Method for artificially inducing triploid percobrachium glehniae

Abstract

The invention discloses a method for artificially inducing triploid percobrachium glehniae, and belongs to the technical field of triploid fish breeding. In order to solve the problems that gonad development affects the growth and meat quality of percobrachium glehniae, reduces the commodity quality of percobrachium glehniae and the like, the invention determines the optimal starting time, the optimal hydrostatic pressure and treatment time, the optimal temperature and the optimal chemical conditions of triploid percobrachium glehniae induction in combination with the triploid proportion of percobrachium glehniae, so that the percobrachium glehniae is induced into the triploid percobrachium glehniae. The method for producing the triploid percobrachium glehniae fish through combined induction of hydrostatic pressure, temperature shock and chemicals is established. The triploid percobrachium glehniae induction method established by the invention has the advantages of clear operation process, strong practicability, high efficiency and the like, and the induction efficiency of the triploid percobrachium glehniae can reach up to 85%, so that the induction method provided by the invention is suitable for artificially and efficiently producing the triploid percobrachium glehniae and has a wide application prospect. And a new technical approach is opened up for polyploidy breeding of the percobrachium glehniae.

Description

Technical Field

[0001] The invention belongs to the technical field of triploid fish breeding, and in particular relates to a method for artificially inducing triploid Channa gordonii. Background Art

[0002] Channa ge's pond bream is a small benthic fish of the genus Channa ge's family Channa ge's order Perciformes. As a native fish of Northeast China, Channa ge's pond bream is increasingly popular among consumers due to its tender meat, delicious taste, few bones and high nutritional value, and has great market development potential. It is generally believed that triploid fish cannot mature their gonads due to unbalanced chromosome composition, which greatly reduces the adverse effects of growth stagnation and decreased fish meat quality during gonad development, and can increase the growth rate of fish and improve meat quality. Therefore, conducting research on triploid fish and cultivating infertile triploid populations will help increase fish farming output and commodity value, and is also crucial to protecting my country's fishery germplasm resources.

[0003] Most fish in nature are diploid. The mature eggs of diploid fish are in the second meiotic metaphase. There are two sets of chromosomes in the eggs. After fertilization, after the sperm (one set of chromosomes) enters the eggs, it stimulates one set of chromosomes in the eggs to be discharged from the eggs as the second polar body, completing the second meiotic division. At this time, the one set of chromosomes in the fertilized egg and the one set of chromosomes brought in by the sperm combine to develop into a normal diploid. If the sperm enters the eggs and the second polar body has not yet been discharged, the fertilized eggs are treated with high temperature, low temperature, high pressure or chemical drugs to prevent the second meiotic division from proceeding, the second polar body just cannot be discharged from the eggs, and then an individual with three sets of chromosomes is induced, which is called artificial induced triploid. At present, there are many reports at home and abroad on the production of triploid fish using methods such as temperature shock, hydrostatic pressure, and chemical treatment. But no matter which method is used for induction, the starting time and duration of the treatment must be strictly controlled. For example, the pressure-sensitive period of crystal crucian carp fertilized eggs is only 4-5 minutes after fertilization. Before this, it is the egg initiation period. Applying pressure will destroy the excitement and trimming process of the egg, resulting in developmental retardation; followed by the pressure refractory period, when the second meiosis tends to be clear, and pressure has no effect on retaining the second polar body. Therefore, in order to obtain triploid crystal crucian carp, hydrostatic pressure induction can only be performed 4-5 minutes after fertilization. For catfish, 5 minutes after the eggs are fertilized at 25±1℃, they are placed in 5℃ seawater for cold shock treatment for 1 hour, and then placed in aerated seawater for cultivation. 100% triploid fry can be obtained, but if the cold shock treatment time is more than 1.5 hours, it will cause 100% mortality. Similarly, hydrostatic pressure induction also requires strict control of the treatment time.

