A method for preventing browning in the process of coconut microspore embryo culture

Abstract

The present application relates to an anti-browning method in the process of coconut microspore embryo culture, and belongs to the field of coconut tissue culture technology. The anti-browning method in the process of coconut microspore embryo culture of the present application comprises the following steps: S1, anther collection and pretreatment; S2, anther observation, disinfection and separation; S3, anther culture and embryo culture. The anti-browning method in the process of coconut microspore embryo culture provided in the present application adds PVP to the culture medium to counteract the browning phenomenon during the induction of coconut microspore embryos; and when cutting the explant, dip it in citric acid solution to reduce the browning caused by physical damage; ascorbic acid (vitamin C) is also added to the culture medium to adjust the pH and counteract the browning phenomenon; in addition, low concentration TDZ (thiadiazole) is used as an induction hormone to improve the efficiency of embryo induction.

Description

Technical Field

[0001] The present application relates to the technical field of coconut tissue culture, and in particular to an anti-browning method in the process of coconut microspore embryo culture. Background Art

[0002] Among the techniques for somatic embryogenesis in coconut, anther culture and microspore culture, anther culture is more commonly used due to its simplicity. In anther culture, the male reproductive organ of the flower, the anther, is used, and the male gamete precursor cells enclosed therein are induced to divide to form multicellular embryos, thereby generating seedlings. Compared with the use of female gamete precursors in ovules, the main advantage of using anthers is that the number of male gamete precursors in anthers is more abundant, and the amount of somatic tissue in anthers is smaller, which can reduce the formation of unnecessary somatic embryos. Chemical stress treatments have been shown to be an important component of male embryogenesis induction, which blocks gametophyte development and triggers embryogenesis in competent microspores. The composition of the culture medium, especially the presence of growth regulators, anti-browning agents and activated carbon, plays a key role in the success of coconut anther culture. Coconut is generally considered to be a species that is extremely difficult to culture in vitro, tissue culture or embryoid, and the success rate of producing haploid plants in anthers or in vitro microspores is extremely low. Currently, browning is the most serious problem affecting the somatic embryo induction process in this process. There is an urgent need to solve this problem and improve the induction rate of coconut microspore embryos. Summary of the invention

[0003] In view of this, the present application provides an anti-browning method in the process of coconut microspore embryo culture, which can resist the browning phenomenon, improve the induction rate of coconut microspore embryos, and effectively overcome the defects of the above-mentioned prior art.

[0004] The present application provides a method for anti-browning during the culture of coconut microspore embryos, comprising the following steps:

[0005] S1, anther collection and pretreatment: select the latest opened inflorescence on the crown of the coconut tree as inflorescence No. 1, and name inflorescence No. 2, No. 3, No. 4 and so on in the order of inflorescence opening. One week after inflorescence No. 1 opens, use a hand saw to completely collect inflorescence No. 4 from the root, and quickly spray citric acid solution, and then wrap the incision with a wet and clean paper towel to prevent rapid browning; use a clean scalpel to cut the inflorescence spathe, pick the spikelet in the middle of the main spike axis of the inflorescence, which contains 2-3 male flower anthers, pretreat the spikelet, wrap all the pretreated spikelets with paper towels sterilized in an autoclave, spray the wet paper towels with sterile water and wrap all the spikelets, and completely cover them with aluminum foil, put them into a polypropylene bag and tie the bag openings at both ends with rubber bands, and put the polypropylene bag in an incubator for pretreatment for one week;

[0006] S2, anther observation, disinfection and separation: after pretreatment, open the polypropylene bag, remove the male flower grains with browning, wilting and cracking of the outer skin, observe the anthers, and after mature spherical microspores are observed, take out healthy, fresh, milky white male flower grains with a length of 5-7 mm for disinfection. After disinfection, put them into a glass culture dish covered with filter paper and dry them, perform explant cutting, dip the wound of the cut anther into citric acid solution again, put them into a glass culture dish covered with filter paper and dry them for standby use;

[0007] S3. Anther culture and embryo culture: Insert 10-20 anthers with the back facing upward and the wound facing downward into a culture dish containing M1 culture medium for the first culture, leaving half of the anthers exposed for easy observation. After the somatic embryos break through the anther wall and expose the surface of the culture medium, transfer the somatic embryos to M2 culture medium for the second culture. After mature opaque buds grow, move them to a light environment for plant culture.

