RU2783891C2 - Simple method for isolating wheat protoplasts for genome editing - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agricultural biotechnology, in particular, to methods for in vitro cultivation of somatic cells and genomic editing of higher plants. The invention constitutes a new method for isolating wheat protoplasts for genome editing. Said method includes effective isolation of wheat protoplasts from 7 to 10-day shoots. The effectiveness of isolation is achieved due to the low-temperature treatment of donor plants and vacuum infiltration of the cooled enzyme solution. In the process of crushing the shoots and prior to processing with enzymes, leaf tissues are sustained on an osmotic medium in order to reduce the cell damage during mechanical impact on tissue cells. Transfection of protoplasts can be executed by a wide range of genetic constructs. The system for transfection and imaging of protoplasts can be used with various marker selection systems, including selection with reporter genes.

EFFECT: invention can be used for comparative estimation of the effectiveness of wheat genome editing by various genetic engineering constructs by means of transfection of protoplasts with an effectiveness of up to 40.5%.

1 cl, 1 dwg, 1 tbl

Description

The field of technology. The invention relates to the field of agricultural biotechnology, in particular to methods for in vitro cultivation of somatic cells and genomic editing of higher plants, for comparative evaluation of the effectiveness of wheat genome editing by various genetic engineering constructs. The invention can be used in the creation of new resistant varieties and hybrids of wheat and is in demand by seed companies and breeding stations, scientific and educational institutions of biotechnological and agricultural profile.

The relevance of the invention. Genome editing is one of the most promising areas of modern molecular biology and biotechnology. The recently developed CRISPR/Cas9 system is a versatile genomic editing tool that allows you to make targeted changes to specific regions of the eukaryotic genome to increase plant resistance to diseases and abiotic stresses, improve grain quality, increase the level of trace elements and vitamins in the plant, and much more (Doudna J. A, 2014). The use of CRISPR/Cas9 technology on plants with a complex polyploid genome requires a high efficiency of the functioning of all its components and is a significant barrier for most monocot species, including corn, rice, oats, barley, and, in particular, wheat. This problem can be solved by creating several variants of genetic constructs that differ from each other in certain parameters, and by comparing their effectiveness. The most convenient way to assess the effectiveness of genetic constructs for genome editing is the transfection of protoplasts, since they lack a cell wall and can be easily transfected with plasmid DNA. Evaluation of editing efficiency is carried out by isolating total DNA from a suspension of protoplasts and targeted sequencing of the genome region containing the editing site. At the same time, the success of the technique is largely determined by the efficiency of protoplast transfection. But due to the fact that the polyploid genome and the increased content of intracellular wheat lipoxygenases reduce the yield of protoplasts and the specificity of genome editing, the task of creating an effective and simple method for isolating domestic wheat protoplasts for genome editing is very relevant.

The level of technology. Plant protoplast research began in the 1960s, when the British botanist Cocking (Cocking, 1960) used a cell wall degrading enzyme to enzymatically extract tomato apical protoplast from roots and fruits and cultivate them under controlled conditions. This discovery was the basis for the development of such important areas of plant biotechnology as cell and genetic engineering. Since then, research on protoplasts has been at a rapid development stage. For example, Nagata and Takeble first reported isolated, cultivated, and regenerated tobacco mesophyll plants obtained in 1970 (Nagata and Takebe, 1970). A known method for obtaining protoplasts of cereal plants selected from the group consisting of Dactylis glomerata Patent US 5596131 A 1997 "Regeneration of graminaceous plants of the subfamily pooideae from protoplasts". The main disadvantage of this method, as, indeed, of most other well-known methods, is that it was developed for model varieties of small grain cereals of foreign breeding and is ineffective for productive varieties of domestic breeding, zoned on the territory of the Russian Federation. In another case, US Patent 5981842 A 1995 "Production of water stress or salt stress tolerant transgenic cereal plants", where the LEA protein gene, HVA1, from barley (Hordeum vulgare L.) was transformed into rice plants (Oryza sativa L.) when using rice protoplasts and is not effective on wheat plants. Closest to this invention is a method for obtaining and transfecting protoplasts described in US Patent 4684611 1983 "Process for the in-vitro transformation of plant protoplasts with plasmid DNA", where the incubation is carried out in the presence of polyethylene glycol and calcium ions.