[0004] Triploid fish have great commercial value and development potential, which can not only improve economic benefits but also avoid interference with natural resources. So far, there has been no report on the production method of triploid Channa georgii. Due to species differences, when the technical parameters for the induction of triploid marine fish and most freshwater fish reported are used for the induction of triploid Channa georgii, the induction rate of triploid Channa georgii is not high, which is difficult to meet the needs of industrial production. Therefore, it is necessary to establish a production method suitable for triploid Channa georgii. Summary of the invention

[0005] In order to solve the problems that gonad development affects the growth and meat quality of Channa grunteri and reduces the commercial quality of Channa grunteri, the present invention combines hydrostatic pressure treatment with temperature shock and colchicine aqueous solution combined induction treatment, determines the appropriate starting time for induction of triploid Channa grunteri, the optimal hydrostatic pressure and treatment time, the optimal temperature and the optimal chemical conditions according to the triploidization ratio of triploid Channa grunteri, and establishes a cultivation method for producing triploid Channa grunteri by using hydrostatic pressure combined with temperature shock and chemical combined induction.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0007] The object of the present invention is to provide a method for artificially inducing triploid Channa gordonii, the method comprising the following steps:

[0008] (1) Artificial insemination of Channa ge'sii: collecting mature eggs and semen of Channa ge'sii, placing the eggs in a dry container, slowly adding semen, slowly operating to mix the sperm and eggs, then adding breeding water, slowly shaking to complete the sperm and egg fertilization process, and obtaining fertilized eggs;

[0009] (2) Induction of triploid Channa ge’sii: The fertilized eggs obtained in step (1) are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, and the fertilized eggs after hydrostatic pressure treatment are subjected to a combined induction treatment of temperature shock and colchicine aqueous solution, and the fertilized eggs are taken out and transferred to aquaculture water for hatching.

[0010] In one embodiment of the present invention, the volume ratio of semen to egg in step (1) is 1 mL:250 mL.

[0011] In one embodiment of the present invention, the temperature of the drying container in step (1) is 25°C.

[0012] In one embodiment of the present invention, the temperature of the aquaculture water in step (1) is 16-18°C, and the amount added is 10 times the volume of the eggs.

[0013] In one embodiment of the present invention, the hydrostatic pressure treatment in step (2) is performed 4 minutes after insemination.

[0014] In one embodiment of the present invention, the pressure of the hydrostatic pressure treatment in step (2) is 5500 PSI.

[0015] In one embodiment of the present invention, the time of the hydrostatic pressure treatment in step (2) is 5 minutes.

[0016] In one embodiment of the present invention, the combined induction treatment of temperature shock and colchicine aqueous solution in step (2) refers to immersing the fertilized eggs in a colchicine aqueous solution with a temperature of 5°C and a concentration of 400 μg / mL.

[0017] In one embodiment of the present invention, it is characterized in that the time of the combined induction treatment of temperature shock and colchicine aqueous solution in step (2) is 30 minutes.

[0018] In one embodiment of the present invention, the incubation in step (2) is carried out in aquaculture water, and the temperature of the aquaculture water is kept constant at 16-18°C.

[0019] Beneficial effects of the present invention:

[0020] The present invention designs typical induction conditions for inducing triploid Channa georgia, including hydrostatic pressure, temperature, chemical substances, etc., and combines the triploid ratio of Channa georgia to determine the appropriate starting time for inducing triploid Channa georgia, the optimal hydrostatic pressure and treatment time, the optimal temperature and the optimal chemical conditions, and establishes a method for producing triploid Channa georgia fish by using hydrostatic pressure combined with temperature shock and chemical combined induction. The triploid Channa georgia induction method established by the present invention has the advantages of clear operation flow and strong practicality, and the method can efficiently induce triploid Channa georgia, and the induction efficiency of the triploid Channa georgia obtained by the method can be as high as 85%. Therefore, the induction method provided by the present invention is suitable for artificial and efficient production of triploid Channa georgia. In addition, the triploid Channa georgia cultivation method provided by the present invention opens up a new technical approach for polyploid breeding of Channa georgia.

