant cell and a gene-modified plant are also provided, as well as a method for obtaining a gene-modified plant and the use of the gene-modified plant in a starch production method or a food production method.

Description

Technical Field

[0001] The techniques presented in this paper generally relate to the field of methods for genetically modified plants. More specifically, the techniques presented in this paper relate to plant cells and plants, particularly potatoes, having a reduced content of at least one steroidal glycoside alkaloid, and methods for obtaining such plant cells and plants. Background Technology

[0002] potato Solanum tuberosum L.) is the world's second most important crop after corn ( Zea mays L.), wheat ( Triticum aestivum ) and rice ( Oryza sativa Potatoes are the fourth most important food crop after starch. They are not only a staple food, but also a common raw material for various industrial applications such as starch-related products, animal feed, and biofuel production.

[0003] Steroidal alkaloids (SA) and their glycosylated forms, known as steroidal glycoside alkaloids (SGA), are nitrogenous secondary metabolites found in various potato species. In cultivated potatoes, α-carboxane and α-solanine are the major SGAs, accounting for over 95% of the total SGA content; while wild potato species exhibit a broader SGA composition, including minor SGAs such as solamarine, solamargine, solasonine, demissine, and leptine. Although the concentration of SGA in potato tubers is relatively low compared to other plant parts (such as flowers, buds, and leaves), its distribution is uneven, with the highest concentration in the pericarp, particularly around the outer buds, and lower concentrations in fresh internal tissues. SGA levels in potato tubers can increase significantly during growth and storage under various abiotic and biotic stresses, such as drought, high temperatures, damage, and light exposure. Although SGAs play a crucial role in plant defense mechanisms, they are generally considered natural toxins with potential adverse effects on human health. Therefore, for food safety reasons, a limit of 200 mg / kg for total SGAs in potato tubers is widely recommended. - ¹Fresh weight (FW).

[0004] Potato starch production generates numerous byproducts, including potato juice (PFJ) and potato pulp, which either require processing or conversion into useful sidestream products. PFJ is rich in economically valuable compounds such as amino acids, minerals, and protein (approximately 2%). Compared to cereal-derived proteins, potato protein is known for its high quality, characterized by its high lysine content. Potato protein, with its hypoallergenic and high nutritional value, has the potential to replace other plant-based and animal-based proteins in food production. Potato pulp, primarily composed of tuber cell wall material and rich in pectin, is a potential source for producing high-value potato dietary fiber. However, the presence of SGAs poses a significant challenge as they accumulate in residual byproducts, complicating the extraction of protein and fiber without affecting their functional properties. Various extraction techniques, such as acid precipitation and thermal coagulation, have been explored for the purification and functional recovery of potato protein from PFJ. However, the functionality of proteins isolated by heat treatment may be compromised, limiting their application to animal feed.

[0005] As mentioned above, there is therefore a need for further methods to manage and / or mitigate the adverse effects of SGA in plants such as potatoes. Summary of the Invention

[0006] This article proposes the purpose of the technology.

[0007] Therefore, the first objective of the technology proposed in this paper is to provide a method for obtaining genetically modified plant cells.

[0008] Another objective of the technology proposed in this paper is to provide a genetically modified plant cell.

[0009] Another objective of the technology proposed in this paper is to provide a method for obtaining genetically modified plants.

[0010] Another objective of the technology proposed in this paper is to provide a genetically modified plant.

[0011] Another objective of the technology presented in this paper is to provide the use of genetically modified plants or parts thereof in starch production methods.

[0012] Another objective of the technology proposed in this paper is to provide the use of genetically modified plants or parts thereof in food production methods.

[0013] Overview According to the corresponding first, second, third, and fourth aspects of the technology proposed herein, at least one of the above-mentioned objectives, or at least one further objective that will become apparent from the following description, is achieved by a method for obtaining genetically modified plant cells, wherein the method comprises the following steps: i. Provide plant cells, and ii. Modify the genome of the plant cell to reduce or suppress the expression of at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 ,in: The gene SMO1-L The sequence having at least 90% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 90% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 The sequence having at least 92% sequence identity with SEQ ID NO: 47, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48, as well as A gene-modified plant cell, wherein the genome of the plant cell has been modified to reduce or suppress the expression of at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 ,in: The gene SMO1-L The sequence having at least 90% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 90% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 The sequence having at least 92% sequence identity with SEQ ID NO: 47, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48, as well as A method for obtaining genetically modified plants includes the following steps: i. Providing genetically modified plant cells obtained by the method according to the first aspect of the present invention or according to the second aspect of the present invention, and ii. Cultivate or regenerate the genetically modified plant cells to obtain a genetically modified plant. as well as A genetically modified plant that comprises or consists of the following: - One or more plant cells obtained by the method according to the first aspect of the technology proposed herein, or - One or more plant cells according to the second aspect of the technology proposed herein; Or it can be obtained by means of the third aspect of the technology proposed in this article.

[0014] Therefore, this invention is based on the inventors' discovery that by reducing or inhibiting the selection of... SMO1-L , DWF7-L and TAM iso 2 The expression of at least one gene in the plant can reduce or prevent the production of SGA in plants.

[0015] As seen in the examples, inactivation of at least one of these genes reduced SGA content in plant cells and plants (including tubers) without significantly affecting plant growth, including tuber yield. In contrast, the other genes tested in Example 1, namely... DWF1-L , 16DOX and CYP88B1 Overall, it offers relatively few advantageous features.

[0016] Genetically modified plant cells and genetically modified plants are not obtained solely through methods that are essentially biological.

[0017] Genetically modified plant cells and genetically modified plants refer to plant cells and the plant genome that have been intentionally genetically modified.

[0018] The genetically modified plant cells have a reduced content of at least one steroidal glycoside alkaloid.

[0019] The reduction in the content of at least one steroidal glycoalkaloid is in comparison to wild-type plant cells or wild-type plants or a subset of wild-type plants. Wild-type plant cells or wild-type plants correspond to plant cells before genome modification and plants obtained from plant cells with unmodified genomes.

[0020] Plant cells can be contained in or contained in plant protoplasts, tissue cultures, and intact plant cells within a plant.

[0021] The term plant encompasses all parts of a plant, including flowers, seeds, leaves, stems, roots, and tubers. A part of a plant may also contain plant callus or plant clumps.

[0022] Therefore, the reduction in the content of at least one steroidal glycoside alkaloid can apply to the whole plant or a part of the plant. A part of the plant may include flowers, seeds, leaves, stems, roots, and tubers. Preferably, the part of the plant is a leaf or a tuber, more preferably a tuber.

[0023] Therefore, the plant can have a reduced content of at least one steroidal glycoside alkaloid by having a reduced content of at least one steroidal glycoside alkaloid in one or more tubers of the plant.

[0024] Alternatively, or additionally, the plant may have a reduced content of at least one steroidal glycoside alkaloid by having a reduced content of at least one steroidal glycoside alkaloid in one or more leaves of the plant.

[0025] Alternatively, or additionally, the plant may have a reduced content of at least one steroidal glycoside alkaloid by having a reduced content of at least one steroidal glycoside alkaloid in one or more flowers, one or more seeds, one or more stems, or one or more roots of the plant.

[0026] Wild-type plants, especially wild-type Solanum tuberosum The plant, namely the potato plant, has a total content of steroidal glycoalkaloids in its leaves of 1500 to 1600 mg / kg, for example 1525 to 1575 mg / kg, for example 1565 mg / kg of the leaves.