Carlson in 1972 used protoplast fusion technology to produce the first interspecific tobacco hybrids (Liu and Deng, 1999). Due to the lack of a cell wall, plant protoplasts are widely used for genetic transformation, which leads to the requirement of a large number of highly active protoplasts. The first report of direct gene transfer in wheat protoplasts was made by Lorz in 1985 (Schreier P. H. et al., 1985) from cultured cells of Triticum toposossite by PEG-mediated transfection. Transformed cells were selected on a medium containing kanamycin and identified by determining the activity of npt II. However, these works have led to low transformation efficiency, on the order of 0.005%, and low regeneration capacity, which makes it difficult to work with protoplasts as explants (Davey, et al., 2005, Peng, et al., 2015). Introduction of the E. coli plasmid, pCGN1055, containing the hpt gene, into wheat protoplasts by cationic liposome-mediated transformation in 1993 resulted in the formation of transgenic albino seedlings.

The presence and activity of transgenes was confirmed by analysis of enzymatic activity and Southern hybridization (Southern, et al. 1975), and the transformation efficiency was about 6%.

Since the discovery of the CRISPR/Cas9 system, several studies have been carried out on plant genome editing in both monocots and dicots, such as Nicotiana benthamiana (Li et al. 2015), Nicotiana tobaccum (Gao et al. 2014), Arabidopsis thaliana (Li et al. al. 2014), Oryza sativa (Shan et al. 2014) and Sorghum bicolor (Jiang et al. 2013) and it was found that a polyploid genome reduces the specificity of genome editing (Peng et al. 2015, Wang et al., 2016, Liang et al., 2018). Therefore, it is necessary to optimize the conditions for isolation and transfection of protoplasts for the maximum number of intact cells. In addition, the optimal conditions for obtaining plant cell protoplasts are individual for different plant species and varieties, and each individual case requires preliminary work to select the composition of enzymes, their concentrations, and the time of treatment with an osmotic stabilizer.

Thus, the task of creating an effective method for isolating wheat protoplasts for genome editing, suitable for a wide range of domestic breeding genotypes, remains relevant.

In addition, additional difficulties may arise in the development of such a method due to the fact that most of the developed methods were carried out on model varieties with a lower probability of membrane rupture as a result of a reduced action of intracellular lipoxygenases using expensive antioxidants (glutathione, trypsin or dithiothriethol) and require additional experimental improvements to implement the results obtained in production. This necessitated the transition from working with model genotypes to the use of productive commercial varieties.

Recently, we have shown the positive effect of low-temperature treatment of wheat plants before manipulations with embryos and increasing their morphogenetic potential (Gaponenko et al., 2018 No. 2646108). In our method, low-temperature treatment was first used to increase the integrity of wheat protoplasts of domestic varieties during the isolation of protoplasts and their further transfection using vacuum infiltration.

In view of all of the above, in the development of the present invention, the task was to create an effective and simple method for obtaining wheat protoplasts for editing the genome, suitable for a wide range of commercially promising domestic spring varieties. The expected technical result was an increase in the efficiency and unification of the protoplast isolation procedure for the efficient delivery of genetic tools for genome editing.

Disclosure of invention

The present invention provides an effective and simple method for isolating wheat protoplasts for genome editing, which can be used for a wide range of domestic breeding genotypes.