[0008] This application first uses polyvinyl pyrrolidone (PVP). PVP generally has no obvious toxicity in coconut tissue culture within the normal concentration range. In this application, it mainly plays an anti-browning role. It can absorb harmful substances such as phenols produced during plant tissue culture, reduce the toxicity of these substances to plant cells, and thus improve the survival rate of explants and the culture effect.

[0009] In coconut tissue culture, the browning phenomenon is mainly caused by polyphenol oxidase acting on natural substrate phenolic substances, oxidizing them into brown quinone substances. Citric acid can bind to the active site of polyphenol oxidase and change its conformation, thereby inhibiting the activity of the enzyme, reducing the chance of phenolic substances being oxidized, and reducing the degree of browning. At the same time, citric acid has a certain antioxidant capacity and can bind to the free radicals generated during the oxidation reaction to prevent the free radicals from damaging plant cells, thereby reducing the browning phenomenon. It can protect the biological macromolecules in plant cells, such as proteins and nucleic acids, from the influence of oxidative stress to a certain extent and maintain the normal physiological functions of cells.

[0010] In view of the fact that acidic substances such as hydrochloric acid are generally used to adjust the pH of plant culture media, this application uses ascorbic acid (vitamin C) as a pH regulator. In this application, ascorbic acid (vitamin C) works synergistically with adsorbents and other antioxidants. Ascorbic acid can remove reactive oxygen species in cells, reduce oxidative damage to explants, and indirectly reduce the occurrence of browning. Adsorbents such as activated carbon and PVP can adsorb browning substances in the culture medium, but may have limited effects on some browning reactions that have already occurred. When ascorbic acid is used in conjunction with other antioxidants and adsorbents, ascorbic acid can inhibit the occurrence of browning reactions in the early stages, while activated carbon can adsorb browning substances that have already been produced. The two work together to more effectively combat browning.

[0011] The phenylurea derivative TDZ (N-phenyl-N′-1,2,3-thiadiazole-5-urea, thidiazuron) of the present application can induce a series of different reactions of explants from callus formation to somatic embryogenesis, and has the special function of dual action of auxin and cytokinin. In addition, TDZ acts by regulating endogenous plant growth hormones, or indirectly by inducing adversity. It can also regulate cell membrane structure, energy level, nutrient absorption and assimilation.

[0012] Preferably, in step S3, the M1 medium contains: TDZ 0.5 mg / L, sucrose 120 g / L or 12% (w / v), ascorbic acid 100 mg / L, PVP 5g / L or 0.5% (w / v), agar 7g / L, potassium nitrate 1900mg / L, ammonium nitrate 1650mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium chloride 440mg / L, potassium iodide 0.83mg / L, boric acid 6.2mg / L, manganese sulfate 22.3mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, cobalt chloride 0.025mg / L, disodium edetate 37.3mg / L, ferrous sulfate 27.8mg / L, inositol 100mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L, its pH value is 5.75.

[0013] Preferably, in step S3, the M2 medium contains: 5 μM 6-benzyladenine, 0.5 μM gibberellin, 100 mg / L ascorbic acid, and PVP. 5g / L or 0.5% (w / v), agar 7g / L, potassium nitrate 1900mg / L, ammonium nitrate 1650mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium chloride 440mg / L, potassium iodide 0.83mg / L, boric acid 6.2mg / L, manganese sulfate 22.3mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, cobalt chloride 0.025mg / L, disodium edetate 37.3mg / L, ferrous sulfate 27.8mg / L, inositol 100mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L, its pH value is 5.75.

