Method for improving induction rate of free microspore derived embryos of autumn radishes

Abstract

The invention discloses a method for improving the inductivity of an MDE (Microspore Derived Embryo) of an autumn radish, which specifically comprises the following steps: (1) soaking radish seeds for 2 hours (25 DEG C), and transferring the radish seeds into a refrigerator at 4-6 DEG C to be treated for 21-25 days if the radish seeds are white; (2) selecting 2-3.5 mm flower buds, and spraying 1.5% mannitol one day ahead of time; (3) after the flower buds are disinfected and cleaned, extracting and purifying microspores by using a B5 liquid culture medium (pH 5.8) added with 1.5% mannitol; (4) suspending the purified microspores in an improved NLN-13 culture medium with the pH value of 5.8-6.0, performing heat shock treatment at the temperature of 32.5 DEG C, and performing standing dark culture for 18-25 days at the temperature of 25 DEG C; (5) after the MDE grows out, transferring the MDE to an MS culture medium and culturing for 4-8 days; transferring the hypocotyls to a strong bud culture medium after the hypocotyls extend, and then transferring to a rooting culture medium; and (6) acclimatizing and transplanting after the root system is developed. According to the method, the seasonal limitation of a radish free microspore culture technology can be broken through, the MDE induction rate in the autumn radish free microspore culture process is increased, the radish genotype is rapidly purified, and the radish variety breeding process is accelerated.

Description

Technical Field

[0001] This invention relates to a method for improving the rate of microspore embryogenesis in radish during autumn, belonging to the field of plant biotechnology. It is used to break the seasonal limitations of microspore culture and improve the induction rate of microspore-derived embryoid (MDE) in radish. Background Art

[0002] Radish (Raphanus sativus L.) is an annual or biennial herbaceous plant belonging to the genus Raphanus in the family Brassicaceae. It is an important root vegetable crop native to my country, with its enlarged fleshy taproot as the main product organ. The annual planting area reaches 18 million mu, and it occupies an important position in vegetable production and supply.

[0003] Radishes are cross-pollinated crops and exhibit a certain degree of self-incompatibility. Conventional breeding techniques for new radish varieties face challenges such as low genotype purification efficiency, difficulty in self-pollination leading to genetic degradation, and long breeding cycles, significantly hindering the radish variety selection process. Haploid breeding technology can significantly accelerate this process. Free microspore culture is one of the effective ways to produce haploids in crops and is an important method for plant haploid breeding.

[0004] Radish is one of the most difficult cruciferous vegetables to culture for free microspores (Chiancone et al., 2016:475-487). Currently, there are few reports on radish free microspore culture, and microspore-derived embryoid (MDE) development is highly seasonal, with difficulties in MDE development and growth, posing a significant challenge to radish microspore culture. This method utilizes radish seed vernalization technology and optimizes the autumn radish free microspore culture system by modifying the types and concentrations of exogenous substances added to the free microspore culture medium. This invention is of great significance for efficiently purifying radish germplasm genotypes and accelerating the haploid breeding process of radish. Summary of the Invention

[0005] This invention addresses the technical problems of seasonal limitations in radish free microspore culture and the low incidence of radish free microspore-derived embryos (MDEs), and provides a method to improve the induction rate of free microspore-derived embryos in autumn radishes to meet actual production needs.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method to improve the induction rate of free microspore-derived embryos in autumn radish, solving the problems of seasonal limitations in the culture of free microspores in radish and the low incidence of MDE in radish. The method includes the following steps: 1) In late July, select disease-free and plump seeds, soak them in water, and germinate them at 25°C in the dark for 12 hours.

[0008] 2) Once the seeds show white sprouts, immediately move them to a refrigerator at 4–6℃ and treat them at low temperature for 21–25 days;

[0009] 3) After vernalization, transfer to plug trays for cultivation (peat moss: vermiculite (V:V) 1:2), and transplant to seedling pots after the two-leaf-one-heart stage; bolting will occur 3-4 weeks after transplanting;

[0010] 4) Select flower buds that are 2.0-3.5 mm long and one week after flowering (peak flowering period) and spray them with 1.5% mannitol for 24 hours as a pretreatment. The size of the flower buds is greatly affected by temperature. When the minimum temperature in autumn is higher than 15℃, select flower buds that are 2.0-3.0 mm long. When the maximum temperature is lower than 15℃, select flower buds that are 2.5-3.5 mm long (the proportion of microspores in the uninucleate marginal stage is the highest).