This method provides an effective yield of protoplasts if 7-10 day old seedlings obtained from donor plants after cold shock are used as mesophyll donors. Wheat seedlings must be grown under aseptic conditions, the occurrence of contamination reduces the yield of intact protoplasts. Immediately prior to isolation, 48 hours before seedlings, it is necessary to place the seedlings in a refrigerator in the dark at +4 C to reduce the effects of intracellular lipoxygenases and peroxidases, leading to a violation of the integrity of cell membranes. For a high yield of protoplasts, a bundle of shoots (30 pcs.) is cut into segments together using a machine with blades in an osmotic solution with a thickness of 0.5 mm. The design of the machine allows you to quickly and evenly cut the shoots without squeezing the tissue. Only the middle part is used - from the beginning of the hypocotyl to the middle of the shoot. After the cut segments must be immediately transferred to a 0.6 M osmotic solution for 10 minutes in the dark to maintain the osmotic pressure. Most often, sugars are used as osmotic agents (glucose, sucrose, xylose, sorbitol, mannitol), sometimes solutions of CaCl2, Na2HPO4, KCI salts at concentrations of 0.3-0.8 M. For the isolation of protoplasts, an enzymatic solution (cellulase and macerozyme) with using a vacuum. It was assumed that vacuum infiltration helps the enzyme solution to penetrate into the plant tissue and even into the intercellular spaces. After enzymatic treatment, the protoplast suspension must be purified from the remnants of intact tissue and cell fragments, and also washed from enzymes for protoplast transfection when editing the wheat genome, depending on the task, various genetic constructs can be used in the method according to the invention. For example, to create plants resistant to powdery mildew, genome editing can be carried out using CRISPR/Cas9 genetic constructs leading to inactivation of the MLO gene (leading to mold resistance).

The presented system for obtaining intact wheat protoplasts takes from 10 to 15 days from the moment of sowing the seeds. The concentration of intact protoplasts is up to 7×10 ⁶ ml ^-1 , which is 1.5-2 times higher than the methods currently used in other laboratories. The frequency of transfection of protoplasts using a genetic construct encoding the green fluorescent protein GFP was 40%.

The present invention relates to a method for producing wheat plant protoplasts, comprising the following steps:

1) Selection or creation of genetic tools (genetically engineered constructs);

2) Preparation of source material;

3) Low-temperature treatment of donor plants;

4) Isolation of protoplasts

5) Osmotic treatment of shoot segments;

6) Isolation of protoplasts in enzyme solution and exposure to vacuum;

7) PEG-mediated transfection of protoplasts;

8) Proliferation and detection of transformed cells;

The technique was used on spring varieties of soft wheat of domestic breeding - Zlata, Amir, Agata, Lyubava. The yield of intact protoplasts was high and ranged from 5×10 ⁵ ml ^-1 to 7×10 ⁶ ml. The transfection efficiency was high for all the studied varieties, regardless of the genotype, up to 32 to 40.5%. This indicator indicates the high efficiency of the proposed method.

Brief description of the drawings

Figure - Stages of isolation of protoplasts from wheat seedlings and transfection.

Implementation of the invention

The use of healthy donor plants is a prerequisite for obtaining protoplasts. Plants must be in an artificial climate, in aseptic conditions. In addition to the obligatory protection of plants from diseases such as powdery mildew, dwarf rust, root rot, etc., optimal growing conditions must be created - high-quality lighting and nutrient medium.

Isolation of protoplasts using a machine with several blades in osmotic solution or with sharp razors (should be done quickly and accurately).

A bioballistics gun - PIG (particle inflow gun) was used to create a vacuum, but another chamber can be used to create a pressure below atmospheric pressure. For enzymatic treatment, the protoplast suspension is kept on an ES 20 BioSan incubator shaker, but another controlled shaker can be used.

Various reporter genes (GFP, DsRed, X-gal, etc.) can be used to detect transfected transgenic cells. The cell concentration is determined using a hematocytometer or under a microscope (×100). In the examples below, the pUBarGFP plasmid vector carrying the bar gene expression cassette under the control of the maize Ubil gene promoter and the Tnos terminator of the nopaline synthase gene and the gfp gene encoding the green fluorescent protein was used.

