A method for extracting protoplasts from forest strawberry callus

Abstract

The invention discloses a method for extracting protoplasts from forest strawberry callus, and belongs to the field of biotechnology. The method comprises the following steps: inducing forest strawberry tissue culture seedling leaves to produce callus, differentiation culture, selecting light green, soft, granular and vigorous growing clustered buds, crushing, filtering, enzymolysis at 23°C and 60rpm for 3h, centrifugation to collect green precipitates, 75μm and 10μm nylon mesh filtering, centrifugation, resuspending precipitation, and obtaining strawberry callus protoplast suspension. The invention is beneficial in that diploid strawberry callus protoplasts are successfully extracted, the problem of difficulty in extracting strawberry leaf protoplasts is overcome, and by optimizing the formula of the enzymolysis solution, the enzymolysis time is shortened from more than 14h to 3h, which greatly improves the extraction efficiency; the extracted strawberry callus protoplasts are in good condition (full body, high activity), and are large in quantity and high in purity.

Description

Technical Field

[0001] The invention relates to a method for extracting protoplasts, in particular to a method for extracting protoplasts from forest strawberry callus, and belongs to the field of biotechnology. Background Art

[0002] Protoplast culture and plant regeneration technology have the advantages of wide application range and strong feasibility, and are the basis of plant bioengineering. Due to the characteristics of simple structure, good development synchronization, large population, easy entry of DNA molecules into cells, and easy acquisition of homozygous transformants, the cross-penetration of its culture technology with related cell, molecular and genetic disciplines has made the research of plant protoplasts more and more valued. Strawberry is a herbaceous plant of the genus Fragaria in the Rosaceae family. It is a perennial fruit tree in horticulture. As an important berry economic crop, it has high economic value, short growth cycle, rich nutrition, strong fruit aroma and other excellent traits. China is the country with the highest strawberry production and the largest planting area in the world, and the forest strawberry genome (about 240Mb) is relatively small, the plant is short, the life cycle is short, and it can be efficiently genetically transformed. The present invention utilizes diploid forest strawberry "Ruegen" as material and optimizes the method for extracting protoplasts from its callus tissue, which has great research significance and application value for subsequent genetic transformation using molecular biological methods or cultivating more cultivars with excellent traits.

[0003] Protoplast fusion is one of the necessary means of genetic engineering. Technical means such as plant protoplast regeneration culture and suspension culture have great application potential in various plants. Compared with other model plants and crops, the research on the molecular mechanism analysis of cell transient transformation, cell fusion, fruit quality, and aroma substance formation in strawberry is not in-depth enough, and the technical system is not perfect enough. It is very difficult to extract protoplasts from forest strawberry leaves. The reason is that diploid strawberry leaves are thin, and in order to adapt to the environment, they have formed developed palisade tissues and guard cells, and the number of mesophyll cells is small. The establishment of a method for extracting protoplasts from forest strawberry callus is of great significance for further carrying out protoplast fusion and analyzing the important life activity mechanism of strawberry by means of gene function. At the same time, with the development of genetic engineering technology, forest strawberry protoplasts will also be further developed and developed in the application of CRISPR / Cas9 technology. Summary of the invention

[0004] In order to solve the problem of difficulty in extracting protoplasts from strawberry leaves, the present invention aims to provide a method for efficiently extracting protoplasts in good condition and sufficient quantity from forest strawberry callus.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for extracting protoplasts from forest strawberry callus, characterized in that it comprises the following steps:

[0007] (1) taking leaves of forest strawberry tissue culture seedlings and inducing callus tissue on an induction medium by in vitro wounding;

[0008] (2) cutting the callus obtained in step (1) and placing it on a differentiation medium for further culture;

[0009] (3) selecting light green, soft, granular and vigorously growing clustered buds from the callus obtained in step (2), placing them in a centrifuge tube containing cold distilled water and crushing them to obtain callus fragments with uniform texture and loosely separated cells;

[0010] (4) filtering the callus tissue fragments obtained in step (3) through a 45 μm nylon mesh, placing the filtered callus tissue into a mixed enzymatic hydrolysis solution, and performing enzymatic hydrolysis at 23° C. and 60 rpm for 3 h to obtain a solution;

[0011] (5) adding an equal volume of extraction buffer to the solution obtained in step (4) to terminate the enzymatic hydrolysis reaction, filtering with a 45 μm nylon mesh, collecting the protoplast filtrate, centrifuging the filtrate at 800 rpm for 5 min, discarding the supernatant, and the green precipitate is the protoplast;

[0012] (6) filtering the protoplasts obtained in step (5) through a 75 μm nylon mesh, filtering the filtrate through a 10 μm nylon mesh, placing the nylon mesh upside down in a centrifuge tube, and washing the protoplasts with an extraction buffer;

[0013] (7) The protoplasts obtained in step (6) were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with a resuspension solution to obtain a strawberry callus protoplast suspension.