[0021] At the same time, the present invention establishes a complete identification method for triploid Channa georgia, including a red blood cell volume identification method, a DNA content identification method, and a chromosome counting analysis method, and determines the range of the red blood cell volume and red blood cell nucleus volume of the triploid Channa georgia under the same breeding conditions; determines the ratio of the relative content of DNA of the diploid Channa georgia and the triploid Channa georgia, determines the absolute content of DNA of the triploid Channa georgia; and determines the chromosome number of the triploid Channa georgia. The identification method for triploid Channa georgia established by the present invention can quickly identify the triploid Channa georgia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of the relative content test of diploid Channa ge’s DNA;

[0023] Figure 2 This is the result of the relative content detection of triploid Channa ge’s DNA;

[0024] Figure 3 This is the result of the relative content test of chicken DNA;

[0025] Figure 4 This is the observation diagram of the metaphase division of the chromosomes of diploid Channa grunteri;

[0026] Figure 5 This is the observation diagram of the metaphase division of the chromosomes of the triploid Channa gei;

[0027] Figure 6 This is the karyotype composition diagram of the diploid Channa grunteri;

[0028] Figure 7 This is the chromosome karyotype composition diagram of the triploid Channa grunter. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0030] The present invention compares the proportions of triploid Channa georgii obtained under different induction conditions, and optimizes the induction method suitable for triploid Channa georgii. Among all the induction methods, the method of artificial insemination of Channa georgii is as follows:

[0031] Selectively mature Ge's perch, collect mature eggs and sperm, place the eggs in a dry container at 25°C, slowly add semen, the fertilization volume ratio of semen to eggs is 1mL:250mL, slowly operate to mix the sperm and eggs, then add 10 times the volume of the eggs and the temperature of 16-18°C breeding water, slowly shake to complete the sperm and egg fertilization process.

[0032] 1. Determination of the induction method of triploid Channa grunteri

[0033] (1) Determination of the start time of triplicate

[0034] Comparative Example 1: 3 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0035] Comparative Example 2: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0036] Comparative Example 3: 5 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0037] Comparative Example 4: 6 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0038] The triploid ratios obtained in the above comparison examples were statistically analyzed. The triploid ratio of comparison example 1 was 58%, the triploid ratio of comparison example 2 was 65%, the triploid ratio of comparison example 3 was 55%, and the triploid ratio of comparison example 4 was 52%. Among them, the triploid ratio of comparison example 2 was the highest. Therefore, the starting time of inducing triploidization was determined to be 4 minutes after fertilization.

[0039] (2) Determination of hydrostatic pressure treatment time

[0040] Comparative Example 5: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 3 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0041] Comparative Example 6: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 4 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0042] Comparative Example 7: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0043] Comparative Example 2: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0044] Comparative Example 8: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 7 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0045] Comparative Example 9: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 8 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0046] The triploid ratios obtained from the above comparative examples were statistically analyzed, and it was found that the triploid ratio of comparative example 5 was 51%, the triploid ratio of comparative example 6 was 63%, the triploid ratio of comparative example 7 was 69%, the triploid ratio of comparative example 8 was 58%, and the triploid ratio of comparative example 9 was 56%. Among them, the triploid ratio of comparative example 7 was the highest, so the time for hydrostatic pressure treatment was determined to be 5 minutes.

[0047] (3) Determination of pressure during hydrostatic pressure treatment

[0048] Comparative Example 10: 4 minutes after fertilization, the hydrostatic pressure was 5000 PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0049] Comparative Example 11: 4 minutes after fertilization, the hydrostatic pressure was 5500 PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0050] Comparative Example 7: 4 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0051] Comparative Example 12: 4 minutes after fertilization, the hydrostatic pressure was 6500PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0052] Comparative Example 13: 4 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0053] The triploid ratios obtained in the above comparative examples were statistically analyzed, and it was found that the comparative example 11 had the highest triploid ratio of 76%. Therefore, the induction conditions were determined as follows: 4 minutes after sperm and egg fertilization, treatment at a hydrostatic pressure of 5500PSI for 5 minutes, and then transfer to aquaculture water to complete hatching.