[0027] The total content of steroidal glycosides and alkaloids in the tubers of such wild-type plants can also be 80-100 mg / kg, for example 85-95 mg / kg or 90.7 mg / kg of the tubers.

[0028] Typically, the total content of steroidal glycoalkaloids in plant cells and plants or parts of plants is less than 1500 mg / kg, preferably less than 1000 mg / kg, more preferably less than 500 mg / kg, and most preferably less than 250 mg / kg of the plant cells, the plant or parts of the plant.

[0029] Alternatively, the total content of the steroidal glycoalkaloids α-solanine and α-carboxine in plant cells and plants or parts of plants is less than 1500 mg / kg, preferably less than 1000 mg / kg, more preferably less than 500 mg / kg, and most preferably less than 250 mg / kg in the plant cells, the plant or parts of the plant.

[0030] Alternatively, the content of the steroidal glycoalkaloid α-solanine in the plant cells and the plant or part of the plant is less than 600 mg / kg, preferably less than 200 mg / kg, more preferably less than 125 mg / kg, and most preferably less than 80 mg / kg of the plant cells, the plant or part of the plant.

[0031] Alternatively, the content of the steroidal glycoalkaloid α-carboxane in the plant cells and the plant or part of the plant is less than 1000 mg / kg, preferably less than 300 mg / kg, more preferably less than 200 mg / kg, and most preferably less than 150 mg / kg of the plant cells, the plant or part of the plant.

[0032] The aforementioned scope particularly pertains to gene modification in plant leaves, and more specifically to the fourth aspect of the technology proposed herein. Solanum tuberosum The content in the leaves of plants, specifically genetically modified potato plants (total content, α-solanine and α-carboxine, α-solanine alone and α-carboxine alone).

[0033] For one or more tubers of a genetically modified plant, the total content of steroidal glycoside alkaloids is preferably less than 90 mg / kg, more preferably less than 45 mg / kg, more preferably less than 25 mg / kg, even more preferably less than 15 mg / kg, and most preferably less than 10 mg / kg of the one or more tubers.

[0034] These areas particularly relate to gene modification based on the fourth aspect of the technology presented in this paper. Solanum tuberosum A plant, specifically one or more tubers of a genetically modified potato plant.

[0035] The plant cells can be obtained from wild-type plants. Sources of plant cells include plant protoplasts, plant cells from tissue cultures from which plants can regenerate, plant callus, plant cell masses, and intact plant cells from plants or plant parts (such as pollen, meristems, flowers, seeds, leaves, stems, etc.).

[0036] The method according to the first aspect of the technique proposed in this paper can be performed on a single plant cell or multiple plant cells. The method can also be applied to one or more plant cells present in a plant.

[0037] Modifying the genome of a plant cell involves altering genes or parts of genes within the plant cell genome. This could include, for example, modifying a portion of a gene, such as a regulatory element or sequence. Regulatory elements include cis-regulatory elements and trans-regulatory elements.

[0038] Cis-regulatory elements are defined as non-coding DNA regions that bind to transcription factors to regulate the transcription of adjacent genes. Trans-regulatory elements are defined as DNA regions that encode regulators (such as transcription factors), which may be located far from the gene they regulate or on a different strand. Transcription factors bind to regulatory sequences and regulate the expression of these adjacent genes.

[0039] Therefore, gene expression regulation can also be achieved through mutations in cis or trans regulatory sequences. Thus, gene downregulation can be achieved by inhibiting or repressing activator transcription factors or by upregulating repressor transcription factors.

[0040] The genome of a plant cell is preferably stably modified such that these modifications are heritable if the plant cell is propagated.

[0041] The at least one gene is selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 As will become apparent below, genes DWF7-L Including genes DWF7-L (a) and DWF7-L (b) .

[0042] These genes all encode proteins that function in the sterol and SGA biosynthetic pathways.

[0043] The genes and their corresponding encoded enzymes are listed below: SMO1-L (Sterol methyl oxidase 1-like, SMO1-1-like) (Gene ID: PGSC0003DMG400012763 – SEQ ID NO: 41).

[0044] It is speculated that SMO1-L uses 24,25-dihydrolanosterol as a substrate to produce 31-nordihydrolanosterol in the pre-cholesterol pathway. Further speculation suggests that SMO1-L uses cycloatinol as a substrate to produce 31-norcycloatinol in the pre-cholesterol pathway.

[0045] DWF7-L (Δ7-C5 sterol desaturase-like) includes two variants: DWF7-L (a) (Δ7-C5 sterol desaturase-like) (Gene ID: PGSC0003DMG400026401 – SEQ ID NO: 43), and DWF7-L (b) (Δ7-C5 sterol desaturase-like) (Gene ID: PGSC0003DMG402026400 – SEQ ID NO: 45) It is speculated that DWF7-L ( DWF7-L (a) and DWF7-L (b) 7-Dehydrocholesterol is produced using 7-encholesterol as a substrate in the pre-cholesterol pathway.

[0046] TAM iso 2 (GABA transaminase isoform 2, GAME12 / PGA4), (Gene ID: PGSC0003DMG400025228 – SEQ ID NO: 47) It is speculated that TAM iso 2 16,22-dihydroxy-26-oxo-cholesterol was produced by using 16,22-dihydroxy-26-amino-cholesterol as a substrate in the post-cholesterol pathway.

[0047] Preferably, the gene SMO1-L It has a sequence having at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 41, or alternatively encodes an amino acid sequence having at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 42.

[0048] Preferably, the gene DWF7-L It has a sequence having at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 43 or 45, or alternatively encodes an amino acid sequence having at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 44 or 46.

[0049] Preferably, the gene TAM iso 2 It has a sequence that has at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 47, or alternatively encodes an amino acid sequence that has at least 95%, preferably at least 98%, for example at least 99% or 100% sequence identity with SEQ ID NO: 48.

[0050] Most preferably, the gene SMO1-L It has the sequence according to SEQ ID NO: 41, or alternatively encodes the amino acid sequence according to SEQ ID NO: 42.

[0051] Most preferably, the gene DWF7-L It has the sequence according to SEQ ID NO: 43 or 45, or alternatively encodes the amino acid sequence according to SEQ ID NO: 44 or 46.

[0052] Most preferably, the gene TAM iso 2 It has the sequence according to SEQ ID NO: 47, or alternatively encodes the amino acid sequence according to SEQ ID NO: 48.

[0053] The reduction or suppression of the expression of the at least one gene may include a decrease in transcription rate or success rate, a decrease or absence of enzyme activity of the protein encoded by the at least one gene, or a partial or complete deletion of the gene sequence.

[0054] The reduction or suppression of the expression of at least one gene can be achieved, for example, by gene silencing, antisense, RNA interference (RNAi), virus-induced gene silencing (VIGS), small RNA-mediated posttranscriptional gene silencing, transcription activator-like effector nuclease (TALEN) gene editing technology, clustered regular-spaced short palindromic repeat (CRISPR / Cas) gene editing technology and / or zinc finger nuclease (ZFN) gene editing technology.

[0055] Therefore, the genome of a plant cell can be modified to achieve reduced or suppressed expression at the gene level, for example, by removing or altering a gene to affect the abundance and function of proteins produced from said at least one gene. Alternatively, reduced or suppressed expression can be provided at the transcriptional stage, for example, by modifying a gene to reduce the likelihood that said at least one gene will be transcribed into mRNA.

[0056] Additionally, reduced or inactivated expression can be provided at the mRNA stage by reducing the likelihood of mRNA being translated into protein (e.g., translational control) or by causing rapid mRNA degradation.

[0057] Genetically modified plant cells can be cultured or regenerated to obtain genetically modified plants.

