Method for improving embryogenesis rate of free microspores of non-heading Chinese cabbage by using 5-AzaC

Abstract

The invention relates to the technical field of plant tissue culture, and provides a method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage by using 5-AzaC, and in the microspore culture stage, 5-AzaC with the concentration of 0.005-0.020 mg is added into a microspore culture medium. The invention designs a method for increasing the embryogenesis rate of free microspores of the non-heading Chinese cabbage by adding 5-AzaC into a microspore culture medium of the non-heading Chinese cabbage. According to the method, the embryogenesis rate of the free microspores of the non-heading Chinese cabbage can be remarkably improved, a non-heading Chinese cabbage microspore culture system is more easily established, the haploid breeding efficiency is improved, and the breeding process is accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage by utilizing 5-AzaC. Background Art

[0002] Chinese cabbage (Brassica chinensis L.), also known as small greens, greens, and rapeseed, belongs to the genus Brassica of the cruciferous family. It is a biennial vegetable with rich nutrition and high economic value. It is mainly eaten with leaves. At present, non-heading cabbage is grown in most parts of the country, among which there are many planting areas in the south, especially in Anhui, Shanghai, Jiangsu and other places, with a large planting area.

[0003] Microspore culture technology is a plant cell culture technology that mainly uses the haploid principle of free microspores to promote the development of microspores into sporophytes under suitable conditions, induce embryoids, and then cultivate microspore pure lines and formulate excellent varieties. This technology can obtain a large number of materials with rich genotypes and genetic stability within 1 to 2 years. On the premise of improving selection efficiency and accuracy, it can quickly select pure lines and shorten the breeding cycle. It has been widely used in crop genetic breeding.

[0004] However, although microspore culture technology has been widely used in current crop breeding, some problems still exist, such as the lack or low efficiency of haploid embryo induction, and the ability to form haploid embryos is highly dependent on the variety and genotype.

[0005] At present, in the process of microspore culture of Brassica, the microspore culture system is mainly optimized by adding different additives to NLN medium. Among them, the addition of activated carbon, silver nitrate, glutathione, etc. is more common, but the effect on microspore culture of non-heading cabbage is not significant. Different plant hormones, such as IAA, 6-BA, BR and other exogenous hormones added to the culture medium also have different degrees of influence on microspore culture of Brassica, but it is difficult to find a system or method with significant effect to improve the embryo emergence rate of microspores of non-heading cabbage. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for improving the embryo emergence rate of free microspores of non-heading cabbage by using 5-AzaC, and the method can significantly improve the embryo emergence rate of free microspores of non-heading cabbage.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] The invention discloses a method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage by using 5-AzaC. In the microspore culture stage, 5-azacytidine (5-AzaC) with a concentration of 0.005 to 0.020 mg is added to the microspore culture medium.

[0009] As one of the preferred embodiments of the present invention, the method comprises the following specific steps:

[0010] S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture;

[0011] S2. After pre-cooling, select flower bud samples at the mononuclear marginal stage for disinfection; then, add B5 culture medium into a glass cup, and crush the flower buds with a glass rod to release microspores; then, filter the microspore solution in the glass cup into a new sterilized glass cup with a wire mesh sieve, and then filter it into a centrifuge tube with a cell mesh sieve for a second time, and centrifuge; finally, resuspend the above-mentioned centrifuged precipitate with B5 culture medium, and centrifuge it again. The final precipitate is the desired purified microspores;

[0012] S3, adding 0.005-0.020 mg of 5-AzaC to the NLN medium, and then using it to resuspend and dilute the purified microspores obtained in step S2; then, adding 0.06-0.1 g / L of activated carbon, and then dispensing into culture dishes;

[0013] S4. After heat shock treatment at 33°C for 24 hours, the culture of step S3 is transferred to dark culture at 25°C. After visible embryoid bodies appear, the culture is placed on a shaker for shaking culture at 25°C.

[0014] As one of the preferred methods of the present invention, in step S2, the specific disinfection method is: first use 70% alcohol to disinfect for 30s, then use 0.1% HgCl 2 Shake and disinfect for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time.

[0015] As one of the preferred embodiments of the present invention, in step S2, the wire mesh sieve is 300 mesh and the cell mesh sieve is 40 um.