[0054] (4) Whether to perform temperature shock and chemical combination induction treatment and the duration of combined induction treatment

[0055] Comparative Example 11: 4 minutes after fertilization, the hydrostatic pressure was 5500 PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0056] Comparative Example 14: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 300 μg / mL for 20 minutes, and then transferred to breeding water to complete hatching.

[0057] Comparative Example 15: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI for 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 300 μg / mL for 30 minutes and then transferred to breeding water to complete hatching.

[0058] Comparative Example 16: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI for 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 300 μg / mL for 40 minutes and then transferred to breeding water to complete hatching.

[0059] The triploid ratios obtained in the above comparative examples were statistically analyzed, and it was found that the highest triploid ratio among the comparative examples was 79% in comparative example 15. Therefore, it was determined that the temperature shock and chemical combination induction treatment was performed after the hydrostatic pressure treatment, and the treatment time of the temperature shock and chemical combination induction was determined to be 30 minutes.

[0060] (5) Determination of the temperature for temperature shock and chemical combination induction treatment

[0061] Comparative Example 17: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 300 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0062] Comparative Example 15: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI for 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 300 μg / mL for 30 minutes and then transferred to breeding water to complete hatching.

[0063] Comparative Example 18: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 20°C and a concentration of 300 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0064] The triploid ratios obtained in the above comparative examples were statistically analyzed, and it was found that the highest triploid ratio among the comparative examples was comparative example 17, which had a triploid ratio of 82%. Therefore, the treatment temperature for the combined induction of temperature shock and chemical substances was determined to be 5°C.

[0065] (6) Determination of drug concentration for temperature shock and chemical combination induction treatment

[0066] Comparative Example 19: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 0 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0067] Comparative Example 20: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 100 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0068] Comparative Example 21: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 200 μg / mL for 30 minutes, and then transferred to breeding water to complete the hatching.

[0069] Comparative Example 17: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 300 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0070] Comparative Example 22: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI, and the action time was 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 400 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0071] Comparative Example 23: 4 minutes after fertilization, the hydrostatic pressure was 5500PSI for 5 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 5°C and a concentration of 500 μg / mL for 30 minutes and then transferred to breeding water to complete hatching.

[0072] The triploid ratios obtained from the above comparative examples were statistically analyzed, as shown in Table 1. Among the comparative examples, the comparative example 22 had the highest triploid ratio, which was 85%.

[0073] Table 1 Triploid ratios obtained in each comparative example

[0074]

[0075] Based on the triploid ratios of the above comparisons, the optimal breeding method for producing triploid Channa ge’sii was finally determined:

[0076] (1) Artificial insemination of Channa ge’sii

[0077] Selectively mature Ge's perch, collect mature eggs and sperm, place the eggs in a dry container at 25°C, slowly add semen, the fertilization volume ratio of semen to eggs is 1mL:250mL, slowly operate to mix the sperm and eggs, then add 10 times the volume of the eggs and the temperature of 16-18°C breeding water, slowly shake to complete the sperm and egg fertilization process.

[0078] (2) Induction of triploid Channa grunniensis

[0079] 4 minutes after fertilization, the fertilized eggs were placed in a hydrostatic pressure chamber for hydrostatic pressure treatment at a pressure of 5500PSI for 5 minutes; after hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment, and the fertilized eggs were immersed in a colchicine aqueous solution with a concentration of 400μg / ml and a temperature of 5°C for 30 minutes; finally, the fertilized eggs were transferred to the breeding water, the temperature was kept constant at 16-18°C, and the hatching was completed. This method has the highest triplicate ratio, which is 85%.