[0011] 5) Disinfect flower buds with 75% alcohol for 1 min, disinfect with sodium hypochlorite with an effective chlorine concentration of 0.296 mol / L for 12 min, and rinse 3 times with sterile water; add to B5 liquid culture medium (with 1.5% mannitol added) at pH 5.8 for purification to obtain purified microspores;

[0012] 6) The purified microspores were suspended in NLN-13 medium (containing 1 mg / L AgNO3, 0.01 μM SAHA, 50 mg / L mp, and 1 g / LAC) at pH 5.8. After heat shock treatment at 32.5℃ for 24–36 h, the microspores were incubated in the dark at room temperature to obtain microspore-derived embryos (MDEs) that are visible to the naked eye.

[0013] 7) MDE static culture to obtain cotyledon-type embryos;

[0014] 8) Transfer the cotyledonous embryos into MS medium at pH 5.8 and incubate statically for 7 days;

[0015] 9) Transfer the plants to MS bud-strengthening medium (with 0.5 mg / L KT, 50 mL / L coconut juice, 3 g / L plant gel, and 60 g / L sucrose added);

[0016] 10) After 3-5 weeks of culture, most plants can be fully developed. Transfer the unrooted haploid seedlings into a rooting medium of pH 5.8 (with 0.5 mg / L IBA and 3 g / L plant gel added) to induce rooting.

[0017] 11) Hardening and transplanting of the obtained seedlings: After opening the tissue culture bottle, add sterile water to cover the roots (operate in a clean bench). After 3 days, take it out and soak the roots in sterile water to remove excess culture medium. Transplant into nutrient pots (culture medium: peat moss: vermiculite (V:V) 1:2) and build a light-transmitting and moisture-retaining cover. Spray water regularly (add 100mg / L cefotaxime). Remove the protective cover one week after transplanting. Attached Figure Description

[0018] Figure 1 Diagram of microspore-derived embryogenesis

[0019] Figure 2 A: The seeds are showing white sprouts; Figure 2 B: Two leaves and one heart stage; Figure 2 C: Bolting and flowering; Figure 2 D: Flower buds at the appropriate time; Figure 2 E: Microspore rupture; Figure 2 F: Normal microspore morphology; Figure 2 G: Microspore-derived embryogenesis; Figure 2 H: Germination of microspore-derived embryos; Figure 2 I: Germination cultivation; Figure 2 J: Inducing rooting; Figure 2 K: Seedling preparation; Figure 2 L: Transplanting

[0020] picture 3: Effect of activated carbon concentration on microspore-derived embryogenesis rate

[0021] Figure 4 Effect of silver nitrate concentration on microspore-derived embryogenesis rate

[0022] Figure 5 Effect of SAHA concentration on microspore-derived embryogenesis rate

[0023] Figure 6 Effect of ampicillin sodium concentration on microspore-derived embryogenesis rate

[0024] This invention addresses the technical problems of seasonal limitations in free microspore culture and the low incidence of microspore-derived embryos (MDE) in radishes by providing a method to improve the induction rate of free microspore-derived embryos in autumn radishes to meet actual production needs.

[0025] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0026] This invention provides a method to improve the induction rate of free microspore-derived embryos in autumn radish, solving the technical problems of seasonal limitations in free microspore culture and low MDE incidence in radish. The method includes the following steps: 1) In late July, select disease-free and plump seeds, soak them in water, and germinate them at 25°C in the dark for 12 hours.

[0027] 2) Once the seeds show white sprouts, immediately move them to a refrigerator at 4–6℃ and treat them at low temperature for 21–25 days;

[0028] 3) After vernalization, transfer to plug trays for cultivation (peat moss: vermiculite (V:V) 1:2). After the two-leaf-one-heart stage, transplant to nutrient pots. The plant will bolt 3-4 weeks after transplanting.

[0029] 4) Select flower buds that are 2.0-3.5 mm long and one week after flowering (peak flowering period) and spray them with 1.5% mannitol for 24 hours as a pretreatment. The size of the flower buds is greatly affected by temperature. When the minimum temperature in autumn is higher than 15℃, select flower buds that are 2.0-3.0 mm long. When the maximum temperature is lower than 15℃, select flower buds that are 2.5-3.5 mm long (the proportion of microspores in the uninucleate marginal stage is the highest).