[0058] Preferably, the at least one steroidal glycoside alkaloid is selected from the group consisting of: α-solanine, α-carboxine, solanine, solanine, tomatine, and tomatidine.

[0059] More preferably, the at least one steroidal glycoside alkaloid includes one or both of α-solanine and α-carboxine, because they are Solanum tuberosum The main steroidal glycoalkaloids in plants. Furthermore, as shown in Example 1, the content of these glycoalkaloids can be specifically reduced.

[0060] Preferably, the gene is selected from the group consisting of: SMO1-L and DWF7-L More preferably, the gene is SMO1-L These genes, especially SMO1-LIt yields the best results in terms of steroidal glycoside alkaloid concentration and growth (e.g., plant height and tuber yield).

[0061] When the gene is SMO1-L At that time, the genome of plant cells can be advantageously modified additionally to reduce gene density. DWF1-L The expression of (gene ID: PGSC0003DMG400021142) is because this provides very low levels of steroidal glycoside alkaloids.

[0062] Preferably, step ii includes mutating or removing at least a portion of the gene.

[0063] Therefore, in genetically modified plant cells, the gene is preferably mutated or at least a portion of the gene has been removed.

[0064] In genetically modified plant cells, the gene has preferably been mutated in vitro. In other words, in genetically modified cells, the gene is not spontaneously mutated.

[0065] When the gene has been mutated, the gene is preferably selected from the group consisting of: SMO1-L and DWF7-L .

[0066] Preferably, step ii is performed using CRISPR / Cas.

[0067] Therefore, in genetically modified plant cells, the plant genome has been modified using CRISPR / Cas.

[0068] This is advantageous because it allows for the removal of at least one of the genes, thereby providing complete inactivation of that gene.

[0069] CRISP / Cas can preferably be CRISPR / Cas9 or CRISPR / Cas12, such as CRISPR / Cas12a or CRISPR / Cas12b.

[0070] The genetically modified plant cells can be derived from the Solanaceae family, preferably from the Solanum genus, and more preferably from the potato species (Potato). Solanum tuberosum In other words, the genetically modified plant cells can be genetically modified potato cells.

[0071] Accordingly, the genetically modified plant can be from the Solanaceae family, preferably from the Solanum genus, and more preferably from the potato species. In other words, the genetically modified plant can be a genetically modified potato plant.

[0072] The fifth aspect of the technology proposed herein relates to the use of genetically modified plants or a portion thereof, preferably their tubers, in a starch production method according to the fourth aspect of the technology proposed herein, wherein preferably, at least one of amino acids, minerals, proteins and dietary fiber is obtained and collected as a byproduct of said method.

[0073] This is advantageous because the reduced content of steroidal glycoalkaloids in the plant makes it easier to obtain and collect the byproducts of the method, particularly amino acids, minerals, proteins, and dietary fiber.

[0074] The use of the fifth aspect can alternatively be regarded as a method for using genetically modified plants, preferably portions thereof, more preferably tubers, according to the fourth aspect of the present invention in a starch production process, wherein preferably, the method includes obtaining and collecting at least one of amino acids, minerals, proteins and dietary fiber as a byproduct of the method.

[0075] The preferred portion of the plant is the starch-containing part, such as the part of the plant with the highest starch content.

[0076] It is preferable to use plant tubers because tubers typically have a high starch content.

[0077] Methods for producing starch may include one or more of the following steps: - Clean the plant, its parts, or its tubers. - To break (grind) the cell walls of the plant, its parts, or its tubers to produce juice and slurry. - Optional separation of juice and pulp, - Starch is extracted from the slurry by washing to produce a starch milk. - Refined starch milk, for example by removing fiber from said starch milk, and - Dehydrates refined starch milk and dries the starch.

[0078] At least one of amino acids, minerals, proteins, and dietary fiber is obtained and collected as a byproduct of the method. At least one of the amino acids, minerals, proteins, and dietary fiber can be obtained and collected from aqueous solutions or suspensions, particularly juices, obtained during the processing of plants, plant parts, or plant tubers to extract starch.

[0079] Preferably, at least one of protein and dietary fiber is obtained and collected.

[0080] The sixth aspect of the technology proposed herein relates to the use of genetically modified plants, preferably portions thereof, more preferably tubers, in a food production method according to the fourth aspect of the technology proposed herein, wherein the food is preferably selected from the group consisting of: peeled potato tubers, mashed potatoes or ready-to-eat mashed potatoes, fried potato products such as French fries or potato chips, and potato chips.

[0081] This is advantageous because the reduced content of steroidal glycoside alkaloids in plants provides safer food.

[0082] The use of the sixth aspect can be alternatively regarded as a method of using genetically modified plants, preferably portions thereof, more preferably tubers, according to the fourth aspect of the technology presented herein in a food production process, wherein the food is preferably selected from the group consisting of: peeled potato tubers, mashed potatoes or ready-to-eat mashed potatoes, fried potato products such as French fries or potato chips, and potato chips.

[0083] Corresponding to the first and second aspects, the seventh aspect of the technology proposed herein relates to the use of plant cell genomes selected from free... SMO1-L , DWF7-L and TAM iso 2 The group consists of at least one gene whose expression is reduced or inhibited to decrease the content of at least one steroidal glycoside alkaloid in plant cells.

[0084] The eighth aspect of the technology proposed in this paper relates to the offspring of plants according to the fourth aspect of the technology proposed in this paper.

[0085] The ninth aspect of the technology proposed herein relates to seeds obtained from plants according to the fourth aspect of the technology proposed herein.

[0086] The tenth aspect of the technology proposed herein relates to cuttings or grafts of plants according to the fourth aspect of the technology proposed herein.

[0087] The eleventh aspect of the technology proposed in this paper relates to plant callus tissue according to the fourth aspect of the technology proposed in this paper.

[0088] The first and second alternative aspects of the technology proposed herein relate to a method for obtaining plant cells with a reduced content of at least one steroidal glycoside alkaloid, wherein the method comprises the following steps: i. Provide plant cells, and ii. Providing at least one reagent in the plant cells, said reagent: a. Reduce or inhibit the enzyme activity of proteins encoded by at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 ,or b. Reduce or suppress the expression of at least one gene selected from the following groups: SMO1-L , DWF7-L and TAM iso 2 , as well as A plant cell having a reduced content of at least one steroidal glycoside alkaloid, wherein the plant cell comprises at least one reagent, said reagent: a. Reduce or inhibit the enzyme activity of proteins encoded by at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 ,or b. Reduce or suppress the expression of at least one gene selected from the following groups: SMO1-L , DWF7-L and TAM iso 2 .

[0089] The first and second alternative aspects of the proposed technology provide plant cells that can be used in the third and fourth aspects of the proposed technology, which are further described above. Attached Figure Description

[0090] A more complete understanding of the foregoing and other features and advantages of the art proposed herein will become apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, wherein: Figures 1A-1B This demonstrates the pre-cholesterol pathway in SGA biosynthesis. Figure 1A ) and post-cholesterol pathway ( Figure 1B The structures of the target key genes in the diagram are shown, with sgRNA and analysis primers represented by vertical and horizontal arrows, respectively.