Methodology:

1) Selection or creation of genetic tools (genetically engineered constructs):

The pUBarGFP plasmid vector carrying the gfp gene encoding the green fluorescent protein was used, since the expression appears as a green glow when illuminated with blue light and is easy to detect.

2) Preparation of source material:

The cleaned wheat seeds are sterilized with 75% ethanol for 1 min. These seeds are then sterilized with 2.5% sodium hypochlorite for 20 minutes, then washed five times with sterile water.

Seeds are incubated on 1/2 MS medium ((Murashige T. & Skoog F. 1962)) with a photoperiod of 12 (16) hours light/dark at 26° C. for 7-10 days. Green seedlings in the amount of 60 pieces are used further.

3) Low-temperature treatment of donor plants:

Test tubes with plants are placed in a refrigerator at 4±2°C for 48 hours.

4) Isolation of protoplasts:

A bunch of shoots (30 pieces) is cut together, only the middle part is used - from the beginning of the hypocotyl to the middle of the shoot. Next, the shoots are cut into strips 0.5 mm wide using a machine with blades in an osmotic solution in a small amount of 0.6 M mannitol + 4 ° C - it is recommended to cut along and across the shoot.

5) Osmotic treatment of shoot segments:

Leaf segments are immediately transferred to a 0.6 M mannitol solution cooled to +4°C for 30 minutes for rapid plasmolysis. After plasmolysis, the leaf tissues are filtered through nylon meshes.

6) Isolation of protoplasts in enzyme solution and exposure to vacuum; The leaf segments are transferred to a 150 ml conical flask containing 50

ml filter-sterilized enzyme solution (1.5% cellulase RS, 0.75% macerozyme R-10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl2 and 0.1% BSA) based on protocol of Shan Q. et al. (Shan Q. et al., 2014) and chilled to +4°C. Next, vacuum infiltrate the leaf segments with enzymes by applying vacuum (~380-508 mmHg) for 30 min. in the dark or immediately wrap the flask in aluminum foil. Then the strips are incubated in the dark for 4-6 hours with gentle mixing (60-80 rpm) at a temperature reduced to +4°C.

After enzymatic digestion, 50 ml of B5 solution (154 MMNaCl, 125 mM CaCl2.5 mM KCl and 2 mM MES at pH 5.7) based on the protocol of Shan Q. et al. (Shan Q. et al., 2014) is added into a conical the flask and then gently shake it by hand for 10 seconds to release the protoplasts. Filter the mixture through 40 µm nylon meshes and wash the leaf strips on the surface of the nylon mesh 3-5 times with solution B5.

The washed protoplasts are collected in three or four round bottom centrifuge tubes with a volume of 30-50 ml, and centrifuged at 80g for 3 min at room temperature, and the supernatant is removed with a pipette. Protoplasts are very fragile, so care must be taken.

After centrifugation, the protoplasts are gently resuspended in 10 ml of B5 solution, collected in one 50 ml round bottom tube and kept on ice for 30 min. Intact protoplasts should settle to the bottom of the tube by gravity after 30 min. Then only the supernatant is removed, directly without centrifugation with a pipette Resuspend the protoplasts in 4 ml calcium and magnesium solution (0.4 M mannitol, 15 mM MgCl2 and 4 mM MES at pH 5.7) at a final concentration of 2.5 x 106 cells per ml. Determine the concentration of protoplasts under a microscope (×100) or using a hemocytometer.

7) PEG-mediated protoplast transfection:

For wheat transfection, add 40 µg of plasmid to a 2 ml microcentrifuge tube, and then add 200 µl of protoplasts (5 x 10 5 cells) and mix gently by tapping the tube or lightly pipetting.). The temperature of the solutions should be +4 degrees.