[0030] 5) Disinfect flower buds with 75% alcohol for 1 min, disinfect with sodium hypochlorite with an effective chlorine concentration of 0.296 mol / L for 12 min, and rinse 3 times with sterile water; add to B5 liquid culture medium (with 1.5% mannitol added) at pH 5.8 for purification to obtain purified microspores;

[0031] 6) The purified microspores were suspended in NLN-13 medium (containing 1 mg / L AgNO3, 0.01 μM SAHA, 50 mg / L MP, and 1 g / L AC) at pH 5.8. After heat shock treatment at 32.5℃ for 24–36 h, they were incubated in the dark at room temperature to obtain MDE that was visible to the naked eye.

[0032] 7) MDE static culture to obtain cotyledon-type embryos;

[0033] 8) Transfer the cotyledon embryos to MS medium at pH 5.8 and incubate statically for 7 days;

[0034] 9) Transfer the plants to MS bud-strengthening medium (with 0.5 mg / L KT, 50 mL / L coconut juice, 3 g / L plant gel, and 60 g / L sucrose added);

[0035] 10) After 3-5 weeks of culture, most plants can be fully developed. Transfer the unrooted haploid seedlings into a rooting medium with pH 5.8 to induce rooting.

[0036] 11) Hardening and transplanting of the obtained seedlings: After opening the tissue culture bottle, add sterile water to cover the roots (operate in an ultra-clean workbench). After 3 days, take it out and soak the roots in sterile water to remove excess culture medium. Transplant into nutrient pots (culture medium: peat moss: vermiculite (V:V) 1:2) and build a light-transmitting and moisture-retaining cover. Spray water regularly (add 100mg / L cefotaxime). Remove the protective cover one week after transplanting. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are only used to explain the present invention, but do not limit the present invention.

[0038] I. Experimental Example

[0039] Example 1: A method for improving the induction rate of embryos derived from free microspores in autumn radishes, comprising the following steps:

[0040] 1) In mid-July, select plump, disease-free 'NAU-SW', 'NAU-LS', and 'NAU-RG' varieties.

[0041] Soak 'NAU-JPCXL' seeds in water at 25℃ for 12 hours. Once the seeds show signs of sprouting, immediately transfer them to a refrigerator at 4-6℃ for vernalization for 21-25 days. (During vernalization, observe the seeds twice a week, record their growth status, and spray water as needed to keep them moist.)

[0042] 2) In August, select seeds with good growth and transfer them to seed trays (substrate is peat moss: vermiculite (V / V) = 1:2). After the two-leaf and one-heart stage, transplant them to nutrient pots. They will bolt and flower in 3-4 weeks.

[0043] 3) Select flower buds one week after flowering (peak flowering period). Spray the flower buds with 1.5% mannitol before the experiment to establish tolerance, which can significantly improve the survival rate of microspores.

[0044] 4) Flower bud size is greatly affected by temperature. When the minimum temperature in autumn is above 15℃, select flower buds with a length of 2.0-3.0 mm. When the maximum temperature is below 15℃, select flower buds with a length of 2.5-3.5 mm (the proportion of microspores in the uninucleate marginal stage is the highest).

[0045] 5) Disinfect with 75% alcohol for 1 minute, disinfect with sodium hypochlorite with an effective chlorine concentration of 0.296 mol / L for 12 minutes, and rinse with sterile water 3 times;

[0046] 6) Microspore isolation and purification: Transfer the above flower buds to an IKA crusher, set to level 3, time 60s, crush and add a small amount of B5 liquid culture medium that has been sterilized at high temperature. Filter the above suspension containing microspores through a 400-mesh nylon screen to remove the residue, collect the suspension in a 15mL centrifuge tube, centrifuge at 1300rpm for 3min, discard the supernatant, add an appropriate amount of B5 liquid culture medium, shake to mix and centrifuge, repeat 3 times, discard the supernatant, and the obtained precipitate is the pure microspores;

[0047] 7) Culture of free microspores: The purified microspores were diluted with NLN-13 culture medium and dispensed into sterile culture dishes with a diameter of 35 mm at 3 mL per dish. The dishes were sealed with paraffin film and then heat-shocked in a constant temperature incubator at 32.5℃ for 24-36 h. After that, they were transferred to a culture room at 25℃ for static dark culture.

