Genome edited grapevine

Abstract

The present invention relates to methods of producing viable grapevine protoplasts with regenerative capacity, and methods of genome editing grapevine protoplasts to regenerate genome edited grapevines.

Description

Title of InventionGENOME EDITED GRAPEVINETechnical Field

[0001] The field of the invention relates to methods of producing viable grapevine protoplasts with regenerative capacity, and methods of genome editing grapevine protoplasts to regenerate genome edited grapevines.Background of Invention

[0002] Grapevine has been commonly viewed as a recalcitrant plant in in vitro methods such as protoplast generation, induction of embryogenesis, tissue culture and regeneration manipulations.

[0003] Only limited grapevine varieties have successfully undergone such manipulations and even fewer have successfully traversed the process to full plantlet regeneration.

[0004] In grapevine, protoplasts can be prepared from a variety of tissues and is reportedly highly genotype dependent (Bertini et al 2019). The reported focussed effort has been on 'Chardonnay' and 'Thompson Seedless' (synonym 'Sultania'), with the latter being regarded as an ideal model and more amenable to surviving the in vitro manipulations (Dhekney et al, 2016; Campos et al 2021). It is widely recognised that efficiencies of plantlet regeneration and protoplasts for grapevine are quite low (Osakabe et al (2018), see citations 28, 29; Reed and Bargmann 2021).

[0005] However, within the limited number of more easily handled varieties successful DNA-free CRISPR based editing has been achieved. Osakabe et al (2018) describe the direct delivery of the Cas9-sgRNA RNPs (ribonucleotide proteins) assembled in vitro into protoplasts derived from Chardonnay grapevine embryonic callus but did not show plant regeneration.

[0006] Bertini et al (2019) reported a method of protoplast isolation and regeneration achieved 0.0054% of viable protoplasts regenerating into plants for the red berry cv Sangioveseand reported difficulties in subculture and regeneration leading to lower recovery of the white variety, Garganega). Malnoy et al (2016) indicated embryogenic calli provided a higher yield of viable protoplasts than leaf tissue in experiments to modify MLO7 (also referred to as MLO17) and showed indel formation in 0.1% in Chardonnay grapevine protoplasts but reportedly were not able to regenerate plants from the protoplasts (Bertini et al 2019).

[0007] There remains a need for methods of preparing viable protoplasts with regenerative capacity that can be genome edited at endogenous genes to provide genome edited grapevine plants.Summary of Invention

[0008] In one aspect, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar, said method comprising; a) contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition, wherein the cell wall digesting composition comprises a culture medium and digestion enzymes; b) isolating protoplasts from the plant cells contacted with the cell wall digesting composition; c) contacting isolated protoplasts with a wash solution; and d) purifying protoplasts from step c) on a sucrose solution of from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

[0009] In one embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the culture medium is a Cl based medium or MS basal medium.

[0010] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the cell wall digestion composition comprises Cellulase, Macerozyme and Pectolyase.

[0011] In a further embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the sucrose solution is about 16% (w / v) sucrose.

[0012] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the plant cells are from embryogenic callus grown in solid or liquid medium.

[0013] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein when the plant cells of a Vitis vinifera cultivar are embryogenic callus in solid medium, manually disrupting the callus when contacted with the cell wall digesting composition.

[0014] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the cell wall digesting composition comprises between about 1 to about 3% cellulase, about 0.5 to about 1%, macerozyme and about 0.05% to about 1% pectolyase.

[0015] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the cell wall digesting composition comprises 1% cellulase, 0.5% macerozyme, and 0.05% pectolyase.

[0016] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the cell wall digesting composition comprises Cl-based medium.

[0017] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the cell wall digesting composition comprises: 1.650 g / L NH4NO3, 370 mg / L MgSO4.7H2O, 1.970 g / L KNO3, 170 mg / L KH2PO4); 100 mg / L myo-inositol, 10 mg / L thiamine HCI, 10 mg / L nicotinic acid, 1 mg / L pyridoxine HCI, 1 mg / L D-pantothenic acid, 0.01 mg / L biotin; 100 mg / L L-glutamic acid, 10 mg / L L-phenylalanine, 2 mg / Lglycine), 3% sucrose; 37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSC>4.7H2O; 6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 22.3 mg / L MnSO4.4H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O; and 5mM MES, lOmM CaCI2, 0.5M mannitol at pH 5.7.

[0018] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the liquid or solid medium comprises a Cl-based medium.

[0019] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the liquid or solid medium comprises a Cl-based medium supplemented with an antioxidant to prevent browning.

[0020] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the liquid or solid medium comprises a Cl-based medium supplemented with polyvinylpyrrolidone (PVP) 40.

[0021] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the wash solution is 4 mM MES, 0.5 M mannitol and 15 mM MgC at pH 5.7

[0022] In another embodiment, the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar as described herein, wherein the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar.

[0023] In another aspect, the present invention provides a Vitis vinifera protoplast produced by a method comprising; a) contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition, wherein the cell wall digesting composition comprises a culture medium and cell wall digestion enzymes; b) isolating protoplasts from the plant cells contacted with the cell wall digesting composition; c) contacting isolated protoplasts with a wash solution; and d) purifying protoplasts from step c) on a sucrose solution of from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

[0024] In one embodiment, the present invention provides a Vitis vinifera protoplast produced by a method as described herein, wherein the culture medium is a Cl based medium or MS basal medium.

[0025] In another embodiment, the present invention provides a Vitis vinifera protoplast produced by a method as described herein, wherein the cell wall digestion composition comprises cellulase, macerozyme and pectolyase.

[0026] In a further embodiment, the present invention provides a Vitis vinifera protoplast produced by a method as described herein, wherein the sucrose solution is about 16% (w / v) sucrose.

[0027] In another aspect, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar, said method comprising genome editing a purified protoplast produced by a method as described herein.

[0028] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein the step of genome editing comprises a step of introducing a ribonucleoprotein complex (RNP) into a purified protoplast produced by a method as described herein.

[0029] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein the step of introducing a ribonucleoprotein complex into a purified protoplast is protoplast transfection comprising contacting a purified protoplast produced by the method of any one of claims 1 to 12 with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaC .

[0030] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, further comprising a step of stopping protoplast transfection by contacting the transfected protoplast with a stop solution at 25 mins to 45mins, preferably at 30 minutes, following contacting the purified protoplast with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaC .

[0031] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein the stop solution is a solution comprising 2 mM MES pH 5.7, 5mM glucose, 154 mM NaCI, 125 mM CaC and 5 mM KCI.

[0032] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, further comprising contacting the genome edited protoplast with a solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7 to wash the genome edited protoplast.

[0033] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, further comprising cultivating the genome edited protoplast.

[0034] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein cultivating the genome edited protoplast comprises a step of (i) incubation for about 1 hour followed by a wash step.

[0035] In another embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein cultivating the genome edited protoplast comprises a step of (i) incubation of about 1 hour, (ii) wash and (iii) followed by forming an alginate bead comprising the genome edited protoplast.

[0036] In another aspect, the present invention provides a genome edited protoplast produced by the method as described herein.

[0037] In another aspect, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, further comprising generating a plant from the genome edited protoplast.

[0038] In another aspect, the present invention provides a plant produced by a method as described herein, or from a protoplast produced by a method as described herein.Brief Description of Drawings

[0039] Figure 1: (A) Germinated Shiraz embryos growing on GS1CA medium in low-light and (B) cotyledonary explants cut from germinated embryos and placed on SM.BA (SM,shooting media; BA, benzyladenine / BAP) media for shoot induction. Pictures were taken 3 months after the transfection of Shiraz protoplasts with DM2c / Cas9 RNP.

[0040] Figure 2: Evidence of target gene editing in Shiraz embryos.

[0041] Figure 3: Shown is a photograph of in vitro-cultured Shiraz plants that represent(from left to right) the non-transfected origin cultivar, transfection WT and Vvdmr6 double mutant. The photos were taken 3-4 weeks after sub-culturing of regenerants that had developed 4-5 months after transfection, with the origin cultivar sub-cultured at the same time.

[0042] Figure 4: Embryo formation from alginate bead-embedded Shiraz protoplasts transfected with GH3-5_3 / Cas9 RNP. Red arrows point to different stages of developing embryos.

[0043] Figure 5: (A) Germinated Shiraz embryos growing on GS1CA medium in low-light and (B) cotyledonary explants cut from germinated embryos and placed on SM.BA media for shoot induction. Pictures were taken 3 months after the transfection of Shiraz protoplasts with GH3-5_3 / Cas9 RNP.

[0044] Figure 6: Editing types of the first six identified VvGH3-5 mutants (E1-E6) as identified by Sanger sequencing of target region amplicon followed by Synthego 'Inference of CRISPR Edits (ICE)' analysis. The reference guide sequence is underlined, PAM sites are highlighted in gray, vertical red bars mark the Cas9 cut site and horizontal red bars represent deleted bases.

[0045] Figure 7: VvDMR6-l (6-1) and VvDMR6-2 (6-2) amplicons before (uc) and after (cut) in vitro treatment with DM2c / Cas9 RNP.

[0046] Figure 8: The coding sequence of VvDMR6-l, aligned with the DM2c guide (shown in green), designed for VvDMR6-2 . The 1 bp mismatch is indicated in orange.

[0047] Figure 9: The coding sequence of VvDMR6-2, aligned with the DM2c guide (shown in blue), designed for this gene.

[0048] Figure 10: (A) Microcallus and (B) embryo formation from alginate bead-embedded Chardonnay protoplasts transfected with DM2c / Cas9 RNP. Red arrows point to microcallus and cotyledonary embryos.

[0049] Figure 11: (A) Germinated Chardonnay embryos growing on GS1CA medium in low- light and (B) cotyledonary explants cut from germinated embryos and placed on SM.BA media for shoot induction. Pictures were taken 3 months after the transfection of Chardonnay protoplasts with DM2c / Cas9 RNP.

[0050] Figure 12: Evidence of target gene editing in Chardonnay embryos.

[0051] Figure 13: Example images of regenerated Chardonnay wild type (WT) line and mutant with double biallelic (dmr6-l / dmr6-l / dmr6-2 / dmr6-2) DMR6 edits.

[0052] Figure 14: Shown is a photograph of representative, glasshouse-grown WT and Vvdmr6 single and double mutant plants used for DM assays. The photo was taken four weeks after pruning, at the time when leaf selection for DM inoculation commenced.

[0053] Figure 15: DM leaf disc assay and defence hormone analysis of Chardonnay Vvdmr6 mutants, (a) Sporangia counted from wildtype plants (WT; 8 lines, 27 leaf replicates), double biallelic Vvdmr6 mutants (dmr6-l / dmr6-2; 22 lines, 72 leaf replicates), and single biallelic Vvdmr6-1 (dmr6-l; 2 lines, 7 leaf replicates) and Vvdmr6-2 (dmr6-2; 9 lines, 29 leaf replicates) mutants at 7 days post inoculation. Tissue from the same leaves was used to quantify (b) salicylic acid and (c) jasmonic acid. P-values are provided for genotypes with a significant difference in sporangia counts or hormone concentrations.

[0054] Figure 16: Shown is a photograph of in vitro-cultured Sauvignon blanc plants that represent (from left to right) the non-transfected origin cultivar, transfection WT and Vvdmr6 double mutant. The photos were taken 3-4 weeks after sub-culturing of regenerants that had developed 4-5 months after transfection, with the origin cultivar sub-cultured at the same time.

[0055] Figure 17: Embryo formation from alginate bead-embedded V4 microvine protoplasts 4 weeks after transfection with GIN1_2 / Cas9 RNP. Red arrows point to different stages of developing embryos.

[0056] Figure 18: (A) Germinated V4 microvine embryos growing on GS1CA medium in low-light and (B) cotyledonary explants cut from germinated embryos and placed on SM.BA media for shoot induction. Pictures were taken 4 months after the transfection of V4 microvine protoplasts with GIN1_2 / Cas9 RNP.

[0057] Figure 19: Evidence of target gene editing in V4 microvine embryos. (A) Schematic of Alel-v2 dCAPS assay to discern WT and edited VvGINl sequences. (B) Agarose gel photo of VvGINl (target of GIN1_2 guide) dCAPS amplicons after restriction digest with Alel-v2. Red stars denote uncut amplicons indicating removal of restriction sites by editing. Results are also reflected in Table 13.

[0058] Figure 20: Shown is a photograph of WT and Vvginl V4 microvine plants one month after transfer to soil and glasshouse environment.

[0059] Figure 21: Chardonnay protoplast purification using 16%-30% sucrose cushions. Photos show (A) 8 mL sucrose cushions loaded with 2 mL protoplast suspension each, (B) protoplast bands after centrifugation, and (C) pelleted cell material after centrifugation.

[0060] Figure 22: Effect of sucrose concentration on Chardonnay protoplast viability and yield. Graphic representation of protoplast viability (A) and recovery data (B) provided in Table14 of Example 6.

[0061] Figure 23: V4 microvine protoplast purification using 16%-30% sucrose cushions. Photos show (A) 8 mL sucrose cushions loaded with 2 mL protoplast suspension each, (B) protoplast bands after centrifugation, and (C) pelleted cell material after centrifugation.

[0062] Figure 24: Effect of sucrose concentration on V4 microvine protoplast viability and yield. Graphic representation of protoplast viability (A) and recovery data (B) provided in Table15 of Example 7.

[0063] Figure 25: Shiraz protoplast purification using 16%-30% sucrose cushions. Photos show (A) 8 mL sucrose cushions loaded with 2 mL protoplast suspension each, (B) protoplast bands after centrifugation, and (C) pelleted cell material after centrifugation.

[0064] Figure 26: Effect of sucrose concentration on Shiraz protoplast viability and yield. Graphic representation of protoplast viability (A) and recovery data (B) provided in Table 16 of Example 8.

