CN108489884B

CN108489884B - Flow identification method for homozygous line progeny obtained by wheat haploid breeding

Info

Publication number: CN108489884B
Application number: CN201810198253.2A
Authority: CN
Inventors: 刘颖; 梁曙炎; 吴泽强; 严星
Original assignee: Wuhan Biological Sample Bank Co Ltd
Current assignee: Wuhan biological sample Bank Co., Ltd
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2020-12-25
Anticipated expiration: 2038-03-12
Also published as: CN108489884A

Abstract

The invention discloses a flow identification method of homozygous line progeny obtained by wheat haploid breeding, belonging to the field of biotechnology. According to the method, the concentrations of citric acid and sodium chloride in the buffer solution mother liquor are optimized to be 400-440 g/L and 160-200 g/L, nonylphenol polyoxyethylene ether is adopted to replace common stabilizers and dispersing agents, Hoechst33258 with the concentration of 100-300 mg/L is adopted to replace DAPI and PI to serve as fluorescent dyes, and the mesh number of a filter screen used for filtering and the centrifugal rotating speed are optimized to be 350-450 meshes and 1000-3000 r/min respectively. Compared with the prior art, the method shortens the identification time, simplifies the identification steps, has more reasonable buffer solution configuration, can obtain more effective cells, removes more cell fragments, avoids detection interference and improves the identification precision.

Description

Flow identification method for homozygous line progeny obtained by wheat haploid breeding

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a flow identification method of homozygous line progeny obtained by wheat haploid breeding.

Background

Wheat is a monocotyledonous gramineous plant and is widely grown around the world. As a staple food for human, the caryopsis of wheat can be ground into flour to make bread, steamed bread, biscuit, noodle and other foods; fermenting to obtain beer, alcohol, Chinese liquor or biomass fuel. Wheat is one of the three major grains and is almost all eaten. The haploid breeding technology in the wheat breeding method has obvious advantages in the aspects of shortening the breeding age and improving the breeding efficiency, and the obtained haploid can be doubled to obtain an advantageous diploid plant or a polyploid plant.

In the existing diploid or polyploid plant identification technology, for identifying whether a plant obtained by wheat haploid breeding is a plant with a required advantage, plant pollen mother cells or young tender root tip cells are generally required to be collected, the plant is required to be cultivated into a complete flowering plant or form seeds, cell chromosomes are drawn and arranged by means of a microscope under the action of reagents such as hydroxyurea, colchicine and the like, and then chromosomes of a standard plant are compared to obtain a final identification result; the method has long time consumption, complicated steps and high requirements on operation and comparison experience, and toxic reagents such as hydroxyurea, colchicine and the like are involved, thereby bringing great potential harm to operators and the environment.

Flow Cytometry (FCM) is a single cell quantitative analysis and sorting technique performed using flow cytometry. The cell/cell nucleus marked by the coloring agent enters the flow chamber under the pushing of certain pressure, the sheath fluid surrounds the cell and flows at high speed to form a circular flow beam, and the cells are arranged in a single row under the wrapping of the sheath fluid and sequentially and rapidly pass through the detection area. The cell stained by the stain is excited by laser to emit fluorescence with a specific wave band, the optical signal is received by an optical system and converted into an electric signal, the electric signal is collected by a computer and then is calculated, and the analysis result is displayed on a computer screen to form storable data. And comparing the peak-to-peak graphs of the standard diploid and the plant cell to be identified to obtain the conclusion that the plant to be identified is haploid, diploid, tetraploid or octaploid and the like. At present, the flow cytometry is not yet used in ploidy identification of homozygous line progeny obtained by wheat haploid breeding.

In the existing ploidy identification method by using a flow cytometer, a callus of a plant is pretreated, cells are subjected to enzymolysis to obtain protoplasts, and then a common staining agent (such as DAPI and PI) is used for staining and analyzing the ploidy relation. The whole identification process has more steps and is more complicated, and the preparation of the used buffer solution and the cell enzymolysis consume long time. For another example, in the invention of patent No. 201610578478.1, "buffer solution suitable for ploidy identification of various plants by flow cytometry, and methods for preparation and use thereof", the concentrations of trisodium citrate and sodium chloride in the buffer solution mother liquor and the diluted buffer solution do not achieve the optimal buffering effect; the dispersing efficiency of the Tween-20 reagent added into the buffer solution to the cell nucleus is relatively low; staining agents such as DAPI and PI have certain damage to the activity of cells, and the proliferation activity of the cells is influenced; the number of meshes of the screened cell suspension is unreasonable, so that the screened filtrate contains more adhered cell nuclei, which is not beneficial to instrument detection and influences the detection precision; the filtrate is directly added into the staining solution, so that the number of effective cells for identification is low, and the detection precision is influenced. These problems all affect the stability and dispersion of the cells and nuclei to be identified, which is not conducive to the final identification.