[0014] Preferably, in step (1), the induction medium is prepared from MS medium, sucrose, agar, indolebutyric acid and thidiazuron, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron are 30 g / L, 9 g / L, 0.2 mg / L and 2.0 mg / L, respectively.

[0015] Preferably, in step (2), the differentiation medium is prepared from MS medium, sucrose, agar, indolebutyric acid and thidiazuron, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron are 30 g / L, 9 g / L, 0.3 mg / L and 1.0 mg / L, respectively.

[0016] Preferably, in step (3), 5 to 8 g of clustered sprouts are selected and the selected clustered sprouts are placed in a 30 mL centrifuge tube containing 5 mL of cold distilled water and crushed.

[0017] Preferably, in step (4), the mixed enzymatic solution is prepared from a mixed solvent, a plurality of enzymes and bovine serum albumin, wherein:

[0018] The mixed solvent is prepared from water, D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride, and the concentrations of D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride are 0.4 mol / L, 20 mmol / L, 10 mmol / L and 20 mmol / L respectively;

[0019] Enzymes included cellulase R-10, macerate R-10, and pectinase Y-23, with concentrations of 10 g / L, 10 g / L, and 1 g / L, respectively;

[0020] The concentration of bovine serum albumin was 1 g / L;

[0021] The pH was adjusted to 5.7 with KOH.

[0022] Preferably, in steps (5) and (6), the extraction buffer is prepared from water, 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose are 4 mmol / L, 150 mmol / L, 0.5 mmol / L, 5 mmol / L and 5 mmol / L, respectively.

[0023] Preferably, in step (7), the resuspension is prepared from water, 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride are 2 mmol / L, 0.4 mol / L and 15 mmol / L, respectively.

[0024] The present invention is beneficial in that:

[0025] (1) The diploid strawberry callus protoplasts were successfully extracted, overcoming the difficulty of extracting strawberry leaf protoplasts. In addition, by optimizing the formula of the enzymatic hydrolysate, the enzymatic hydrolysis time was shortened from more than 14 h to 3 h, greatly improving the extraction efficiency.

[0026] (2) The extracted strawberry callus protoplasts are in good condition (full in shape and high in activity), and are large in quantity and high in purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1is a diagram of the enzymatic hydrolysis process of strawberry callus, where CK is a diagram of the cell morphology of the unenzymatically hydrolyzed tissue under a microscope, Bar = 10 μm;

[0028] Figure 2 are morphological images of protoplasts extracted from different strawberry tissue materials, wherein A is a morphological image of protoplasts extracted from mature strawberry leaves, B is a morphological image of protoplasts extracted from strawberry regeneration cluster buds, and C is a morphological image of protoplasts extracted from strawberry callus, Bar=2μm;

[0029] Figure 3 This is a diagram showing the FDA staining observation result of strawberry callus protoplasts extracted by the method of the present invention, Bar=10 μm;

[0030] Figure 4 3 are morphological diagrams of strawberry callus protoplasts before and after purification, wherein A is the morphological diagram of strawberry callus protoplasts before purification, and B is the morphological diagram of strawberry callus protoplasts after purification. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] 1. Prepare induction medium, differentiation medium, mixed enzymatic solution, extraction buffer and resuspension solution

[0033] (1) Preparation of induction medium

[0034] Sucrose, agar, indolebutyric acid and thidiazuron were added into MS medium, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron were 30 g / L, 9 g / L, 0.2 mg / L and 2.0 mg / L, respectively.

[0035] (2) Preparation of differentiation medium

[0036] Sucrose, agar, indolebutyric acid and thidiazuron were added into MS medium, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron were 30 g / L, 9 g / L, 0.3 mg / L and 1.0 mg / L, respectively.