[0091] Figure 2A- Figure 2E This shows mutant potato events generated by targeting key genes in the pre- and post-cholesterol pathways of SGA via DNA-free CRISPR / Cas9. Figure 2A: Height of four-month-old wild-type and selected 36 mutant events grown under controlled conditions. Figure 2B : Tuber yield of wild type and 36 mutation events harvested after six months of cultivation. Figure 2C : The general appearance of wild-type plants (three months old) and tubers at harvest. Figure 2D and Figure 2E Pre-cholesterol pathway ( Figure 2D ) and post-cholesterol pathway ( Figure 2E Representative mutation events targeting different genes (3 months old) and the general appearance of their corresponding tubers (at harvest). Figure 2A and Figure 2BThe data in the table represent the mean ± standard deviation, n = 3–27, depending on the number of mutation events in each mutant group for each gene. P-values ​​were calculated using one-way ANOVA combined with the Dunnett test and are indicated by an asterisk (*). P<0.05, P<0.01, P<0.001, P<0.0001). The scale bar in the tuber is 5 cm.

[0092] Figures 3A-3B Detailed information on plant height and tuber yield for all 36 independent events is displayed. Statistical analysis was performed using one-way ANOVA combined with Dunnett's test. P-values ​​are indicated by an asterisk (p-value). P<0.05, P<0.01, P<0.001, P<0.0001).

[0093] Figure 4A The TGA level of independent mutation events is shown. Statistical analysis was performed using one-way ANOVA to compare with wild-type untreated tuber samples; p-values ​​<0.05 are indicated by an asterisk.

[0094] Figure 4B The figures show the results in tubers at harvest (untreated), tubers that underwent damage treatment for 2 days (damaged), and tubers exposed to light for 8 days (light). DWF7-L , SMO1-L and TAM iso 2 TGA levels of genes.

[0095] Figures 5A-5B The average SGA level of the selected 36 mutation events is shown. Figure 5A The levels of major SGA (α-solanine and α-carboxane) and TGA (total SGA) in leaves of mutant potato events (8 weeks old). Figure 5B The levels of TGA in tubers at harvest (untreated), tubers that underwent damage treatment for 2 days (damage), and tubers exposed to light for 8 days (light). Figure 5A and Figure 5B The data in the table represent the mean ± standard deviation, n = 2–16, depending on the number of mutation events for each gene. One-way ANOVA combined with Dunnett's post-hoc test was used for statistical analysis. P-values ​​are indicated by an asterisk (ns, not significant). P<0.05, P<0.01, P<0.001, P<0.0001).

[0096] Figure 6 The sterol profiles in the mutation events are shown. The relative metabolite levels of eight sterols in the leaves of the mutant plants are also presented. Values ​​are expressed as mean ± standard deviation, n = 2–16, depending on the number of mutation events per gene. Statistical analysis was performed using one-way ANOVA combined with the Dunnett test. P-values ​​are indicated by an asterisk (p-value). P<0.05, P<0.01, P<0.001, P<0.0001).

[0097] Figure 7 This shows the process of obtaining starch. Solanum tuberosum L. Total glycoalkaloid levels in starch, fiber, and protein components obtained from typical processing of tubers of wild-type and cultivated varieties of the Kuras mutant potato.

[0098] Figure 8 Showing Solanum tuberosum L. Total glycoalkaloid levels in tubers of wild-type and cultivated variety Kuras mutant potatoes after two postharvest treatments (damage, light exposure). Detailed Implementation

[0099] Example 1 – Producing glycoalkaloid-free potato plants by CRISPR / Cas9-mediated mutation of key genes in the glycoalkaloid biosynthesis pathway. 1.1 Materials and Methods 1.1.1 Plant materials and growth conditions This study adopted Solanum tuberosum L. kuras, a potato starch cultivar. In vitro shoots were cultured in a controlled culture chamber under LD (16 hours light / 8 hours dark) conditions, with a photosynthetic photon flux density of 80 μmol photons / m². - ²s - ¹, The culture temperature was 24 °C (18 °C during the dark period). Three copies of both the mutant and wild-type plants were cultured in the greenhouse under the same conditions as in vitro, but with natural light supplemented by mercury lamps (approximately 70 μmol / m²). - ²s - ¹). All mutation events were cultivated simultaneously and subjected to uniform growth conditions in a greenhouse.

[0100] 1.1.2 Obtaining CRISPR / Cas9 mutant plants Genomic DNA was extracted from leaf tissues of the cultivar Kuras using the GeneJet Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham, USA) and used to amplify target regions in different genes to identify homologous regions among all four alleles. For each gene, CRISPR RGEN Cas-Designer was used. http: / / www.rgenome.net / cas-designer / ) and CRISPOR ( http: / / crispor.tefor.net / crispor.py Two sgRNAs targeting different exons in the homologous regions of alleles were designed. Mutations were induced in the cultivar Kuras via polyethylene glycol (PEG)-mediated protoplast transfection. In vitro propagation, protoplast isolation, and shoot regeneration were performed according to the methods described by Nicolia et al. (2015; 2021). RNPs were transfected into freshly isolated protoplasts according to the methods described by Andersson et al. (2018), using 0.1 nmol of synthetic sgRNA (Synthego, Redwood City, CA) and 5 µg TrueCut™ Cas9 v.2 (Thermo Fisher, Waltham, USA) for each target. Protoplast transfection was performed by treating with 25% PEG (PEG 4000, Sigma-Aldrich, Germany) at room temperature for 3 min. Only one regenerated shoot was selected from a single callus and transferred to root regeneration. Preliminary screening of mutants in regenerated shoots was performed using high-resolution fragment analysis (HRFA), followed by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) according to the method described by Andersson et al. (2017). The primers and sgRNAs used in this study are listed in Tables 1 and 2 below, with the SEQ ID NO (NO) of the corresponding sequence also given in the last column.

[0101] Table 1. Primers used

[0102] The forward primer has a FAM for labeling HRFA ​​( ) or HEX ( ).

[0103] Table 2. sgRNAs used

[0104] The sequences of the target genes and their expression products are shown in Table 3 below: Table 3. Sequences of target genes and their expression products

[0105] 1.1.3 Glycoside and Alkaloid Analysis Referring to Simonovska & Vovk (2000) and adapting the in vitro plant materials, α-carboxane and α-solanine (Sigma) were used as internal standards, and thin-layer chromatography (TLC) was employed to rapidly screen for SGA levels in 5-week-old in vitro mutation events.

[0106] Leaf and tuber samples were collected from greenhouse-grown plants at 8 weeks and 6 months of age, respectively. Treated tuber samples were prepared under SGA-induced conditions, with and without damage and light exposure, as described by Petersson et al. (2013b). All prepared samples were immediately frozen in liquid nitrogen and stored at -70 °C for subsequent analysis, including SGA quantification, sterol analysis, and metabolomics analysis.

[0107] The determination of SGA (α-carboxane and α-solanine) in leaf and tuber samples from both the mutant and wild-type plants was performed by LC-MS analysis, basically as described by Merino et al. (2023).

[0108] 1.1.4 Sterol Analysis The determination of sterols was performed by GC-MS analysis, basically as described by Merino et al. (2023).

[0109] 1.2 Results and Discussion 1.2.1 Producing potatoes by inducing mutations in key genes of SGA biosynthesis using DNA-free CRISPR / Cas9 event To generate SGA-free potato mutants, CRISPR / Cas9-mediated mutagenesis targeted six genes encoding enzymes that play key roles in the pre-cholesterol or post-cholesterol portions of the SGA biosynthesis pathway. The target genes functioning in the pre-cholesterol pathway are: DWF1-L , SMO1-L and DWF7-L ; TAM Type 2 ( GAME12 ), 16DOX ( GAME11 )and CYP88B1 It plays a role in the post-cholesterol pathway of SGA biosynthesis.