Therefore, a new flow identification method for homozygous line progeny obtained by wheat haploid breeding needs to be invented, the step of preparing protoplast in advance in the traditional method is avoided, the identification process is simplified, and the identification efficiency is improved; the damage of the staining agent to cells is reduced, and the reproductive activity of the cells is ensured; improve the stability of cells/free nuclei in buffer.

Disclosure of Invention

The invention provides a flow identification method of homozygous line progeny obtained by wheat haploid breeding, which is realized by the following technology.

A flow identification method for homozygous line progeny obtained by wheat haploid breeding comprises the following steps:

s1, adding a certain amount of citric acid and sodium chloride into double distilled water, and uniformly mixing to ensure that the concentrations of the citric acid and the sodium chloride are 400-440 g/L and 160-200 g/L respectively, so as to obtain a buffer solution mother solution for later use;

s2, uniformly mixing double distilled water and the buffer solution mother liquor according to the volume ratio of 19:1 to obtain a buffer solution for later use;

s3, adding 0.01-0.1 ml of nonylphenol polyoxyethylene ether and 0.01-0.09 ml of beta-mercaptoethanol into each 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use;

s4, adding a certain amount of Hoechst33258 into the buffer solution, dissolving and uniformly mixing to ensure that the concentration of the Hoechst33258 is 100-300 mg/L, and obtaining a dyeing mother solution for later use;

s5, mixing the dyeing mother liquor and the buffer solution according to the volume ratio of 1-2: 500, uniformly mixing to obtain a dyeing working solution;

s6, taking a fresh wheat callus sample to be identified, removing surface culture medium components, and placing the sample in a culture dish; adding the buffer working solution to ensure that the tissue sample is soaked in the buffer working solution, and cutting up the tissue sample by a sharp blade; adding the buffer working solution to a constant volume, slightly oscillating, and standing for 10-30 minutes;

s7, filtering the solution obtained in the step S6 by a 350-450-mesh filter screen, centrifuging the obtained filtrate for 5-15 minutes at the rotating speed of 1000-3000 r/min, discarding the supernatant, and adding the dyeing working solution to the dark to dye for 10-30 minutes for later use;

s8, loading the solution dyed in the dark place in the step S7, taking a scattergram of an area parameter HOECHST-A and a height parameter HOECHST-H to remove the adhesive body, taking HOECHST-A as a straight peak graph, maintaining the G0/G1 peak of the standard wheat diploid used for comparison at 350, and comparing the positions of the peaks of the wheat tissue sample to be identified to identify the multiple.

Preferably, the concentrations of citric acid and sodium chloride in the buffer mother liquor prepared in step S1 are respectively 420g/L and 180 g/L.

Preferably, in step S3, 0.06ml nonylphenol polyoxyethylene ether and 0.06ml β -mercaptoethanol are added to each 100ml of the buffer.

Preferably, the concentration of Hoechst33258 in the mother liquor for dyeing prepared in step S4 is 200 mg/L.

Preferably, step S6 is: taking 20mg of a fresh wheat callus sample to be identified, removing surface culture medium components, and placing the sample in a culture dish; adding 200-500 mu l of the buffer working solution to ensure that the tissue sample is soaked in the buffer working solution, and cutting up the tissue sample by a sharp blade; and adding the buffer working solution to a constant volume of 1.5ml, slightly oscillating, and standing for 10-30 minutes.

Preferably, in step S7, the solution obtained in step S6 is filtered through a 400-mesh screen.

Preferably, in step S7, the obtained filtrate is centrifuged at 2000r/min for 5-15 minutes.

Preferably, in step S7, the dyeing working solution is added in an amount of 200 μ l.

Preferably, in step S8, the sample application rate is 10 μ l/min.