[0065] Figure 27: Protoplast, alginate bead, plant regeneration and editing data for Chardonnay, Cabernet Sauvignon, Shiraz and Sauvignon blanc transfections with Cas9 / DM2c- RNP of cells derived from embryogenic callus cultured on agar plates (EC) or in suspension (SC). + Mix of globular, heart-shaped and cotyledonary embryos, t This number is an underestimation as prolific embryo germination continued but was not further captured. § Includes all edits of VviDMR6-2 and / or VviDMR6-l as determined by PCR-CAPS analysis.Detailed Description

[0066] The present invention is based in part on the characterisation of methods of preparing protoplasts of a Vitis vinifera cultivar with increased viability and / or decreased irregular cells. Importantly, the present inventors have demonstrated that protoplasts from different cultivars of grapevine sediment differently in the presence of sucrose, and the protoplasts produced are viable and have regenerative capacity.

[0067] The present inventors have also demonstrated DNA free gene editing and plantlet regeneration for Chardonnay, Cabernet Sauvignon (data not shown), and Shiraz from protoplasts.

[0068] In particular, Examples 1 and 2 demonstrate the generation of viable protoplasts with regenerative capacity from Shiraz. Example 2 demonstrates the generation of viable protoplasts with regenerative capacity from Shiraz. Example 3 demonstrates the generation of viable protoplasts with regenerative capacity from Chardonnay. Example 4 demonstrates the generation of viable protoplasts with regenerative capacity from Sauvignon Blanc. Example 5demonstrates the generation of viable protoplasts with regenerative capacity from microvines. Examples 6 to 9 demonstrate the effect of sucrose concentration used for protoplast purification cushions on viability and yield of protoplasts.

[0069] The present inventors propose that while yield of purified protoplasts is important, and yield can be altered using the methods described herein, in a preferred embodiment, increased viability and / or regenerative capacity of the purified protoplasts is preferred relative to increased yield.

[0070] Accordingly, in one aspect the present invention provides a method for producing protoplasts of a Vitis vinifera cultivar, said method comprising: a) contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition, wherein the cell wall digesting composition comprises a culture medium and digestion enzymes; b) isolating protoplasts from the plant cells contacted with a cell wall digesting enzyme; c) contacting isolated protoplasts with a wash solution; and d) purifying protoplasts from step c) on a sucrose solution of from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

[0071] In another embodiment, the sucrose solution is about 16% (w / v) to 27% (w / v) sucrose.

[0072] In another embodiment, the sucrose solution is about 16% (w / v) sucrose.

[0073] The present inventors have demonstrated that methods described herein can be used to produce viable protoplasts from embryogenic callus cultured in either solid or liquid medium. Protoplast yield ( / g fw callus) and viability (%) from liquid and solid medium are similar. However, embryogenic callus growing as a suspension in liquid culture proliferates more quickly than that on solid medium and is significantly quicker to subculture than that grown on solid medium.

[0074] As used herein, the term "Vitis vinifera" includes cultivars of Vitis vinifera, including Chardonnay, Sauvignon Blanc, shiraz, pinot noir, and Cabernet Sauvignon and other winegrape or tablegrape cultivars such as Thompson seedless. As used herein the term variety is usedinterchangeably with the term cultivar, and variety is not intended to refer to the taxonomic rank.

[0075] As used herein, the term "protoplast" refers to plant cells which have had their cell wall removed, and are usually of spherical shape. As described herein, in one aspect the present invention provides methods of producing protoplasts that have fewer irregular protoplasts.

[0076] As described herein, protoplasts can be prepared in in a number of ways.

[0077] In one embodiment, embryogenic callus was initiated from stamens dissected from immature flower buds. In the examples, depending on the variety, stamens were placed first onto either PIV or Harst medium. Embryogenic callus that developed was transferred to Cl proliferation medium where it was maintained by regular 4-6 weekly subcultures.

[0078] In other embodiments, embryogenic callus was also grown as a suspension culture by harvesting established callus from Cl medium plates into a small volume of Cl liquid medium and gradually increasing the volume of culture as the callus proliferated. Routinely, 120ml cultures were established and subcultured weekly by a 50% exchange of medium, splitting or sieving. New suspension cultures were regularly initiated from callus growing on agar medium.

[0079] Accordingly, in one embodiment, protoplasts are produced from solid cultures. In another embodiment, protoplasts are produced from liquid cultures (e.g. suspension cultures).

[0080] In one aspect, the plant cells are from a plant tissue. Suitable plant tissues include whole flowers, stamens, filaments and pistils.

[0081] In one embodiment, the plant cells are from embryogenic plant tissue.

[0082] In one embodiment, the embryonic plant tissue is embryogenic callus.

[0083] In another embodiment, the plant cells are from embryogenic callus grown in solid or liquid medium. In a further embodiment, the plant cells of a Vitis vinifera cultivar areembryogenic callus in solid medium, manually disrupting the callus when contacted with the cell wall digesting composition.

[0084] Plant media for growth of cultures of plant cells are known in the art.

[0085] In a preferred embodiment, the plant cells are cultured in medium that is to be used as the basis of the medium to be used in the step of contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition.

[0086] In a preferred embodiment, the plant cells are cultured in a Cl based medium, such as Cl based proliferation medium.

[0087] In a preferred embodiment, the Cl based proliferation medium comprises:1.650 g / L NH4NO3, 440 mg / L CaCI2.2H2O, 370 mg / L MgSO4.7H2O, 1.970 g / L KNO3, 170 mg / L KH2PO4);100 mg / L myo-inositol, 10 mg / L thiamine HCI, 10 mg / L nicotinic acid, 1 mg / L Pyridoxine HCI, 1 mg / L D-pantothenic acid, 0.01 mg / L biotin; 100 mg / L L-glutamic acid, 10 mg / L L- phenylalanine, 2 mg / L blycine), 5.0 pM 2,4-dichlorophenoxyacetic acid (2,4D), 1.0 pM 6- benzylaminopurine (BAP or BA), 0.1% casein enzymatic hydrolysate, 3% sucrose, pH 5.8, 0.5% Phytagel;37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O; and6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 22.3 mg / L MnSO4.4H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O.

[0088] In one embodiment, the liquid or solid medium comprises a Cl-based medium.

[0089] In another embodiment, the liquid or solid medium comprises a Cl-based medium supplemented with an antioxidant to prevent browning.

[0090] In another embodiment, the antioxidant is any antioxidant suitable for culture medium. In a further embodiment, the antioxidant is selected from the group consisting of tocopherol, ascorbic acid, glutathione, cysteine, lipoic acid, dithiothreitol, selenite, phenoxane, citric acid, vitamins, polyvinylpyrrolidone (PVP) 40 and capsinoids.

[0091] In a further embodiment, the liquid or solid medium comprises a Cl-based medium supplemented with polyvinylpyrrolidone (PVP) 40.

[0092] In another embodiment, the plant cells are from plant tissue that has been treated to increase access of the cell wall digesting composition to the plant cell wall. For example, by physical disruption of the tissue, vacuum infiltration of plant tissue, or other means.

[0093] As used herein the term "contacting" includes mixing plant cells of a Vitis vinifera cultivar with a suitable cell wall digesting composition to form a mixture, and retaining the mixture for a period of time and at a temperature suitable to digest the cell walls of the plant cells.

[0094] In addition to temperature and / or duration, light conditions and / or agitation of the mixing during this step of contacting can impact the yield, viability and / or regenerative capacity of protoplasts.

[0095] In one embodiment, contacting is performed at conditions to allow for release of protoplasts, while maintaining viability and / or regenerative capacity of the protoplasts released.

[0096] The duration of contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition typically ranges from 5 to 24 h. In one embodiment, contacting is performed for between 10 and 24 hours. In another embodiment, contacting is performed for between about 16 and 22 hours.

[0097] In one embodiment, the step of contacting includes contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition under more than one set of conditions. For example, the step of contacting can include sub-steps of contacting for one duration of time in one set of conditions, followed by treatment of the mixture (e.g. manual disruption of callus) as another sub-step, followed by contacting for a further duration of time in another set of conditions as a further sub-step.

[0098] In one embodiment wherein the plant cells are callus, contacting can include manual disruption when the step of contacting begins (e.g. at the time of addition of the cellwall digesting composition), or at a period of time following the addition of the cell wall digesting composition (e.g. within the first hour of contacting).

[0099] The duration of contact needs to be long enough to release sufficient numbers of protoplasts, but not too long as to decrease the viability due to cell damage or the lack of nutrients and growth regulators in the cell wall digesting composition. For onward experimentation, in one embodiment, protoplast cell numbers from at least 1 xlO6protoplasts / g callus are required if higher recovery is desired.

[0100] In one embodiment, contacting is performed at a temperature that allows for activity of cell wall digesting composition and release of protoplasts, while maintaining viability and / or regenerative capacity of the protoplasts released.

[0101] In one embodiment, contacting is performed at room temperature.

[0102] In another embodiment, contacting is performed at between 20 and 27°C.

[0103] In a preferred embodiment, contacting is performed at around 25°C.

[0104] In one embodiment, contacting is performed in dark conditions to allow for activity of cell wall digesting composition and release of protoplasts, while maintaining viability and / or regenerative capacity of the protoplasts released.

[0105] In one embodiment, contacting is performed with agitation (e.g. using a rotary shaker).

[0106] In one embodiment, agitation is performed at a speed of from 10 to 90 rpm.

[0107] In a preferred embodiment, agitation is performed at a speed of around 30 rpm.

[0108] Digestion medium was added at 5ml / 0.5g callus and incubated in the dark. The present inventors have demonstrated the nutrient medium in which the embryogenic callus was growing, when used as a basis for the enzymes (and buffers) used for the methods described herein, increases the viability of the isolated protoplasts. Without wishing to be bound by theory, the present inventors propose that the use of the culture medium used forpreparing the plant cells to be treated (e.g. growing callus) as a basis for the cell wall digesting composition supports the yield, viability and regenerative capacity of the protoplasts produced.

[0109] Accordingly, in one embodiment, the cell wall digesting composition is based on the medium the plant cells are cultured in.

[0110] For example, in one embodiment when the culture (e.g. nutrient) medium in which the embryogenic callus was growing is a Cl based medium, the cell wall digesting composition is Cl medium without plant grown regulators or casein hydrolysate, and with the addition of MES, mannitol and CaCI2. For example, 5mM MES, lOmMCaCb, 0.5M mannitol pH5.8.

[0111] In another embodiment, the culture medium in which the which the embryogenic callus was grown in is used as a basis for the cell wall digesting composition, without plant growth regulators or casein hydrolysate.

[0112] In one embodiment, the osmolarity of the cell wall digesting composition wash is osmotically adjusted to substantially correspond to the osmotic values of the protoplasts to be isolated.

[0113] In some embodiments the cell wall digesting composition includes osmolytes, pH buffers, alternate targets for proteases, and reducing agents, in addition to an enzyme or enzymes.

[0114] In another embodiment, the cell wall digesting composition is a Cl based composition.

[0115] In another embodiment, the cell wall digesting composition is a Cl based composition without plant growth regulators and casein hydrolysate.

[0116] In a preferred embodiment, the cell wall digesting composition comprises:1.650 g / L NH4NO3, 370 mg / L MgSO4.7H2O, 1.970 g / L KNO3, 170 mg / L KH2PO4);100 mg / L myo-inositol, 10 mg / L thiamine HCI, 10 mg / L nicotinic acid, 1 mg / L pyridoxine HCI, 1 mg / L D-pantothenic acid, 0.01 mg / L biotin; 100 mg / L L-glutamic acid, 10 mg / L L- phenylalanine, 2 mg / L glycine), 3% sucrose;37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O; and6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 22.3 mg / L MnSO4.4H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O; and5mM MES, lOmM CaCI2, 0.5M mannitol at pH 5.7.

[0117] In one embodiment, the culture medium is a Cl based medium or MS basal medium.

[0118] As used herein the term "cell wall digesting composition" includes an enzyme or enzymes ('digestion enzymes') to digest the carbohydrate polymers of the cell wall of the plant cells to result in protoplasts. Suitable enzymes are known in the art, for example, cellulase, pectolyase, hemicellulase, and / or macerozyme (macerating enzyme from Rhizopus).

[0119] In a one embodiment, the cell wall digesting composition comprises cellulase, macerozyme and / or pectolyase.

[0120] In another embodiment, the cell wall digesting composition comprises between about 1 to about 3% cellulase, about 0.5 to about 1% macerozyme and / or about 0.05 to about 1% pectolyase.

[0121] In a preferred embodiment, the cell wall digesting composition comprises between about 1 to about 3% cellulase, about 0.5 to about 1% macerozyme and about 0.05 to about 1% pectolyase.

[0122] In a preferred embodiment, the cell wall digesting composition comprises 1% cellulase, 0.5% macerozyme, and 0.05% pectolyase.

[0123] The present inventors have demonstrated (data not shown) that the yield of protoplasts produced is reduced if the plant cells are contacted with the cell wall digesting composition prepared more than 2 hours before contacting with cells. Accordingly, to furtherincrease yield of protoplasts produced, the plant cells are contacted with the cell wall digesting composition within two hours of the preparation of the cell wall digesting composition. Without wishing to be bound by theory, the present inventors propose that the concentration of the enzymes cellulase and macerozyme used allows for contacting to be performed overnight (e.g. about 16 hours) without the risk of over-digestion and excessive contamination from free-floating organelles and other cellular debris, and contacting with plant cells within two hours of preparation increases yield.

[0124] In one embodiment, the plant cells are contacted with a cell wall digesting composition prepared no more than 2 hours before contacting with cells. In another embodiment, the plant cells are contacted with a cell wall digesting composition prepared no more than 90 minutes before contacting with cells. In a further embodiment, the plant cells are contacted with a cell wall digesting composition prepared no more than 60 minutes before contacting with cells. In a further embodiment, the plant cells are contacted with a cell wall digesting composition prepared no more than 30 minutes before contacting with cells.

[0125] As used herein the term "isolating" in the context of isolating protoplasts from the plant cells contacted with a cell wall digesting composition refers to separating the plant cells contacted with the cell wall digesting composition from said cell wall digesting composition and undigested cell wall material, dead cells, and / or debris, to isolate the protoplasts formed.