[0110] For each gene, two different sgRNAs were designed, each targeting a different exon, and used as single targets to knock out the corresponding key gene (Figure 1). Furthermore, a dual-gene knockout strategy was employed for SMO1-L + DWF1-L, in which the two sgRNAs were multiplexed in different combinations. HRFA ​​was used for preliminary screening of mutations in regenerated shoots, and editing efficiency was calculated based on the number of shoots with at least one mutated allele. Mutation types were divided into three groups: Group 1, complete knockout, where all alleles had insertions / deletions outside the frame; Group 2, complete knockout, where at least one allele was an insertion / deletion within the frame; Group 3, partial mutation, where at least one wild-type allele was not edited.

[0111] The editing efficiency of each sgRNA targeting these six genes is shown in Table 4. The highest editing efficiency was found in TAM(iso 2) regenerated shoots, followed by DWF7-L and DWF1-L, with efficiencies of 49.5%, 48.5%, and 37.1%, respectively. For SMO1-L and CYP88B1 regenerated shoots, the editing efficiencies were only 20.4% and 19.9%, respectively. In particular, for SMO1-L, the majority of the results obtained were partial mutations that retained two or three wild-type alleles (Group 3). Given the limited potency of both sgRNAs in SMO1-L, only 57 (17.4%) of the 328 events in both SMO1-L and DWF1-L showed double mutations, and none of those events had all alleles carrying both genes with out-of-frame mutations (Group 1).

[0112] Table 4. Editing efficiency of regenerated shoots targeting different genes

[0113] 1.2.2 Preliminary screening of SGA level of in vitro mutation events Based on the selection of mutant groups, a total of 96 events targeting different genes in the pre-cholesterol and post-cholesterol pathways were selected, with at least 2-3 events in each mutant group, thus covering mutations ranging from complete knockout to partial mutation (mutations in the selected events are shown in Table 5).

[0114] Preliminary screening of α-solanine and α-carboxine levels in extracts from five-week-old in vitro rooting mutant events was performed using TLC analysis. TLC results showed that for the pre-cholesterol mutant, SGA levels were reduced in 30 out of 50 events (60%), while for the post-cholesterol mutant, SGA levels were reduced in 32 out of 46 events (70%) (Table 12). Differences in the frequency of SGA reduction were observed among the mutant genes obtained. For example, for both the SMO1-L + DWF1-L double mutant and 16DOX, SGA levels were reduced in 100% of the selected events, while in the case of the CYP88B1 mutant, only one out of seven events showed a reduced SGA level; this event was the only complete out-of-frame knockout obtained (Table 5).

[0115] Table 5. Mutations and TLC results at the TGA level for the selected 96 events.

[0116] Insertions / deletions in mutation events are represented by positive values ​​for insertions and negative values ​​for deletions. "0" indicates an unedited wild-type allele / multiple alleles. In events lacking "0", the wild-type allele is absent. Note that two or more events may have insertions / deletions of the same size.

[0117] TGA levels were estimated using TLC analysis, ranging from the highest level defined as "+++++" (e.g., TGA levels in wild-type plants) to the lowest level "-" (TGA not detected or below the detection level of TLC analysis).

[0118] 1.2.3 Phenotypic changes between potato mutation events Based on the low TGA levels obtained from TLC screening (mainly group 1 and group 2 mutation events), we subsequently selected 36 events listed in Table 6 below and monitored plant growth and tuber yield under greenhouse conditions. Plant height of the mutant events was in most cases similar to that of the wild-type plants (Figure 2A). Figure 3A Exceptions include those that are 30%–40% significantly shorter than wild-type plants. SMO1-L + DWF1-L Double mutant (Group 2) DWF7-L (Group 1) and 16DOX Complete knockout events (Group 1) significantly impacted tuber yield, particularly those in Group 1 mutations, with reductions ranging from 27% to 87%. Figure 2B ,Replenish Figure 3BThe largest decrease in tuber yield was in [specific region / situation]. SMO1-L + DWF1-L Double mutant, in which both genes are completely mutated (Group 2).

[0119] Whether pre-cholesterol or post-cholesterol mutants, the overall appearance and phenotype were normal in most events compared to the wild type. Figure 2C – Figure 2E However, a few events exhibited anomalous phenotypes, including dwarf growth models with small or irregular leaflets. For example... DWF1-L Event 4h033 in the mutant DWF7-L Events 9e019 and 8f080 in the mutant ( Figure 2C ), SMO1-L + DWF1-L Event 7g026 in the double mutant, and 16DOX Event 9b046 in the mutant. It is noteworthy that in the tubers of the mutant events, except for a few events such as 6d003 ( TAM iso 2 mutant) and 7g026 ( SMO1-L + DWF1-L Apart from the slightly elongated tubers, no abnormal phenotypes were observed in the double mutant.

[0120] Table 6. 36 selected events targeting key upstream and downstream genes in the SGA biosynthesis pathway via CRISPR / Cas9 mutagenesis

[0121] Insertions / deletions in mutation events are represented by positive values ​​for insertions and negative values ​​for deletions, with "0" representing the unedited wild-type allele. In events lacking "0", there is no wild-type allele / multiple alleles. Note that two or more events may have insertions / deletions of the same size.

[0122] 1.2.4 Decreased SGA levels in pre- and post-cholesterol mutation events To further analyze the impact of mutations on SGA metabolism, total SGA (TGA) levels in leaves and tubers from 36 selected mutant events in greenhouse cultivation were quantified using LC-MS analysis. In leaf samples, significantly reduced TGA levels were observed in all 36 events, falling to less than 10% of wild-type levels. The most dramatic reduction was observed in the 16DOX mutant, which showed almost complete absence of TGA, followed by the TAM iso 2 mutant and the SMO1-L + DWF1-L double mutant. Figure 5A , Figure 4AThe LC-MS results for TGA levels in leaves were consistent with the initial TLC screening. In tuber samples, except for the CYP88B1 mutant, TGA levels were also significantly reduced in most events. In the CYP88B1 mutant, except for one event (9d050) which showed no TGA, Figure 4B TGA levels remained similar to those of the wild type. A total of 16 events were identified in which TGA was significantly reduced to below 4 mg / kg FW in tuber samples. These 16 events included five from the DWF1-L mutant (7c028, 7e014, 7d025, 7f023, and 4h033) (not in...). Figure 4B (as shown in the image), three events (7f043, 4h024, and 7f037) from the SMO1-L + DWF1-L double mutant (not shown in the image). Figure 4B (As shown in the image), two events (6d012 and 6d031) from the TAM iso 2 mutant, see Figure 4B All five events in the 16DOX mutant (8b026, 9b014, 9b023, 9b046, and 9b055) (not in Figure 4B (as shown in the image), and a knockout event (9d050) from the CYP88B1 mutant (not shown in the image). Figure 4B (As shown in the image). All five events from 16DOX showed almost no TGA in both leaves and tubers, and all originated from mutant group 1.

[0123] To investigate the effects of SGA-induced conditions on SGA levels, tubers from selected mutation events were subjected to damage and light exposure treatments, respectively. Figure 5B Results labeled "damage" and "light" (from individual events of DWF7-L, SMO1-L, and TAM iso 2) are shown in [the table / image / etc.]. Figure 4B Wild-type tubers showed statistically significant approximately 3-fold SGA accumulation under both damage and light exposure treatments. In contrast, the mutation event showed no increase in TGA levels or only a slight increase in TGA levels in treated tubers, and the final TGA levels were statistically indistinguishable from the corresponding untreated controls in all cases.