Compared with the prior art, the invention has the advantages that:

1. the flow identification method is applied to ploidy identification of homozygous line progeny obtained by wheat haploid breeding, and the blank of the prior art is filled;

2. the free cell nucleus is obtained by a mechanical crushing mode, and the protoplast is not prepared in advance for ploidy analysis of the callus, so that the identification process is simplified;

3. the whole identification process does not need to wait for the growth process of the wheat plants, so that the identification time is shortened, the detection and analysis time of each sample is not more than ten minutes, and the sensitivity, the accuracy and the reproducibility are good;

4. the Hoechst33258 is adopted to replace DAPI and PI to be used as a dye as a non-insertion fluorescent dye, the activity damage of the Hoechst33258 to cells is small, the dyed cells still keep the proliferation activity and can be continuously cultured;

5. the composition of the buffer solution is optimized, toxic and harmful reagents such as hydroxyurea, colchicine and the like are avoided, the operation is safe and simple, and the cell integrity is better;

6. compared with the common stabilizing agent and dispersing agent such as Tween-20 and the like, the adopted nonylphenol polyoxyethylene ether improves the dispersing efficiency of the cell nucleus and obtains more effective cells;

7. a more appropriate screen and a low-speed centrifugal rotating speed are selected, so that not only can the effective cell number be obtained as much as possible, but also redundant cell fragments can be removed, the detection interference is prevented, and the precision is improved;

8. by optimizing the concentration, volume, instrument sample loading speed and the like of each solution in the identification step, the tissue sample is effectively ensured to be fully and uniformly minced and dispersed, cells of all the tissue sample are fully dyed, and the measurement precision of the instrument is not influenced by too low effective cell concentration during sample loading.

Drawings

FIG. 1 is a scatter plot of a wheat tissue sample to be identified of example 1;

FIG. 2 is a histogram of the wheat tissue samples to be identified of example 1;

FIG. 3 is a scattergram of a standard diploid sample of wheat used in examples 1 to 7 and comparative examples 1 to 6;

FIG. 4 is a direct peak view of a standard diploid sample of wheat used in examples 1 to 7 and comparative examples 1 to 6;

FIGS. 5 and 6 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 2;

FIGS. 7 and 8 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 3;

FIGS. 9 and 10 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 4;

FIGS. 11 and 12 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 5;

FIGS. 13 and 14 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 6;

FIGS. 15 and 16 are a scatter plot and a straight-peak plot, respectively, of the wheat tissue sample to be identified of example 7;

FIGS. 17 and 18 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 1;

FIGS. 19 and 20 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 2;

FIGS. 21 and 22 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 3;

FIGS. 23 and 24 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 4;

FIGS. 25 and 26 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 5;

FIGS. 27 and 28 are a scatter plot and a straight-peak plot, respectively, of a wheat tissue sample to be identified in comparative example 6.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, 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 fresh wheat callus samples (hereinafter referred to as wheat tissue samples) selected in the following examples 1 to 7 and comparative examples 1 to 6 were all taken from the same wheat plant to be identified.

Example 1

The flow identification method of homozygous line progeny obtained by haploid breeding of wheat adopted by the embodiment comprises the following steps:

s1, adding a certain amount of citric acid and sodium chloride into double distilled water, mixing uniformly to make the concentrations of the citric acid and the sodium chloride respectively 420g/L and 180g/L to obtain a buffer solution mother solution, and storing at 4 ℃ for later use;

s3, adding 0.06ml of nonylphenol polyoxyethylene ether and 0.06ml of beta-mercaptoethanol into each 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use;

s4, adding a certain amount of Hoechst33258 into the buffer solution, dissolving and uniformly mixing to ensure that the concentration of the Hoechst33258 is 200mg/L to obtain dyeing mother liquor, and storing at 4 ℃ for later use;

s5, adding 200 mu l of the staining mother liquor into 50ml of the buffer solution, and uniformly mixing to obtain a staining working solution;

s6, taking 20mg of a fresh wheat callus sample to be identified, removing surface culture medium components, and placing the sample in a culture dish; adding 400 mu l of the buffer working solution to ensure that the tissue sample is soaked in the buffer working solution, and cutting up the tissue sample by a sharp blade; adding the buffer working solution to make the final volume be 1.5ml, slightly oscillating, and standing for 20 minutes;

s7, filtering the solution obtained in the step S6 by a 400-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 2000r/min, discarding the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use;

s8, loading the solution dyed in the dark place in the step S7 at the speed of 10 mul/min, taking a scattergram of an area parameter HOECHST-A and a height parameter HOECHST-H to remove the adhesive body, taking a direct peak image of HOECHST-A, maintaining the G0/G1 peak of the standard wheat diploid used for comparison at 350, and comparing the positions of the peaks of the wheat tissue samples to be identified to identify the multiple.

Example 2

The flow identification method of homozygous line progeny obtained by haploid breeding of wheat adopted in this example is basically the same as that of example 1, except for the following aspects:

step S3 is: and adding 0.1ml of nonylphenol polyoxyethylene ether and 0.09ml of beta-mercaptoethanol into each 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use.

Example 3

step S3 is: and adding 0.01ml of nonylphenol polyoxyethylene ether and 0.01ml of beta-mercaptoethanol into every 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use.