[0126] In one embodiment, the isolated protoplasts are contacted with a wash solution to allow for separation of the cell wall digesting composition and undigested cell wall material, dead cells, and / or debris, from the protoplast released.

[0127] In a preferred embodiment, the isolated protoplasts are contacted with a wash solution then pelleted by centrifugation once, or more than once.

[0128] In a preferred embodiment, the wash solution is MMG.

[0129] In one embodiment, the method comprises a step of filtering the plant cells contacted with a cell wall digesting composition.

[0130] In another embodiment, the method comprises a step of filtering the plant cells contacted with a cell wall digesting composition through a 100pm filter and or 70pm mesh.

[0131] As used herein "wash solution" refers to a solution that is suitable for washing protoplasts formed to remove any residual cell wall digesting composition and digested cell wall material, while maintaining viability and / or regenerative capacity of the protoplasts formed. In one embodiment, the wash solution is osmotically adjusted to substantially correspond to the osmotic values of the isolated protoplasts.

[0132] In one embodiment, following washing, the isolated protoplasts are purified on a sucrose solution from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

[0133] In another embodiment, the sucrose solution is about 16% (w / v) to 27% (w / v) sucrose. In another embodiment, following washing, the sucrose solution is about 16% (w / v) sucrose.

[0134] Alternatively, the isolated protoplasts are purified on a suitable density gradient medium with an equivalent density to the % (w / v) sucrose solutions described herein.

[0135] For example, 13% (w / v) sucrose is equivalent to a density of 1.05064 g / cm3. Accordingly, a solution with a specific gravity of about 1.05064 g / cm3 can be used to purify protoplasts. 14% (w / v) sucrose is equivalent to a density of 1.05487 g / cm3. Accordingly, a solution with a specific gravity of about 1.05487 g / cm3 can be used to purify protoplasts. 15% (w / v) sucrose is equivalent to a density of 1.05914 g / cm3. Accordingly, a solution with a specific gravity of about 1.05914 g / cm3 can be used to purify protoplasts. 16% (w / v) sucrose is equivalent to a density of 1.06343 g / cm3. Accordingly, a solution with a specific gravity of about 1.06343 g / cm3 can be used to purify protoplasts. 17% (w / v) sucrose is equivalent to a density of 1.06776 g / cm3. Accordingly, a solution with a specific gravity of about 1.06776 g / cm3 can be used to purify protoplasts. 18% (w / v) sucrose is equivalent to a density of 1.07212 g / cm3. Accordingly, a solution with a specific gravity of about 1.07212 g / cm3 can be used to purify protoplasts. 19% (w / v) sucrose is equivalent to a density of 1.07651 g / cm3. Accordingly, a solution with a specific gravity of about 1.07651 g / cm3 can be used to purify protoplasts. 20% (w / v) sucrose is equivalent to a density of 1.08093 g / cm3. Accordingly, asolution with a specific gravity of about 1.08093 g / cm3 can be used to purify protoplasts. 21% (w / v) sucrose is equivalent to a density of 1.08538 g / cm3. Accordingly, a solution with a specific gravity of about 1.08538 g / cm3 can be used to purify protoplasts. 22% (w / v) sucrose is equivalent to a density of 1.08987 g / cm3. Accordingly, a solution with a specific gravity of about 1.08987 g / cm3 can be used to purify protoplasts. 23% (w / v) sucrose is equivalent to a density of 1.09439 g / cm3. Accordingly, a solution with a specific gravity of about 1.09439 g / cm3 can be used to purify protoplasts. 24% (w / v) sucrose is equivalent to a density of 1.09894 g / cm3. Accordingly, a solution with a specific gravity of about 1.09894 g / cm3 can be used to purify protoplasts. 25% (w / v) sucrose is equivalent to a density of 1.10353 g / cm3. Accordingly, a solution with a specific gravity of about 1.10353 g / cm3 can be used to purify protoplasts. 26% (w / v) sucrose is equivalent to a density of 1.10814 g / cm3. Accordingly, a solution with a specific gravity of about 1.10814 g / cm3 can be used to purify protoplasts. 27% (w / v) sucrose is equivalent to a density of 1.1128 g / cm3. Accordingly, a solution with a specific gravity of about 1.1128 g / cm3 can be used to purify protoplasts. 28% (w / v) sucrose is equivalent to a density of 1.11748 g / cm3. Accordingly, a solution with a specific gravity of about 1.11748 g / cm3 can be used to purify protoplasts. 29% (w / v) sucrose is equivalent to a density of 1.1222 g / cm3. Accordingly, a solution with a specific gravity of about 1.1222 g / cm3 can be used to purify protoplasts. 30% (w / v) sucrose is equivalent to a density of 1.12695 g / cm3. Accordingly, a solution with a specific gravity of about 1.12695 g / cm3 can be used to purify protoplasts.

[0136] The term "about" and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 1-2%, more preferably 1%, of the particular term.

[0137] As used herein "purifying protoplasts on a sucrose solution" refers to adding protoplasts to a sucrose solution, centrifuging and purifying protoplasts at the interphase.

[0138] If an alternative suitable density gradient medium is used, the term "purifying protoplasts on a density gradient solution" as used herein, refers to adding protoplasts to a density gradient solution, centrifuging and purifying protoplasts at the interphase.

[0139] The present inventors have demonstrated herein that protoplasts produced from different varieties of grapevine sediment differently in the presence of sucrose. The use of sucrose gradients in the methods described herein allows for a high yield of protoplasts and with high viabilities and regenerative capacity. To test the most appropriate concentration of sucrose to use for the purification of grape protoplasts a sucrose gradient was set up by layering sucrose in concentrations of 35, 30, 25, 20, 15 and 10% sequentially into a 15ml centrifuge tube and layering 2ml of protoplast digest in MMG on top. Sucrose was freshly prepared and filtered through a 0.22pm filter before use. This tube was then centrifuged at 90g for 4mins (no brake) and bands of protoplasts collected. Bands were kept separate, washed in 20ml and then 10ml MMG (100g for 3 mins with no braking) and then counts performed to determine the number and viability of protoplasts from each band (Moon et al., 2021).

[0140] Importantly, the present inventors have demonstrated the production of high yields of protoplasts with high viability. For example, Examples 1 to 5 all demonstrate the production of a high yield of protoplasts - with high viabilities of at least 80% - and pivotally, regenerative capacity.

[0141] Examples 1 to 9 demonstrate the production of a high yield of protoplasts - with high viabilities of at least 80% - and pivotally in some examples, regenerative capacity. Examples 1 to 4 also all demonstrate the production of a high yield of protoplasts - with high viabilities of at least 85% - and pivotally, regenerative capacity, when 20, 22, 25 and 30% (w / v) sucrose is used.

[0142] For example, Examples 6 to 10 demonstrate that when the Vitis vinifera cultivar is Chardonnay, the protoplasts contacted with a wash solution with the highest viability were purified on a sucrose solution of 20%, that when the Vitis vinifera is a microvine (V4), the protoplasts contacted with a wash solution with the highest viability were purified on a sucrosesolution of 25%, that when the Vitis vinifera cultivar is Shiraz, the protoplasts contacted with a wash solution with the highest viability were purified on a sucrose solution of 22%, and that that when the Vitis vinifera cultivar is Sauvignon blanc the protoplasts contacted with a wash solution with the highest viability were purified on a sucrose solution of 27%.

[0143] Accordingly, in one another embodiment, following washing, the isolated protoplasts are purified on a sucrose solution of about 21% (w / v) sucrose to about 30% (w / v) sucrose.

[0144] In another embodiment, following washing, the isolated protoplasts are purified on a sucrose solution of about 25% (w / v) sucrose to about 30% (w / v) sucrose.

[0145] In another embodiment, following washing, the isolated protoplasts are purified on a sucrose solution of about 25% (w / v) sucrose.

[0146] In another embodiment, following washing, the isolated protoplasts are purified on a sucrose solution of about 22% (w / v) sucrose.

[0147] In another embodiment, following washing, the sucrose solution is about 20% (w / v) sucrose. Without wishing to be bound by theory, the present inventors propose that a % sucrose level can be used to provide a controlled viability of purified protoplasts at a controlled yield of purified protoplasts.

[0148] For example, in one embodiment, protoplasts contacted with a wash solution are purified on a sucrose solution of about 16% (w / v) sucrose to provide viable protoplasts at a controlled yield.

[0149] Accordingly, in one embodiment, when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a sucrose solution of about 16% (w / v) sucrose. Without wishing to be bound by theory, the present inventors propose that a % sucrose level can be used to provide a controlled viability of purified protoplasts at a controlled yield of purified protoplasts.

[0150] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a sucrose solution of from about 13 (w / v) sucrose to about 30% (w / v) sucrose.

[0151] In another embodiment, the isolated protoplasts are purified on a suitable density gradient medium with an equivalent density to the % (w / v) sucrose solutions described herein. For example, in one embodiment, the present invention provides a method as described herein, wherein when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a suitable density gradient medium of from about 1.05064 g / cm3 to about 1.12695 g / cm3.

[0152] In another embodiment, the present invention provides a method as described herein, wherein when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a sucrose solution of about 13% (w / v) sucrose, about 13.5% (w / v) sucrose, about 14% (w / v) sucrose, about 14.5% (w / v) sucrose, about 15% (w / v) sucrose, about 15.5% (w / v) sucrose, about 16% (w / v) sucrose, about 16.5% (w / v) sucrose, about 17% (w / v) sucrose, about 17.5% (w / v) sucrose, about 18% (w / v) sucrose, about 18.5% (w / v) sucrose, about 19% (w / v) sucrose, 19.5% (w / v) sucrose, about 20% (w / v) sucrose, about 20.5% (w / v) sucrose, about 21% (w / v) sucrose, about 21.5% (w / v) sucrose, about 22% (w / v) sucrose, about 22.5% (w / v) sucrose, about 23% (w / v) sucrose, about 23.5% (w / v) sucrose, about 24% (w / v) sucrose, about 24.5% (w / v) sucrose, about 25% (w / v) sucrose, about 25.5% (w / v) sucrose, about 26% (w / v) sucrose, about 26.5% (w / v) sucrose, about 27% (w / v) sucrose, about 27.5% (w / v) sucrose, about 28% (w / v) sucrose, about 28.5% (w / v) sucrose, about 29% (w / v) sucrose, about 29.5% (w / v) sucrose, or about 30% (w / v) sucrose.

[0153] In preferred embodiment, the present invention provides a method as described herein, wherein when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or isa microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a sucrose solution of about 16% (w / v) sucrose.

[0154] In preferred embodiment, the present invention provides a method as described herein, wherein when the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar, the protoplasts contacted with a wash solution are purified on a sucrose solution of about 16% (w / v) sucrose.

[0155] In one embodiment, centrifugation of the protoplasts added to the sucrose solution, are centrifuged at about 90 x g.

[0156] In one embodiment, centrifugation of the protoplasts added to the sucrose solution, are centrifuged at about 90 x g for about 4 minutes.

[0157] In one embodiment, centrifugation of the protoplasts added to the sucrose solution, are centrifuged and then the protoplasts at the interphase are removed.

[0158] In a preferred embodiment, the purified protoplasts from the interphase of the sucrose solution are contacted with a wash solution then pelleted by centrifugation once, or more than once.

[0159] In a preferred embodiment, the wash solution is MMG.

[0160] An example method of producing protoplasts from plant cells is detailed below. In brief, embryogenic callus was initiated from stamens dissected from immature flower buds. Depending on the variety stamens were placed first onto either PIV or Harst medium. Embryogenic callus that developed was transferred to Cl proliferation medium where it was maintained by regular 4-6 weekly subcultures. Embryogenic callus was also grown as a suspension culture by harvesting established callus from Cl plates into a small volume of Cl liquid medium and gradually increasing the volume of culture as the callus proliferated. 120ml cultures were established and subcultured weekly by a 50% exchange of medium, splitting or sieving. New suspension cultures were regularly initiated from callus growing on agar medium. Callus growing on solid medium at about 4-5 weeks post-subculture was harvested directly into empty petri dishes. Callus in liquid medium, that had received 50% fresh medium 24hprior, was harvested by first decanting off as much medium as possible, removing the excess by absorption on to a stack of filter paper and then transferring to petri dishes. A cell wall digesting composition was added at 5ml / 0.5g callus and petri dishes placed in the dark at 25°C shaking at 30rpm overnight. The cell wall digesting composition comprised Cl basal medium (without plant growth regulators and casein hydrolysate) 0.5% Macerozyme, 1% Cellulase, 0.05% Pectolyase, 5 mM MES (pH5.7), 10 mM CaC (total) and 0.5 M Mannitol adjusted to pH 5.7. Callus from agar plates was manually disrupted immediately when the cell wall digesting composition was added or within the first hour of incubation. Undigested callus was removed by pouring the digest through a filter stack of 100pm and 60pm mesh with 15ml MMG. The digest was centrifuged at 120g for 3 minutes was then washed twice more with 20ml MMG solution. The final pellet was resuspended in MMG and 2ml aliquots layered on to an 8ml sucrose gradient. After centrifugation (90g, 4 minutes, no brake), purified protoplasts were harvested as a band from the interface and washed twice with MMG (centrifugation at 100g, 3 minutes, no brake). Protoplasts were resuspended in a final volume of 1-2.5ml MMG and protoplast concentration and viability determined using a haemocytometer after Trypan blue staining.

[0161] Importantly, the present inventors have demonstrated that when a cell wall digesting composition comprising a culture medium based on the culture medium in which the embryogenic callus is grown in is used, the yield and viability of protoplasts is increased relative to the use of a cell wall digesting composition not based on the culture medium in which the embryogenic callus is grown in is used.

[0162] For example, in the case of Cl based medium, when a cell wall digesting composition comprising a Cl based medium (without plant growth regulators) is used, compared to a cell wall digestion buffer without the Cl based medium base, viability of the protoplast produced was increased, and the number of irregular cells decreased, as is shown below.

[0163] Both digestion compositions contained the following enzymes 0.5% Macerozyme, 1% Cellulase, 0.05% Pectolyase. The cell wall digesting composition comprising a Cl basedmedium (without plant growth regulators and casein hydrolysate) is referred to herein as MS Basal Digestion Buffer.

