An open - sugar - free culture method for Fengtang plums

Abstract

The present invention discloses a method for open sugar-free culture of Fengtang plums, using WPM + 0.5 mg / L 6-BA + 0.1 mg / L IAA; and a bacteriostatic agent as the culture medium. The culture medium is selected as vermiculite sterilized for 30 minutes. Add 3% NaClO4 that covers the bottom of the bottle to the sterilized culture container, shake it and then let it stand for sterilization, and set aside. Select semi-lignified Fengtang plum stem segments or Fengtang plum tissue culture seedlings. Disinfect by soaking in 2% NaClO4 for 6 minutes, rinse several times and then soak in 75% alcohol for 30 seconds. Tissue culture seedlings do not need to be disinfected. Finally, insert the materials into the container, perform dark treatment for 4 days and then light treatment for 8 days. When the cultured Fengtang plum seedlings grow to 7-8 cm, transplant them to the field. The sugar-free open culture adopted by the present invention is simple to operate in the rapid propagation process of Fengtang plums, greatly reduces the cost, the cultured seedlings are stronger, do not need hardening-off, can be directly transplanted and applied, and is simple and effective.

Description

Technical Field

[0001] The present invention relates to the technical field of asexual propagation of nursery stocks, and particularly relates to a method for open-type sugar-free culture of Fengtang plums. Background Art

[0002] Fengtang plum is a new bud mutation variety screened from local plum varieties in Guizhou. It is currently mainly planted in China. Due to its high sugar and low acid content, and its sweet taste similar to honey, it is popular in the market and in short supply. However, in actual production, it has also been found that many orchards planted with Fengtang plums have a high incidence of virus diseases, often resulting in the death of seedlings and the destruction of orchards, causing great losses; moreover, for Fengtang plums in the full fruit period, the branches of the tree body are prone to aging, and the tree vigor declines severely; in the propagation of nursery stocks, currently, Fengtang plums are mostly propagated by cutting and dividing, not only with a slow propagation speed, but also exacerbating the spread of virus diseases and the decline of tree vigor in the later stage; the above problems have become the main factors restricting the rapid and healthy development of Fengtang plum production.

[0003] Plant tissue culture technology can rapidly propagate a large number of uniform, highly genetically fidelity and virus-free plants by using the tissues and organs of research materials, and it has been widely applied in production. However, there are also problems in using traditional tissue culture methods, such as high requirements for operating equipment and technology, cumbersome operating procedures, serious pollution, high production costs, time-consuming and laborious transplanting and low survival rate, which limit the application of this technology in production. For this reason, researchers have developed two methods: open tissue culture and sugar-free tissue culture. Open tissue culture refers to preparing the culture medium in a completely open natural bacteria-containing environment without autoclaving, and the inoculation process is separated from the laminar flow bench, and finally achieving the same effect as traditional tissue culture. The technical principle of the sugar-free cultivation and rapid propagation seedling technology is mainly based on the premise that the culture medium does not contain processed sugar. By means of biological environment regulation, etc., the tissue culture seedlings are made to strengthen the rapid photosynthesis of their own cells, converting heterotrophic seedlings into highly efficient photosynthetic autotrophic types, so as to effectively reduce the production cost of nursery stocks and achieve the technical purpose of cultivating a high-quality seedling population with rapid and efficient growth. This seedling cultivation technology mainly emphasizes using a special cultivation container with a large enough volume, and the produced high-quality seedlings will be more neat and robust. If the open tissue culture technology and the sugar-free culture technology can be integrated, it will be able to more effectively reduce the production cost and improve the efficiency of nursery stock propagation to a greater extent, which is a major development trend in the future, but there is currently no report on this type of technology in plums (including Fengtang plums).

[0004] Therefore, taking Fengtang Plum as the material, according to the respective characteristics of open tissue culture and sugar-free tissue culture techniques, the culture method of sugar-free tissue culture is adopted, combined with the operation method of open tissue culture, to screen and study the culture medium, disinfection method of tissue culture utensils, antibacterial agent in the culture system, optimal medium formula, and culture conditions, and establish an open, efficient, and low-cost open sugar-free tissue culture system for Fengtang Plum, providing technical support for the industrial production of Fengtang Plum and also providing reference for the breeding of other woody plants. Summary of the Invention

[0005] In view of the above deficiencies, according to the characteristics of Fengtang Plum, the present invention conducts the following research to screen the sugar-free culture technology system for Fengtang Plum:

[0006] The present invention is achieved by the following technical means:

[0007] A culture medium for Fengtang Plum:

[0008] Establishment and optimization of the conventional tissue culture rapid propagation system for Fengtang Plum:

[0009] Taking Fengtang Plum as the object, study the explant sampling time, disinfection method, selection of basic medium, selection of the most suitable medium for adventitious bud induction, rooting technology, etc., establish the conventional tissue culture rapid propagation system for Fengtang Plum, and obtain virus-free tissue culture seedlings of Fengtang Plum.