[0124] 1.2.5 Changes in the sterol profile during pre- and post-cholesterol mutation events Sterol profiles were analyzed from leaves of potato mutant events to investigate the impact of mutations in key genes involved in sterol biosynthesis. The sterol profiles were altered in most events, particularly in the cholesterol pre-mutant. Figure 6Early cholesterol intermediates cycloatinol and cycloatinanol showed significant accumulation in the SMO1-L event, with levels 174-fold and 12-fold higher, respectively, than in wild-type plants. A similar trend was observed in the SMO1-L + DWF1-L double mutant event, although the accumulation of cycloatinol and cycloatinanol was less. 24,25-dihydrolanosterol was significantly increased in the SMO1-L and SMO1-L + DWF1-L double mutant events, with levels 154-fold and 18-fold higher, respectively, than in wild-type plants, indicating that 24,25-dihydrolanosterol is also a substrate of SMO1-L.

[0125] Compared to the post-cholesterol mutant, the reduction in cholesterol was more pronounced in the pre-cholesterol mutant, with 17% and 54% of cholesterol retained in the pre-cholesterol and post-cholesterol mutants, respectively. This indicates that mutations in genes along the pre-cholesterol pathway effectively block cholesterol synthesis. Notably, a significant increase in lanosterol was only observed in the DWF7-L event, supporting the use of lanosterol as a substrate in DWF7-L. Interestingly, regarding phytosterol levels, only the DWF1-L event showed a significant increase in campesterol, while mutations in DWF7-L resulted in a corresponding decrease in sitosterol and the end product stigmasterol.

[0126] 1.2.6 Discussion Here, we used CRISPR / Cas9 DNA-free technology to target and mutagenesis six key genes involved in the pre- or post-cholesterol biosynthesis pathways of SGA. TLC analysis of 96 mutation events revealed considerable SGA variation both within and between different gene mutants. Nevertheless, most gene mutants exhibited reduced SGA levels in both leaves and tubers, with several events showing tubers with near-SGA-free levels (SGA levels <0.1 mg / kg FW). Figures 5A-5B (Table 5). A correlation was found between the number of mutated alleles (mutant groups) and the degree of SGA reduction in mutational events. Generally, mutations in all four alleles are necessary to achieve significant reductions in SGA levels for mutational events targeting different genes, regardless of whether the mutation is out-of-frame (Group 1) or includes an in-frame inducible mutation (Group 2). Exceptions are the TAM iso 2 and 16DOX mutations, some of which also resulted in significant reductions in SGA levels due to Group 3 mutations. Conversely, in the CYP88B1 mutation, only completely out-of-frame mutations (Group 1) resulted in significant reductions in SGA levels. This variability collectively suggests that these genes encode proteins that restrict SGA biosynthesis to varying degrees.

[0127] Of the 36 mutations selected for further investigation, the degree of SGA reduction correlated with the location of the target gene in the pre-cholesterol or post-cholesterol pathway. Post-cholesterol mutations showed significantly lower SGA levels in both leaves and tubers, with tubers exhibiting a more pronounced reduction. Figures 5A-5B This is likely because the genes in the post-cholesterol pathway are single-copy genes and are more specific for SGA biosynthesis. Knocking out these genes leads to a significant decrease in SGA levels. In contrast, some pre-cholesterol genes are duplicates, such as DWF1-L and DWF1, SMO1-L and SMO1, and DWF7-L and DWF7, which are involved in SGA and sterol biosynthesis, respectively. It can be inferred that when selected pre-cholesterol genes are knocked out, the duplicate counterparts involved in sterol biosynthesis can, to some extent, compensate for the knocked-out genes involved in SGA biosynthesis, thereby maintaining a certain level of both sterols and SGA in the mutant.

[0128] Compared to untreated wild-type tubers, all mutation events (primarily Group 1 or Group 2 mutations) maintained persistently low SGA levels after damage and light exposure. Exceptions were noted in two CYP88B1 mutation events (9d050 and 9d040), where SGA levels in untreated tubers were comparable to wild-type, reflecting the wild-type pattern and showing significant increases after both damage and light exposure. This suggests that different key gene mutations have the ability to block both basal and inducible SGA synthesis.

[0129] Most mutant plants and tubers retained similar morphology to the wild type, with some events showing a slight reduction in height and / or tuber yield (Figure 2A-). Figure 2E However, no correlation was observed between decreased SGA content or even the absence of SGA and plant height or tuber yield. Overall, the distribution and low frequency of phenotypic changes among mutants suggest that the observed phenotypic effects are more likely related to random secondary effects arising from mutation generation or regeneration processes than to the decrease in their SGA levels themselves.

[0130] Both SMO1-L and DWF7-L mutations resulted in a significant decrease in SGA levels, confirming their involvement in the SGA biosynthetic pathway. The accumulation of 24,25-dihydrolanosterol, cycloartenol, and cycloartenanol in the SMO1-L mutation event suggests their role as substrates for SMO1-L. Figure 6 Similarly, the accumulation of lanosterol in DWF7-L mutation events confirms the position of DWF7-L in the cholesterol biosynthesis pathway. Figure 6 ).

[0131] Compared to single mutants of SMO1-L or DWF1-L, the SMO1-L + DWF1-L double mutant showed a greater degree of reduction. Combined with the accumulation of cycloatinol and cycloatinanol in both the SMO1-L mutant and the double mutant, our results suggest that both SMO1-L and DWF1-L contribute to the conversion of cycloatinol to 31-norcycloatinol. The complete in-frame knockout event 7g026 in both SMO1-L and DWF1-L was the only event we obtained that resulted in severe dwarfing and significantly reduced tuber yield compared to wild-type plants. This finding suggests that complete out-of-frame knockout in SMO1-L can be lethal, as indicated by the absence of such mutants. In conclusion, the SMO1-L + DWF1-L double knockout strategy appears to produce a more robust reduction in SGA compared to mutants targeting either SMO1-L or DWF1-L. However, this method has side effects on mutant phenotypes, possibly due to the key role of the duplicated gene in sterol biosynthesis.

[0132] In all nine TAM iso2 knockout events analyzed (mutant group 1 and mutant group 2), SGA levels in leaves and tubers were significantly reduced, while the mutant plants and tubers exhibited normal morphology. This was true regardless of whether the mutation occurred in an early stage (exon 2) or a later stage (exon 16) of the event. This indicates that TAM plays an important and indispensable role in SGA biosynthesis.

[0133] 16DOX is the first gene identified as playing a role in the SGA biosynthesis pathway, encoding 2-oxoglutarate-dependent dioxygenase (2OGD), and 16DOX-silenced potato and tomato plants showed reduced SGA content. The 16DOX mutations obtained in this study showed significantly reduced SGA levels in leaves and complete absence of SGA in tubers. After greenhouse harvest, tubers from both 16DOX events were observed to exhibit inhibited germination and prolonged germination time (not shown) compared to wild-type tubers.

[0134] In the CYP88B1 (GAME4) out-of-frame knockout (event 9d050), SGA levels were significantly reduced in both leaves and tubers. However, this event exhibited a dwarfing phenotype and a significantly reduced tuber yield. Figure 2E These observed phenotypic differences may be attributed to the accumulation of unknown compounds, which was particularly significant in out-of-frame knockouts compared to other mutation events.

[0135] In summary, the obtained SMO1-L, DWF7-L(a, b), and TAM iso 2 mutants, characterized by low SGA levels in tubers and normal phenotype and tuber yield, provide promising genetically modified plants for future industrial applications. This includes developing novel food-grade protein and fiber products from industrial byproducts, as well as new foods with low SGA levels.