[0014] Preferably, in step S1, the specific process of pre-treating the spikelets is: rinsing the spikelets under running tap water and adding detergent to remove dust and impurities; then dividing the spikelets into top, middle and bottom parts, taking only 30% of the middle part, spraying it with 75% ethanol and then drying it.

[0015] Preferably, in step S1, the temperature of the incubator is set to 38°C and the humidity is set to 90%.

[0016] Preferably, in step S2, the specific process of the observation is: taking a small amount of anthers, pressing them with 4% acetic acid carmine under an optical microscope, and observing the development stage of microspores released by the anthers.

[0017] Preferably, in step S2, the specific process of the disinfection treatment is: clean the stains on the surface of the male flower grains with detergent without damaging the calyx, and then rinse with tap water for 5 minutes, then move to the sterilized clean bench, put every 10 male flower grains into 30 mL of 75% ethanol solution, shake and disinfect for 90 seconds, and place them in 21g / L or 2.1% (w / v) calcium hypochlorite solution for oscillation disinfection for 12 minutes, rinse three times with sterile deionized water, filter with a filter, put into a glass culture dish covered with filter paper and dry.

[0018] Preferably, in step S2, the specific process of the explant cutting treatment is: placing a 200 mg / L sterile citric acid solution on a glass culture dish with the liquid level 2 mm from the bottom, under a 60x microscope, using a disposable scalpel to carefully open and remove the three sepals of the male flower and open the buds, using fine-tipped tweezers to separate the anther from the filament, cutting the filament tissue at the root of the anther, and leaving as little wound as possible.

[0019] Preferably, in step S3, the specific conditions of the first culture are: sealing the culture dish with a sealing film and culturing at 27-28° C. in the dark for 8-10 months; or

[0020] In step S3, the specific conditions of the second culture are: culture at 27-28°C in the dark for 4 weeks, and subculture every 4 weeks.

[0021] Preferably, the specific conditions for the plant culture are: 16 hours of light and 8 hours of dark treatment per day (i.e., 16 / 8 photoperiod), 25 μmol m - ² s -1 The plants were cultured under light intensity of

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] The present application adds PVP to the culture medium to counteract the browning phenomenon during the induction of coconut microspore embryos; and dips the explants in a citric acid solution when cutting to reduce the browning caused by physical damage; ascorbic acid (vitamin C) is also added to the culture medium to adjust the pH and counteract the browning phenomenon; in addition, a low concentration of TDZ (thidiazuron) is used as an induction hormone to increase the induction rate of coconut microspore somatic embryos. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of this application clearer, the technical solution in this application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0025] Unless otherwise specified, the experimental methods used in the examples of this application are all conventional methods.

[0026] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0027] In the following examples and comparative examples, the specific calculation method of the browning rate is as follows:

[0028] Browning rate (%) = browning explants / total number of explants * 100%

[0029] It should be noted that in order to reduce the competition of explants for nutrition and facilitate counting, through experiments, the present application found that in general, 10 explants are placed in a culture dish with a diameter of 90 mm and a height of 15 cm, and the best culture effect can be obtained. At the same time, this method is also convenient for counting. For example, if 10 culture dishes (which can be considered as 10 repetitions) are used, and 10 explants are placed in each dish, a total of 100 explants are used in the experiment. How to identify browned explants is another difficulty. Generally, according to the document published by the inventor of the present application, Mu, Zhihua, et al. "The Influence of Maturity, Storage, and EmbryoSize on Coconut Callus Induction Success." Forests 15.5 (2024): 764., the browning judgment criteria therein are adopted. Under a 40x microscope, by comparing the colorimetric card, explants with a browning degree greater than 7 are considered to be browned explants.

[0030] All data discussed in this application are the browning rates during embryoid induction.

[0031] This application focuses on the process of induction from pollen microspores to embryoids, which is the first stage of culturing microspores taken from the inflorescence, that is, the somatic embryo induction process. After this stage, there are also somatic embryos to produce seedlings, tissue culture seedlings, seedling hardening processes, etc., which are not within the scope of this application.