[0010] Establishment and optimization of the open sugar-free tissue culture rapid propagation system for Fengtang Plum:

[0011] Taking the virus-free tissue culture seedlings of Fengtang Plum as the material, establish and optimize the open sugar-free tissue culture system for Fengtang Plum. The main contents include screening of the culture medium, screening of the disinfection method of tissue culture utensils, screening of antibacterial agents, screening of the best medium formula for rapid propagation, and screening of culture conditions.

[0012] 1. Research Methods

[0013] 1.1 Establishment and optimization of the conventional tissue culture rapid propagation system for Fengtang Plum

[0014] ① Establishment of aseptic culture: Using the semi-lignified stem segments of Fengtang Plum as explants, selecting mercuric chloride and alcohol as surface disinfectants, and screening out the best combination of disinfectants and treatment time.

[0015] ② Establishment of the conventional tissue culture and rapid propagation culture system: Using the aseptic explants obtained by treating with the best surface disinfectant screened in the experiment as materials, inoculate them onto the special culture medium composed of the above various basic plant culture medium formulas and various ratios of plant growth regulators, and screen out the most suitable types of basic culture media and the best combinations of plant growth regulators at each stage above. Screening and biotoxicity identification of the component combinations of the basic bacteriostatic agents in the open-type sugar-free culture. Select four bacteriostatic agents, namely NaClO4, potassium sorbate, streptomycin sulfate for agricultural use, and chlorothalonil, evaluate the biotoxicity of the bacteriostatic agents and the pollution situation after inoculating the materials, and screen the best compound combination.

[0016] 1.2 Establishment and optimization of the open-type sugar-free tissue culture and rapid propagation system for Fengtang plum

[0017] ① Screening of the culture medium: Using a sugar-free culture container, with vermiculite and agar as the matrix, screen out the best medium suitable for the sugar-free culture of Fengtang plum.

[0018] ② Screening of the disinfectant for tissue culture utensils: Select NaClO4 as the disinfectant for the culture container, and screen the best treatment and combination.

[0019] ③ Screening of the culture medium for the open-type sugar-free rapid propagation of Fengtang plum: Based on the culture media for each stage of Fengtang plum obtained by conventional tissue culture, combined with the culture medium and the disinfection method of tissue culture utensils screened above, screen the best basic culture medium, combination of plant growth regulators and culture conditions for the rapid propagation of Fengtang plum under open-type sugar-free culture conditions.

[0020] 2. Materials and methods

[0021] 2.1 Experimental materials

[0022] The experimental materials were 4-year-old grafted seedlings of Fengtang plum introduced from the experimental farm of the Department of Agriculture, Forestry and Food Engineering of Yibin University.

[0023] 2.2 Experimental methods

[0024] 2.2.1 Establishment of the primary system for the conventional tissue culture and rapid propagation of Fengtang plum

[0025] When the weather is clear, select the stem segments of Fengtang Plum with good growth, no pests and diseases, and semi-lignification. Remove 2 / 3 of the leaves, soak them in a solution with washing powder for 10 minutes, clean the attached dirt on the stem segments with a brush, then rinse with running water for 2 hours. After that, rinse with sterile water 3 times in sequence on the ultra-clean workbench, soak in 75% alcohol for 45 seconds, rinse with sterile water 3 times, soak in 0.1% mercuric chloride (for 7 minutes, 8 minutes, and 9 minutes respectively), rinse with sterile water 4 times, 30 seconds each time. After the disinfection, dry the moisture of the materials with sterile absorbent paper, remove the remaining leaves and petioles, cut the materials into single-bud stem segments, and inoculate them into the primary culture medium. The basic medium for primary culture is MS, 1 / 2MS, and WPM, supplemented with 0.5 mg / L 6-BA + 0.1 mg / L IAA, 20 g / L sucrose, and 6.5 g / L agar, with the pH adjusted to 5.8. After inoculation, the culture conditions are light flux density of 70 umol.m-2.s-1, temperature of 25°C ± 1°C, and relative humidity of 80%. For each treatment, inoculate 20 bottles, inoculate 1 stem segment in each bottle, repeat 3 times, and count the contamination rate, mortality rate, and germination rate after 15 days.