After the plasmid, freshly prepared polyethylene glycol (PEG) solution (40%, PEG 4000, 0.2 M mannitol and 0.1 M CaCl2) is added and mixed thoroughly by gentle tapping or light pipetting.

Then the mixture is incubated for 15-20 minutes in the dark, be sure to wrap the test tubes in aluminum foil. Add 880 µl of solution B5 to the tube and mix well by inverting to stop the transformation process. Wheat protoplasts are centrifuged at 80 g for 3 min and the supernatant is removed. Resuspend the protoplasts in 2 ml of solution B5.

8) Proliferation and detection of transformed cells:

Transfer the protoplasts to 6-well plates, wrap the plates in aluminum foil and incubate them at 23° C. for at least 48 hours (maximum 72 hours).

Check the condition of the protoplasts under a microscope, healthy cells should appear full and round. Transfection efficiency can be determined by counting the number of fluorescent GFP cells.

The following are specific examples of the invention, which can not be considered as limiting its scope.

Example 1. Obtaining transfected protoplasts of spring wheat cv. Zlata containing the gfp gene.

Cleaned wheat seeds of the Zlata variety in the amount of 50 pieces were sterilized with 75% ethanol for 1 min. These seeds were further sterilized with 2.5% sodium hypochlorite for 20 minutes, then washed five times with sterile water. Seeds were incubated on medium with 1/2 MS with a photoperiod of 12 (16) h light/dark at 26°C for 9 days. Green seedlings in test tubes of 30 pieces were placed in a refrigerator at 4±2°C for 48 hours. +4°C 0.6 M mannitol. Leaf segments were immediately transferred to a 0.6 M mannitol solution cooled to +4°C for 30 min for rapid plasmolysis. After plasmolysis, leaf tissues were filtered through nylon meshes and transferred to a 150 ml conical flask containing 50 ml of filter-sterilized enzyme solution cooled to +4°C. Leaf segments of cv. Zlata were subjected to vacuum infiltration (~500 mmHg) for 30 min. in the dark at +4°C, and immediately wrap the flask with aluminum foil. The strips were then incubated in the dark for 5 h with gentle mixing (80 rpm) at +4°C. Then 50 ml of solution B 5 was added to the conical flask and shaken by hand for 10 seconds to release the protoplasts. Filter the mixture through 40 µm nylon meshes and 3 times with solution B5. Then the washed protoplasts were collected in three 30 ml round bottom centrifuge tubes and centrifuged at 80g for 3 min at room temperature, the supernatant was removed with a pipette. They were then resuspended in 10 ml of solution B5, collected in one 50 ml round bottom tube and kept on ice for 30 minutes. Intact protoplasts were deposited on the bottom of the tube under the action of gravity after 25 min. After 30 minutes, the supernatant was removed. The protoplasts were resuspended in 4 ml of calcium and magnesium solution. Determined the concentration of protoplasts using a hemocytometer - 7×10 ⁶ . The PEG solution was prepared 3 h before transfection. The protoplasts were brought to the desired concentration of 5×10 ⁵ cells. For wheat transfection, 40 μg of plasmid was added to a 2 ml microcentrifuge tube, followed by 200 μl of protoplasts. The polyethylene glycol solution was added after the plasmid. All transfection solutions were +4°C. The mixture was then incubated for 20 min in the dark in aluminum foil. 880 μl of solution B5 was added to the tube and mixed by inverting. Zlata wheat protoplasts were centrifuged at 80 g for 3 min, and the supernatant was removed. The protoplasts were resuspended in 2 ml of solution B5. The transfection efficiency was 40.5%.

Example 2. Obtaining transfected protoplasts of spring wheat cv. Agata containing the gfp gene.