[0037] (3) Preparation of mixed enzymatic solution

[0038] D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride are added into water, and the concentrations of D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride are 0.4 mol / L, 20 mmol / L, 10 mmol / L and 20 mmol / L, respectively, to prepare a mixed solvent. In this mixed solvent, 2-(N-morpholine)ethanesulfonic acid, D-mannitol, calcium ions and potassium ions play a role in stabilizing the plasma membrane and osmotic pressure.

[0039] Cellulase R-10, macerate R-10, pectinase Y-23 and bovine serum albumin were added to a mixed solvent, the concentrations of cellulase R-10, macerate R-10, pectinase Y-23 and bovine serum albumin were 10 g / L, 10 g / L, 1 g / L and 1 g / L respectively, and the pH value was adjusted to 5.7 with KOH.

[0040] (4) Preparation of extraction buffer

[0041] 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose were added into water, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose were 4mmol / L, 150mmol / L, 0.5mmol / L, 5mmol / L and 5mmol / L respectively.

[0042] (5) Preparation of resuspension

[0043] 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride were added into water, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride were 2mmol / L, 0.4mol / L and 15mmol / L respectively.

[0044] 2. Induce callus

[0045] Leaves of forest strawberry tissue culture seedlings with good growth were taken and callus tissue was induced on the induction medium by in vitro wounding. The culture conditions were: 24±2℃, 16h photoperiod, light intensity 500-1000lux, and culture for 8w.

[0046] 3. Differentiation and culture of callus tissue

[0047] The callus obtained in the previous step was cut and then placed on a differentiation medium for further culture. The culture conditions were: 24±2°C, 16h photoperiod, light intensity 500-1000lux, and culture for 8 weeks.

[0048] 4. Broken callus tissue

[0049] From the callus obtained in the previous differentiation culture, light green, soft, granular and vigorously growing clustered buds were selected, and 5 to 8 g were placed in a 30 mL centrifuge tube containing 5 mL of cold distilled water and crushed to obtain callus fragments with uniform texture and loosely separated cells.

[0050] 5. Enzymatic hydrolysis of callus

[0051] The callus tissue fragments obtained in the previous step were filtered through a 45 μm nylon mesh, and the filtered callus tissue was placed in a mixed enzymatic hydrolysis solution and enzymatically hydrolyzed at 23° C. and 60 rpm for 3 h to obtain a solution.

[0052] 6. Collect protoplasts

[0053] Add an equal volume of extraction buffer to the solution obtained in the previous step to terminate the enzymatic reaction, filter with a 45 μm nylon mesh, collect the protoplast filtrate, centrifuge the filtrate at 800 rpm for 5 minutes, discard the supernatant, and the green precipitate is the protoplast.

[0054] 7. Preliminary purification of protoplasts

[0055] The protoplasts obtained in the previous step were filtered through a 75 μm nylon mesh, the nylon mesh was discarded, and other cell interference was removed. The filtrate was filtered through a 10 μm nylon mesh, and the protoplasts were retained on the nylon mesh. The nylon mesh was turned upside down in a 50 mL centrifuge tube, and rinsed 2 to 3 times with extraction buffer to rinse the extracted protoplasts.

[0056] 8. Further purification of protoplasts

[0057] The protoplasts obtained in the previous step were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with 1 mL of resuspension solution to obtain a strawberry callus protoplast suspension, which was stored at 4°C.

[0058] The FDA staining observation results of the strawberry callus protoplasts extracted by the method of the present invention are shown in Figure 3 .

[0059] Depend on Figure 3 It can be seen that the strawberry callus protoplasts finally extracted are in good condition (full shape, high activity) and in large quantity.

[0060] The morphology of strawberry callus protoplasts before and after purification is shown in Figure 4 .

[0061] Depend on Figure 4 It can be seen that by adding preliminary purification and further purification operations, the purity of the finally extracted strawberry callus protoplasts is significantly improved.

[0062] Before determining the aforementioned method for extracting protoplasts from forest strawberry callus provided by the present invention, we studied the effects of the composition of the mixed enzymatic hydrolysis solution, the enzymatic hydrolysis method and time, and the selection of tissue materials on the separation of protoplasts.

[0063] 1. Effect of the composition of the mixed enzymatic solution on the separation of protoplasts

[0064] (1) Preparation of mixed solvent

[0065] D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride, potassium chloride and water were mixed, and the concentrations of D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride were 0.4 mol / L, 20 mmol / L, 10 mmol / L and 20 mmol / L, respectively, to prepare a mixed solvent.

