1. A preparation method of chimonanthus nitens protoplasts is characterized by comprising the following steps:

culturing wintersweet anther in dark condition, and inducing callus;

placing the callus in an enzymolysis solution, and culturing in a shaking incubator in the dark to obtain an enzymolysis solution containing protoplasts; wherein the enzymatic solution comprises: the cellulase and the eductase, wherein the concentration of the cellulase is 1.0-2.0%, and the concentration of the eductase is 0.1-0.5%;

adding a proper amount of W5 solution into the enzymolysis solution for dilution, and filtering by using a cell filter; wherein the W5 solution is a mixed solution of sodium chloride, calcium chloride, potassium chloride, 2- (N-morpholinyl) ethanesulfonic acid and water;

the obtained filtrate was centrifuged, and the supernatant was discarded to obtain a solution containing wax plum protoplasts.

2. The method for preparing a chimonanthus nitens protoplast according to claim 1, wherein a culture medium for inducing the chimonanthus nitens anther is MS +1.0 mg/L6-BA + 1.0-2.0 mg/L2, 4-D.

3. The method for preparing chimonanthus nitens protoplasts according to claim 1, wherein the anther callus is cultured in the dark at a temperature of 24 ± 2 ℃ and an air humidity of 44%.

4. The method for preparing a chimonanthus nitens protoplast according to claim 1, wherein the chimonanthus nitens anther is a bract of chimonanthus nitens selected from the gene types of red heart, plain heart or crinkled flap, and after cleaning and sterilizing, the anther is removed; wherein the washing and disinfecting steps include washing with water, soaking in 70% alcohol, washing with sterile water, soaking in 0.1% mercuric chloride, and washing with sterile water.

5. The method of claim 1, wherein the cellulase concentration is 1.0% and the macerase concentration is 0.1%.

6. The method for preparing chimonanthus nitens protoplasts according to claim 1, wherein the enzymatic solution further comprises: mannitol, potassium chloride, 2- (N-morpholinyl) ethanesulfonic acid, calcium chloride, and bovine serum albumin; wherein the concentration of the mannitol is 0.4mol/L, the concentration of the potassium chloride is 0.02mol/L, the concentration of the 2- (N-morpholinyl) ethanesulfonic acid is 0.02mol/L, the concentration of the calcium chloride is 0.02mol/L, and the concentration of the bovine serum albumin is 1.0%.

7. The method for preparing chimonanthus nitens protoplasts according to claim 1, wherein the callus is cultured in an enzymolysis solution in a shaking incubator in the dark at a temperature of 22 ℃ and a rotation speed of 70 rpm/min.

8. The method for producing a chimonanthus nitens protoplast according to claim 1, wherein the callus is cultured with shaking for 4 to 6 hours.

9. The method for preparing chimonanthus nitens protoplasts according to claim 1, wherein the cell filter is a 75 μm cell filter.

10. The method for preparing chimonanthus nitens protoplasts according to claim 1, wherein the centrifugation of the filtrate is: centrifuge at 200Xg for 2 min.

Background

Chimonanthus praecox (Chimonanthus praecox) is a rare flower and tree of the genus Chimonanthus of the family Chimonadaceae, and is a rare species of flowering tree with winter flowering. In recent years, the demand of people on the wintersweet is increasing day by day, the application and the market of the wintersweet are continuously expanded, the character heredity and the improvement of the wintersweet become key points of scientific research and industry, the wintersweet is used as a woody plant, a genetic transformation system is difficult to establish, the establishment of the genetic transformation system of the wintersweet has great significance on the genetic functions of the wintersweet such as flower color, flower fragrance, stress resistance and the like, and the extraction of protoplast is just a basic technology for instantaneous expression and genetic character improvement.

Plant protoplasts refer to the portion of the protoplast encapsulated by the exposed cell membrane of a plant cell after the cell wall has been removed. Since the protoplast has no cell wall to isolate the external environment, the protoplast can easily absorb the introduced exogenous gene, chromosome and other genetic materials, can overcome the sexual reproduction barrier, has short preparation time, can quickly observe the phenotype, realizes the expression of the gene, and becomes an ideal receptor in molecular biology related experiments.

The chimonanthus nitens as woody plants have long growth period and complex plant structure, and related research on preparation of chimonanthus nitens protoplasts does not exist at present and is still in the starting stage. The technology for extracting the chimonanthus nitens protoplast is researched, the optimal material for extracting the chimonanthus nitens protoplast and the preparation method are obtained, and the method has great significance for establishing a chimonanthus nitens transient transformation system, genetic transformation, somatic cell hybridization and the like.

Disclosure of Invention

The invention aims to provide a preparation method of chimonanthus nitens protoplast, which prepares the protoplast by taking enzymolysis liquid combination of cellulase and eductase and calli as materials, is not influenced by the material drawing time and quantity, and can obtain average 1.11 multiplied by 10 by enzymolysis⁶The yield of individual calyx canthus protoplast is high-efficient and quick.