[0048] 8) After MDE is visible to the naked eye, the embryoids are statically cultured to obtain cotyledon-type embryos, and the incidence of MDE is counted.

[0049] 9) The cotyledonary embryos were transferred to MS medium at pH 5.8 without added hormones and cultured for 7 days until the hypocotyl elongated.

[0050] 10) After being transferred to a bud-strengthening medium at pH 5.8 and cultured for 2-3 weeks, most plants have fully developed.

[0051] 11) Transfer the unrooted plants to a rooting medium with a pH of 5.8 for further culture;

[0052] 12) Hardening off and transplanting: After opening the tissue culture bottle, add sterile water to cover the roots (operate in a clean bench). After 3 days, take it out and soak the roots in sterile water to remove excess culture medium. Transplant into nutrient pots (culture medium: peat moss: vermiculite (V:V) 1:2) and build a light-transmitting and moisture-retaining cover. Spray water regularly (add 100mg / L cefotaxime). Remove the protective cover one week after transplanting.

[0053] Experiment Example 2: Preparation of Experimental Materials and Pre-reagents

[0054] Radishes for which flower buds are to be harvested: In autumn, select seeds of 'NAU-SW', 'NAU-LS', 'NAU-RG', and 'NAU-JPCXL', treat them according to steps 1) and 3), and plant them in a plastic greenhouse. Field management is the same as usual.

[0055] Reagent preparation

[0056] Silver nitrate mother liquor: Weigh 0.5g of silver nitrate powder, add deionized water to make up to 50mL, filter, and store in a sterile 50mL centrifuge tube at 4℃ protected from light.

[0057] Deacetylase inhibitor (SAHA) stock solution: Weigh 0.1g, add DMSO to make up to 10mL, mix well, then take 1mL of the solution and dilute it 10 times with sterile water. Store at -20℃.

[0058] Ampicillin sodium (Amp): Weigh 5g of ampicillin sodium, add deionized water to a final volume of 50mL, filter, and store in a sterile 50mL centrifuge tube at 4℃.

[0059] Activated carbon (AC): Weigh 1g of activated carbon and 0.05g of agarose, and add deionized water to a final volume of 100mL.

[0060] KT stock solution: Weigh 0.05g of KT powder and add water to make up to 100mL.

[0061] IBA stock solution: Weigh 0.05g of IBA powder and add water to make up to 100mL.

[0062] Preparation of B5 medium: Weigh 3.21g of B5 powder, 15g of mannitol, and 130g of sucrose, add water to a final volume of 1000mL, adjust the pH to 5.8, and autoclave at high temperature.

[0063] Preparation of NLN-13 medium: Weigh 1.27g NLN powder, 0.05g calcium nitrate, 130g sucrose, 1mg / L AgNO3, 0.01μM SAHA, 50mg / L MP, and 1g / L AC, add water to make up to 1000mL, adjust pH to 5.8, and sterilize by filtration.

[0064] MS medium preparation: Weigh 4.74g MS powder, add 60g sucrose, 7g agar, and deionized water to a final volume of 1000mL. Adjust the pH to 5.8 and autoclave.

[0065] Preparation of bud-strengthening culture medium: 4.74 g / L MS powder, 60 g / L sucrose, 0.5 mg / L KT, 50 mL / L coconut juice, 3 g / L plant gel, adjust pH to 5.8, and autoclave at high temperature;

[0066] Preparation of rooting medium: 2.47 g / L 1 / 2 MS powder, 30 g / L sucrose, 0.25 mg / L IBA, deionized water to a final volume of 1000 mL, pH adjusted to 5.8, and autoclaved at high temperature;

[0067] B5 culture medium: purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: B8831-250g

[0068] MS medium: purchased from Nanjing Bochang Biotechnology Co., Ltd., catalog number: HB8469

[0069] SAHA: Purchased from Nanjing Youqing Biotechnology Co., Ltd., Product No.: SC0231-10mM

[0070] NLN culture medium: purchased from Nanjing Junyiyu Trading Co., Ltd., item number: HB8514

[0071] 'NAU-SW', 'NAU-LS', 'NAU-RG', and 'NAU-JPCXL' are radish cultivation resources preserved in this laboratory.