[0026] 2.2.2 Screening of the combination of basic bacteriostatic agents for open-type sugar-free culture and identification of biological toxicity

[0027] Select four bacteriostatic agents, namely NaClO4, potassium sorbate, streptomycin sulfate for agricultural use, and chlorothalonil. Through the compound combinations with different concentration gradients set pairwise (see Tables 3 - 6), add them into the MS medium (added with 20 g / L sucrose) respectively, boil and then pour into 350 ml tissue culture bottles. After cooling and solidifying, observe and evaluate indexes such as the performance of the medium and the contamination rate. At the same time, select seeds and plant stem segments with simple disinfection to evaluate the biological toxicity of the bacteriostatic agents and the contamination situation after inoculating the materials, and screen the best compound combination.

[0028] 2.2.3 Comparison of sterilization methods for the culture medium vermiculite in open-type sugar-free culture

[0029] After slightly crushing the purchased vermiculite, sieve it through a 2 mm sieve, put it into a special sterilization cloth bag, and adopt high-pressure moist heat sterilization, conventional steaming sterilization (steam for 30 minutes after boiling), and high-temperature dry heat sterilization (dry at 150°C for 1 hour) respectively. After cooling, add it to the sugar-free culture box, add the sugar-free MS medium, inoculate the disinfected test materials, and observe and count the contamination situation of the substrate.

[0030] 2.2.4 Screening of bacteriostatic agents and culture media for open-type sugar-free culture of Fengtang Plum

[0031] Rescreening of bacteriostatic agents and screening of culture media

[0032] Based on the best basal medium screened by the above conventional tissue culture, using Fengtang plum as the culture material, combined with the best bacteriostatic agent formula screened above, different amounts of nano-cinnamomum longepaniculatum essential oil and nano-artemisia argyi essential oil are added on this basis to evaluate the toxicity and bacteriostatic effect on the simply disinfected Fengtang plum stem segments under open-type sugar-free culture conditions. At the same time, agar and vermiculite are used as the culture media to verify the effect of different disinfectants on different media and the bacteriostatic effect, so as to screen out the optimal culture medium.

[0033] 2.2.5 Operation process of open-type sugar-free culture

[0034] Disinfection of culture bottles: Add 2% NaClO4 to the culture bottles to cover the bottom of the bottles. After covering the bottle caps and shaking a few times, let it stand for 20 minutes, and then pour out the NaClO4 for standby.

[0035] Prepare agar medium culture medium: Add NaClO4, chlorothalonil, mancozeb, nano-cinnamomum longepaniculatum essential oil and artemisia argyi essential oil into a beaker according to the formula and concentration. Then add the basal medium with agar that has been boiled, stir evenly, add hormones, adjust the pH to 5.8, and quickly dispense it into the above-mentioned disinfected culture bottles. Wait for it to cool and solidify for standby.

[0036] Divide it into four groups, namely:

[0037] Group A: Cinnamomum longepaniculatum essential oil agar group: 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-cinnamomum longepaniculatum essential oil;

[0038] Group B: Artemisia argyi essential oil agar group: 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-artemisia argyi essential oil.

[0039] Prepare vermiculite medium culture medium: The operation is the same as above, and the only difference is that agar is not added to the basal medium.

[0040] Group C: Cinnamomum longepaniculatum essential oil vermiculite group: 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-cinnamomum longepaniculatum essential oil;

[0041] Group D: Artemisia argyi essential oil vermiculite group: 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-artemisia argyi essential oil.

[0042] Disinfection treatment of test materials: Take fresh semi-lignified Fengtang plum branches on a sunny day. After cutting off 2 / 3 of the leaves, rinse them with running tap water for 30 minutes, then soak them in 2% NaClO4 for 6 minutes, rinse them three times with sterile water, then soak them in 75% alcohol for 30 seconds, and finally rinse them 4 times with sterile water.

[0043] Inoculation: Prepare an alcohol lamp, three pairs of forceps, scissors, and a cup of absolute ethanol. Soak the forceps and scissors in alcohol first. After taking them out, burn them on the alcohol lamp for simple disinfection, and set them aside after cooling. Cut the stem segments into small stem segments with two buds and insert them into the prepared culture medium, 3 in each bottle.

[0044] Cultivation conditions: After inoculation, place it in a dark place for 4 days of dark treatment, and then carry out light cultivation. The light intensity is 40 μmol·m -2 ·s -1 from 0 to 2 days, 120 μmol·m -2 ·s -1 from 3 to 6 days, and 200 μmol·m -2 ·s -1 from 6 to 8 days. The day / night time is 14 h / 10 h; while irradiating with light, introduce CO2, and the CO2 concentration (v / v) is 0.8 - 1.6×10 -3 , and it increases gradually; the cultivation temperature is 25 °C ± 1 °C, and the relative humidity is 80 ± 5%.