[0032] The original data of this application comes from the preliminary experimental data of Hainan University, Hainan Seed Industry Laboratory, and Sri Lanka Coconut Research Institute. The experiment used three coconut varieties from China and Sri Lanka (Sri Lanka tall variety, yellow dwarf coconut and Hainan tall variety). Example 1

[0033] 1. Experimental Preparation

[0034] 1. Plant materials

[0035] Coconut trees with an age of 12 to 15 years are selected as donors of explants, regardless of species.

[0036] 2. Required equipment and experimental apparatus

[0037] Controlled environment incubator, 40x optical microscope, clean bench, pH meter, analytical balance (one thousandth accuracy), autoclave, disposable culture dish (120×20mm), measuring cylinder, beaker, conical flask, test tube, tweezers, scalpel with replaceable blade, hand saw, filter, pruning shears, autoclave tape, sealing film, sterile filter paper, aluminum foil, cotton plugs, rubber bands, sterile water, 30μm filter, transparent polypropylene bag, etc.

[0038] 3. Solutions and culture media

[0039] Explant fungicide: commercial calcium hypochlorite bleaching powder tablets (solid purity 95%, single tablet 1g), 75% ethanol (v / v).

[0040] Sterile distilled water: Autoclave at 121°C for 20 minutes.

[0041] Embryoid induction medium (M1): modified MS medium (Table 1), added with 0.5 mg / L TDZ and 12% (w / v) sucrose. After adding 100 mg / L ascorbic acid, adjust the pH to 5.7, add 0.5% (w / v) PVP and 7 g / L agar, sterilize at 121°C for 20 minutes, aseptically dispense into culture dishes under a clean bench, and seal after solidification.

[0042] Embryoid expansion medium (M2): modified MS medium (Table 1), added with 5 μM 6-benzyladenine (6-BA) and 0.5 μM gibberellin (GA3), added with 100 mg / L ascorbic acid, adjusted to pH 5.7, added with the same concentration of PVP and agar as in M1, sterilized at 121°C for 20 minutes, dispensed into culture dishes under a laminar flow clean bench, and sealed after solidification.

[0043] Citric acid working solution: 200 mg / L citric acid was dissolved in deionized water and sterilized by high pressure at 121°C for 20 minutes before use.

[0044]

[0045] 2. Anther collection and culture

[0046] 1. Anther collection and pretreatment

[0047] Generally, there are 3-5 inflorescences of different sizes on a coconut tree at the same time. The latest inflorescence on the crown of the coconut tree is selected as the reference inflorescence (inflorescence No. 1), and named 2, 3, 4 and so on from large to small. The larger the number, the smaller the inflorescence. One week after the reference inflorescence (inflorescence No. 1) opens, use a hand saw to completely collect inflorescence No. 4 from the root, quickly spray it with citric acid solution, and wrap the cut with a wet and clean paper towel to prevent rapid browning.

[0048] After returning to the laboratory, use a clean scalpel to cut the inflorescence spathe and pick the spikelet in the middle of the main spike axis of the inflorescence (containing 2-3 male flower anthers inside). Rinse the spikelet under running tap water and add detergent to remove dust and impurities. Divide the spikelet into the top, middle and bottom parts, and only take 30% of the middle part, spray it with 75% ethanol spray and dry it. Wrap the spikelet with a paper towel sterilized in an autoclave, spray the wet paper towel with sterile water, wrap all the spikelets, and cover them completely with aluminum foil, put them in a polypropylene bag and tie the bag openings at both ends with rubber bands. Pre-treat the polypropylene bag in an incubator for one week, set the temperature to 38°C, and set the humidity to 90%.