Agata wheat seeds in the amount of 70 pieces were sterilized with ethanol for 1 min, then sterilized with 2.5% sodium hypochloride for 20 min, then washed five times with sterile water. Seeds of cv. Agata were incubated on medium with 1/2 MS with a photoperiod of 12 (16) h light/dark at 26°C for 7 days. Green seedlings in test tubes of 33 pieces were placed in a refrigerator at 4 ± 2°C for 48 hours. +4°C 0.6 M mannitol. Leaf segments were immediately transferred to 0.6 M mannitol solution for 30 min for rapid plasmolysis. After plasmolysis, leaf tissues were filtered through nylon meshes and transferred to a 150 ml conical flask containing 50 ml of filter-sterilized enzyme solution. The leaf segments were subjected to vacuum infiltration (~450 mm Hg) for 30 min and then the strips were incubated in the dark for 5 h with gentle mixing (60 rpm) +4°C. Then 50 ml of solution B5 was added to the conical flask and shaken by hand for 10. Filter the mixture through 40 μm nylon meshes and 4 times with solution B5. Then the washed protoplasts were collected in three 50 ml round bottom centrifuge tubes and centrifuged at 80g for 3 min at room temperature, the supernatant was removed with a pipette. Then they were suspended in 10 ml of B5 solution, collected in one 50 ml round bottom tube and kept on ice for 30 minutes. Intact protoplasts were deposited on the bottom of the tube under the action of gravity after 30 min. After 30 minutes, the supernatant was removed. The protoplasts were resuspended in 4 ml of a solution of calcium and magnesium. The concentration of protoplasts was determined using a hemocytometer - 6.6×10 ⁵ . The PEG solution was prepared 5 h before transfection. The protoplasts were brought to a concentration of 5×10 ⁵ cells. For wheat transfection, 40 μg of plasmid was added to a 2 ml microcentrifuge tube, followed by 200 μl of protoplasts. The polyethylene glycol solution was added after the plasmid. The mixture was then incubated for 15 min in the dark in aluminum foil. 880 μl of solution B5 was added to the tube and mixed by inverting. Agata wheat protoplasts were centrifuged at 80 g for 3 min, and the supernatant was removed. The protoplasts were resuspended in 2 ml of solution B5. The transfection efficiency was 36%.

Example 3. Obtaining transfected protoplasts of spring wheat cv. Amir containing the gfp gene.

Wheat seeds of the Amir variety in the amount of 45 pieces were sterilized with ethanol for 1 min, then sterilized with 2.5% sodium hypochloride for 20 min, then washed five times with sterile water. Seeds of cv. Amir were incubated on medium with 1/2 MS with a photoperiod of 12 (16) h light/dark at 26°C for 7 days. Green seedlings in test tubes of 30 pieces were placed in a refrigerator at 4±2°C for 48 hours. After a cold shock, the shoots were cut along and across the shoot into strips 0.5 mm wide in 30 ml of 0.6 M mannitol. Leaf segments were immediately transferred to 100 ml of 0.6 M mannitol solution for 30 minutes for rapid plasmolysis. After plasmolysis, leaf tissues were filtered through nylon meshes and transferred to a 150 ml conical flask containing 50 ml of filter-sterilized enzyme solution. The leaf segments were subjected to vacuum infiltration (~400 mm Hg) for 30 min and then the strips were incubated in the dark for 5 h with gentle mixing (80 rpm) at +4°C. Then 50 ml of solution B5 was added to the conical flask and shaken by hand for 10. Filter the mixture through 40 μm nylon meshes and 3 times with solution B5. Then the washed protoplasts were collected in four 50 ml round bottom centrifuge tubes and centrifuged at 80g for 3 min at room temperature, the supernatant was removed with a pipette. Then they were suspended in 10 ml of B5 solution, collected in one 50 ml round bottom tube and kept on ice for 30 minutes. Intact protoplasts were deposited on the bottom of the tube under the action of gravity after 30 min. After 30 minutes, the supernatant was removed. The protoplasts were resuspended in 4 ml of calcium and magnesium solution. The concentration of protoplasts was determined using a hemocytometer - 6.2×10 ⁶ . The PEG solution was prepared 4 h before transfection. The protoplasts were brought to a concentration of 5×10 ⁵ cells. For wheat transfection, 40 μg of plasmid was added to a 2 ml microcentrifuge tube, followed by 200 μl of protoplasts. The polyethylene glycol solution was added after the plasmid. The mixture was then incubated for 15 min in the dark in aluminum foil. 880 μl of solution B5 was added to the tube and mixed by inverting. Agata wheat protoplasts were centrifuged at 80 g for 3 min, and the supernatant was removed. The protoplasts were resuspended in 2 ml of solution B5. The transfection efficiency was 34%.