[0045] 3. Result Analysis

[0046] Establishment of the Primary Tissue Culture and Rapid Propagation System for Fengtang Plum

[0047] 3.1 Screening of Disinfection Methods for Fengtang Plum Explants

[0048] The treatment time of the disinfectant mercuric chloride has different effects on the disinfection effect of explants. As can be seen from Table 1, when the mercuric chloride concentration is 0.1%, the fungal contamination rate of the T3 sterilization treatment method is the highest, reaching 78.7%, but its bacterial contamination rate is the lowest at zero. Secondly, for the T2 sterilization treatment method, the fungal contamination rate decreases slightly, but bacterial contamination begins to appear. For the T1 sterilization treatment method, the fungal contamination rate is significantly lower than that of T3 and T2, but the bacterial contamination rate reaches 38.1%, which is significantly higher than that of T3 and T2 (as shown in Table 1); the browning rates of Fengtang plum explants under the three sterilization treatment methods are T1 > T2 > T3 (as shown in Table 1), and the survival rates of the plants are T2 > T3 > T1. Considering the comprehensive contamination rate, browning rate, and survival rate, the T2 sterilization treatment method is the best disinfection method for Fengtang plum explants, that is, the treatment time with 75% alcohol is 45 s, and the treatment with 0.1% mercuric chloride is 8 min.

[0049] Table 1 Comparison of Different Sterilization Methods

[0050]

[0051]

[0052] 3.2 Influence of Basic Culture Medium on the Germination and Growth of Fengtang Plum Explants

[0053] As can be seen from Table 2, after the combined disinfection treatment, the highest browning rate of the materials inoculated on the MS medium was 19.40%, followed by the WPM medium, and the lowest browning rate of the materials on the 1 / 2MS medium was 11.90%, with significant differences among the treatments; the basal medium also had a significant impact on the germination survival rate of the explants of Fengtang plums. Among them, the highest germination survival rate of the materials inoculated on the WPM medium reached 29.9%, which was significantly higher than that of the 1 / 2MS and MS medium treatments. Followed by the MS medium, the lowest germination survival rate of the inoculated materials on the 1 / 2MS medium was 9.00%. The basal medium also had a significant impact on the growth of the buds on the germinated explants of Fengtang plums. According to Table 2 and Table 1, from the bacterial and fungal contamination after disinfection, bud damage and browning, and the gradual germination of the explants and the growth status of the materials in different media, it can be seen that the buds of the Fengtang plum explants on the WPM medium germinated the fastest and had the best growth. Followed by the 1 / 2MS medium treatment, and the MS medium treatment had the worst effect. The buds germinated the slowest within the same time, and the growth rate and growth were also the weakest.

[0054] Table 2 Effects of basal media on browning, germination survival and growth of Fengtang plums

[0055] Basal medium Browning rate Germination survival rate Growth vigor MS 19.40％ 11.90％ + 1 / 2MS 11.90％ 9.00％ +++ WPM 13.40％ 29.90％ ++++++

[0056] 3.3 Screening of antibacterial effects of different compound combinations of basic bactericides

[0057] As can be seen from Tables 3 to 5, in the blank medium treatments of each combination, after 30 days of cultivation, the treatments of chlorothalonil + potassium sorbate all had serious contamination, while the compounding and single treatments of NaClO4 and mancozeb had a contamination rate of 0. Under the compounding treatment of NaClO4 and potassium sorbate, only 2 combinations were contaminated, and the antibacterial effects of the remaining combinations were all good; under the compounding treatment of NaClO4 and mancozeb, the contamination rate was 0; in the compounding treatment of NaClO4 and streptomycin sulfate, the single treatment of streptomycin sulfate was 100% contaminated. In the compounding treatment, only the contamination rates of 0.1% NaClO4 with 0.05 g / L and 0.1 g / L streptomycin sulfate were 20%, and the rest were 0.

[0058] Table 3 Antibacterial effects and biological toxicity of the compound treatment of potassium sorbate and chlorothalonil

[0059]

[0060]

[0061] Table 4 Antibacterial agent effects and biological toxicity of the compound treatment of potassium sorbate and NaClO4

[0062]

[0063] In the treatment of inoculated sorghum seeds, in the compound combination of chlorothalonil and potassium sorbate, when the concentration of chlorothalonil was fixed, with the increase of the addition amount of potassium sorbate, the germination rate of sorghum seeds gradually decreased, while the pollution rate gradually increased, indicating that potassium sorbate not only had a poor antibacterial effect, but also had certain biological toxicity. Therefore, the treatment of potassium sorbate could be excluded from the subsequent experiments. In the compound treatment of NaClO4 and mancozeb, under the treatment of mancozeb alone, with the increase of the addition concentration of mancozeb, the germination rate of sorghum seeds gradually increased, and the differences between treatments were significant. More importantly, the pollution rate of each treatment was 0, and the effect was good.