[0049] (II) Anther observation, disinfection and separation

[0050] After pretreatment, open the package and remove the male flower grains (buds are rice-shaped) with browned, wilted, and cracked outer skin. Take a small amount of it and press it with 4% acetic acid carmine under an optical microscope to observe the development stage of microspores released by anthers. Mature spherical microspores need to be observed. If it meets the requirements, take out healthy, fresh, milky white, and about 5-7mm long male flower grains, clean the surface stains with detergent (do not damage the calyx), and then rinse with tap water for 5 minutes. Then move it to the sterilized ultra-clean workbench, put every 10 male flowers in 30mL of 75% ethanol solution, shake and disinfect for 90 seconds, and put the anthers in 2.1% (w / v) calcium hypochlorite solution for 12 minutes. Oscillate and disinfect, rinse three times with sterile deionized water, filter with a filter, and put it in a glass culture dish covered with filter paper (all the utensils and consumables in this section are sterilized at high temperature and high pressure) and dry. When cutting explants, prepare a sterile citric acid solution containing 200 mg / L and place it in a glass culture dish, with the liquid level about 2 mm from the bottom. Under a 60x microscope, use a disposable scalpel to carefully open and remove the three calyxes (triangular cross-section) of the male flower and open the buds. Use fine-tipped tweezers to separate the anthers from the filaments, cut the filament tissue at the root of the anther, and leave as few wounds as possible. Dip the wound of the cut anthers in the citric acid solution again and place it in a glass culture dish covered with filter paper to dry for later use.

[0051] (III) Anther culture and embryo culture

[0052] Insert 10-20 anthers with the back facing up and the wound facing down into a culture dish containing 30 ml of M1 medium, leaving half of the anthers exposed for easy observation. Seal the culture dish with a sealing film and culture it in the dark at 27-28°C for 8-10 months (no subculture). When the embryoid (somatic embryo) breaks through the anther wall and exposes the surface of the culture medium, transfer the embryoid to 30 ml of M2 medium and culture it for 4 weeks, subculture it every 4 weeks. After mature opaque buds grow, move them to a light environment with a 16 / 8 photoperiod and 25 μmol m -2 s -1 The plant culture is further developed and cultivated. The development and cultivation method can refer to patent CN116250482A. The comprehensive browning rate of this embodiment is about 46%, and the embryoid production efficiency is that 120 embryoids can be produced in every 100 anthers for subsequent seedling formation. Comparative Example 1

[0053] Other experimental conditions can refer to Example 1, except that 0.5 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) was used in M1 medium instead of 0.5 mg / L LTDZ as an induction hormone, the browning rate reached 85.0%, and the somatic embryo generation rate was as low as 10.8%.

[0054] It should be noted that the counting method of the somatic embryo generation rate in Comparative Example 1 is described as follows:

[0055] As mentioned above, the best culture effect can be obtained by placing every 10 explants in a culture dish with a diameter of 90mm and a height of 15cm. After 4-8 weeks, check whether the microspore volume can be observed to be significantly enlarged and embryoids appear after the first subculture. In each culture dish, each explant with embryoids among the 10 explants placed is counted as 1, and no matter how many embryoids grow from a single explant, it is only counted as 1. The following is the calculation formula for the somatic embryo generation rate:

[0056] Somatic embryo generation rate (%) = number of explants that generated somatic embryos / total number of explants * 100%.

[0057] As mentioned above, every 100 anthers can produce 120 embryoids for subsequent seedling formation, which is the final efficiency obtained by integrating the loss rate of each subculture, contamination loss, etc. Comparative Example 2

[0058] Other experimental conditions can refer to Example 1, except that: both M1 and M2 culture media use 0.1% (w / v) activated carbon instead of 0.5% (w / v) PVP, and the browning rate reaches 83.9%. Comparative Example 3

[0059] Other experimental conditions can refer to Example 1, except that: when the explants were cut directly in the air without being dipped in citric acid solution, browning occurred near the wound after an average of 1.5 weeks, and the wound browning rate was 90.3% after three weeks.

[0060] It should be noted that the wound browning rate after three weeks in Comparative Example 3 refers to the browning phenomenon near the cut surface after cutting. At the end of the experiment, if browning is found near the cut surface of the explant, it is counted as browning of the explant, and the value obtained by dividing it by the total number of explants. This browning rate is specifically used to define the browning condition after the use of anti-browning operation (citric acid).

[0061] Browning phenomenon during embryo induction: the comprehensive browning rate of explants mentioned above.