Example 4. Preparation of transfected spring wheat cv. Lyubava protoplasts containing the gfp gene.

Seed material of wheat variety Lyubava in the amount of 80 pieces was sterilized with ethanol for 1 min, then sterilized with 2.5% sodium hypochloride for 20 min, then washed six times with sterile water. Seeds were incubated on medium with 1/2 MS with a photoperiod of 12 (16) h light/dark at 26°C for 8 days. Green seedlings in test tubes of 34 pieces were placed in a refrigerator at 4±2°C for 48 hours. After a cold shock, the shoots were cut along and across the shoot into strips 0.5 mm wide in 20 ml of 0.6 M mannitol +4°C. Leaf segments were immediately transferred to 100 ml of 0.6 M mannitol solution for 30 min for rapid plasmolysis at +4°C. After plasmolysis, leaf tissues were filtered through nylon meshes and transferred to a 150 ml conical flask containing 50 ml of filter-sterilized enzyme solution. The leaf segments were subjected to vacuum infiltration (~380 mm Hg) for 30 min and then the strips were incubated in the dark for 5 h with gentle mixing (70 rpm) at +4°C. Then 50 ml of solution B5 was added to the conical flask and shaken by hand for 10. Filter the mixture through 40 μm nylon meshes and 5 times with solution B5. Then the washed protoplasts were collected in four 50 ml round bottom centrifuge tubes and centrifuged at 80g for 3 min at room temperature, the supernatant was removed with a pipette. Then suspended in 10 ml of solution B5, they were collected in one 50 ml round bottom tube and kept on ice for 30 minutes. Intact protoplasts were deposited on the bottom of the tube under the action of gravity after 20 min. After 30 minutes, the supernatant was removed. The protoplasts were resuspended in 4 ml of calcium and magnesium solution. The concentration of protoplasts was determined using a hemocytometer - 5×10 ⁵ . The PEG solution was prepared 4.5 hours before transfection. For wheat transfection, 40 μg of plasmid was added to a 2 ml microcentrifuge tube, followed by 200 μl of protoplasts. The polyethylene glycol solution was added after the plasmid. The mixture was then incubated for 20 min in the dark in aluminum foil. 880 μl of solution B5 was added to the tube and mixed by inverting. The protoplasts were centrifuged at 80 g for 3 min, and the supernatant was removed. The protoplasts were resuspended in 2 ml of solution B5. The transfection efficiency of Lyubava protoplasts was 32%.

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Claims (1)

A method for isolating wheat protoplasts for genome editing, including cold shock of donor plants, crushing of shoots from the hypocotyl to the middle of the leaf and isolation of protoplasts in an osmotic solution, vacuum infiltration of leaf segments in an enzyme solution at a low temperature, filtration and washing with a salt solution, selection whole protoplasts by centrifugation and sedimentation of protoplasts on ice under the action of the force of the center of gravity, PEG-mediated transfection of protoplasts, and characterized in that cold shock is used for shoots of soft spring wheat plants in test tubes on days 7-9 after sowing at a temperature of +4°C C for 48 hours, the isolation of protoplasts during the grinding of shoots is carried out in a chilled osmotic solution to +4°C, the leaf segments are vacuum-infiltrated for 30 minutes in an enzyme solution at a temperature of +4°C.

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