[0064] Under the treatment of 0.1% NaClO4, the germination rate of sorghum seeds was significantly lower than that of the treatment with mancozeb alone, but the pollution rate increased significantly. However, under the treatment of 0.1% NaClO4, the germination rate and pollution rate of the seeds were both 0. Although the antibacterial effect was good, the biological toxicity was the highest. After the compound treatment of 0.1% NaClO4 and mancozeb, with the increase of the treatment concentration of mancozeb, the germination rate of the seeds gradually decreased, but was extremely significantly lower than that of the 0.01% mancozeb treatment, while the pollution rate increased significantly, indicating that the compound treatment of NaClO4 and mancozeb produced toxicity. The treatment with better antibacterial effect was the 0.1% NaClO4 + 0.03% mancozeb treatment. After the compound treatment of 0.2% NaClO4 and mancozeb, greater toxicity was produced, and the germination rate of each treatment of seeds was 0. However, with the increase of the compound treatment concentration of mancozeb, the pollution rate increased significantly, indicating that the comprehensive antibacterial ability of the compound treatment of high-concentration NaClO4 and high-concentration mancozeb was worse.

[0065] Table 5 Antibacterial effect and biological toxicity of the compound treatment of NaClO4 and mancozeb

[0066]

[0067]

[0068] Table 6 Antibacterial effect and biological toxicity of the compound treatment of NaClO4 and streptomycin agricola

[0069]

[0070] As shown in Table 6, in the treatment of NaClO4 and agricultural streptomycin, under a single treatment, with the increase of the concentration of agricultural streptomycin added, the germination rate and contamination rate of sorghum seeds gradually increased; after the treatment of 0.1% NaClO4 and agricultural streptomycin, with the increase of the concentration of mancozeb treatment, the seed germination rate of each compound treatment gradually increased, but all were lower than 30%, while the contamination rate gradually increased. Among them, the treatment with better antibacterial effect was 0.1% NaClO4 + 0.2g / L agricultural streptomycin, and after the treatment of 0.2% NaClO4 and agricultural streptomycin, greater toxicity was produced, and the seed germination rate of each treatment was 0, but with the increase of the concentration of agricultural streptomycin treatment, the contamination first increased and then decreased, but all were lower than 20%, among which 0.1% NaClO4 + 0.2g / L agricultural streptomycin treatment had a better antibacterial effect.

[0071] 3.4 Effects of different sterilization methods on the sterilization of open sugar-free culture medium vermiculite

[0072] As shown in Table 7, different sterilization methods have different effects on the sterilization effect of open sugar-free culture medium vermiculite. After sterilization, under the condition of no inoculation of test materials, after 20 days of culture in sugar-free culture container, the vermiculite treated with high temperature dry heat sterilization had the highest contamination rate, reaching 17.4%, while the high pressure wet heat sterilization had the best effect, only 10.1%, but the contamination rate under conventional steaming sterilization was 11.3%, which was no different from the high pressure wet heat sterilization. Under the condition of culture with inoculated materials, the contamination rate of all sterilization treatment methods was higher than that of the uninoculated materials. The contamination rate under the treatment was high, but compared with each treatment, the contamination rate of high-temperature dry heat sterilization treatment was the highest, reaching 26.8%, indicating that the bacteria in the vermiculite were not killed well under this treatment temperature. The second was high-pressure wet heat sterilization treatment, with a contamination rate of 21.9%. This may be because all bacteria were killed after the matrix was sterilized with high-pressure wet heat, but it was easy to cause contamination by bacteria brought into the inoculated material during the culture process; the contamination rate was the lowest when conventional steaming and sterilization was used, which may be related to the antagonistic inhibition of residual bacteria on bacteria brought into the material, but the relevant mechanism should be further studied.