[0136] Example 2 – Glycoside Alkaloid Levels in Wild-Type Potato (Cultural Variety Desiree) and RNAi or Antisense Transformers 2.1 Materials and Methods In this embodiment, transformants were obtained using RNAi and antisense methods, and their concentrations of steroidal glycoalkaloids in leaves and tubers were compared with those of wild-type potato (cultivar Desiree). Specifically, the antisense gene TAM iso2 was affected, and RNAi was used to affect the gene SMO1-L.

[0137] 2.1 Results The following results were obtained: Table 7: HPLC glycoside alkaloid content (mg / kg) in leaves of wild-type and antisense transformants – Experiment A

[0138] Table 8: LC-MS glycoside alkaloid content (mg / kg) in leaves of wild-type and RNAi transformants – Experiment B

[0139] Table 9: LC-MS glycoalkaloid content (mg / kg) in leaves of wild-type and RNAi transformants – Experiment C

[0140] Table 10: LC-MS glycoside alkaloid content (mg / kg) in tubers of wild-type and RNAi-transformed strains – Experiment C

[0141] Therefore, as shown in Example 2, in addition to CRISPR / Cas9, other methods can also be used to obtain the genetically modified plant cells according to the first aspect of the present invention.

[0142] Example 3 – Quantification of total glycoside alkaloid levels in different components of potato starch flow products 3.1 Materials and Methods As described in Example 1, the cultivated variety Kuras was obtained through CRISPR / Cas9-mediated mutation. Solanum tuberosum L. potato mutants. Seven types of mutations were prepared, each reducing the expression of the following genes: TAMiso 2. DWF1-L , SMO1-L , SMO1-L and DWF1-L Double mutant 2OGD , GAME4 and GAME9 . GAME9 (GLYCOALKALOIDMETABOLISM 9, ethylene-responsive transcription factor 1-like), (Gene ID: 102597083). It is speculated that GAME9 is an APETALA2 / ethylene-responsive factor that regulates SGA biosynthesis and several upstream mevalonate and cholesterol pre-pathway genes by directly acting on or synergistically binding to downstream target gene promoters with the SlMYC2 transcription factor.

[0143] Mutant and wild-type (Kuras) plants were harvested after being cultivated for XXX time in an outdoor field trial (coordinates).

[0144] The potato tubers from both wild-type and mutant strains were then processed to separate and extract the starch, fiber, and protein components. After washing, the tubers were milled to convert them into a mixture of pulp and potato juice. The pulp contained most of the fiber and starch components, while the potato juice contained the protein components. The pulp was then washed using a steam-guided scrubber to separate the starch and fiber from each other, where a strong water flow washed the starch milk (starch component) out of the pulp (fiber component). The total SGA content in each component of the mutant or wild-type tubers was quantified using LC-MS analysis, as described by Merino et al. (2023).

[0145] 3.2 Results The levels of total glycoalkaloids in the starch, fiber, and protein components of the mutant were lower than those in the wild type, see [link to relevant documentation]. Figure 7 .

[0146] The starch fractions of all mutant genotypes and wild-types contained low levels of glycoalkaloids, below 3 mg / kg fresh weight.

[0147] The measured values ​​can be compared with the maximum content of edible tubers of 200 mg / kg and the maximum protein content of 150 mg / kg as stipulated by the Swedish government.

[0148] The wild-type genotype showed a higher content of glycoalkaloids in its fiber fraction, approximately 1100 mg / kg fresh weight. In contrast, all mutants maintained extremely low levels of glycoalkaloids in their fiber fraction. GAME4The mutant is an exception, with a content of approximately 400 mg / kg fresh weight. Nevertheless, GAME4 The glycoalkaloid content in the fiber component of the mutant is still about one-third that of the wild-type starch component.

[0149] The wild-type genotype had the highest content of glycosidic alkaloids in its protein fraction, approximately 5800 mg / kg fresh weight. Compared to the wild-type, the glycosidic alkaloid content in the protein fraction of all mutants remained consistently at extremely low levels, but... GAME4 The mutant is an exception, with a content of approximately 400 mg / kg fresh weight.

[0150] Similar to the fibrous component, the protein component maintained extremely low levels of glycoalkaloids relative to the wild type in all mutants.

[0151] The protein component with the least reduction in glycosidic alkaloids is GAME4 The mutant contains about a quarter of the protein components found in the wild type.

[0152] This experiment clearly demonstrates that the distribution of glycoalkaloids among different components is not uniform. The starch component has the lowest glycoalkaloid content, the fiber component has a higher content, and the protein component has the highest observed content.

[0153] It is equally clear that mutations in these genes lead to a decrease in the content of glycoalkaloids in all components of starch, fiber, and protein.

[0154] Furthermore, different gene mutations exhibit varying efficiencies in reducing glycoalkaloids. For example, GAME4 The decrease in glycoalkaloids caused by mutation was significantly smaller than that caused by mutation. TAMiso 2 or SMO1-L Other genes.

[0155] Example 4 – Quantification of total glycoside and alkaloid content in CRISPR mutant tubers after two post-harvest treatments in field cultivation 4.1 Materials and Methods Cultivation and harvesting were carried out as described in Example 1 and according to Example 3, and the cultivar Kuras was obtained through CRISPR / Cas9-mediated mutation. Solanum tuberosum L. potato mutants. Seven types of mutants were prepared, each reducing the expression of the following genes: TAMiso 2. DWF1-L , SMO1-L , SMO1-L and DWF1-L Double mutant 2OGD , GAME4 and GAME9 .

[0156] Prepared treated tuber samples and subject them to SGA-induced conditions, including damage and light exposure, as described by Petersson et al. (2013b).

[0157] The total SGA content in tubers of the control group or the SGA-induced treatment group exposed to light or damage was quantified using LC-MS analysis, as described by Merino et al. (2023).

[0158] 4.2 Results The lowest levels of glycoalkaloids in wild-type tubers were observed in the control group, with light-induced stress leading to a significant increase, while damage-induced stress resulted in a more significant increase (see [reference needed]). Figure 8 As shown, the levels of glycoalkaloids in wild-type tubers increased two to three times under damage and light treatment.

[0159] The total levels of glycoalkaloids in all mutant genotypes were lower than those in the wild-type control group, regardless of treatment.

[0160] Some mutants produce very low amounts of glycoalkaloids under wound and light treatment, for example TAM iso 2 , DWF1-L , SMO1-L and DWF1-L Double mutant 2OGD as well as GAME9 .

[0161] Similar to the wild type, in the TAM iso 2 and GAME4 mutants, damage-induced stress resulted in a greater increase in glycoalkaloid content compared to light-induced stress.

[0162] In the SMO1-L mutant, light-induced stress resulted in a greater increase in glycoalkaloid content compared to damage-induced stress, which is the opposite of the wild type.

[0163] The fact that mutants had lower levels of glycoalkaloids than wild-type controls after exposure to damage or light suggests that the reduction in glycoalkaloids is a general effect of mutations.

[0164] Example 5 – Effects of CRISPR / Cas9-mediated glycoside-free alkaloids on potato tuber yield 5.1 Materials and Methods Cultivation and harvesting were carried out as described in Example 1 and as described in Examples 3 and 4, and the cultivar Kuras was obtained through CRISPR / CAS9-mediated mutation. Solanum tuberosum L. Potato mutants. Eight types of mutants were prepared that reduced the expression of the following genes: TAM iso 2, DWF1-L, SMO1-L, Include SMO1-L and DWF1-L Double mutants DWF7-L, 2OGD, GAME4 and GAME9 .