[0016] As one of the preferred embodiments of the present invention, in step S2, the centrifugal speed of the two centrifugal treatments is 1200 r / min, and the centrifugal time is 3 min.

[0017] As one of the preferred embodiments of the present invention, in step S3, the density of the microspores to be purified is diluted to 1×10 5 ~5×10 5 When the concentration is 0.06-0.1 g / L activated carbon is added and dispensed into 60 mm culture dishes.

[0018] As one of the preferred embodiments of the present invention, in step S4, the rotation speed of the shaking culture is 50 r / min.

[0019] As one of the preferred embodiments of the present invention, when used for the cultivation of "Golden Brilliant F1 Variety Non-heading Cabbage", the specific added concentration of 5-AzaC in the NLN culture medium is 0.015 mg / L.

[0020] Design principle of the present invention:

[0021] Free microspore culture technology refers to the process of using the omnipotence of cells, combining modern cell culture technology, separating microspore cells by squeezing and grinding, changing the development process of their gametophytes through temperature, laser and other stimulations, transforming them into sporophytes, propagating them on a liquid culture medium that can meet the conditions for cell growth and development, and finally cultivating them into a complete plant. Microspore culture technology is mainly divided into two processes: one is free microspore culture. Two is the culture of microspore plants. At present, there are many factors that affect microspore culture, the most important of which is genotype. In addition, there are also various external factors, including hormones, temperature, culture medium, etc. Plant hormone gibberellin (GA3) controls many physiological processes, including cell differentiation, cell elongation, seed germination, flower organ formation, etc., especially the regulation of anther development. 6-BA has a high cytokinin activity and is mainly used to promote plant cell division. Both GA3 and 6-BA have been used to induce embryogenesis. 5-AzaC is a specific DNA methylation inhibitor that can induce cell autophagy and may affect the omnipotence of plant cells. Therefore, the present invention explores the effect of 5-AzaC on embryogenesis by comparing it with GA3 and 6-BA.

[0022] The advantages of the present invention over the prior art are as follows: the present invention designs a method for improving the embryo emergence rate of free microspores of non-heading cabbage by adding 5-AzaC to a spore culture medium; the method can significantly improve the embryo emergence rate of free microspores of non-heading cabbage, make it easier to establish a microspore culture system for non-heading cabbage, improve the haploid breeding efficiency, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a state diagram of free microspore embryoids of non-heading Chinese cabbage after shaking culture in a shaking table in Example 1. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the plant materials and reagents used in the following embodiments are all conventional commercially available materials and reagents in the field unless otherwise specified; the experimental methods used are all conventional methods in the field unless otherwise specified, and will not be repeated.

[0025] Example 1

[0026] A method for improving the embryo generation rate of free microspores of non-heading Chinese cabbage by using 5-AzaC in this embodiment comprises the following specific steps:

[0027] S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture;

[0028] S2. After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2 Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0029] S3, add 0.015 mg / L 5-AzaC to NLN medium, and use it to resuspend the purified microspores obtained in step S3. When the microspore density reaches 1×10 5 ~5×10 5 When the concentration was 0.05 g / mL, 0.08 g / L of activated carbon was added and the mixture was dispensed into culture dishes.

[0030] S4. After heat shock treatment at 33°C for 24 hours, the culture of step S3 is transferred to dark culture at 25°C. After visible embryoids appear, the culture is placed on a shaker for shaking culture at 25°C with a shaking speed of 50 r / min. Figure 1 This is a picture of the state of free microspore embryoids of non-heading cabbage after shaking culture.

[0031] Example 2

[0032] A method for improving the embryo generation rate of free microspores of non-heading Chinese cabbage by using 5-AzaC in this embodiment comprises the following specific steps:

[0033] S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture;

[0034] S2. After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0035] S3, add 5-AzaC at a concentration of 0.005 mg / L to NLN medium, and then use it to resuspend the purified microspores obtained in step S3. When the microspore density reaches 1×10 5 ~5×10 5 When the concentration was 0.06 g / L, activated carbon was added and the mixture was dispensed into culture dishes.

[0036] S4. After heat shock treatment at 33°C for 24 hours, the culture of step S3 is transferred to dark culture at 25°C. After visible embryoids appear, the culture is placed on a shaker for shaking culture at 25°C with a shaking speed of 50 r / min.