[0073] Table 7 Sterilization effect of different sterilization methods on open sugar-free culture medium vermiculite

[0074]

[0075] 3.5 Screening of compound antibacterial agents for open sugar-free culture of honey plum

[0076] Based on the above-screened optimal bacteriostatic agent combination and using nano essential oil as the compounding base material, the bacteriostatic agent formulation for open sugar-free culture of Fengtang plums was further optimized. As shown in Table 8, after 22 days of culture, the contamination rates of Group A and Group C were 23.3% and 16.7% respectively, which were higher than 6.7% and 3.3% of Group B and Group D, indicating that the bacteriostatic effect of nano Artemisia argyi essential oil was better than that of nano Cinnamomum longepaniculatum essential oil. In terms of the contamination rate, comparing Group A with Group C and Group B with Group D, it can be seen that regardless of whether it is Artemisia argyi essential oil or Cinnamomum longepaniculatum essential oil, the contamination rate of the culture medium with agar as the medium was significantly higher than that of the culture medium with vermiculite as the medium; in terms of the browning rate, the browning rates of 50.0% and 63.3% in Group A and Group B were significantly higher than 16.7% and 26.7% in Group C and Group D, indicating that the browning rate of the culture medium with vermiculite as the medium was significantly lower than that with agar; however, the browning rates of 63.3% and 26.7% in Group B and Group C were significantly higher than 50.0% and 16.7% in Group A and Group C, indicating that nano Artemisia argyi essential oil was more likely to cause browning than nano Cinnamomum longepaniculatum essential oil; in terms of the germination rate, it was generally very low, which might be because CO2 was not introduced and the plants had no C source to produce enough organic matter to meet the germination conditions. However, the germination rate of Group A was higher than that of Group B, and the germination rates of Group C and Group D were the same, indicating that there was no significant difference in the germination rate between Cinnamomum longepaniculatum essential oil and Artemisia argyi essential oil; overall, the germination rates of Group C and Group D were higher than those of Group A and Group B, indicating that the germination rate of the vermiculite medium was higher than that of the agar medium.

[0077] Table 8 Bacteriostatic effects of Cinnamomum longepaniculatum essential oil and Artemisia argyi essential oil and the effects of vermiculite and agar on the effects

[0078] Group Contamination rate Browning rate Germination rate Group A 23.3％ 50.0％ 3.3％ Group B 6.7％ 63.3％ 0％ Group C 16.7％ 16.7％ 3.3％ Group D 3.3％ 26.7％ 3.3％

[0079] 3.6 Open sugar-free culture method system for Fengtang plums

[0080] Conventional tissue culture for mass propagation of materials

[0081] Select the stem segments of Fengtang plums with good growth, no diseases and pests, and semi-lignification. Remove 2 / 3 of the leaves, soak them in a solution containing washing powder for 10 minutes, clean the attached dirt on the stem segments with a brush, then rinse with running water for 2 hours, and then rinse 3 times with sterile water in sequence on the ultra-clean workbench, soak in 75% alcohol for 45 seconds, rinse 3 times with sterile water, soak in 0.1% mercuric chloride for 8 minutes, rinse 4 times with sterile water, 30 seconds each time. After disinfection, dry the moisture of the materials with sterile absorbent paper, remove the remaining leaves and petioles, cut the materials into single-bud stem segments, and inoculate them into the primary culture medium. The primary culture medium is WPM, supplemented with 0.5 mg / L 6-BA + 0.1 mg / L IAA, 20 g / L sucrose and 6.5 g / L agar, the pH is adjusted to 5.8, and the culture conditions after inoculation are light flux density 80 umol.m -2 .s -1 , temperature 25°C ± 1°C, relative humidity 80%.

[0082] Open sugar-free culture

[0083] Using the tissue culture seedlings bred above as the open tissue culture materials, no disinfection treatment is required.

[0084] Disinfection of culture containers: Add 2% NaClO4 to the culture containers to cover the bottom of the containers, shake a few times after covering the lids, let stand for 20 min, and then pour out the NaClO4 for standby.

[0085] Disinfection of the medium; Steam the vermiculite in a steamer conventionally for 30 min.

[0086] Prepare the vermiculite medium culture medium: Add 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-Artemisia argyi essential oil to a beaker, then add the WPM basal medium, stir evenly, add hormones, adjust the pH to 5.8, and quickly dispense it into the above-mentioned disinfected vermiculite culture containers, and wait for cooling and solidification for standby.

[0087] Inoculation: Prepare an alcohol lamp, three pairs of forceps, scissors, and a cup of absolute ethanol. Soak the forceps and scissors in alcohol first, take them out and burn them on the alcohol lamp for simple disinfection treatment, and wait for cooling for standby. Connect the bred Fengtang plum materials into the containers at appropriate intervals.

[0088] Cultivation conditions: After connection, place it in a dark place for dark treatment for 4 days.

[0089] 4. Conclusion

[0090] This invention has studied the establishment of the primary culture system of Fengtang plum and the disinfection methods of bacteriostatic agents and culture media in the open sugar-free culture system, and obtained the following results:

[0091] 1) In the disinfection treatment of explants, the fungal contamination of the treatment with 75% alcohol for 45 s + 0.10% mercuric chloride for 9 min is the highest, the bacterial contamination and browning rate are the lowest, and the survival rate is in the middle. The contamination rate, browning rate and survival rate of the treatment method with 75% alcohol for 45 s + 0.10% mercuric chloride for 8 min are significantly better than other treatments, and the sterilization effect is the best.