[0080] Identification of triploid Channa grunniensis

[0081] Whether the individuals induced by triploidization are truly triploid still needs to be strictly identified. The present invention uses the following method to identify triploid Channa ge'sii:

[0082] (1) Identification of red blood cell volume

[0083] It is generally believed that cell size is related to chromosome and DNA content. In a single cell, because the number of triploid chromosomes is more than that of diploid, in order to maintain the constant nuclear-cytoplasmic ratio, the volume of triploid cells is generally larger than that of diploid cells. There are large differences in the volume of triploid and diploid red blood cells and the volume of red blood cell nuclei in different fish. Theoretically, the volume of red blood cell nuclei of diploid fish and triploid fish is 1:1.5. The present invention uses a blood collection needle to collect blood from diploid Ge's perch Channa sphenanthera and triploid Ge's perch Channa sphenanthera obtained by the induction method provided by the present invention, prepares blood smears, HE stains, and observes under an optical microscope. The length of the long and short diameters of 10 red blood cell nuclei is measured for each smear, and the volume of red blood cells and nuclei is calculated. The values ​​of red blood cells and red blood cell nuclei obtained from diploid Ge's perch Channa sphenanthera and triploid Ge's perch Channa sphenanthera are shown in Table 2.

[0084] As can be seen from Table 2, all the values ​​of triploid Channa ge's were significantly higher than those of diploid Channa ge's. The long diameter, short diameter, area and volume of erythrocytes of triploid Channa ge's were 1.62, 1.43, 1.64 and 1.52 times of those of diploid, respectively, and the long diameter, short diameter, area and volume of erythrocyte nucleus of triploid Channa ge's were 1.66, 1.56, 1.60 and 1.50 times of those of diploid, respectively. All the parameters of diploid Channa ge's and triploid Channa ge's were significantly different (P < 0.05).

[0085] The red blood cell volume of diploid Channa grunniensis ranges from 53.25±7.23 μm 3 The range of red blood cell nuclear volume is 36.28±3.11μm 3 The red blood cell volume of triploid Channa grunniensis ranged from 81.11±12.42μm 3 The range of red blood cell nuclear volume is 54.41±9.16μm 3 between.

[0086] Table 2 Erythrocyte and erythrocyte nucleus values ​​of diploid and triploid Channa grunniensis

[0087]

[0088]

[0089] (2) DNA content identification

[0090] Blood was collected from the tail vein of the diploid Channa grunteri and the triploid Channa grunteri obtained by the induction method of the present invention, and the blood was fully mixed with 0.75% saline in a ratio of 1:5. Lymphocytes were separated with lymphocyte separation fluid, stained with PI in the dark at 4°C for 30 minutes, and filtered with a PARTEC ploidy meter with 400 mesh before ploidy identification. 100 samples were tested, and chicken blood with known DNA content was used as a control. Flow cytometry was used to analyze the relative content of DNA in the diploid Channa grunteri, the triploid Channa grunteri obtained by the induction method of the present invention, and the chicken to determine the ploidy. The flow cytometry experimental results are shown in Figure 1 , Figure 2 and Figure 3 .

[0091] The experimental results showed that the relative DNA content of diploid Channa georgii was 132.25±19.54, and the relative DNA content of triploid Channa georgii was 206.23±14.27. The DNA content of triploid Channa georgii was about 1.56 times that of diploid, which was consistent with the results of red blood cell size determination (see Table 3). It is known that the DNA content of chicken red blood cells is 2.3pg / N -1, the absolute DNA content of diploid Channa ge's somatic cells is 3.19 pg.N -1 The absolute DNA content of triploid Channa ge’s somatic cells is 4.98 pg.N -1 .