[0072] Experiment Example 3: Seed vernalization treatment breaks the seasonal limitations of radish free microspore culture

[0073] In late July, plump, disease-free seeds of 'NAU-SW', 'NAU-LS', 'NAU-RG', and 'NAU-JPCXL' were selected and soaked in water at 25℃ for 12 hours. Once the seeds showed signs of sprouting, they were immediately transferred to a refrigerator at 4-6℃ for vernalization for 21-25 days. Healthy seedlings were then transferred to plug trays (substrate: peat moss:vermiculite (V / V) = 1:2). After reaching the two-leaf-one-heart stage, they were transplanted into seedling pots. Flowering rates were recorded 3-4 weeks later (Table 1). The seed vernalization method proposed in this invention resulted in higher radish survival and flowering rates.

[0074] Table 1. Vernalization survival rate and flowering rate of different radish varieties

[0075]

[0076] Experiment Example 4: Mannitol treatment promotes the development of free microspore-derived embryos

[0077] Mannitol can increase the osmotic pressure of microspores, preventing them from rupturing and dying due to excessive osmotic pressure in the B5 liquid culture medium. Experiments were conducted using flower buds treated with 1.5% mannitol and untreated buds (CK), and the survival rate of microspores after heat shock treatment was observed (Table 2). It was found that the improved treatment method described in this invention can improve the survival rate of microspores.

[0078] Table 2. Survival rate of radish microspores under different pretreatments.

[0079]

[0080] Experiment Example 5: Effect of activated carbon treatment on the development of free microspore-derived embryos

[0081] Activated carbon can adsorb secondary metabolites produced during microspore development, increasing the rate of free microspore-derived embryos. Taking 'NAU-SW' as an example, 0, 0.33, 0.67, 1.00, and 1.33 g / LAC were added to the culture medium, and the number of derived embryos was counted. Figure 3 It was found that using the improved concentration described in this invention can increase the number of MDE occurrences.

[0082] Experimental Example 6: Effects of AgNO3 treatment on the development of free microspore-derived embryos

[0083] Adding AgNO3 to the culture medium can reduce the harmful effects of oxidation during the culture of free microspores. Taking 'NAU-SW' as an example, 0.0, 0.5, 1.0, 2.0, and 5.0 mg / L AgNO3 were added to the culture medium, and the number of derived embryos was counted. Figure 4 It was found that using the modified concentration described in this invention can increase the number of microspore-derived embryos.

[0084] Experimental Example 7: Effects of SAHA treatment on the development of free microspore-derived embryos

[0085] Adding SAHA to the culture medium can promote the transcriptional expression of genes related to derived embryogenesis. Taking 'NAU-SW' as an example, 0.00, 0.01, 0.03, 0.05, and 0.10 μM SAHA were added to the culture medium, and the number of derived embryos was counted. Figure 5 It was found that using the improved concentration described in this invention can increase the number of MDE occurrences.

[0086] Experiment Example 8: Effect of Amp Treatment on the Development of Microspore-Derived Embryogenesis

[0087] Adding the antibiotic Amp to the culture medium significantly reduced the contamination rate of free microspore cultures. Taking 'NAU-SW' as an example, 0, 50, 100, 200, and 300 mg / L Amp were added to the culture medium, and the number of derived embryos was counted. Figure 6 It was found that using the improved concentration described in this invention can increase the number of MDE occurrences.

[0088] Experimental Example 9: The effect of simultaneous application of multiple exogenous additives on the occurrence of MDE.

[0089] The microspore-derived embryogenesis (MDE) rate was tested by simultaneously adding the above additives to NLN-13 medium. It was found that the highest MDE rate (1.78 embryos / bud) was achieved when 1 g / LAC, 1.0 mg / L silver nitrate, 0.03 μM SAHA, and 50 mg / LAmp were applied simultaneously, significantly higher than when only a single exogenous additive was added.

[0090] Experimental Example 10: Effects of MS medium on the growth and development of free microspore-derived embryos

[0091] Not all derived embryos develop normally; albinism, browning, and vitrification may occur during embryo growth. Taking 'NAU-SW' as an example, derived embryos were cultured using 1 / 2 MS, MS, and B5 media, and the survival rate of derived embryos was statistically analyzed (Table 3). It was found that using the MS medium proposed in this invention can improve the survival rate of microspore-derived embryos during germination.