[0164] This 'MS Basal Digestion Buffer' is a Cl based buffer:1.650 g / L NH4NO3, 370 mg / L MgSO4.7H2O, 1.970 g / L KNO3, 170 mg / L KH2PO4);100 mg / L myo-inositol, 10 mg / L thiamine HCI, 10 mg / L nicotinic acid, 1 mg / L pyridoxine HCI, 1 mg / L D-pantothenic acid, 0.01 mg / L biotin; 100 mg / L L-glutamic acid, 10 mg / L L- phenylalanine, 2 mg / L glycine), 3% sucrose;37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O; and6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 22.3 mg / L MnSO4.4H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O; and500mM MES, lOmM CaCI2, 0.5M mannitol at pH 5.7.

[0165] The comparative 'Digestion Buffer' is:5mM MES, 10mMCaCI2, 0.5M mannitol pH5.8

[0166] Viability was determined using trypan blue staining. As can be seen, in MS Basal Digestion Buffer, the yield and viability of protoplasts is increased using MS Basal Digestion Buffer, both prior to and following purification of Shiraz protoplasts on a 16% sucrose solution (e.g. sucrose cushion), and the number of irregular cells is decreased:Cell Viability IrregularDigestion Buffer count % cells %Before Sucrose cushion 6480000 79.02% 10.87%After Sucrose cushion 2120000 70.48% 9.18%MS Basal DigestionBuffer Before Sucrose cushion 10880000 75.77% 8.65%After Sucrose cushion 6160000 84.06% 5.18%

[0167] The present inventors have also demonstrated in Examples 1 to 5 that the methods of the present invention provide protoplasts that can be genome edited, and plants regenerated from the protoplast.

[0168] Accordingly, in one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar, said method comprising genome editing a purified protoplast produced by a method as described herein.

[0169] In a preferred embodiment, the present invention relates to the methods described herein, wherein genome editing is performed using a CRISPR-Cas system, preferably CRISPR-Cas9. For example, genome editing can be performed by RNPs and single gRNA or by means of transient expression of a vector encoding the nuclease (i.e. Cas9) and the sgRNA.

[0170] In one embodiment, genome editing comprises introducing into a protoplast one or more selected from the group consisting of CRISPR-Cas components, ribonucleic protein (RNP), single guide RNA (sgRNA), a vector encoding a Cas nuclease and / or the sgRNA. Preferably the Cas nuclease is Cas9, Casl2a, Cpfl, or Cmsl, or the like.

[0171] In another embodiment, genome editing comprises introducing into a protoplast a single gRNA or by means of transient expression of a vector encoding the nuclease (i.e. Cas9) and / or the sgRNA.

[0172] In a preferred embodiment, genome editing comprises introducing into a protoplast a guide RNA (gRNA) and Cas9 nuclease. The guide RNA comprises CRISPR RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and a tracr RNA (tracrRNA).

[0173] As used herein the term "regeneration" refers to the growth of a multicellular structure, such as shoot meristem, shoots, somatic embryos, embryogenic callus, somatic meristems, and / or organogenic callus from a protoplast. Regenerating can also include the growth of a plantlet or plant, including fertile plants, from a protoplast.

[0174] The protoplasts and methods described herein can also be used in preparing protoplasts that are suitable for genome editing using other suitable means, such as ZFN (ZincFinger Nucleases), TALEN (Transcription activator-like effector nucleases) systems, or protoplasts suitable for transformation by other genomic modification methods such as Agrobacterium DNA transfer etc.

[0175] In one aspect, the present invention provides a method as described herein, wherein the step of genome editing comprises a step of introducing a ribonucleoprotein complex (RNP) into a purified protoplast produced by the method as described herein.

[0176] As used herein, the term "ribonucleoprotein complex" (RNP) refers to a guide RNA (gRNA) combined with a Cas9 nuclease. In one embodiment, the Cas nuclease is a Cas9 nuclease, and the guide RNA comprises CRISPR RNA (crRNA), typically a 17-20 nucleotide sequence complementary to the target DNA, and a tracr RNA (tracrRNA).

[0177] In one embodiment, the step of introducing a ribonucleoprotein complex (RNP) into a purified protoplast transfection is performed using a concentration of about lxlO6protoplasts / ml.

[0178] In one embodiment, the step of introducing a ribonucleoprotein complex (RNP) into a purified protoplast transfection is performed using a concentration of lxlO6protoplasts / ml.

[0179] As used herein, the term "introducing" refers to delivery of biological material, such as a ribonucleoprotein complex into a protoplast by any suitable means involving intervention by man. In one embodiment, the RNP is introduced using liposome- or polyethylene glycol-(PEG) transfection, electroporation or lipofection.

[0180] In one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar, wherein the step of introducing a ribonucleoprotein complex into a purified protoplast is protoplast transfection comprising contacting a purified protoplast produced by a method as described herein with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaC .

[0181] In another embodiment, the method for producing a genome edited protoplast of a Vitis vinifera cultivar further comprises a step of stopping protoplast transfection by addition of a suitable stop solution.

[0182] In another embodiment, the method for producing a genome edited protoplast of a Vitis vinifera cultivar further comprises a step of stopping protoplast transfection by addition of a suitable stop solution 25 mins to 45mins, preferably at 30 minutes, following contacting the purified protoplast with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaCI2.

[0183] In another embodiment, the method for producing a genome edited protoplast of a Vitis vinifera cultivar further comprises a step of stopping protoplast transfection by contacting the transfected protoplast with a solution comprising 2 mM MES pH 5.7, 5mM glucose, 154 mM NaCI, 125 mM CaC and 5 mM KCI.

[0184] In a preferred embodiment, the method for producing a genome edited protoplast of a Vitis vinifera cultivar further comprises contacting the genome edited protoplast with a solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7 to wash the genome edited protoplast.

[0185] In a further embodiment, the method for producing a genome edited protoplast of a Vitis vinifera cultivar further comprises contacting the genome edited protoplast with a wash solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7 to for about an hour to allow for recovery of the genome edited protoplast, followed by washing the recovered genome edited protoplasts in further wash solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7.

[0186] In the Examples, the present inventors have used PEG transfection to transfect protoplasts produced by the methods as described herein. In brief, in one embodiment 200 pl of protoplasts at a concentration of lx 106cells / ml were gently mixed with 12pl of RNP and 210pl 40% PEG4000 solution and incubated for 30 minutes at 25°C on a shaking platform (30 rpm) in darkness. The incubation was stopped by the addition of 950pl of W5 solution and centrifugation lOOxg for 3 minutes at room temperature (no brake). This wash was repeatedwith 500 pl of W5 and the pellet resuspended in 1 ml of W1 solution. Transfected protoplasts were then incubated for a further 1 hour in W1 in the conditions or for 48 hours for testing of guides.

[0187] As used herein, "W5" refers to a solution comprising 2 mM MES pH 5.7, 5mM glucose, 154 mM NaCI, 125 mM CaC and 5 mM KCI.

[0188] As used herein, "Wl" refers to a solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7 to wash the genome edited protoplast.

[0189] The present inventors have demonstrated in the Examples that transfected protoplasts can be regenerated into embryos following cultivation of the genome edited protoplasts.

[0190] Accordingly, in one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar, further comprising cultivating the genome edited protoplast.

[0191] In one embodiment, the embryos are cultivated at a concentration of around 5xl05protoplasts / ml to around lxlO6protoplasts / ml.

[0192] In one embodiment, the embryos are cultivated at a concentration of around 5xl05protoplasts / ml or greater.

[0193] In a further embodiment, embryos detected during cultivation are further incubated for at least 1 or 2 weeks in the dark, and then transferred to low-light conditions.

[0194] For example, in the Examples, the present inventors have used encapsulation and cultivation of protoplasts in alginate beads. In brief, in one embodiment, protoplasts were washed again in Wl to remove all calcium then resuspended in the same medium. An equal volume of 3.2% NaAlginate in 0.4M mannitol was added so that the final protoplast plating density was 5xl05 / ml or greater. Beads were formed by expelling the alginate:protoplast solution through a 27G syringe needle into a 50 mM CaC , 0.4M mannitol solution and allowed to set for a minimum of 30 minutes. Beads were washed with reservoir medium containing0.3M glucose without charcoal and decanted into petri dishes. Reservoir medium with 0.3M glucose and 0.3% activated charcoal was added to each petri dish and these incubated in the dark at 27°C. Reservoir medium was exchanged fortnightly with the glucose concentration halved each time until it was absent. Depending on the variety of grapevine, embryos are found as early as 4 weeks post-transfection. When embryos become visible within beads, reservoir medium that had been removed as part of the fortnightly exchange, was spread onto GS1CA plates. Embryos were shown to be present in this liquid and to continue to develop on these plates. Larger more developed free-floating embryos were picked from around the beads and placed in a grid pattern directly onto GS1CA plates. Embryos were incubated for a further 2 weeks in the dark and then transferred to low-light for further development.

[0195] As used herein 'reservoir medium' comprises:1 x NN (1969) macronutrients (750 mg / L NH4NO3; 220 mg / L CaCI2.2H2O; 185 mg / L MgSO4.7H2O; 950 mg / L KNO3; 68 mg / L KH2PO4), 1 x NN (1969) micronutrients (10 mg / L H3BO3, 0.025 mg / L CuSO4.5H2O, 25 mg / L MnSO4.H2O, 0.25 mg / L Na2MoO4.2H2O, 10 mg / L ZnSO4.2H2O), 1 x NN (1969) vitamins (100 mg / L myo-inositol, 0.5 mg / L thiamine-HCI, 5 mg / L nicotinic acid, 0.5 mg / L pyridoxine HCI, 2 mg / L glycine, 0.5 mg / L folic acid, 0.05 mg / L D-biotin), 1 x Fe EDTA (37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O), 10.75 pM NAA, 2.25 pM BAP, 0.09 M sucrose, 0.3M or 0.15M or no glucose, pH 5.8, 0.3 % activated charcoal.

[0196] In one embodiment, cultivating the genome edited protoplast comprises a step of (i) incubation for about 1 hour followed by a wash step.

[0197] In another embodiment, cultivating the genome edited protoplast comprises a step of (i) incubation of about 1 hour, (ii) wash and (iii) followed by forming an alginate bead comprising the genome edited protoplast.

[0198] In one embodiment, the genome edited protoplasts are cultivated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks post transfection.

[0199] In one embodiment, the genome edited protoplasts are cultivated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks post transfection in the dark.

[0200] In one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein cultivating the genome edited protoplast comprises a step of forming an alginate bead comprising the genome edited protoplast. Other suitable alginate forms can be used.

[0201] In a preferred embodiment, the alginate forms (e.g. alginate bead) are in a reservoir medium.

[0202] In one embodiment, the medium referred to as protoplast culture medium (PCM) comprises NN basal medium with NN vitamins 10.75pM 1-naphthaleneacetic acid (NAA) and 2.25pM BA, 3.08% sucrose and glucose, with glucose decreasing fortnightly over 6 weeks until absent (e.g. 0.3M, 0.15M, 0M).

[0203] In one embodiment, activated charcoal (0.3% w / v) is added to the reservoir medium 1-2 days before use.

[0204] In another embodiment, activated charcoal (0.3% w / v) is added to the reservoir medium immediately before use.

[0205] In one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, wherein a solution comprising the genome edited protoplast is contacted with alginate and formed into beads by expelling the alginate solution through a needle.

[0206] In one embodiment, the alginate is a solution of 3.2% Na Alginate.

[0207] In another embodiment, the 3.2% Na Alginate solution and the solution comprising the genome edited protoplast are contacted at a ratio of 1:1.

[0208] In a further aspect, the present invention provides a genome edited protoplast produced by a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein.

[0209] Importantly, the present inventors have demonstrated that genome edited plants can be regenerated from the methods for producing a genome edited protoplast of a Vitis vinifera cultivar described herein.

[0210] For example, in the Examples, the present inventors have germinated embryos and formed plantlets. Cytokinins and auxins may be added to the medium to support the growth of microcalli from protoplasts. In brief, in one embodiment, germinated embryos were harvested, cut 5mm below the apical meristem to form hypocotyl explants and these transferred to a modified MS-based medium containing cytokinin for shoot development, 1.25 - 5.00pM BA dependant on grape variety and incubated under the same conditions. Shoots were excised and transferred to screw-capped vessels containing a modified MS-based medium with or without auxin (NAA) for root development. Rooted plantlets were produced within 2 weeks of transfer to this medium. Leaves were harvested from these plantlets for genetic testing and / or they were further propagated by tip cuttings to create clones for transfer to the glasshouse.

[0211] In one embodiment cytokinins and / or auxins are added to the medium to support the growth of microcalli from protoplasts. In another embodiment, the cytokinin or auxin is any cytokinin or auxin suitable for culture medium. In a further embodiment, the cytokinin is selected from the group consisting of 6-furfurylaminopurine (kinetin), trans-zeatin (tZ) and isopentenyladenine (iP).

[0212] For embryo germination, in one embodiment, embryos are grown in GS1CA medium (lOpM |3-naphthoxyacetic acid (NOA), lpM BA).

[0213] For embryo germination, in another embodiment, embryos are grown in the dark for two weeks, followed by 16 hours of low light and 8 hours of dark per day.

[0214] For shoot formation, in one embodiment, germinated embryos are grown in a shoot promoting medium. In another embodiment, germinated embryos are grown in SM with BA medium (1.25-5pM) in 16 hours of low light and 8 hours of dark per day.

[0215] As used herein, SM medium comprises:1 x SM macronutrients (160 mg / L NH4NO3; 440 mg / L CaCI2.2H2O; 370 mg / L MgSO4.7H2O; 2,325 mg / L KNO3; 340 mg / L KH2PO4), 1 x Fe EDTA (37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O); l x MS micronutrients (6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 16.9 mg / L MnSO4.H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O); 1 x B5 vitamins (100 mg / L myo-inositol, 10 mg / L thiamine-HCI, 1 mg / L nicotinic acid, 1 mg / L pyridoxine HCI), 1.5 % sucrose, 1.25 or 2.5 pM BAP, pH 5.7, 1% bacto agar.