[0066] (2) Preparation of mixed enzymatic hydrolysates with different compositions

[0067] Different masses of cellulase R-10, macerate R-10, pectinase Y-23 and bovine serum albumin were added to the mixed solvent to prepare mixed enzymatic hydrolysates (hereinafter referred to as enzymatic hydrolysates) with different compositions, as follows:

[0068] Table 1 Composition of enzymatic hydrolysate

[0069]

[0070]

[0071] (3) Extraction of protoplasts from forest strawberry callus

[0072] Protoplasts were extracted from forest strawberry callus using the four enzymatic hydrolysates with different compositions given in Table 1 according to the method for extracting protoplasts from forest strawberry callus given in the present invention.

[0073] (4) Results

[0074] The yield of protoplasts can reflect the effect of the composition of the enzymatic hydrolysate on the extraction of protoplasts.

[0075] Table 2 Protoplast yield

[0076] <![CDATA[Yield of protoplasts (×10 7 / gfw)]]> Enzyme solution 1 3.90 Enzyme solution 2 5.42 Enzyme solution 3 3.21 Enzyme solution 4 12.70

[0077] From Table 2, we can see that:

[0078] (i) At the same cellulase concentration, as the concentrations of pectinase and macerate increased, the yield and activity of protoplasts increased accordingly. This may be because the callus tissue contains a relatively high amount of pectin, which requires a correspondingly higher concentration of pectinase and macerate enzyme for enzymatic hydrolysis;

[0079] (ii) Among the four enzymatic hydrolysates tested, enzymatic hydrolysate 4 had the best protoplast separation effect, with a yield of up to 12.70×10 7 / gfw, after testing, its activity was as high as 60.2%.

[0080] The experiment found that at the same cellulase concentration, when the hydrolysate contained 4g / L cleavage enzyme R-10 and 0.2g / L pectinase Y-23 (hydrolysate 1), a large amount of fragments existed in the hydrolysate; at the same pectinase concentration, when the cellulase concentration was in the range of 5g / L to 10g / L, the yield and activity of the protoplasts increased with the increase of the cellulase concentration.

[0081] Therefore, it was finally decided to use enzymatic solution 4 to extract protoplasts from forest strawberry callus.

[0082] 2. Effects of enzymatic hydrolysis method and time on protoplast separation

[0083] (1) Extraction of protoplasts from forest strawberry callus

[0084] There are two types of enzymatic hydrolysis: oscillation and static.

[0085] According to the method for extracting protoplasts from forest strawberry callus provided by the present invention, protoplasts are extracted from forest strawberry callus by using two enzymatic hydrolysis methods, namely oscillation and static, and samples are taken at different enzymatic hydrolysis times to detect the yield and activity of the protoplasts.

[0086] (2) Results

[0087] The yield and activity of protoplasts can reflect the effects of enzymatic hydrolysis method and time on the separation of protoplasts.

[0088] Table 3 Protoplast yield and viability

[0089]

[0090] From Table 3, we can see that:

[0091] (i) Enzymatic hydrolysis method and time are important factors affecting protoplast yield and vitality;

[0092] (ii) Under the same enzymatic hydrolysis time conditions, the protoplast yield of shaking enzymatic hydrolysis was higher than that of static enzymatic hydrolysis, but the activity was not as high as that of static enzymatic hydrolysis;

[0093] (iii) As the enzymatic hydrolysis time gradually increased, the yield and activity of protoplasts released by shaking enzymatic hydrolysis gradually decreased, reaching the highest value at the 3rd hour of culture;

[0094] (iv) The yield of protoplasts released by static enzymatic hydrolysis first increased and then decreased, and the activity gradually decreased with the extension of enzymatic hydrolysis time;

[0095] (v) Regardless of whether the enzymatic hydrolysis was performed on a shaking table or at rest, the yield and activity of protoplasts decreased rapidly after 12 h of enzymatic hydrolysis, as evidenced by the breakage and death of a large number of protoplasts. No protoplasts survived after 15 h of enzymatic hydrolysis.

[0096] (vi) The time required for reaching the peak yield of the shaking enzymatic hydrolysis and the static enzymatic hydrolysis was also different. The shaking enzymatic hydrolysis reached the best yield and activity at 3 hours, while the static enzymatic hydrolysis reached the best yield and activity at 6 hours. This may be because the shaking enzymatic hydrolysis released protoplasts relatively quickly, while the static enzymatic hydrolysis released protoplasts slowly.