[0092] Table 3. Survival rate of radish microspore-derived embryos during germination in different culture media.

[0093]

[0094] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make corresponding modifications and adjustments within the scope of the claims. Therefore, the present invention is not limited to the experimental examples shown herein.

Claims

1. A method for improving the induction rate of embryos derived from free microspores in autumn radishes, characterized in that... Includes the following steps: 1) In the latter part of the month, select disease-free, plump seeds, soak them in water, and germinate them at 25℃ in the dark for 12 hours; 2) Once the seeds show white sprouts, immediately transfer them to a refrigerator at 4–6 ℃ and treat for 21–25 days; 3) After vernalization, the seeds are transplanted into plug trays (peat moss: vermiculite (V:V) = 1:2). When the seedlings grow to two leaves and one bud, they are transferred to seedling pots. The seedlings will bolt 3-4 weeks after transplanting. 4) One week after flowering, spray 1.5% mannitol on flower buds of different lengths one day in advance, depending on the temperature. When the minimum temperature is above 15℃, the proportion of microspores at the edge of the mononuclear stage of radish flower buds with a length of 2.0-3.0 mm is the highest. When the maximum temperature in autumn is below 15℃, select flower buds with a length of 2.5-3.5 mm. 5) First, disinfect the flower buds from step 4) with 75% alcohol for 1 min, then disinfect with sodium hypochlorite with an effective chlorine concentration of 0.296 mol / L for 12 min, and then wash them 3 times with sterile water; isolate and purify the microspores using B5 liquid medium (pH 5.8) with 1.5% mannitol added. 6) The purified microspores from step 5) were suspended in NLN-13 medium (containing 1 mg / L AgNO3, 0.01 μmol / L histone deacetylase inhibitor (SAHA), 50 mg / L ampicillin sodium (Amp), 1 g / L activated charcoal (AC), and 13% sucrose) at pH 5.

8. After heat shock treatment at 32.5℃ for 24–36 h, the microspores were cultured in the dark at 25℃ for 18–25 days to obtain microspore-derived embryoids (MDEs) visible to the naked eye. 7) Place the MDE from step 6) in a static environment at 25℃, light intensity of 4000 Lx, and photoperiod of 16 h / d to culture and obtain cotyledon embryos; 8) Transfer the cotyledonary embryos to MS medium (pH 5.8, with 60 g / L sucrose and 8 g / L agar) and incubate statically for 7 days; 9) Transfer the plants obtained in step 8) into a bud-strengthening medium at pH 5.8 (with 0.5 mg / L KT, 50 mL / L coconut juice, and 3 g / L plant gel added) for culture. 10) Transfer the seedlings that have not yet rooted in step 9) into a rooting medium at pH 5.8 (with 0.5 mg / L IBA and 3 g / L plant gel added) to induce rooting; 11) Transplant the hardened-off seedlings obtained in step 10): In a clean bench, open the tissue culture bottle and add sterile water to cover the roots. After 3 days, remove the bottle and soak the roots in sterile water to remove excess culture medium. Transfer the seedlings to nutrient pots (culture medium: peat moss: vermiculite (V:V) = 1:2), and build a light-transmitting and moisture-retaining cover. Spray water regularly (add 100 mg / L cefotaxime (Cef)). Remove the protective cover one week after transplanting.

2. The method for improving the induction rate of free microspore-derived embryos in autumn radish according to claim 1, characterized in that: Step 2) After the seeds show white sprouts, immediately transfer them to a refrigerator at 4-6℃ to prevent excessive elongation of the hypocotyl and generally improve the vernalization success rate.

3. The method for improving the induction rate of free microspore-derived embryos from autumn radishes according to claim 1, characterized in that: Steps 4) and 5) use B5 liquid medium supplemented with 1.5% mannitol to isolate and purify microspores.

4. The method for improving the induction rate of free microspore-derived embryos from autumn radishes according to claim 1, characterized in that: In step 6), 1 g / L AC, 1 mg / L AgNO3, 0.01 μmol / L SAHA, and 50 mg / L Amp are simultaneously administered in NLN-13 medium at pH 5.8.

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