Example 4

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 450-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 2000r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Example 5

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 350-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 2000r/min, discarding the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Example 6

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 400-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 3000r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Example 7

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 400-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 1000r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Comparative example 1

The flow identification method of homozygous line progeny obtained by haploid breeding of wheat adopted by the comparative example comprises the following steps:

s3, adding 0.15ml of nonylphenol polyoxyethylene ether and 0.15ml of beta-mercaptoethanol into each 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use;

s8, loading the solution dyed in the dark place in the step S7 at the speed of 10 mul/min, taking a scattergram of an area parameter HOECHST-A and a height parameter HOECHST-H to remove the adhesive body, taking a direct peak image of HOECHST-A, maintaining the G0/G1 peak of the standard wheat diploid used for comparison at 350, and comparing the positions of the peaks of the wheat tissue samples to be identified to identify the multiple. Comparative example 2

The flow identification method of homozygous line progeny obtained by haploid breeding of wheat adopted in the comparative example is basically the same as that of comparative example 1, and the differences are as follows:

step S3 is: and adding 0.005ml of nonylphenol polyoxyethylene ether and 0.005ml of beta-mercaptoethanol into every 100ml of buffer solution, and uniformly mixing to obtain buffer working solution for later use.

Comparative example 3

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 500-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 2000r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Comparative example 4

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 300-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 2000r/min, discarding the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Comparative example 5

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 400-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 3500r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Comparative example 6

step S7 is: and (4) filtering the solution obtained in the step S6 by using a 400-mesh filter screen, centrifuging the obtained filtrate for 10 minutes at the rotating speed of 500r/min, removing the supernatant, adding 200 mu l of the dyeing working solution, and dyeing for 20 minutes in a dark place for later use.

Application example 1: influence of nonylphenol polyoxyethylene ether and mercaptoethanol content in buffer working solution on flow-type identification of ploidy of wheat callus

As shown in FIGS. 1 to 4, the flow identification was carried out according to the method of example 1, and a scatter plot and a straight-peak plot were obtained for the wheat tissue sample to be identified of example 1 and the standard diploid used in examples 1 to 7 and comparative examples 1 to 6. By adjusting parameters such as current, voltage and the like of the flow cytometer, the peak value in the straight peak graph of the standard diploid corresponds to 350 of the abscissa axis. As shown in FIGS. 5 to 8, the flow identification was performed according to the methods of examples 2 and 3, and the corresponding scattergram and direct peak pattern of the wheat tissue sample to be identified were obtained. As shown in FIGS. 17 to 20, flow identification was performed according to the methods of comparative examples 1 and 2, respectively, to obtain a scatter plot and a peak plot of the corresponding wheat tissue sample to be identified.

By observing the scattergrams of the wheat tissue samples to be identified of examples 1 to 3 and comparative examples 1 and 2, the distribution of the effective cells and debris is indicated by the P1 value, and the higher the P1 value is, the greater the number of effective cells is. The P1 values in the scattergrams corresponding to examples 1-3 are all high, namely 53.10%, 47.76% and 38.06%, which means that enough effective cells can be obtained, wherein the P1 value in the scattergram of example 1 is the highest, namely the most effective cells can be obtained, and the measurement result can be ensured to be more accurate. The scatter plots of comparative examples 1 and 2 each had a low P1 value of 11.22% and 8.22%, respectively, indicating that sufficient effective cells could not be obtained.

By observing the peak-to-peak graphs of the wheat tissue samples to be identified of examples 1 to 3 and comparative examples 1 and 2, the P3 value indicates the relative content of effective cells, and the higher the P3 value, the higher the relative content of effective cells. The P3 values in the straight peak graphs corresponding to the embodiments 1-3 are respectively 54.82%, 53.03% and 51.05%, and the main peaks are obvious and located near 175, and the comparison of the straight peak graphs of the standard diploid of the wheat tissue sample can prove that the wheat tissue sample to be identified is a wheat haploid. On the other hand, the histogram of comparative examples 1 and 2 shows signal dispersion, and the ploidy thereof cannot be judged.

From the above results, it can be seen that the flow-type identification performed by using the nonylphenol polyoxyethylene ether and mercaptoethanol contents of examples 1 to 3 has higher accuracy and better accuracy.

Application example 2: influence of mesh number of filter screen on flow type identification of ploidy of wheat callus

As shown in FIGS. 9 to 12, flow identification was performed according to the methods of examples 4 and 5, and a scatter plot and a peak plot of the corresponding wheat tissue sample to be identified were obtained. As shown in FIGS. 21 to 24, flow identification was performed according to the methods of comparative examples 3 and 4, and a scatter plot and a peak plot of the corresponding wheat tissue sample to be identified were obtained.