[0165] The tubers were counted, weighed, and the total SGA content was quantified using LC-MS in the same manner as in Examples 3 and 4. This experiment was repeated twice in two different growing seasons in 2023 and 2024.

[0166] 5.2 Results Some mutants exhibited severely reduced tuber yields, while others were less adversely affected (see Table 11 below). Compared to the wild type, some mutants showed more severe impacts on tuber yield, such as TAM iso 2 (12-35%), DWF7-L (2-3%), and GAME4 (21-22%). Some mutants maintained relatively high yields compared to the wild type, such as DW1F-L (50-59%), SMO1-L (92-81%), and GAME9 (85-61%).

[0167] All mutants produced less glycoalkaloids than the wild type, although the degree of reduction varied. Some mutants showed a significant impact on yield, but a substantial reduction in total glycoalkaloids, such as TAM iso 2, 2OGD, and GAME4. Some mutants, such as DW1F-L, SMO1-L, and GAME9, maintained high yields while significantly reducing total glycoalkaloids.

[0168] Clearly, mutations in glycoalkaloid genes reduce the amount of glycoalkaloids present in tubers with varying degrees of efficiency, but they also affect the plant's adaptability, resulting in different levels of yield maintenance.

[0169] Table 11. Yield results of field trials of mutant and wild-type plants. WT yield is calculated relative to wild-type tubers harvested in each year.

[0170]

[0171] a and b This indicates which experiment and year the results were derived from.

[0172] c TGA levels were not analyzed in the tubers harvested in 2023.

[0173] References Andersson, M., Turesson, H., Nicolia, A., Fält, A.S., Samuelsson, M.&Hofvander, P. (2017). Efficient targeted multiallelic mutagenesis intetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression inprotoplasts. Plant Cell Reports , vol. 36 (1), pp. 117-128. Andersson, M., Turesson, H., Olsson, N., Fält, A.S., Ohlsson, P.,Gonzalez, M.N., Samuelsson, M.&Hofvander, P. (2018). Genome editing in potatovia CRISPR-Cas9 ribonucleoprotein delivery. Physiologia Plantarum , vol. 164(4), pp. 378-384. Merino, I., Guasca, A.O., Krmela, A., Arif, U., Ali, A., Westerberg,E., Jalmi, S.K., Hajslova, J., Schulzova, V.&Sitbon, F. (2023). Metabolomicand transcriptomic analyses identify external conditions and key genesunderlying high levels of toxic glycoalkaloids in tubers of stress-sensitivepotato cultivars. Frontiers in Plant Science , vol. 14 (October), pp. 1-16. Nicolia, A., Fält, A.S., Hofvander, P.&Andersson, M. (2021).Protoplast-Based Method for Genome Editing in Tetraploid Potato. Methods Mol Biol. 2021;2264:177-186. doi: 10.1007 / 978-1-0716-1201-9_12. Nicolia, A., Proux-wéra, E., Åhman, I., Onkokesung, N., Andersson,M., Andreasson, E.&Zhu, L. (2015). Targeted gene mutation in tetraploidpotato through transient TALEN expression in protoplasts. Journal of Biotechnology , vol. 204, pp. 17--24 Elsevier B.V. DOI: https: / / doi.org / 10.1016 / j.jbiotec.2015.03.021 . Petersson, E. V, Arif, U., Schulzova, V., Krtková, V., Hajšlová, J.,Meijer, J., Andersson, H.C., Jonsson, L.&Sitbon, F. (2013b). Glycoalkaloidand calystegine levels in table potato cultivars subjected to wounding,light, and heat treatments. Journal of agricultural and food chemistry , vol.61 (24), pp. 5893--5902 United States. Simonovska, B.&Vovk, I. (2000). High-performance thin-layerchromatographic determination of potato glycoalkaloids. Journal of Chromatography A , vol. 903 (1--2), pp. 219-225.

Claims

1. A method for obtaining genetically modified plant cells, wherein the method comprises the following steps: i. Provide plant cells, and ii. Modify the genome of the plant cell to reduce or suppress the expression of at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 , in: The gene SMO1-L The sequence having at least 90% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 90% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 It has a sequence that has at least 92% sequence identity with SEQ ID NO: 47, or alternatively encodes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:

48.

2. The method according to claim 1, wherein: The gene SMO1-L Having a sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 It has a sequence that has at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 47, or alternatively encodes an amino acid sequence that has at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO:

48.

3. The method according to claims 1-2, wherein the gene is selected from the group consisting of: SMO1-L and DWF7- L Preferably, the gene is SMO1-L .

4. The method according to any one of the preceding claims, wherein step ii comprises mutating or excising at least a portion of the gene.

5. The method according to any one of the preceding claims, wherein step ii is performed using CRISPR / Cas.

6. The method according to any one of the preceding claims, wherein the genetically modified plant cells are derived from the Solanaceae family, preferably from the Solanum genus, and more preferably from the potato species.

7. A gene-modified plant cell, wherein the genome of said plant cell has been modified to reduce or suppress the expression of at least one gene selected from the group consisting of: SMO1-L , DWF7-L and TAM iso 2 ,in: The gene SMO1-L The sequence having at least 90% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 90% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 It has a sequence that has at least 92% sequence identity with SEQ ID NO: 47, or alternatively encodes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:

48.

8. The gene-modified plant cell according to claim 7, wherein: The gene SMO1-L Having a sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 41, or alternatively encoding an amino acid sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 42, The gene DWF7-L Having a sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 43 or 45, or alternatively encoding an amino acid sequence having at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 44 or 46, and The gene TAM iso 2 It has a sequence that has at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO: 47, or alternatively encodes an amino acid sequence that has at least 90%, preferably at least 95%, for example at least 99%, or 100% sequence identity with SEQ ID NO:

48.

9. The gene-modified plant cell according to any one of claims 7-8, wherein the gene is selected from the group consisting of: SMO1-L and DWF7-L Preferably, the gene is SMO1-L .

10. The gene-modified plant cell according to any one of claims 7-9, wherein the gene has been mutated or at least a portion of the gene has been removed.

11. The gene-modified plant cell according to any one of claims 7-10, wherein the genome of the plant cell has been modified using CRISPR / Cas.

12. The genetically modified plant cell according to any one of claims 7-11, wherein the plant cell is derived from the Solanaceae family, preferably from the Solanum genus, and more preferably from the potato species.

13. A method for obtaining genetically modified plants, comprising the following steps: i. Providing genetically modified plant cells obtained by the method according to any one of claims 1-6, or genetically modified plant cells according to any one of claims 7-12, and ii. Cultivate or regenerate the genetically modified plant cells to obtain the genetically modified plant.

14. A genetically modified plant comprising or consisting of: - One or more gene-modified plant cells obtained by the method according to any one of claims 1-6, or - Plant cells modified with one or more genes according to any one of claims 7-12; Or obtained by the method according to claim 13.

15. The genetically modified plant according to claim 14, wherein the plant is from the Solanaceae family, preferably from the Solanum genus, and more preferably from the potato species.

16. The use of the genetically modified plant, or a portion thereof, preferably its tuber, according to any one of claims 14-15, in a starch production method, wherein preferably, at least one of amino acids, minerals, proteins, and dietary fiber is obtained and collected as a byproduct of the method.

17. Use of the genetically modified plant, preferably a portion thereof, more preferably its tuber, in a food production method according to any one of claims 14-15, wherein preferably, the food is selected from the group consisting of: peeled potato tubers, mashed potatoes or ready-to-eat mashed potatoes, fried potato products such as French fries or potato chips, and potato chips.

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