[0216] In one embodiment, shooting medium + 1.25 pM BAP is used for microvines, and shooting medium + 2.5 pM is used for other cultivars.

[0217] For plantlet formation, in one embodiment, germinated embryos are grown in SM with or without NAA.

[0218] Accordingly, in one embodiment, the present invention provides a method for producing a genome edited protoplast of a Vitis vinifera cultivar as described herein, further comprising generating a plant from the genome edited protoplast.

[0219] In another aspect, the present invention provides a plant produced by a method as described herein, or from a protoplast produced by a method as described herein.

[0220] In another aspect, the RNP targets one or more genes selected from the group consisting of VvDMR6-l, VvDMR6-2, VvGH3-5, VvOMT3, and VvGINl.EXAMPLESExample 1: RNP / Cas9 transfection of Shiraz cv. protoplasts and regeneration of genome edited plantsProtoplast isolation:

[0221] For transfection of protoplasts with the intent to regenerate edited plants, protoplasts from Shiraz cultivar (BVRC12) embryogenic callus (5 weeks post last sub-culturing) produced from anthers in 2021 were isolated as follows:

[0222] Fresh cell wall digesting composition was prepared in MS Basal Digestion Medium. Three samples of callus (#1 = 0.63 g, #2 = 0.63 g, #3 = 0.68 g) were harvested into 55 mm petri dishes within 30 min each, cell wall digesting composition equivalent to 10 mL / g callus was added immediately after harvest of each sample. Placed on rotary shaker at 25°C and 30 rpm for 1 h (#1), 45 min (#2) or 30 min (#3) before mechanically disrupting the callus by pipetting up and down with a P1000 with 1 mL cut-tip and then with a 1 mL fine-tip. Petri dishes were then placed back on to the rotary shaker at 25°C and 30 rpm for 22 h in the dark.

[0223] After the overnight incubation the callus solutions were assessed for cell wall digestion using an inverted microscope which confirmed all three samples showed a large number of released protoplasts. Samples were kept separate throughout the following filtering process:

[0224] Protoplast samples were washed by filtering through a nylon mesh filter stack (Greiner; 100 pm (top) and 70 pm (bottom)), pre-wetted with 3 mL wash solution MMG, into a Falcon tube. Using a 10 mL pipette (Nunc) the protoplast digests were transferred to the filter stack, each petri dish was rinsed with 5 mL MMG, which was added to the strainers. Strainers were rinsed with 5 mL MMG. The 100 pm cell strainers were carefully removed and discarded and the 70 pm cell strainers were rinsed with an additional 5 mL MMG before discarding. The Falcon tubes were gently rolled to mix the digest, which was then carefully poured into a round-bottomed 50 mL centrifuge tube. After centrifugation for 3 min at 120xg with an acceleratiombrake setting of 1:1 (swing-out rotor), the supernatants were decanted anddiscarded. Each protoplast pellet was resuspended in 10 mL of MMG by gentle rolling of the tubes, then another 10 mL MMG was added and mixed by gentle rolling. Protoplasts were pelleted and supernatants discarded as described above and the wash step repeated once. Protoplasts were then resuspended and pooled as follows: 1 mL MMG was added to the samplletl pellet and resuspended cells were transferred to the sample# pellet. After resuspension the combined solution was added to the sample#3 pellet. The first tube was then rinsed with 1 mL MMG, which was used to rinse the second tube, after which the rinse solution was added to the pool tube and the total volume adjusted to 4 mL.

[0225] The cell suspension was carefully layered onto 2x 8 mL of a 16% sucrose solution (15 mL centrifuge tube, 2 mL cell suspension each) using a transfer pipette. After a centrifugation for 4 min at 90xg (no brake; swing-out rotor) thick protoplast bands were visible, which were carefully transferred using a pipetted P1000 with a cut-tip to a 50 mL round- bottomed centrifuge tube. Protoplasts were washed in 20 mL MMG and pelleted by centrifugation for 3 min at lOOxg (no brake; swing-out rotor), resuspended in 10 mL MMG, pelleted as above, the supernatant discarded, and the pellet resuspended in 1 mL MMG. Cell count (5.60xl06cells / mL) and viability (88.0%) were determined using a haemocytometer and Trypan Blue staining.Guide design, RNP assembly, and guide test in protoplasts:Transfection with RNPs, and alginate bead embedding

[0226] Purified Shiraz protoplasts were diluted in MMG to 1.2xl06cell / mL, and 12 pL of DM2c / Cas9 RNP and 210 pL of 40% PEG solution were added to 5x 200 pL cell aliquots in 2 mL Eppendorf tubes. After incubation at 25°C on a rotary shaker at 30 rpm for 30 min in the dark, 950 pL W5 solution was carefully added to each tube, followed by centrifugation for 3 min at lOOxg (no brake). The supernatant was removed (P1000), 500 pL W5 was added to each pellet and the centrifugation repeated, followed by removal of the supernatant and careful resuspension in 1 mL W1 solution. After incubation at 25°C on a rotary shaker at 30 rpm for either 1 h in the dark, one sample was merged evenly (250 pL each) with the other 4 samples, then protoplasts were pelleted as above, resuspended in 500 pL W1 solution and pooled toobtain 2x 1 mL samples, which were pelleted again and resuspended in 500 pL W1 solution each. Results for cell count and viability assays are listed in Table 1.

[0227] The W1 protoplast solution was gently mixed with 3.2% Na Alginate solution in a 1:1 (vol / vol) ratio, the mix was loaded into a 3 mL syringe and expelled as drops from a 27G needle into petri dishes containing a 50 mM CaC , 0.4 M mannitol solution. Beads were left to set for 30 min. Set beads were strained in a stainless-steel sieve and washed with 50 mL PCM_0.3IVI glucose (without activated charcoal) using a serological pipette. Beads were then placed into 35 mm petri dishes with 3 mL PCM_0.3l\ / l glucose (with 0.3% activated charcoal) and incubated in the dark at 27°C.

[0228] Table 1: RNP transfection details.Plant regeneration and detection of edits in regenerated plants:

[0229] Two weeks post alginate bead plating, the level of glucose in the reservoir medium was reduced to 0.15 M, and from one month post alginate bead plating onwards glucose was completely eliminated from the reservoir medium, which was refreshed every two weeks. 6 weeks after transfection embryos had started to develop in the beads and have continued to emerge 4 months after the transfection (Table 2).

[0230] Table 2: Genotypes of Shiraz embryos / plants from RNP-transfected protoplasts 3 months post transfection. Edit type for each target gene is indicated as wildtype (- / -), monoallelic edit (- / +) or biallelic edit (+ / +).At the transfer stage of embryos onto SM.BA plates for induction of shoot formation (Figure 1), root tissue was sampled for DNA extractions and assessed for target gene editing via restriction digest (Figure 2). Editing of the DMR6 target genes as indicated by digest results (summarized in Table 2) was confirmed by Sanger sequencing.

[0231] Out of the 16 tested embryos, 15 showed evidence of editing in at least one of the two target genes and 7 embryos showed biallelic edits in both DMR6 genes (Table 2, Figure 2).

[0232] Agarose gel photo of VvDMR6-2 (target of DM2c guide) and VvDMR6-l (target of DM2c guide) amplicons after restriction digest with Bgl I. Red stars denote uncut amplicons indicating removal of restriction sites by editing. Results are also shown in Table 2.Summary of genotypes and phenotypes of regenerated plants:

[0233] A total of 23 Shiraz regenerants, confirmed as diploid by flow cytometry assay, were selected for further studies. These include 4 WT plants, 8 plants with biallelic edits in both VvDMR6 genes and 11 plants with various combinations of VvDMR6-l and VvDMR6-2 edits.

[0234] In-vitro, no phenotypic differences between Vvdmr6 mutant plants and WT plants were observed (Figure 3).

[0235] A selection of 13 plants has been established in the glasshouse. Plants have been growing for over 10 months in the glasshouse. No phenotypic abnormalities have been observed.Example 2: Editing of Shiraz cv. protoplasts and regeneration of genome edited plantsProtoplast isolation:

[0236] For transfection of protoplasts with the intent to regenerate edited plants, protoplasts from Shiraz cv (BVRC12) embryogenic callus (4 weeks post last sub-culturing) produced from anthers in 2021 were isolated as follows:

[0237] Fresh cell wall digesting composition was prepared in MS Basal Digestion Medium. Three samples of callus (#1 = 0.66 g, #2 = 0.53 g, #3 = 0.58 g) were harvested into 55 mm petri dishes within 30 min each, cell wall digesting composition equivalent to 10 mL / g callus was added immediately after harvest of each sample. Then the following treatments were applied: Samples #1 and #2 were placed on to the shaker at 25°C and 30 rpm for 1 h in the dark then an additional treatment was applied as outlined below. Samples #2 and #3 were treated immediately after collection.

[0238] #1: lots of large cell clumps were visible, manual disruption by gentle pipetting until the digest appeared cloudy with lots of small groups of cells, then placed back on to the shaker for 16 h.

[0239] #2: less clumps than #1, applied vacuum of -0.6 bar (= -0.06 MPa) for 20 min (plate was placed in vacuum desiccator in flow as plate was vented left lid on but removed parafilm) with occasional swirling of the desiccator. Air was reintroduced slowly, and the plate resealed and placed back on to the shaker for 16 h.

[0240] #3: similar to #1 in appearance, applied vacuum of -0.7 bar (= -0.07 MPa) for 20 min (plate was placed in vacuum desiccator in flow as plate was vented left lid on but removedparafilm) with occasional swirling of the desiccator. Air was reintroduced slowly, and the plate resealed and placed back on to the shaker for 16 h.

[0241] After 16 h incubation the callus solutions were assessed for cell wall digestion using an inverted microscope. As digestion appeared most complete for sample #1 protoplast sample (which also contained the most callus starting weight), samples were kept separate throughout the following filtering process.

[0242] Protoplast samples were washed by filtering through a nylon mesh filter stack (Greiner; 100 pm (top) and 70 pm (bottom)), pre-wetted with 3 mL wash solution MMG, into a Falcon tube. Using a 10 mL pipette (Nunc) the protoplast digests were transferred to the filter stack, each petri dish was rinsed with 5 mL MMG, which was added to the strainers. Strainers were rinsed with 5 mL MMG. The 100 pm cell strainer was carefully removed and discarded and each 70 pm cell strainer was rinsed with an additional 5 mL MMG before discarding. The Falcon tubes were gently rolled to mix the digest, which was then carefully poured into a round-bottomed 50 mL centrifuge tube. After centrifugation for 3 min at 120xg with an acceleratiombrake setting of 1:1 (swing-out rotor), the supernatants were decanted and discarded. Samples #2 and #3 had the smaller and cleaner looking pellets than sample #1. Each protoplast pellet was resuspended in 10 mL of MMG by gentle rolling of the tubes (tube roller at 3.5 setting), then another 10 mL MMG were added and mixed by gentle rolling. Protoplasts were pelleted and supernatants discarded as described above and the wash step repeated once. Protoplast pellets for all samples looked a lot cleaner after the wash steps. Protoplasts were then resuspended as follows: 1 mL MMG was added to sample #2 pellet and resuspended cells were transferred to and pooled with the sample #3 pellet. The first tube was rinsed with 500 pL MMG and the rinse solution was added to the pool tube for a total volume of 2 mL. The pellets from sample #1 were resuspended in 1 mL MMG each and the volume adjusted to 2 mL.

[0243] The three cell suspensions were carefully layered onto 8 mL of a 16% sucrose solution (15 mL centrifuge tube) each using transfer pipettes. After a centrifugation for 4 min at 90xg (no brake; swing-out rotor) thick protoplast bands were visible for all three samples, which were carefully transferred (P1000 and cut-tip) to 50 mL round-bottomed centrifugetubes. Protoplasts were washed in 20 mL MMG and pelleted by centrifugation for 3 min at lOOxg (no brake; swing-out rotor) with sampletl yielding a bigger pellet than the combined sample #2 and #3 sample. The pellets were sequentially resuspended in 10 mL MMG, pelleted as above and the supernatant discarded, and the pellet resuspended in 1.5 mL MMG. Cell count and viability of the pooled protoplasts was assessed. Cell count (16.7xl06cells / mL) and viability (85.0%) were determined using a haemocytometer and Trypan Blue staining.

[0244] For transfection of protoplasts to test guides for their target gene editing effectiveness, protoplasts from a 5 month old Shiraz cv (BVRC12) suspension culture produced from embryogenic callus derived from anthers in 2020 were isolated as above, with the following modifications:

[0245] Six samples of cells (0.53 g - 0.65 g) were harvested and filtered and washed separately. Each pellet was resuspended in 1.5 mL MMG, all resuspended cells were pooled, and each tube rinsed with 500 pL MMG to reach a final volume of 12 mL. The solution was split (2 mL each) across 6x 8 mL of a 16% sucrose solution (15 mL centrifuge tube) using transfer pipettes, yielding thick protoplast bands, which were pooled, washed, and resuspended in MMG (final volume was 2 mL). The final pooled sample cell viability was determined to be 89.3% and cell count was 8.0xl06cells / mL.Guide design, RNP assembly, and guide test in protoplasts:

[0246] For the editing of the VvGH3-5 (Vitvil2g00593) gene, encoding for an auxinconjugating acyl transferase, with the aim to produce grapevine mutants with increased water use efficiency, three guides for testing in Shiraz protoplasts were selected based on the following process: the most suitable exon for guide placement as determined by Synthego 'Knockout Guide Design' tool was selected for guide identification using the Benchling 'CRISPR Guide RNA design' tool and guides ranked highly in both guide design programs were further assessed for their GC content, CRISPOR), WU-CRISPR , and TUSCAN scores, as well as predicted RNA-fold and sequence identity across multiple winegrape cultivars. The three guides that most closely fulfilled all desired sequence characteristics were selected for testing in Shiraz protoplasts (Table 3).