[0037] Example 3

[0038] A method for improving the embryo generation rate of free microspores of non-heading Chinese cabbage by using 5-AzaC in this embodiment comprises the following specific steps:

[0039] S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture;

[0040] S2. After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2 Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0041] S3, add 5-AzaC at a concentration of 0.010 mg / L to NLN medium, and use it to resuspend the purified microspores obtained in step S3. When the microspore density reaches 1×10 5~5×10 5 When the concentration was 0.05 g / mL, 0.08 g / L of activated carbon was added and the mixture was dispensed into culture dishes.

[0042] S4. After heat shock treatment at 33°C for 24 hours, the culture of step S3 is transferred to dark culture at 25°C. After visible embryoids appear, the culture is placed on a shaker for shaking culture at 25°C with a shaking speed of 50 r / min.

[0043] Example 4

[0044] A method for improving the embryo generation rate of free microspores of non-heading Chinese cabbage by using 5-AzaC in this embodiment comprises the following specific steps:

[0045] S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture;

[0046] S2. After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2 Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0047] S3, add 5-AzaC at a concentration of 0.020 mg / L to NLN medium, and use it to resuspend the purified microspores obtained in step S3. When the microspore density reaches 1×10 5 ~5×10 5 When the concentration was 0.1 g / mL, 0.1 g / L activated carbon was added and the mixture was dispensed into culture dishes.

[0048] S4. After heat shock treatment at 33°C for 24 hours, the culture of step S3 is transferred to dark culture at 25°C. After visible embryoids appear, the culture is placed on a shaker for shaking culture at 25°C with a shaking speed of 50 r / min.

[0049] Comparative Example 1

[0050] A method for improving the embryo emergence rate of free microspores of non-heading cabbage by using 5-AzaC in this comparative example is compared with GA3 and 6-BA, and comprises the following specific steps:

[0051] S1. During the flowering period, the flower buds of non-heading cabbage plants were picked for subsequent microspore culture.

[0052] S2. Pre-cool the flower buds at 4°C for 24 hours.

[0053] S3. After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2 Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0054] S4, add 0.015 mg / L 5-AzaC or 0.2 mg / L 6-BA or 0.4 mg / L GA3 to NLN medium, and then use it to resuspend the purified microspores obtained in step S3. When the microspore density reaches 1×10 5 When the concentration of exogenous hormone was 0.0147 / mL, 0.08 g / L of activated carbon was added and the mixture was dispensed into 60 mm culture dishes. Each different exogenous hormone was dispensed into 20 dishes and the experiment was repeated 3 times.

[0055] S5. After the culture of step S3 is subjected to heat shock treatment at 33°C for 24 hours, it is transferred to dark culture at 25°C. After visible embryoid bodies appear, it is placed on a shaker for shaking culture at 25°C with a shaking speed of 50 r / min.

[0056] Experimental Example 1

[0057] This experiment is used to verify the effect of adding different concentrations of 5-AzaC to the free microspore NLN culture medium on the embryo emergence rate of free microspores of non-heading Chinese cabbage:

[0058] 1. Experimental methods:

[0059] Ten existing varieties of non-heading cabbage (W1, W2, W3, W4, W5, W6, xbc1, xbc2, xbc3, xbc4; see Table 1 for details) were used as experimental materials, with 50 copies of each variety numbered 1 to 50 respectively; and 500 copies of the materials were subjected to conventional field management until bolting and flowering.

[0060] Table 1 Detailed information of existing varieties of non-heading cabbage

[0061]

[0062]

[0063] During the flowering period, 10 varieties of flower buds with good growth and no diseases or insect pests were picked, marked, and placed in 4°C cold shock for 24 hours for the subsequent isolation and cultivation of microspores.