[0092] Table 3 DNA content of diploid, triploid Channa grunniensis and chicken

[0093]

[0094] (3) Chromosome counting analysis

[0095] The metaphase of chromosomes was prepared by in vivo culture of kidney cells, stained with Giemsa, and observed under a microscope. The number of chromosomes in 100 metaphases of diploid and triploid Channa grenadiers was counted. The counting results showed that the number of chromosomes of diploid Channa grenadiers was 2n=44, n=22, and the karyotype formula was 2n=36sm+2st+6t. The number of chromosomes of triploid Channa grenadiers was 3n=66, n=22, and the karyotype formula was 3n=54sm+3st+9t. The metaphase of chromosomes of diploid and triploid Channa grenadiers is shown in Figure 1. Figure 4 , Figure 5 As shown in Figure 2, the karyotype composition of diploid and triploid Channa grunniensis chromosomes is as follows: Figure 6 , Figure 7 As shown in the above experimental results, it can be seen that the chromosome numbers of diploid Channa grunniensis and triploid Channa grunniensis are consistent with the ploidy relationship.

[0096] The above identification methods were combined to find that polyploidization increases the number of chromosomes, the DNA content also increases proportionally, and the cell nucleus volume also increases. There are significant differences in the red blood cells and red blood cell nuclear area and volume, DNA content, and chromosome number between the diploid and triploid Channa grunniensis, and the data differences are consistent with the ploidy relationship.

[0097] In this experiment, the identification of triploid Channa ge’s perch was mainly based on the determination of red blood cells and red blood cell nuclear volume, assisted by the identification methods of chromosome counting and DNA content detection. Red blood cell determination can be conveniently and quickly identified, and the experimental process is relatively simple, which is suitable for large-scale determination at the breeding site. Chromosome karyotype analysis can very intuitively observe the ploidy of cells, but the operation is relatively complicated and time-consuming. The flow cytometer method for identifying DNA content requires professional experimental instruments and is relatively limited. In summary, the red blood cell determination method can be used as the preferred method for large-scale identification of ploidy, and the accuracy of identification can be improved by identification methods such as chromosome counting and DNA content detection, so that large quantities of Channa ge’s perch can be quickly and effectively identified.

[0098] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for artificially inducing triploid Channa gordonii, characterized in that: The following steps are involved: (1) Artificial insemination of Channa ge'sii: collecting mature eggs and semen of Channa ge'sii, placing the eggs in a dry container, slowly adding semen, slowly operating to mix the sperm and eggs, then adding breeding water, slowly shaking to complete the sperm and egg fertilization process, and obtaining fertilized eggs; (2) Induction of triploid Channa ge’sii: The fertilized eggs obtained in step (1) are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, and the fertilized eggs after hydrostatic pressure treatment are subjected to a combined induction treatment of temperature shock and colchicine aqueous solution, and the fertilized eggs are taken out and transferred to aquaculture water for hatching.

2. The cultivation method according to claim 1, characterized in that The volume ratio of semen to egg in step (1) is 1 mL:250 mL.

3. The cultivation method according to claim 1, characterized in that The temperature of the drying container in step (1) is 25°C.

4. The cultivation method according to claim 1, characterized in that The temperature of the aquaculture water in step (1) is 16-18° C., and the amount added is 10 times the volume of the eggs.

5. The cultivation method according to claim 1, characterized in that: The hydrostatic pressure treatment in step (2) is performed 4 minutes after insemination.

6. The cultivation method according to claim 1, characterized in that: The pressure of the hydrostatic pressure treatment in step (2) is 5500 PSI.

7. The cultivation method according to claim 1, characterized in that: The time of the hydrostatic pressure treatment in step (2) is 5 minutes.

8. The cultivation method according to claim 1, characterized in that: The combined induction treatment of temperature shock and colchicine aqueous solution in step (2) refers to immersing the fertilized eggs in a colchicine aqueous solution with a temperature of 5° C. and a concentration of 400 μg / mL.

9. The cultivation method according to claim 1, characterized in that: The time for the combined induction treatment of temperature shock and colchicine aqueous solution in step (2) is 30 minutes.

10. The cultivation method according to claim 1, characterized in that: In step (2), the incubation is carried out in aquaculture water, and the temperature of the aquaculture water is kept constant at 16-18°C.

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