[0247] The assembly of each of the three GH3-5 / Cas9 RNP (Table 3) was performed according to IDT guidelines as follows: the duplex between guide RNA (crRNA) and RNA scaffold (tracrRNA) was formed by resuspending crRNA and tracrRNA to 2mM in IDTE buffer, pH 8.0, combining equal volumes of the two RNAs in a 0.2 mL PCR tube, heating to 95°C for 5 minutes, cooling to room temperature and incubating in the dark for 1 h at room temperature. For RNP formation with Cas9, ratios of 2 nmole of the crRNA / tracrRNA duplex (sgRNA) and 10 pg of IDT Cas9 (diluted to 1 pg / pL in PBS buffer, pH 7.4) were combined in a 0.2 mL PCR tube, incubated at room temperature for 10 min and stored at -20°C for less than a month prior to protoplast transfection.

[0248] Table 3: Sequences of three tested guides for editing of VvGH3-5 and evaluation of editing competence in Shiraz protoplasts.*Sanger sequencing of target region amplicon followed by Synthego 'Inference of CRISPR Edits (ICE)' analysis)

[0249] For guide testing, purified Shiraz protoplasts (suspension culture, see section above) were diluted in MMG to lxlO6cell / mL, and for each of the three GH3-5 / Cas9 RNPs 12 pL RNP solution and 210 pL of 40% PEG solution were added to 2x 200 pL cell aliquots in 2 mL Eppendorf tubes. After incubation at 25°C on a rotary shaker at 30 rpm for 30 min in the dark, 950 pL W5 solution was carefully added to each tube, followed by centrifugation for 3 min at lOOxg (no brake). The supernatant was removed (P1000), 500 pL W5 were added to each pellet and the centrifugation repeated, followed by removal of the supernatant and careful resuspension in 1 mL W1 solution. After incubation at 25°C on a rotary shaker at 30 rpm for either 1 hr or 48 h, protoplast DNA was extracted and used for target region amplification followed by Sanger sequencing for edit detection (Table 3).

[0250] Guide GH3-5_3 was selected for transfection of Shiraz protoplasts with the intent to regenerate genome edited plants.Transfection with RNPs, and alginate bead embedding

[0251] Three 200 pL aliquots of purified Shiraz protoplasts (derived from solid embryogenic callus, see section above) were transfected with the GH3-5_3 / Cas9 RNP as described above. After incubation at 25°C on a rotary shaker at 30 rpm for 1 h in the dark protoplasts were washed and pelleted as above, resuspended in a total of 400 pL W1 solution (450 pL resuspension volume) and cell count / viability assayed as described above. Results are shown in Table 4.

[0252] The W1 protoplast solution was gently mixed with 3.2% Na Alginate solution in a 1:1 (vol / vol) ratio, the mix was loaded into a 3 mL syringe and expelled as drops from a 27G needle into petri dishes containing a 50 mM CaC , 0.4 M mannitol solution. Beads were left to set for 30 min. Set beads were strained in a stainless-steel sieve and washed with 50 mL PCM_0.3IVI glucose (without activated charcoal) using a serological pipette. Beads were then placed into a 35 mm petri dish with 3 mL PCM_0.3l\ / l glucose and 3% activated charcoal and incubated in the dark at 27°C.

[0253] Table 4: RNP transfection details.Plant regeneration and detection of edits in regenerated plants:

[0254] Two weeks post alginate bead plating, the level of glucose in the reservoir medium was reduced to 0.15 M, and from one month post alginate bead plating onwards glucose was completely eliminated from the reservoir medium, which was refreshed every two weeks.Seven weeks after transfection embryos had started to develop in the beads and continue to develop 6 months after the transfection (Figure 4, Table 5).

[0255] Table 5: Genotypes of Shiraz embryos / plants from RNP-transfected protoplasts 6 months post transfection. Edit type for each target gene is indicated as wildtype (- / -), monoallelic edit (- / +) or biallelic edit (+ / +)■

[0256] At the transfer stage of embryos onto SM.BA plates for induction of shoot formation (Figure 5), root tissue was sampled for DNA extractions and assessed for target gene editing via Sanger sequencing (Figure 6). The detected genotypes are summarized in Table 5.

[0257] A high number of germinated embryos were recovered in this experiment, 16 were selected for additional testing. Out of the 16 tested embryos, 6 showed evidence of editing (El-6), with 4 monoallelic (E3-E6) and 2 biallelic (El, E2) genotypes identified (Table 5, Figure 6). A total of 9 Shiraz regenerants, confirmed as diploid by flow cytometry assay, were selected for further studies. These include 4 WT plants and 5 plants with biallelic edits in the VvGH3-5 gene.Example 3: RNP / Cas9 transfection of Chardonnay protoplasts and regeneration of edited plantsProtoplast isolation:

[0258] Protoplasts from Chardonnay (clone V1011) embryogenic callus (5 weeks post last sub-culturing) produced from anthers in 2021 were isolated as follows:

[0259] Fresh cell wall digesting composition was prepared in MS Basal Digestion Medium. Two samples of 0.5 g callus were harvested into 55 mm petri dishes within 30 min each, cellwall digesting composition equivalent to 10 mL / g callus was added immediately after harvest of each sample. Callus was broken up by pipetting up and down with a P1000 with 1 mL cuttip and then with a 1 mL fine-tip, then placed on to the shaker for 16 h in the dark (25°C, 30 rpm).

[0260] After 16 h incubation the disrupted callus solutions were assessed for cell wall digestion using an inverted microscope. As digestion appeared incomplete for both samples, due to presence of visible cell clumps, samples were kept separate throughout the following filtering process.

[0261] Protoplast samples were washed by filtering through a nylon mesh filter stack (Greiner; 100 pm (top) and 70 pm (bottom)), pre-wetted with 3 mL wash solution MMG, into a Falcon tube. Using a 10 mL pipette (Nunc) the protoplast digests were transferred to the filter stack, each petri dish was rinsed with 5 mL MMG, which was added to the strainers. Strainers were rinsed with 5 mL MMG. The 100 pm cell strainers were carefully removed and discarded and the 70 pm cell strainers were rinsed with an additional 5 mL MMG before discarding. The Falcon tubes were gently rolled to mix the digest, which was then carefully poured into a round-bottomed 50 mL centrifuge tube. After centrifugation for 3 min at 120xg with an acceleratiombrake setting of 1:1 (swing-out rotor), the supernatants were decanted and discarded. Each protoplast pellet was resuspended in 10 mL of MMG by gentle rolling of the tubes, then another 10 mL MMG were added and mixed by gentle rolling. Protoplasts were pelleted and supernatants discarded as described above and the wash step repeated once. Protoplasts from the two samples were then pooled as follows: 1 mL MMG was added to one pellet and resuspended cells were transferred to the second pellet. The first tube was rinsed twice with 200 pL MMG and the rinse solutions were added to the pool tube.

[0262] The total pool volume was adjusted to 2 mL and carefully layered onto 8 mL of a 16% sucrose solution (15 mL centrifuge tube) with a transfer pipette. After a centrifugation for 4 min at 90xg (no brake; swing-out rotor) a thick protoplast band was visible, which was carefully transferred using a pipetted P1000 and cut-tip, to a 50 mL round-bottomed centrifuge tube. Protoplasts were washed twice in 20 mL MMG with centrifugations for 3 min at lOOxg(no brake; swing-out rotor) and resuspended in 2 mL MMG. Cell count (8.7xl06cells / mL) and viability (86.1%) were determined using a haemocytometer and Trypan Blue staining.Guide design, in vitro guide test and RNP assembly:

[0263] For the editing of two VvDMR6 (VvDMR6-l, VvDMR6-2) genes with the aim to produce grapevine mutants with increased resistance to downy mildew, several guides were designed based on outputs from the CRISPOR guide design tool in combination with scores from the WU-CRISPR and TUSCAN guide design tools, and were shown to elicit cleaving of either VvDMR6-l or VvDMR6-2 as expected using in vitro and in planta test systems (data not shown). A previously published dsgRNA guide, DM2c (Table 6), designed and reported to edit VvDMR6-2 in table grapes using a DNA-free protoplast based editing system was also tested.

[0264] Table 6: Guide sequence for VvDMR6-l / VvDMR6-2.

[0265] In the in vitro assay, this guide successfully cleaves both, the VvDMR6-l, and VvDMR6-2 genes in the isolated Chardonnay protoplasts (Figure 7). The current inventors observed that the VvDMR6-l sequence only differs by one nucleotide across the region complementary to the 20 bp DM2c guide sequence when compared with VvDMR6-2 (Figures 8 and 9). In this experiment, the DM2c guide was used for the transfection and successful editing of the endogenous genes in isolated Chardonnay protoplasts. This dual activity of DM2c was recently reported for Crimson seedless and Sugraone table grape cultivar, where DMR6 gene editing was achieved using agrobacterium-mediated callus transformation. In contrast, here the DM2c guide was used for the transfection of the isolated Chardonnay protoplasts.

[0266] The assembly of the DM2c / Cas9 RNP (Table 7) was performed according to IDT guidelines as follows: the duplex between guide RNA (crRNA) and RNA scaffold (tracrRNA) was formed by resuspending crRNA and tracrRNA to 2mM in IDTE buffer, pH 8.0), combining equal volumes of the two RNAs in a 0.2 mL PCR tube, heating to 95°C for 5 minutes, cooling to roomtemperature and incubating in the dark for 1 h at room temperature. For RNP formation with Cas9, ratios of 2 nmole of the crRNA / tracrRNA duplex (sgRNA) and 10 pg of IDT Cas9 (diluted to 1 pg / pL in PBS buffer, pH 7.4) were combined in a 0.2 mL PCR tube, incubated at room temperature for 10 min and stored at -20°C for less than a month prior to protoplast transfection.Transfection with RNPs, and alginate bead embedding

[0267] Purified Chardonnay protoplasts were diluted in MMG to lxlO6cell / mL and 13x 200 pL cell aliquots in 2 mL Eppendorf tubes were prepared. 12 pL of DM2c / Cas9 RNP prepared gRNA / Cas9 mixture was added to each tube along with 210 pL of 40% PEG solution. After incubation at 25°C on a rotary shaker at 30 rpm for 30 min in the dark, 950 pL W5 solution was carefully added to each tube, followed by centrifugation for 3 min at lOOxg (no brake). The supernatant was removed by pipette (P1000), 500 pL W5 was added to each pellet and the centrifugation repeated, followed by removal of the supernatant and careful resuspension in 1 mL of W1 solution. After incubation at 25°C on a rotary shaker at 30 rpm for either 1 h (10 transfection samples) or 22 h (3 transfection samples) in the dark, protoplasts were pelleted as above, resuspended in 500 pL W1 solution and pooled, followed by cell count and viability assays as described above. Results are summarised in Table 7.

[0268] The W1 protoplast solution (4 mL (1 h), 1 mL (22 h)) was gently mixed with 3.2% Na Alginate solution in a 1:1 (vol / vol) ratio, the mix was loaded into a 3 mL syringe and expelled as drops from a 27G needle into petri dishes containing a 50 mM CaC , 0.4 M mannitol solution. Beads were left to set for 30 min. Set beads were strained in a stainless-steel sieve and washed with 50 mL PCM_0.3l\ / l glucose (without activated charcoal) using a serological pipette. For the 1 h sample, beads were then placed into 3x 55 mm petri dishes (280 beads / petri dish) with 4 mL PCM_0.3IVI glucose (without activated charcoal) and incubated in the dark at 27°C. An additional 424 beads (140-144 / well) were placed into 3 wells of a 6-well plate with 3 mL PCM_0.3l\ / l glucose (without activated charcoal) per well and incubated in the same way. For the 22 h sample, beads were placed into 3x 35 mm petri dishes (126-138 beads / petri dish), 2.5 mLof PCM_0.3l\ / l glucose (without activated charcoal) was added to each dish and the incubation occurred as above.

[0269] Table 7: RNP transfection details.Plant regeneration and detection of edits in regenerated plants:

[0270] Two weeks post alginate bead plating, the level of glucose in the reservoir medium was reduced to 0.15 M, and from one month post alginate bead plating onwards glucose was completely eliminated from the reservoir medium, which was refreshed every two weeks. Seven weeks after transfection microcalli formation was documented (Figure 10A) followed by embryo emergence a week later (Figure 10B). Thousands of embryos continued to develop in the beads until six months after transfection (Table 7).

[0271] At the transfer stage of embryos onto SM.BA plates for induction of shoot formation (Figure 11), root tissue was sampled for DNA extractions and assessed for target gene editing via restriction digest (Figure 12). Editing of the DMR6 target genes as indicated by digest results was confirmed by Sanger sequencing.

[0272] Agarose gel photos of VvDMR6-2 and VvDMR6-l (both targets of DM2c guide) amplicons after restriction digest with Bgl I. Stars denote uncut amplicons (= biallelic edits), # indicate partially cut amplicons (= monoallelic edits) and Chard -ve represents a non-transfected, fully digested wildtype sample. Aligned lanes in both panels correspond to the same embryo DNA sample.

[0273] Out of the 443 germinated embryos that have been tested for editing of the VvDMR6-2 and VvDMR6-l genes as describe above, 204 of the developing plants were confirmed as containing a genome edit (46%), with all possible combinations of mono- and biallelic edits detected (Table 8).

[0274] Table 8: Genotypes of Chardonnay embryos / plants regenerated from protoplasts after RNP transfection with the DM2c guide.

[0275] Selected lines for each genotype have been regenerated in tissue culture, with no obvious phenotypic differences between WT lines and edited plants (Figure 13).

[0276] Plants of all eight genotypes are assessed for their susceptibility to downy mildew (DM), as well as powdery mildew (PM) in the glass house and selected lines tested under field conditions.Summary of genotypes and phenotypes of regenerated plants:

[0277] Chardonnay WT plants and Vvdmr6 mutants displaying apparent diploid allelic profiles by Sanger sequencing (Table 9) were transferred to potting mix and cultivated in theglasshouse for a year, where all plants developed without any noticeable phenotypic abnormalities (Figure 14).

[0278] Table 9: Edit types of phenotyped Chardonnay plants. Protospacer Adjacent Motif (PAM) sites are indicated in bold and nucleotide insertions and deletions are shown in underline.