[0064] After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2 Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0065] Different concentrations of 5-AzaC were added to NLN medium and mixed thoroughly. The purified microspores were then resuspended and diluted. When the microspore density reached 1×10 5 ~5×10 5 When the concentration of 5-AzaC was 0.08 g / L, 0.08 g / L of activated carbon was added, and the mixture was then dispensed into 60 mm culture dishes (the concentration of purified microspores in each dish was counted using a cell counting plate); the microspore suspensions of each variety and different concentrations of 5-AzaC were dispensed into 5 culture dishes on average, and numbered A, B, C, D, and E according to the concentration of 5-AzaC added, and the treatment was as follows:

[0066] Treatment 1: Add sterile water to NLN medium A as a control;

[0067] Treatment 2: 0.005 mg / L 5-AzaC was added to NLN medium B;

[0068] Treatment 3: 0.010 mg / L 5-AzaC was added to NLN medium C;

[0069] Treatment 4: 0.015 mg / L 5-AzaC was added to NLN medium D;

[0070] Treatment 5: Add 0.020 mg / L 5-AzaC to NLN medium E.

[0071] Five groups of cultures were obtained, each of which was heat-shocked at 33°C for 24 hours and then transferred to dark culture at 25°C; after visible embryoids appeared, they were placed on a shaker at 25°C for shaking culture (speed 50 r / min). The entire culture process was carried out in the dark.

[0072] 2. Experimental Results

[0073] The results are shown in Table 2.

[0074] Table 2 Effect of adding different concentrations of 5-AzaC to NLN medium on microspore embryo rate (embryo / bud)

[0075]

[0076] As shown in Table 2, compared with the control group, for most non-heading cabbage varieties, adding 5-AzaC to NLN medium can increase the microspore embryo emergence rate, and the materials that cannot embryonate in conventional medium can embryonate after adding 5-AzaC; however, excessively high concentrations of 5-AzaC have an inhibitory effect on microspore embryo emergence, and the optimal concentration is 0.010-0.015 mg / L.

[0077] Experimental Example 2

[0078] This experimental example is used to verify the effect of adding 0.015 mg / L 5-AzaC or 0.2 mg / L 6-BA or 0.4 mg / L GA3 (Comparative Example 1) to the microspore culture medium on promoting the embryo emergence rate of free microspores of non-heading cabbage:

[0079] 1. Experimental methods:

[0080] 150 samples of three non-heading Chinese cabbages, namely W1, W5 and W2, which are difficult to produce embryos, were used as experimental materials (see Table 1 for details). They were numbered a (1-50), b (51-100) and c (101-150) respectively, and conventional field management was performed on the 150 samples until they bolted and bloomed.

[0081] Pick the flower buds that are free of pests and diseases and growing well, and place them in a cold shock at 4℃ for 24 hours.

[0082] After precooling, select the flower bud samples at the single-core edge stage, first disinfect them with 70% alcohol for 30 seconds, then disinfect them with 0.1% HgCl 2Shake and sterilize for 6 minutes, and finally rinse with sterile water 3 times, 5 minutes each time; then, add B5 culture medium into the glass, and crush the flower buds with a glass rod to release the microspores; then, filter the microspore solution in the glass into a new sterilized glass with a 300-mesh steel wire mesh sieve, and then filter it into a centrifuge tube with a 40um cell mesh sieve for a second time, and centrifuge it at 1200r / min for 3 minutes; finally, resuspend the precipitate after the above centrifugation with B5 culture medium, and centrifuge it again at 1200r / min for 3 minutes. The final precipitate obtained is the purified microspore required for the target.

[0083] Add 0.015 mg / L 5-AzaC or 0.2 mg / L 6-BA or 0.4 mg / L GA3 to NLN medium and mix thoroughly. Then resuspend the purified microspores obtained by dilution. When the microspore density reaches 1×10 5 ~5×10 5 When the concentration of purified microspores per dish is 10, add 0.08 g / L activated carbon and dispense into 60 mm culture dishes (use a cell counting plate to count the concentration of purified microspores per dish). 2 ); each variety and the microspore suspension with different exogenous hormones were evenly distributed into 10 culture dishes, and numbered A, B, C, and D according to the type of exogenous hormones added, and the treatment was as follows:

[0084] Treatment 1: No exogenous hormones were added to NLN medium A to serve as a control;

[0085] Treatment 2: 0.015 mg / L 5-AzaC was added to NLN medium B;

[0086] Treatment 3: 0.2 mg / L 6-BA was added to NLN medium C;

[0087] Treatment 4: Add 0.4 mg / L GA3 to NLN medium D.