By observing the scattergrams of examples 1, 4, and 5 and comparative examples 3 and 4, the P1 values in the scattergrams of examples 4 and 5 were high, 43.39% and 34.89%, respectively, but both were lower than those in example 1, i.e., examples 4 and 5, and a large number of effective cells were obtained. In contrast, the dot plots of comparative examples 3 and 4 both had low P1 values of 29.47% and 30.28%, respectively, and thus sufficient effective cells could not be obtained.

By observing the peak plots of the wheat tissue samples to be identified of examples 1, 4 and 5 and comparative examples 3 and 4, the P3 values in the peak plots corresponding to examples 4 and 5 are higher, respectively 49.35% and 46.91%, but are smaller than those in example 1; the main peak is obvious and is positioned near 175, and the comparison of a straight peak diagram of a standard diploid of the wheat tissue sample can prove that the wheat tissue sample to be identified is a wheat haploid. On the other hand, the histogram of comparative examples 3 and 4 shows signal dispersion, and the ploidy thereof cannot be judged.

From the above results, it can be seen that the filter screens used in examples 4 and 5 can obtain more effective cells, and the accuracy of identification is higher and better, and the accuracy and precision of the identification result of the filter screen used in example 1 are the best.

Application example 3: influence of rotational speed of low-speed centrifugation on flow identification of ploidy of wheat callus

As shown in FIGS. 13 to 16, flow identification was performed according to the methods of examples 6 and 7, and a scatter plot and a peak plot of the corresponding wheat tissue sample to be identified were obtained. As shown in FIGS. 25 to 28, flow identification was performed according to the methods of comparative examples 5 and 6, and a scatter plot and a peak plot of the corresponding wheat tissue sample to be identified were obtained.

By observing the scattergrams of examples 1, 6, and 7 and comparative examples 5 and 6, the P1 values in the scattergrams of examples 6 and 7 were high, 34.82% and 35.35%, respectively, but both were lower than those in example 1, i.e., examples 6 and 7, and a large number of effective cells were obtained. In contrast, the dot plots of comparative examples 5 and 6 both had low P1 values of 19.30% and 13.33%, respectively, and thus sufficient effective cells could not be obtained.

By observing the peak plots of the wheat tissue samples to be identified of examples 1, 6 and 7 and comparative examples 5 and 6, the P3 values in the peak plots corresponding to examples 6 and 7 are higher, 42.76% and 40.45% respectively, but are smaller than those in example 1; the main peak is obvious and is positioned near 175, and the comparison of a straight peak diagram of a standard diploid of the wheat tissue sample can prove that the wheat tissue sample to be identified is a wheat haploid. On the other hand, the histogram of comparative examples 5 and 6 shows signal dispersion, and the ploidy thereof cannot be judged.

From the above results, it can be seen that the rotation speed of the low-speed centrifugation used in examples 6 and 7 can obtain more effective cells, and the identification precision is higher and the accuracy is better, and the rotation speed of the low-speed centrifugation used in example 1 makes the identification result precision and accuracy the best.

Claims

1. A flow identification method for homozygous line progeny obtained by wheat haploid breeding is characterized by comprising the following steps:

s1, adding a certain amount of citric acid and sodium chloride into double distilled water, and uniformly mixing to ensure that the concentrations of the citric acid and the sodium chloride are respectively 420g/L and 180g/L, so as to obtain a buffer solution mother solution for later use;

2. The method of claim 1, wherein in step S3, 0.06ml nonylphenol polyoxyethylene ether and 0.06ml β -mercaptoethanol are added to each 100ml buffer solution.

3. The method of claim 1, wherein the concentration of Hoechst33258 in the mother solution of the stain obtained in step S4 is 200 mg/L.

4. The flow identification method of homozygous line progeny obtained by haploid breeding of wheat according to claim 1, wherein in step S6, 20mg of a fresh wheat callus sample to be identified is taken, and placed in a culture dish after surface culture medium components are removed; adding 200-500 mu L of the buffer working solution to ensure that the tissue sample is soaked in the buffer working solution, and cutting up the tissue sample by a sharp blade; and adding the buffer working solution to a constant volume of 1.5mL, slightly oscillating, and standing for 10-30 minutes.

5. The method of claim 1, wherein in step S7, the solution obtained in step S6 is filtered through a 400-mesh screen.

6. The flow identification method of homozygous line progeny obtained by haploid breeding of wheat according to claim 1, wherein in step S7, the obtained filtrate is centrifuged at 2000r/min for 5-15 minutes.