[0279] Changes to DM susceptibility conferred by edits in Chardonnay VvDMR6 genes were assessed by detached leaf disc assays, involving the quantification of DM sporangia produced within 7 days of inoculation. A significant reduction in sporangia counts of about 50% was observed for the double mutant lines as well as for Vvdmr6-2 single mutants, whereas Vvdmr6-1 single mutants displayed WT infection severity (Figure 15A).

[0280] The DM - and broader - resistance phenotype often observed for dmr6 mutants is based on the hydroxylase activity of functional DMR6 enzymes, which leads to the inactivation of the plant hormone salicylic acid (SA), thereby reducing the concentration of this essential signalling component in defence pathways against biotrophic pathogens. Consequentially, increased SA levels are typically observed in leaves of dmr6 mutants with reduced DM / pathogen susceptibility. However, the expected elevated concentration of SA in Vvdmr6 Chardonnay plants with decreased DM sporangia counts was only detected in double mutant leaves (Figure 15B), reflecting previous findings by Giacomelli et al. (https: / / doi.org / 10.3389 / fpls.2023.1242240) in two table grape cultivars. Except for three outlier data points stemming from one of the nine analysed Vvdmr6-2 mutant lines, leaf SA concentrations of Vvdmr6-2 plants were comparable to WT and Vvdmr6-1 lines (Figure 15B).

[0281] Whilst SA is the predominant plant hormone associated with defence responses against biotrophic pathogens, a second hormone, jasmonic acid (JA), has also been linked to the grapevine DM response in previous studies. In their role as defence hormones, SA and JA are most commonly known for their antagonistic interactions in response to biotrophic and necrotrophic pathogens, but extensive cooperative gene regulation in Arabidopsis, are suggestive of a potential synergistic relationship between the two hormones in the grapevine response to DM. Measurements of leaf JA content in Chardonnay WT and Vvdmr6 mutants revealed increased concentration of this defence hormone in both Vvdmr6-l / Vvdmr6-2 double mutants as well as Vvdmr6-2 single mutants (Figure 15C), thereby providing a possible causal link to the observed reduced DM susceptibility of Vvdmr6-2 lines (Figure 15A).

[0282] That glasshouse-grown Chardonnay plants were shown to be significantly less susceptible to downy mildew (DM) infection when carrying edits in either VviDMR6-2 alone, or in combination with VviDMR6-l, represents a novel finding regarding cultivar-specific contributions of VviDMR6 genes to DM susceptibility. Leaf hormone measurements in the mutants confirmed the known link of SA with DM susceptibility reduction, but also uncovered a potential, VviDMR6-2-associated, role for jasmonic acid (JA) in grapevine defence against this pathogen.Example 4: Isolation and Editing of Sauvignon Blanc protoplasts; regeneration of edited plantsSauvignon Blanc Protoplast isolation

[0283] Protoplasts were prepared from Sauvignon Blanc (clone F4V6) embryogenic callus produced from anthers in 2022 and then grown in suspension culture as described in Example 3 with the following modifications. The culture medium for Sauvignon Blanc was modified by the addition of 1% (w / v) polyvinylpyrrolidone (PVP) 40 to prevent browning, which was an issue with this callus when growing on Cl medium only. The Sauvignon Blanc callus that was used had been grown and sub-cultured on solid Cl-PVP1plates to improve health of the callus. Cells were incubated with the cell wall digestion composition overnight.

[0284] Since Sauvignon Blanc protoplast isolation and density requirements was unknown a sucrose gradient was prepared. Protoplasts were handled as per Example 3 except that samples were layered onto either 25%, 20% or 16% sucrose (w / v) gradients. Clear protoplast bands were obtained from each tested sucrose gradient. Similar to the previous method of Example 3, the recovered protoplasts were pooled and resuspended in 1.5 mL MMG. Cell count were determined using a haemocytometer and Trypan Blue staining. The obtained cell count of 10.2x106 cells / mL with an exception viability of 94.0%.Transfection with RNPs, alginate bead embedding and detection of gene edits in Sauvignon Blanc protoplasts

[0285] DM2c guide and RNP transfection process as per Chardonnay, Cabernet Sauvignon and Shiraz examples. The inventors assessed the viability and cell count lh after the transfection, resulting viability was 96%, cell count was 2.16xl06cells / mL.Plant regeneration and detection of genome edits in regenerated plants

[0286] Following the method described in Example 3, developing embryos were observed in beads six weeks post alginate bead plating and eight months after transfection 514 embryos have been moved to GS1CA plates and 53 regenerants have been moved to RM medium and have developed into plants.

[0287] Out of the first 53 tested regenerants, 44 showed evidence of editing in at least one of the two target genes and 15 regenerated plants appear to have biallelic edits in both target genes (Table 10). For 24 regenerants the genotypes as determined by CAPS assays have also been confirmed by Sanger sequencing.

[0288] Table 10: Genotypes of Sauvignon Blanc embryos regenerated from RNP- transfected protoplasts 8 months post transfection. Edit type for each target gene is indicated as wildtype (- / -), monoallelic edit (- / +) or biallelic edit (+ / +).

[0289] Summary of genotypes and phenotypes of regenerated plants:

[0290] A total of 59 Sauvignon blanc regenerants, confirmed as diploid by flow cytometry assay, were selected for further studies. These include 6 WT plants, 37 plants with biallelic edits in both VvDMR6 genes and 16 plants with various combinations of VvDMR6-l and VvDMR6-2 edits.

[0291] In-vitro, no phenotypic differences between Vvdmr6 mutant plants and WT plants were observed (Figure 16).

[0292] A selection of 21 plants has been established in the glasshouse. Plants have been growing for over 7 months in the glasshouse. No phenotypic abnormalities have been observed.

[0293] The present inventors found that protoplasts from different varieties of grapevine sediment differently in the presence of sucrose prepared in purified water. To test the most appropriate concentration of sucrose to use for the purification of grape protoplasts, a sucrose gradient was set up by layering sucrose in concentrations of 35, 30, 25, 20, 15 and 10% sequentially into a 15ml centrifuge tube and layering 2ml of protoplast digest in MMG on top.Sucrose was freshly prepared and filtered through a 0.22pm filter before use. This tube is then centrifuged at 90g for 4mins (no brake) and bands of protoplasts collected. Bands were kept separate, washed in 20ml and then 10ml MMG (100g for 3 mins with no braking) and then counts done to determine the number and viability of protoplasts from each band (Moon et al., 2021). For grapevine protoplasts we have identified that the most suitable concentration of sucrose varies between varieties. Interestingly the recalcitrant Cabernet Sauvignon was difficult to recover from lower concentrations of sucrose compared to concentrations above about 20%. Using the higher concentrations of sucrose permitted recovery and plantlet regeneration without expensive growth support nutrients and difficult methods.Example 5: Editing of V4 microvine protoplasts and regeneration of edited plantsProtoplast isolation:

[0294] Protoplasts from a 4 months old microvine (V4) suspension culture produced from embryogenic callus derived from anthers in 2021 were isolated as follows.

[0295] Fresh cell wall digesting composition n was prepared in MS Basal Digestion Medium. Eight batches of cells (#1 = 0.40 g, #2 = 0.47 g, #3 = 0.40 g, #4 = 0.38 g, #5 = 0.46 g, #6 = 0.52 g, #7 = 0.47 g, #8 = 0.43 g) were harvested from 120 mL suspension cultures as follows: the culture flask was placed on a stand at 45° and most of the culture medium was pipetted off, placing the first 50 mL in a Falcon tube. The culture was poured into an autoclaved 150 mL beaker, the flask rinsed with the reserved 50 mL medium and added to the beaker. After allowing the cells to settle the liquid was removed as much as possible (pipette), then the cells were gently scraped from the beaker on to a stack of filter paperto absorb the remaining liquid and weighed into 55 mm petri dishes. Cell wall digesting composition solution (10 mL / g) was added immediately after harvest of each batch. Dishes were then placed on the rotary shaker at 25°C and 30 rpm for 22 h in the dark.

[0296] The callus solutions were assessed for cell wall digestion (inverted microscope) and protoplasts as well as some undigested materials were observed. The 8 samples were kept separate throughout the following filtering process.

[0297] Protoplast samples were washed by filtering through a nylon mesh stack (Greiner; 100 pm (top) and 70 pm (bottom)), pre-wetted with 3 mL wash solution MMG, into a Falcon tube. Using a 10 mL pipette (Nunc) the protoplast digests were transferred to the filter stack, each petri dish was rinsed with 5 mL MMG, which was added to the strainers. Strainers were rinsed with 5 mL MMG. The 100 pm cell strainers were carefully removed and discarded and the 70 pm cell strainers were rinsed with an additional 5 mL MMG before discarding. The Falcon tubes were gently rolled to mix the digest, which was then carefully poured into a round-bottomed 50 mL centrifuge tube. After centrifugation for 3 min at 120xg with an acceleratiombrake setting of 1:1 (swing-out rotor), the supernatants were decanted and discarded. Each protoplast pellet was resuspended in 10 mL MMG by gentle rolling ofthe tubes (tube roller at 3.5 setting), then another 10 mL MMG were added and mixed by gentle rolling. Protoplasts were pelleted and supernatants discarded as described above and the wash step repeated once. Protoplast pellets for all batches looked to be a good size and colour. Protoplasts were then resuspended sequentially in two batches of four pellets using 1 mL MMG as the starting resuspension solution each. This was followed by a 500 pL MMG rinse performed in the same way. The volume for each batch was adjusted to 4 mL each.

[0298] The cell suspensions were carefully layered in 2 mL aliquots onto 4x 8 mL of a 16% sucrose solution (15 mL centrifuge tube) using transfer pipettes. After a centrifugation for 4 min at 90xg (no brake; swing-out rotor) protoplast bands were visible for all four samples, which were carefully pooled (P1000 and cut-tip) in a 50 mL round-bottomed centrifuge tube. Protoplasts were washed in 20 mL MMG and pelleted by centrifugation for 3 min at lOOxg (no brake; swing-out rotor), resuspended in 10 mL MMG, pelleted as above, the supernatant discarded, and the pellet resuspended in 2 mL MMG. Cell count (10.6xl06cells / mL) and viability (88.0%) were determined using a haemocytometer and Trypan Blue staining.Guide design, RNP assembly, and guide test in protoplasts:

[0299] Forthe editing ofthe VvGINl (Vitvil6g00713) gene, encodingforthe main vacuolar invertase in grape, with the aim to produce grapevine mutants with reduced hexose sugar levels, three guides for testing in V4 microvine protoplasts were selected based on the following process: the most suitable exon for guide placement as determined by Synthego'Knockout Guide Design' tool was selected for guide identification using the Benchling 'CRISPR Guide RNA design' tool and guides ranked highly in both guide design programs were further assessed for their GC content, CRISPOR, WU-CRISPR, and TUSCAN scores, as well as predicted RNA-fold and sequence identity across multiple winegrape cultivars. The three guides that most closely fulfilled all desired sequence characteristics were selected for testing in V4 microvine protoplasts (Table 11).

[0300] The assembly of each of the three GIN1 / Cas9 RNP (Table 11) was performed according to IDT guidelines as follows: the duplex between guide RNA (crRNA) and RNA scaffold (tracrRNA) was formed by resuspending crRNA and tracrRNA to 2 mM in IDTE buffer, pH 8.0, combining equal volumes of the two RNAs in a 0.2 mL PCR tube, heating to 95°C for 5 minutes, cooling to room temperature and incubating in the dark for 1 h at room temperature. For RNP formation with Cas9, ratios of 2 nmole of the crRNA / tracrRNA duplex (sgRNA) and 10 pg of IDT Cas9 (diluted to 1 pg / pL in PBS buffer, pH 7.4) were combined in a 0.2 mL PCR tube, incubated at room temperature for 10 min and stored at -20°C for less than a month prior to protoplast transfection.

[0301] Table 11: Sequences of top three selected guides for editing of VvGINl and evaluation of editing competence in V4 microvine protoplasts.*Sanger sequencing of target region amplicon followed by Synthego 'Inference of CRISPR Edits (ICE)' analysis

[0302] For guide testing, purified V4 microvine protoplasts were diluted in MMG to lxlO6cell / mL, and for each of the three GIN1 / Cas9 RNPs 12 pL RNP solution and 210 pL of 40% PEG solution were added to 3x 200 pL cell aliquots in 2 mL Eppendorf tubes. After incubation at 25°C on a rotary shaker at 30 rpm for 30 min in the dark, 950 pL W5 solution was carefullyadded to each tube, followed by centrifugation for 3 min at lOOxg (no brake). The supernatant was removed (P1000), 500 pL W5 were added to each pellet and the centrifugation repeated, followed by removal of the supernatant and careful resuspension in 1 mL W1 solution. After incubation at 25°C on a rotary shaker at 30 rpm for 22 h, protoplast DNA was extracted and used for target region amplification followed by Sanger sequencing for edit detection (Table 11).

[0303] Guide GIN1_2 was the best performing guide and was selected for transfection of V4 protoplasts with the intent to regenerate edited plants.Transfection with RNPs, and alginate bead embedding

[0304] A 200 pL aliquot of purified V4 microvine protoplasts (see first section above) was transfected with the GIN1_2 / Cas9 RNP as described above. After incubation at 25°C on a rotary shaker at 30 rpm for 1 h in the dark protoplasts were washed and pelleted as above, resuspended in 200 pL W1 solution (230 pL resuspension volume) and cell count / viability assayed as described above. Results are listed in Table 12.

[0305] The W1 protoplast solution was gently mixed with 3.2% Na Alginate solution in a 1:1 (vol / vol) ratio, the mix was loaded into a 3 mL syringe and expelled as drops from a 27G needle into petri dishes containing a 50 mM CaC , 0.4 M mannitol solution. Beads were left to set for 30 min. Set beads were strained in a stainless-steel sieve and washed with 50 mL PCM_0.3IVI glucose using a serological pipette. Beads were then placed into a 35 mm petri dish with 2.5 mL PCM_0.3IVI glucose and incubated in the dark at 27°C.