[0088] Four groups of cultures were obtained, each of which was heat-shocked at 33°C for 24 hours and then transferred to dark culture at 25°C; after visible embryoids appeared, they were placed on a shaker at 25°C for shaking culture (speed 50 r / min). The entire culture process was carried out in the dark.

[0089] 2. Experimental Results

[0090] The results are shown in the table.

[0091] Table 3 Effects of adding different exogenous hormones to NLN on the embryo emergence rate (embryo / bud) of free microspores of non-heading Chinese cabbage W1

[0092]

[0093]

[0094] As shown in Table 3, the three different additives all have a promoting effect on the embryo emergence rate of free microspores of non-heading cabbage. Adding different types of exogenous hormones with suitable concentrations has different promoting effects on the embryo emergence rate of free microspores of non-heading cabbage. 5-AzaC with a concentration of 0.015 mg / L has the best promoting effect, especially for W1 and W5 materials that are extremely difficult to embryo.

[0095] In summary, the optimal concentration varies among different non-heading cabbage varieties. However, in general, "adding 5-AzaC during the culture medium cultivation process" is more conducive to the embryo formation of free microspores in non-heading cabbage, especially for the non-heading cabbage variety of Golden Splendid F1.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage using 5-AzaC, characterized in that: During the microspore culture stage, 5-AzaC was added to the microspore culture medium at a concentration of 0.005 to 0.020 mg.

2. The method for improving the free microspore embryo generation rate of non-heading Chinese cabbage using 5-AzaC according to claim 1, characterized in that: The specific steps include: S1. During the flowering period, the flower buds of non-heading cabbage plants with good growth and no pests and diseases were picked and pre-cooled at 4°C for 24 hours for subsequent microspore culture; S2. After pre-cooling, select flower bud samples at the mononuclear marginal stage for disinfection; then, add B5 culture medium into a glass cup, and crush the flower buds with a glass rod to release microspores; then, filter the microspore solution in the glass cup into a new sterilized glass cup with a wire mesh sieve, and then filter it into a centrifuge tube with a cell mesh sieve for a second time, and centrifuge; finally, resuspend the above-mentioned centrifuged precipitate with B5 culture medium, and centrifuge it again. The final precipitate is the desired purified microspores; S3, adding 0.005-0.020 mg of 5-AzaC to the NLN medium, and then using it to resuspend the purified microspores obtained in the diluted step S2; then, adding 0.06-0.1 g / L of activated carbon, and then dispensing into 60 mm culture dishes; S4. After the culture of step S3 is subjected to heat shock treatment at 33°C for 24 hours, it is transferred to dark culture at 25°C. After visible embryoid bodies appear, it is placed on a shaker for shaking culture at 25°C.

3. The method for improving the free microspore embryo generation rate of non-heading Chinese cabbage using 5-AzaC according to claim 2, characterized in that: In step S2, the specific disinfection method is: first disinfect with 70% alcohol for 30 seconds, then shake and disinfect with 0.1% HgCl2 for 6 minutes, and finally rinse with sterile water for 3 times, each time for 5 minutes.

4. The method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage using 5-AzaC according to claim 2, characterized in that: In step S2, the wire mesh sieve is 300 mesh and the cell mesh sieve is 40 um.

5. The method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage using 5-AzaC according to claim 2, characterized in that: In step S2, the centrifugal speed of the two centrifugal treatments is 1200 r / min, and the centrifugal time is 3 min.

6. The method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage using 5-AzaC according to claim 2, characterized in that: In step S3, the density of the microspores to be purified is diluted to 1×10 5 ~5×10 5 When the concentration is 0.06-0.1 g / L activated carbon is added and dispensed into 60 mm culture dishes.

7. The method for improving the free microspore embryo generation rate of non-heading Chinese cabbage using 5-AzaC according to claim 2, characterized in that: In step S4, the rotation speed of the shaking culture is 50 r / min.

8. The method for improving the embryo emergence rate of free microspores of non-heading Chinese cabbage using 5-AzaC according to any one of claims 2 to 7, characterized in that: When used for the cultivation of "Golden Brilliant F1 Variety Non-heading Cabbage", the specific added concentration of 5-AzaC in the NLN medium is 0.015 mg / L.

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