[0062] Calculation formula: Browning rate (%) = browning explants / total number of explants * 100%. Comparative Example 4

[0063] Other experimental conditions can refer to Example 1, except that: 1 mMol / L hydrochloric acid was used in both M1 and M2 culture media instead of 100 mg / L ascorbic acid, and the browning rate reached 87.3%.

[0064] Test example

[0065] (1) Adding PVP to the culture medium to counteract the browning phenomenon during coconut microspore embryo induction

[0066] First, in the field of coconut tissue culture, there is no report of using PVP as an anti-browning agent. In coconut tissue culture, activated carbon is generally used as the main anti-browning agent. The anti-browning principle of activated carbon is physical adsorption, which adsorbs harmful substances into activated carbon particles. PVP is different. The carbonyl group therein has a strong ability to bind polyphenol compounds. Its binding ability is strengthened with the increase of the number of aromatic ring hydroxyl groups in the polyphenol compounds, so that the polyphenol compounds cannot become substrates of polyphenol oxidase, thereby inhibiting the occurrence of browning. The application found in the study that activated carbon, in the process of absorbing harmful substances, can adsorb and slowly release the nutrients and hormones in the culture medium, causing the concentrations of various nutrients in the culture medium to be extremely unstable and uncontrollable. PVP can avoid this problem, so PVP solves the problem of browning in the coconut tissue culture process and the problem of uncontrollable concentration of culture medium components in the process of using activated carbon.

[0067] (2) Dip the explants in citric acid solution when cutting to reduce browning caused by physical damage

[0068] At present, in the field of coconut pollen tissue culture, the main means of preventing browning is to change the composition of the culture medium, and there is still no means to prevent browning during the explant cutting process. The process of explant processing will cause wounds to the plants in the air, which is also the stage most likely to cause browning. This application uses the process of explant processing for the first time to fill the operating space with anti-browning agents (antioxidants), reducing the chance of browning.

[0069] (3) Add ascorbic acid (vitamin C) to the culture medium to adjust the pH and combat browning

[0070] In the current coconut tissue culture technology system, hydrochloric acid is mainly used as a pH auxiliary regulator, which cannot prevent oxidation and browning. However, vitamin C, as an antioxidant, can replace hydrochloric acid to adjust pH to a certain extent, and at the same time produce antioxidant and anti-browning effects.

[0071] (4) Use low concentration of TDZ (thiadiazole) as an induction hormone to improve embryo induction efficiency