In order to achieve the above object, the present invention provides a method for preparing a chimonanthus nitens protoplast, comprising: culturing wintersweet anther in dark condition, and inducing callus; placing the callus in an enzymolysis solution, and culturing in a shaking incubator in the dark to obtain an enzymolysis solution containing protoplasts; wherein the enzymatic solution comprises: the cellulase and the eductase, wherein the concentration of the cellulase is 1.0-2.0%, and the concentration of the eductase is 0.1-0.5%; adding a proper amount of W5 solution into the enzymolysis solution for dilution, and filtering by using a cell filter; wherein the W5 solution is a mixed solution of sodium chloride, calcium chloride, potassium chloride, 2- (N-morpholinyl) ethanesulfonic acid and water; the obtained filtrate was centrifuged, and the supernatant was discarded to obtain a solution containing wax plum protoplasts.

Preferably, the culture medium for inducing the wintersweet anther is MS +1.0 mg/L6-BA + 1.0-2.0 mg/L2, 4-D.

Preferably, the anther callus is cultured under dark conditions at a temperature of 24 ± 2 ℃ with an air humidity of 44%.

Preferably, the wintersweet anther is a calyx of wintersweet selected from red heart, vegetarian heart or crinkled petal gene types, and the anther is taken down after cleaning and disinfection; wherein the washing and disinfecting steps include washing with water, soaking in 70% alcohol, washing with sterile water, soaking in 0.1% mercuric chloride, and washing with sterile water.

Preferably, the concentration of the cellulase is 1.0% and the concentration of the macerozyme is 0.1%.

Preferably, the enzymatic solution further comprises: mannitol, potassium chloride, 2- (N-morpholinyl) ethanesulfonic acid, calcium chloride, and bovine serum albumin; wherein the concentration of the mannitol is 0.4mol/L, the concentration of the potassium chloride is 0.02mol/L, the concentration of the 2- (N-morpholinyl) ethanesulfonic acid is 0.02mol/L, the concentration of the calcium chloride is 0.02mol/L, and the concentration of the bovine serum albumin is 1.0%.

Preferably, the callus is cultured in an enzymolysis solution in a shaking incubator in the dark at 22 ℃ and at a rotation speed of 70 rpm/min.

Preferably, the shaking culture time of the callus is 4-6 h.

Preferably, the cell filter is a 75 μm cell filter.

Preferably, the centrifugation of the filtrate is: centrifuge at 200Xg for 2 min.

The preparation method of the chimonanthus nitens protoplast has the following advantages:

the invention takes the calli of Chimonanthus fragrans as the material to prepare the protoplast, and is not influenced by the time and the quantity of the materials. The invention uses the enzymolysis liquid combination of cellulase and eductase to carry out enzymolysis for 4 hours, thus obtaining the average output of the chimonanthus nitens protoplast of 1.11 multiplied by 106/g, which is high-efficient and quick. The genotypes of different chimonanthus nitens used by the invention have obvious difference on the yield of protoplasts, wherein the yield of the protoplasts with plain cores can be as high as 1.56 multiplied by 10⁶Per gram. The invention opens up a preparation technology of the chimonanthus nitens protoplast, can carry out operations such as protoplast culture, instantaneous transformation, cell fusion and the like on the basis, and has important significance for the research of chimonanthus nitens gene functions and the improvement of genetic characters.

Drawings

FIG. 1 shows the materials for preparing protoplasts according to an embodiment of the invention.

FIG. 2 is a photograph (10-fold) of a protoplast cell prepared according to an example of the present invention.

FIG. 3 is a photograph (40-fold) of a protoplast cell prepared according to an example of the invention.

FIG. 4 shows the relationship between the yield of protoplasts and cellulase in examples of the present invention.

FIG. 5 shows the relationship between protoplast yield and the amount of eductases in the examples of the present invention.

FIG. 6 shows the relationship between the protoplast yield and the enzymolysis time in the example of the present invention.

FIG. 7 shows the relationship between protoplast yield and plant genotype in examples of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The reagents used in the following examples were prepared as follows:

0.8M mannitol (100 mL): weighing 15.18g of mannitol powder, dissolving in distilled water, diluting to 100mL, and storing in a refrigerator at 4 ℃.

10% BSA (10 mL): 1.0g of BSA powder was weighed, dissolved in distilled water, and then diluted to 10mL to be stored in a refrigerator at 4 ℃.

0.2M MES (10 mL): 0.3g MES (2- (N-morpholinyl) ethanesulfonic acid) powder is weighed, dissolved in distilled water, and then the volume is determined to 10mL, and the mixture is stored in a refrigerator at 4 ℃.

1M Potassium chloride (10 mL): 0.75g of potassium chloride powder is weighed, dissolved in distilled water, and then the solution is added to 10mL to be constant volume and stored in a refrigerator at 4 ℃.

1M calcium chloride (10 mL): weighing 11.1g of calcium chloride powder, dissolving in distilled water, diluting to 10mL, and storing in a refrigerator at 4 ℃.