[0092] 2) In the treatment of different basal media, the browning rate of the explants under the treatment of WPM medium is the lowest, and the germination survival rate and growth potential are the best.

[0093] 3) Under open conditions, among different combinations of bacteriostatic agents, when the materials were not inoculated, single streptomycin agricola treatment and the combination of potassium sorbate and chlorothalonil had almost no bacteriostatic effect, while other combinations had significant bacteriostatic effects. After inoculating the materials, the optimal treatment was single 0.03% mancozeb treatment, with the highest germination rate and the lowest contamination rate. Secondly, the germination rate of the treatment with 0.1 g / L potassium sorbate + 0.1 g / L chlorothalonil was relatively high and the contamination rate was relatively low. Although the contamination rates of all other combinations were low, their biotoxicity to sorghum seeds was relatively high and further tests should be conducted.

[0094] 4) The best sterilization method for the open culture medium vermiculite is conventional steaming for 30 min.

[0095] 5) The best combined bacteriostatic agent for open sugar-free culture of Fengtang plum is 0.1 g / L chlorothalonil + 0.2 g / L mancozeb + 2 ml / L NaClO4 + 3 ml / L nano-artemisia argyi essential oil. The bacteriostatic effect and germination rate with vermiculite as the medium are better than those with agar.

[0096] 6) The basic operation process for open sugar-free culture of Fengtang plum is as follows: Vermiculite as the medium is steamed conventionally for 30 min. Add 3% NaClO4 to the culture container to cover the bottom of the bottle, shake and then let it stand for 20 min. After pouring it out, add the culture medium formula, that is, the combination of bacteriostatic agent and basic medium: WPM + 0.5 ml / L 6-BA + 0.1 ml / L IAA, 0.2 g / L mancozeb + 0.1 g / L chlorothalonil + 2 ml / L NaClO4 + 3 ml / L artemisia argyi essential oil, and adjust the pH to 5.8. Select semi-lignified Fengtang plum stem segments, soak them in 2% NaClO4 for 6 min, rinse several times and then soak them in 75% alcohol for 30 s. Finally, rinse off the residual alcohol. Finally, trim the stem segments and insert them into the container, and conduct dark treatment for 4 days.

[0097] The beneficial effects of the present invention are as follows:

[0098] The present invention uses the conventional tissue-cultured aseptic adventitious buds of the characteristic fruit Fengtang plum as explant materials, and a compound of mancozeb, chlorothalonil, NaClO4 and nano-artemisia argyi essential oil as an antibacterial agent to establish an open-type sugar-free rapid propagation culture system for Fengtang plum. Although the open-type sugar-free culture method has been applied in the rapid propagation of other fruit trees, there has been no report in the rapid propagation process of Fengtang plum. The application of the open-type sugar-free culture can greatly reduce the cost and simplify the operation in the in vitro culture system of Fengtang plum. Therefore, the application of the open-type tissue culture system in the propagation of Fengtang plum seedlings is very promising and is expected to be an important method for the in vitro culture of Fengtang plum. An effective culture method for the open-type sugar-free culture of Fengtang plum was screened out: the culture medium vermiculite was sterilized by steaming, the culture container was sterilized by soaking in 3% NaClO4, the culture medium was WPM + 0.5 ml / L 6-BA + 0.1 ml / L IAA + 0.2 g / L mancozeb + 0.1 g / L chlorothalonil + 2 ml / L NaClO4 + 3 ml / L artemisia argyi essential oil + vermiculite. After 4 days of dark culture, the light intensity was gradually increased to 200 μmol·m -2 ·s -1 , CO2 was introduced as a carbon source, and the CO2 concentration (v / v) was increased to 1.6×10 -3 . Under the premise of considering cost, in the application process of this open-type sugar-free culture of Fengtang plum, the best rapid propagation effect of Fengtang plum is achieved. This open-type sugar-free culture method is simple to operate, greatly reduces the cost, the cultured seedlings are stronger, and there is no need for acclimatization, and they can be directly transplanted and applied. This method can be used as a simple and effective method in the rapid propagation process of Fengtang plum. Specific embodiments

[0099] Example 1

[0100] The preparation of adventitious buds of Fengtang plum includes:

[0101] 1) Selection of conventional tissue-culture explants of Fengtang plum: Select the stem segments of Fengtang plum with good growth, no pests and diseases and semi-lignification;