[0306] Table 12: RNP transfection details.Plant regeneration and detection of edits in regenerated plants:

[0307] Two weeks post alginate bead plating, the level of glucose in the reservoir medium was reduced to 0.15 M. After one month and prior to the elimination of glucose from the reservoir medium, embryos had started to develop in the beads (Figure 17) and continue to develop 4 months after the transfection (Table 13).

[0308] Table 13: Regeneration of edited plants from RNP-transfected protoplasts 4 months post transfection. Edit type for each target gene is indicated as wildtype (- / -), monoallelic edit (- / +) or biallelic edit (+ / +)■

[0309] At the transfer stage of embryos onto SM.BA plates for induction of shoot formation (Figure 18), root tissue was sampled for DNA extractions and assessed for target gene editing via Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assay as shown in Figure 19.

[0310] Out of the 16 tested embryos, 13 showed evidence of editing, with 2 monoallelic and 11 biallelic genotypes identified (Table 13, Figure 19B).Summary of genotypes and phenotypes of regenerated plants:

[0311] A total of 9 V4 microvine regenerants, confirmed as diploid by flow cytometry assay, were selected for further studies. These include 3 WT plants and 6 plants with biallelic edits in the VvGINl gene.

[0312] In-vitro, and at early maturation stage in the glasshouse (Figure 20), no phenotypic differences between Vvginl mutant plants and WT plants were observed.

[0313] Using the methods described herein the inventors have been able to improve the recovery of robust protoplasts which are useful for methods of plant regeneration and genome modification for example as demonstrated herein by CRISPR Cas9 genome editing of endogenous genes. The methods have generally shortened the time for embryo emergence which is demonstrated here as short as 4-6 weeks and up to 8 weeks for key winegrape cultivars. Plants have been regenerated and no obvious phenotypic differences are observed. The method has been demonstrated across multiple cultivars and may address genotype dependent effects observed in grapevine research.Example 6: Effect of sucrose concentration used for protoplast purification cushions on viability and yield of Chardonnay protoplasts

[0314] Protoplasts were isolated from suspension cultures as per the method described in Example 3 with the following modifications. After wash and resuspension in MMG, 2 mL of protoplast suspension each was loaded onto 8 mL of six sucrose cushions (16-30% (w / v)) and spun (see Figure 21). Each cushion yielded one protoplast band, which was harvested into a 14 mL tube. MMG was added to each tube to a volume of 8 mL and protoplasts pelleted by centrifugation. Supernatants were removed and pellets washed with 5 mL MMG. Protoplasts were resuspended in a small volume of MMG for cell counts and viability tests.

[0315] The higher viability results were recorded across the 20 to 27% (w / v) sucrose cushion, although the highest yield for the 30% (w / v) sucrose cushion which had a fuzzy, darker coloured protoplast band, and included more debris. The viability and yield of protoplasts for each tested sucrose concentration is shown in Figure 22, and in Table 14.

[0316] Table 14: Chardonnay protoplast viability and yield prior to and after centrifugation through sucrose cushions. Except for single pre-sucrose measurements, the data represents mean / standard deviation of n = 3 technical replicates.Example 7: Effect of sucrose concentration used for protoplast purification cushions on viability and yield of microvine (V4) protoplasts

[0317] Protoplasts were isolated from suspension cultures as per the method described in Example 5 with the following modifications. After wash and resuspension in MMG, 2 mL of protoplast suspension each was loaded onto 8 mL of six sucrose cushions (16-30% (w / v)) and spun (see Figure 23). Each cushion yielded one protoplast band, which was harvested into a 14 mL tube. MMG was added to each tube to a volume of 8 mL and protoplasts pelleted by centrifugation. Supernatants were removed and pellets washed with 5 mL MMG. Protoplasts were resuspended in a small volume of MMG for cell counts and viability tests.

[0318] The highest viability was recorded for the 25% (w / v) sucrose cushion, with the highest yield for the 20% (w / v) sucrose cushion. The 22% (w / v) sucrose cushion provides a suitable balance of viability and yield useful for protoplast isolation, editing and plantlet regeneration. The viability and yield of protoplasts for each tested sucrose concentration is shown in Figure 24, and in Table 15. Accordingly, sucrose solutions of 20% (w / v) or more can be used to purify protoplasts.

[0319] Table 15: V4 protoplast viability and yield prior to and after centrifugation through sucrose cushions. Except for single pre-sucrose measurements, the data represents mean / standard deviation of n = 3 technical replicates.Example 8: Effect of sucrose concentration used for protoplast purification cushions on viability and yield of Shiraz protoplasts

[0320] Protoplasts were isolated from suspension cultures as per the method described in Example 1 with the following modifications. After wash and resuspension in MMG, 2 mL of protoplast suspension each was loaded onto 8 mL of six sucrose cushions (16-30% (w / v)) and spun (see Figure 25). Each cushion yielded one protoplast band, which was harvested into a 14 mL tube. MMG was added to each tube to a volume of 8 mL and protoplasts pelleted by centrifugation. Supernatants were removed and pellets washed with 5 mL MMG. Protoplasts were resuspended in a small volume of MMG for cell counts and viability tests.

[0321] The highest viability was recorded for the 25% (w / v) sucrose cushion, and similar viability also found for 22%, 27% and 30% (w / v) sucrose. The highest yield for the 20% (w / v) sucrose cushion. The viability and yield of protoplasts for each tested sucrose concentration is shown in Figure 26, and in Table 16.

[0322] Table 16: Shiraz protoplast viability and yield prior to and after centrifugation through sucrose cushions. Except for single pre-sucrose measurements, the data represents mean / standard deviation of n = 3 technical replicates.Example 9: Effect of sucrose concentration used for protoplast purification cushions on viability and yield of Sauvignon blanc protoplasts

[0323] Protoplasts were isolated from suspension cultures as per the method described in Example 4 with the following modifications.

[0324] After wash and resuspension in MMG, 2 mL of protoplast suspension each was loaded onto 8 mL of six sucrose cushions (16-30% (w / v)) and spun (see Figure 27). Each cushion yielded one protoplast band, which was harvested into a 14 mL tube. MMG was added to each tube to a volume of 8 mL and protoplasts pelleted by centrifugation. Supernatants were removed and pellets washed with 5 mL MMG. Protoplasts were resuspended in a small volume of MMG for cell counts and viability tests.

[0325] The highest viability was recorded for the 27% (w / v) sucrose cushion, with the highest yield for the 22% (w / v) sucrose cushion. Fuzzy, darker coloured protoplast bands were observed for 20% (w / v) sucrose, and smaller pellets were observed for 27% and 30% sucrose. The viability and yield of protoplasts for each tested sucrose concentration is shown in Table 17.

[0326] Table 17: Sauvignon blanc protoplast viability and yield prior to and after centrifugation through sucrose cushions. Except for single pre-sucrose measurements, the data represents mean / standard deviation of n = 3 technical replicates.

[0327] Figure 27 presents a summary of Protoplast, alginate bead, plant regeneration and editing data for Chardonnay, Cabernet Sauvignon, Shiraz and Sauvignon blanc transfections with Cas9 / DM2c-RNP of cells derived from embryogenic callus cultured on agar plates (EC) or in suspension (SC).

[0328] Protoplast viability is a key parameter for any transfection and regeneration protocol, and the present inventors have demonstrated herein that centrifugation through a sucrose solution yielded protoplast preparations with high numbers of viable cells (86-94%). The four elite winegrape cultivars used in this study, encompass cultivars amenable to in vitro culturing and regeneration (Chardonnay and Sauvignon blanc) as well as those classed as moderately (Shiraz) or highly recalcitrant (Cabernet Sauvignon). Plant survival rates once cultured in NAA supplemented medium for root development were >95% for Chardonnay and Cabernet Sauvignon, and >70% for Shiraz and Sauvignon blanc. The microvine was also demonstrated to show the effectiveness of the broader concentrations of sucrose isolation across the various grapevines.

Claims

The claims defining the invention are as follows1. A method for producing protoplasts of a Vitis vinifera cultivar, said method comprising; a) contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition, wherein the cell wall digesting composition comprises a culture medium and digestion enzymes; b) isolating protoplasts from the plant cells contacted with the cell wall digesting composition; c) contacting isolated protoplasts with a wash solution; and d) purifying protoplasts from step c) on a sucrose solution of from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

2. A method according to claim 1, wherein the culture medium is a Cl based medium or MS basal medium.

3. A method according to claim 1 or claim 2, wherein the cell wall digestion composition comprises cellulase, macerozyme and pectolyase.

4. A method according to any one of claims 1 to 3, wherein the sucrose solution is about 16% to about 27% (w / v) sucrose.

5. A method according to any of the preceding claims 1 to 4, wherein the plant cells are from embryogenic callus grown in solid or liquid medium.

6. A method according to any one of any of the preceding claims 1 to 5, wherein when the plant cells of a Vitis vinifera cultivar are embryogenic callus in solid medium, manually disrupting the callus when contacted with the cell wall digesting composition.7 A method according to any one of claims 1 to 6, wherein the cell wall digesting composition comprises between about 1 to about 3% cellulase, about 0.5 to about 1%, macerozyme and about 0.05% to about 1% pectolyase.

8. A method according to any one of claims 1 to 7, wherein the cell wall digesting composition comprises 1% cellulase, 0.5% macerozyme, and 0.05% pectolyase.

9. A method according to any one of claims 2 to 8, wherein the cell wall digesting composition comprises Cl-based medium.10 A method according to any one of claims 1 to 6, wherein the cell wall digesting composition comprises;1.650 g / L NH4NO3, 370 mg / L MgSO4.7H2O, 1.970 g / L KNO3, 170 mg / L KH2PO4);100 mg / L myo-inositol, 10 mg / L thiamine HCI, 10 mg / L nicotinic acid, 1 mg / L pyridoxine HCI, 1 mg / L D-pantothenic acid, 0.01 mg / L biotin; 100 mg / L L-glutamic acid, 10 mg / L L- phenylalanine, 2 mg / L glycine), 3% sucrose;37.2 mg / L Na2EDTA.2H2O, 9.3 mg / L FeSO4.7H2O;6.2 mg / L H3BO3, 0.025 mg / L CoCI2.6H2O, 0.025 mg / L CuSO4.5H2O, 22.3 mg / L MnSO4.4H2O, 0.25 mg / L Na2MoO4.2H2O, 0.83 mg / L KI, 8.6 mg / L ZnSO4.2H2O; and5mM MES, lOmM CaCI2, 0.5M mannitol at pH 5.7.

11. A method according to any one of claims 2 to 10 wherein the liquid or solid medium comprises a Cl-based medium.

12. A method according to any one of claims 2 to 11 wherein the liquid or solid medium comprises a Cl-based medium supplemented with an antioxidant to prevent browning.

13. A method according to any one of claims 2 to 12 wherein the liquid or solid medium comprises a Cl-based medium supplemented with polyvinylpyrrolidone (PVP) 40.

14. A method according to any one of claims 1 to 13, wherein the wash solution is 4 mM MES, 0.5 M mannitol and 15 mM MgC at pH 5.

715. A method according to any one of claims 1 to 14, wherein the Vitis vinifera cultivar is Sauvignon Blanc, Chardonnay or Shiraz, or is a microvine of a Vitis vinifera cultivar.

16. A Vitis vinifera protoplast produced by a method comprising; a) contacting plant cells of a Vitis vinifera cultivar with a cell wall digesting composition, wherein the cell wall digesting composition comprises a culture medium and cell wall digestion enzymes; b) isolating protoplasts from the plant cells contacted with the cell wall digesting composition; c) contacting isolated protoplasts with a wash solution; and d) purifying protoplasts from step c) on a sucrose solution of from about 13% (w / v) sucrose to about 30% (w / v) sucrose.

17. A Vitis vinifera protoplast produced by a method according to claim 16, wherein the culture medium is a Cl based medium or MS basal medium.

18. A Vitis vinifera protoplast produced by a method according to claim 16, wherein the cell wall digestion composition comprises cellulase, macerozyme and pectolyase.

19. A Vitis vinifera protoplast produced by a method according to any one of claims 16 to 18, wherein the sucrose solution is about 16% (w / v) sucrose.

20. A method for producing a genome edited protoplast of a Vitis vinifera cultivar, said method comprising genome editing a purified protoplast produced by the method of any one of claims 1 to 19.

21. A method according to claim 20, wherein the step of genome editing comprises a step of introducing a ribonucleoprotein complex (RNP) into a purified protoplast produced by the method of any one of claims 1 to 19.

22. A method according to claim 21, wherein the step of introducing a ribonucleoprotein complex into a purified protoplast is protoplast transfection comprising contacting a purified protoplast produced by the method of any one of claims 1 to 15 with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaC .

23. A method according to claim 22, further comprising a step of stopping protoplast transfection by contacting the transfected protoplast with a stop solution at 25 mins to 45mins, preferably at 30 minutes, following contacting the purified protoplast with a RNP in the presence of 20% PEG4000, 0.1M mannitol, and 50mM CaC .

24. A method according to claim 23, wherein the stop solution is a solution comprising 2 mM MES pH 5.7, 5mM glucose, 154 mM NaCI, 125 mM CaCb and 5 mM KCI,25. A method according to claim 24, further comprising contacting the genome edited protoplast with a solution comprising 0.5 M mannitol, 20 mM KCI, 4 mM MES, pH 5.7 to wash the genome edited protoplast.

26. A method according to any one of claims 20 to 25, further comprising cultivating the genome edited protoplast.

27. A method according to claim 26, wherein cultivating the genome edited protoplast comprises a step of (i) incubation for about 1 hour followed by a wash step.

28. A method according to claim 26, wherein cultivating the genome edited protoplast comprises a step of (i) incubation of about 1 hour, (ii) wash and (iii) followed by forming an alginate bead comprising the genome edited protoplast.

29. A genome edited protoplast produced by the method of any one of claims 16 to 28.

30. A method according to any one of claims 16 to 29, further comprising generating a plant from the genome edited protoplast.

31. A plant produced by the method of claim 30, or from a protoplast produced by the method of any one of claims 1 to 29.

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