[0072] Currently, 2,4-D is used as the main source of hormones in all coconut tissue culture studies. However, in the previous study, the applicant found that high concentrations of 2,4-D are too stimulating to explants, and due to the slow release effect of activated carbon, 2,4-D will bring continuous high-concentration stimulation to the explants, leading to necrosis, browning, etc. In the later experiments, the applicant found that TDZ, as an inducing hormone, used in combination with PVP, has excellent anti-browning and callus induction effects.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preventing browning in a microsporophyte embryo culture process of coconut, characterized in that: The following steps are involved: S1, anther collection and pretreatment: select the latest opened inflorescence on the crown of the coconut tree as inflorescence No. 1, and name inflorescence No. 2, No. 3, No. 4 and so on in the order of inflorescence opening. One week after inflorescence No. 1 opens, use a hand saw to completely collect inflorescence No. 4 from the root, and quickly spray citric acid solution, and then wrap the incision with a wet and clean paper towel to prevent rapid browning; use a clean scalpel to cut the inflorescence spathe, pick the spikelet in the middle of the main spike axis of the inflorescence, which contains 2-3 male flower anthers, pretreat the spikelet, wrap all the pretreated spikelets with paper towels sterilized in an autoclave, spray the wet paper towels with sterile water and wrap all the spikelets, and completely cover them with aluminum foil, put them into a polypropylene bag and tie the bag openings at both ends with rubber bands, and put the polypropylene bag in an incubator for pretreatment for one week; S2, anther observation, disinfection and separation: after pretreatment, open the polypropylene bag, remove the male flower grains with browning, wilting and cracking of the outer skin, observe the anthers, and after mature spherical microspores are observed, take out healthy, fresh, milky white male flower grains with a length of 5-7 mm for disinfection. After disinfection, put them into a glass culture dish covered with filter paper and dry them, perform explant cutting, dip the wound of the cut anther into citric acid solution again, put them into a glass culture dish covered with filter paper and dry them for standby use; S3, anther culture and embryo culture: 10-20 anthers are placed with their backs facing upwards and their wounds facing downwards into a culture dish containing M1 medium for the first culture, and half of the anthers are exposed for easy observation. After the somatic embryos break through the anther wall and are exposed on the surface of the medium, the somatic embryos are transferred to M2 medium for the second culture. After mature opaque buds grow out, they are moved to a light environment for plant culture. In step S3, the M1 medium contains: TDZ 0.5 mg / L, sucrose 120 g / L, ascorbic acid 100 mg / L, PVP5 g / L, agar 7g / L, potassium nitrate 1900mg / L, ammonium nitrate 1650mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium chloride 440mg / L, potassium iodide 0.83mg / L, boric acid 6.2mg / L, manganese sulfate 22.3mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, cobalt chloride 0.025mg / L, disodium ethylenediaminetetraacetic acid 37.3mg / L, ferrous sulfate 27.8mg / L, inositol 100mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L, its pH value is 5.75; In step S3, the M2 medium contains: 5 μM 6-benzyladenine, 0.5 μM gibberellin, 100 mg / L ascorbic acid, 5 g / L, agar 7g / L, potassium nitrate 1900mg / L, ammonium nitrate 1650mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium chloride 440mg / L, potassium iodide 0.83mg / L, boric acid 6.2mg / L, manganese sulfate 22.3mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, cobalt chloride 0.025mg / L, disodium ethylenediaminetetraacetic acid 37.3mg / L, ferrous sulfate 27.8mg / L, inositol 100mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L, its pH value is 5.75; In step S2, the specific process of the explant cutting treatment is: placing a 200 mg / L sterile citric acid solution on a glass culture dish with the liquid level 2 mm from the bottom, under a 60x microscope, using a disposable scalpel to carefully open and remove the three sepals of the male flower and open the buds, using fine-tipped tweezers to separate the anther from the filament, cutting the filament tissue at the base of the anther, and leaving as little wound as possible.

2. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that: In step S1, the specific process of pre-treating the spikelets is: rinse the spikelets under running tap water and add detergent to remove dust and impurities; then divide the spikelets into top, middle and bottom parts, take only 30% of the middle part, spray it with 75% ethanol spray and then dry it.

3. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that, In step S1, the temperature of the incubator is set to 38°C and the humidity is set to 90%.

4. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that, In step S2, the specific process of the observation is: taking a small amount of anthers, pressing them with 4% acetic acid carmine under an optical microscope, and observing the development stage of microspores released by the anthers.

5. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that, In step S2, the specific process of the disinfection treatment is: clean the stains on the surface of the male flower grains with detergent without damaging the calyx, and then rinse with tap water for 5 minutes, then move to the sterilized clean bench, put every 10 male flower grains into 30mL of 75% ethanol solution, shake and disinfect for 90 seconds, and place them in 21g / L calcium hypochlorite solution for oscillation disinfection for 12 minutes, rinse three times with sterile deionized water, filter with a filter, put into a glass culture dish covered with filter paper and dry.

6. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that: In step S3, the specific conditions of the first culture are: sealing the culture dish with a sealing film and culturing at 27-28° C. in the dark for 8-10 months; or In step S3, the specific conditions of the second culture are: culture at 27-28°C in the dark for 4 weeks, and subculture every 4 weeks.

7. The anti-browning method in the coconut microsporophyte embryo culture process according to claim 1, characterized in that: The specific conditions for the plant culture are: a photoperiod of 16 hours of light and 8 hours of dark treatment per day, 25 μmol m - ² s -1 Plants were cultured under light intensity of

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