1M sodium chloride (100 mL): 5.84g of sodium chloride powder is weighed, dissolved in distilled water, and then the volume is determined to be 100mL, and the mixture is stored in a refrigerator at 4 ℃.

W5 solution (100 mL): weighing 15.4mL of 1M sodium chloride, adding 12.5mL of 1M calcium chloride, 0.5mL of 1M potassium chloride and 1mL of 0.2M MES, mixing uniformly, diluting with distilled water to 100mL, and storing in a refrigerator at 4 ℃.

Example 1

Preparing chimonanthus nitens protoplasts according to the following steps:

(1) orthogonal tests at the design 4-factor 3 level table 1 is as follows:

TABLE 1 orthogonal experimental design of different enzymatic conditions (L)₉4³)

Note: these genotypes were all collected at the university of agriculture in Huazhong.

(2) Preparing 5mL of enzymolysis liquid: the water bath was opened and set at 55 ℃ for use, and appropriate amounts of cellulase and macerozyme were weighed out as in Table 1, and 2.5mL of 0.8M mannitol, 0.1mL of 1M potassium chloride, and 0.5mL of 0.2M MES were added. After mixing uniformly, incubating in a water bath for 10min, cooling to room temperature, adding 0.1mL of 1M calcium chloride and 0.5mL of 10% BSA (bovine serum albumin), diluting to 5mL with distilled water, filtering with a 0.45 μ M filter into a conical flask, and preparing for use.

(3) Respectively weighing 0.2g of red heart, plain heart and wrinkled plum anther-induced callus which has been subcultured for 10 months, adding prepared 5mL of enzymolysis solution, and gently mincing with bamboo stick. Selecting calyx of Chimonanthus nitens of red heart, vegetarian heart or crinkled petal gene type, cleaning and sterilizing, and taking off anther, wherein the cleaning and sterilizing comprises soaking in washing powder water, washing with water, soaking in 70% alcohol, washing with sterile water, soaking in 0.1% mercuric chloride, and washing with sterile water. Inducing wintersweet anther with induction culture medium of MS +1.0 mg/L6-BA +1.0 mg/L2, 4-D or MS +1.0 mg/L6-BA +2.0 mg/L2, 4-D, culturing anther callus in dark at 24 + -2 deg.C and air humidity of 44%.

(4) The conical flask filled with the enzymolysis liquid is fixed on a shaking bed and incubated for 4, 5 and 6 hours at 22 ℃ and 70r/min respectively under the dark condition.

(5) 1/2 volumes (2.5mL) of W5 solution was slowly added to the enzymatic solution near the wall, the enzymatic solution was slowly aspirated with a disposable plastic pipette, filtered on a 75 μm cell filter, and the filter was washed with W5 solution.

(6) The resulting filtrate was centrifuged at 200g for 2min, placed on ice and the supernatant discarded. And adding the W5 solution with the volume 2 times that of the residual enzymolysis solution again, centrifuging for 2min under the same centrifugation condition, discarding the supernatant, and performing ice bath on the residual about 1mL of enzymolysis solution to obtain the solution containing the waxberry protoplast.

The extracted protoplast cells were observed under an inverted microscope and the number of protoplast cells was recorded using a hemacytometer (see FIG. 1, FIG. 2) and repeated 3 times.

Identification of protoplast yield: the protoplast yield was calculated using a blood cell counting plate of a cross-grid type, a counting chamber of 25 middle grids × 16 cells, and the number of cells counted for the four-corner middle grids + the middle grid of 5 middle grids (5 × 16 ═ 80 cells).

Big square (0.1 mm)³) Total number of protoplasts is the average number of protoplast cells in middle lattice × 25.

Of protoplastsDensity (one/mL) is large square (0.1 mm)³) Total number of protoplasts X10⁴。

Protoplast yield (per g) ═ protoplast density x total volume of protoplast suspension)/fresh weight of calli used.

In the step (2), the concentrations of the cellulase are 1.0%, 1.5% and 2.0%, and the results show that the cellulase can achieve better effect when the concentration is 1.0%, and the yield is 1.17 multiplied by 10⁶Per g (see FIG. 4).

In the step (2), the concentrations of the eductases were set to 0.1%, 0.3%, and 0.5%, and the results showed that the concentration of the eductases was 0.1%, which resulted in the best results, and the yield of protoplasts was 1.37X 10 on average⁶Per g (see FIG. 5).

In the step (3), the enzymolysis time is set to be 4, 5 and 6 hours, and the result shows that the yield of the protoplast has no significant difference when the enzymolysis time is 4 to 6 hours, the enzymolysis time is 4 hours optimal, and the yield can reach 1.16 multiplied by 10⁶Per g (see FIG. 6).

In the step (3), the results of 3 different chimonanthus nitens genotypes show that the yield of the vegetarian core protoplast is the highest and can reach 1.56 multiplied by 10⁶Per g (see FIG. 7).

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.