[0102] 2) The disinfection method of the conventional tissue-culture explants of Fengtang plum is: soak in a washing powder aqueous solution for 10 min, rinse with running water for 2 h, rinse 3 times with sterile water in sequence on a super-clean workbench, the treatment time with 75% alcohol is 45 s, the treatment with 0.1% mercuric chloride is 8 min, and rinse 4 times with sterile water, 30 s each time;

[0103] 3) The adventitious bud induction medium for culturing Fengtang plum: WPM + 0.5 mg / L 6-BA + 0.1 mg / L IAA; among them, 20 g / L of sucrose and 6.5 g / L of agar are added to the WPM medium, and the pH is adjusted to 5.8;

[0104] 4) The culture conditions for inducing adventitious buds of Fengtang Plum by tissue culture are as follows: light flux density of 70 μmol·m-2·s-1, temperature of 25°C ± 1°C, and relative humidity of 80 ± 5%, and adventitious buds of Fengtang Plum are obtained.

[0105] Example 2

[0106] Preparation of a culture medium for Fengtang Plum, including:

[0107] 1) Preparation of the culture medium bacteriostatic agent: Mancozeb 0.2 g / L + Chlorothalonil 0.1 g / L + NaClO4 2 ml / L + Nano Artemisia argyi essential oil 3 ml / L;

[0108] 2) Preparation of the culture medium culture medium: Vermiculite sterilized by conventional steaming for 30 min is used;

[0109] 3) Disinfection of the culture container: Add 3% NaClO4 covering the bottom of the container, shake and then let it stand for disinfection for 30 - 60 min;

[0110] 4) Preparation of the culture medium for Fengtang Plum: WPM + 6-BA 0.5 mg / L + IAA 0.1 mg / L, adding the bacteriostatic agent prepared in step 1); Add the sterilized vermiculite in step 2) to the sterilized culture container in step 3), with a thickness of 3 - 4 cm, and then add the culture medium until it just submerges the culture medium to obtain the culture medium for Fengtang Plum.

[0111] Example 3

[0112] A method for open-type sugar-free culture of Fengtang Plum, including:

[0113] 1) Inoculation method: Cut the adventitious buds of the conventional tissue culture of Fengtang Plum prepared in Example 1, with a length of 2 - 3 cm and 1 - 2 leaves, and inoculate them into the culture container;

[0114] 2) Culture conditions: After the connected Fengtang Plum stem segments are dark-treated in a dark place for 4 days, they are cultured under illumination. The light intensity is: from 0 - 2 days, the light intensity is 40 μmol·m -2 ·s -1 ; from 3 - 6 days, the light intensity is 120 μmol·m -2 ·s -1 ; from 6 - 8 days, the light intensity is 200 μmol·m -2 ·s -1 ; the day / night time is 14 h / 10 h; CO2 is introduced during illumination, and the CO2 concentration (v / v) is 0.8 - 1.6×10 -3 , and it gradually increases; the culture temperature is 25°C ± 1°C, and the relative humidity is 80 ± 5%;

[0115] 3) Transplanting of open-type sugar-free cultured seedlings of Fengtang Plum: When the Fengtang Plum seedlings to be cultured grow to 7-8 cm, they can be directly transplanted to the field.

[0116] The above content does not determine that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the art, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the patent protection scope determined by the claims submitted for the present invention.

Claims

1. A honey plum culture medium, which consists of the following: WPM + 0.5 mg / L 6-BA + 0.1 mg / L IAA and a bacteriostatic agent; wherein: The bacteriostatic agent is: 0.2 g / L mancozeb + 0.1 g / L chlorothalonil + 2 ml / L NaClO4 + 3 ml / L nano-artemisia argyi essential oil.

2. A preparation method of a honey plum culture medium, comprising: 1) Prepare the bacteriostatic agent for the culture medium: 0.2 g / L mancozeb + 0.1 g / L chlorothalonil + 2 ml / L NaClO4 + 3 ml / L nano-artemisia argyi essential oil; 2) Prepare the culture medium culture medium: vermiculite sterilized by conventional steaming for 30 min; 3) Disinfect the culture container: Add 3% NaClO4 that covers the bottom of the container, shake it, and then let it stand for disinfection for 30 - 60 min; 4) Prepare the honey plum culture medium: Prepare the basic medium according to WPM + 0.5 mg / L 6-BA + 0.1 mg / L IAA, and then add the bacteriostatic agent prepared in step 1); Add the sterilized vermiculite in step 2) to the sterilized culture container in step 3), with a thickness of 3 - 4 cm; Then add the above basic medium added with the bacteriostatic agent until it just submerges the culture medium to obtain the honey plum culture medium.

3. A honey plum culture medium prepared by the preparation method according to claim 2.

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