[0097] When enzymatic hydrolysis was performed using a shaking table, the morphology of strawberry callus at different enzymatic hydrolysis times was Figure 1 .

[0098] Taking into account the morphology of strawberry callus as well as the yield and vitality of protoplasts, it was determined that enzymatic hydrolysis with shaking at 60 rpm for 3 h was the optimal solution.

[0099] 3. Effect of tissue material selection on protoplast isolation

[0100] We studied three types of strawberry tissue materials, namely: mature strawberry leaves, strawberry regenerated cluster buds, and strawberry callus tissue.

[0101] According to the above method for extracting protoplasts from forest strawberry callus provided by the present invention, protoplasts were extracted from mature strawberry leaves, strawberry regeneration buds, and strawberry callus, and the morphology of protoplasts extracted from different strawberry tissue materials was observed. Figure 2 .

[0102] Therefore, it was finally decided to use strawberry callus for protoplast extraction.

[0103] It should be noted that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A method for extracting protoplasts from forest strawberry callus, characterized in that: The following steps are involved: (1) Taking leaves of forest strawberry tissue culture seedlings, inducing callus tissue on an induction medium by in vitro wounding, wherein the induction medium is prepared from MS medium, sucrose, agar, indolebutyric acid and thidiazuron, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron are 30 g / L, 9 g / L, 0.2 mg / L and 2.0 mg / L, respectively; (2) cutting the callus obtained in step (1) and placing it in a differentiation medium for further culture, wherein the differentiation medium is prepared from MS medium, sucrose, agar, indolebutyric acid and thidiazuron, and the concentrations of sucrose, agar, indolebutyric acid and thidiazuron are 30 g / L, 9 g / L, 0.3 mg / L and 1.0 mg / L, respectively; (3) Selecting light green, soft, granular and vigorously growing clustered buds from the callus obtained in step (2), placing them in a centrifuge tube containing cold distilled water and crushing them to obtain callus fragments with uniform texture and loosely separated cells; (4) filtering the callus tissue fragments obtained in step (3) through a 45 μm nylon mesh, placing the filtered callus tissue into a mixed enzymatic hydrolysis solution, and performing enzymatic hydrolysis at 23° C. and 60 rpm for 3 hours to obtain a solution, wherein the mixed enzymatic hydrolysis solution is prepared from a mixed solvent, multiple enzymes and bovine serum albumin, wherein the mixed solvent is prepared from water, D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride, and the concentrations of D-mannitol, 2-(N-morpholine)ethanesulfonic acid, calcium chloride and potassium chloride are 0.4 mol / L, 20 mmol / L, 10 mmol / L and 20 mmol / L, respectively; the enzymes include cellulase R-10, macerate R-10 and pectinase Y-23, and the concentrations are 10 g / L, 10 g / L and 1 g / L, respectively; the concentration of bovine serum albumin is 1 g / L; and the pH value is adjusted to 5.7 with KOH; (5) adding an equal volume of extraction buffer to the solution obtained in step (4) to terminate the enzymatic hydrolysis reaction, filtering with a 45 μm nylon mesh, collecting the protoplast filtrate, centrifuging the filtrate at 800 rpm for 5 min, discarding the supernatant, and the green precipitate is the protoplast, wherein the extraction buffer is prepared from water, 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, sodium chloride, calcium chloride, potassium chloride and glucose are 4 mmol / L, 150 mmol / L, 0.5 mmol / L, 5 mmol / L and 5 mmol / L, respectively; (6) Filter the protoplasts obtained in step (5) through a 75 μm nylon mesh, filter the filtrate through a 10 μm nylon mesh, place the nylon mesh upside down in a centrifuge tube, and rinse the protoplasts with extraction buffer; (7) The protoplasts obtained in step (6) were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with a resuspending solution to obtain a strawberry callus protoplast suspension.

2. The method for extracting protoplasts from forest strawberry callus according to claim 1, characterized in that: In step (3), 5 to 8 g of clustered buds are selected and crushed in a 30 mL centrifuge tube containing 5 mL of cold distilled water.

3. The method for extracting protoplasts from forest strawberry callus according to claim 1, characterized in that: In step (7), the resuspension is prepared from water, 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride, and the concentrations of 2-(N-morpholine)ethanesulfonic acid, D-mannitol and magnesium chloride are 2 mmol / L, 0.4 mol / L and 15 mmol / L, respectively.

Images