Application of bacteriostatic agent in open tissue culture and rapid propagation of polyploidy bamboo reed

Abstract

The invention provides application of a bacteriostatic agent in open type tissue culture and rapid propagation of polyploidy bamboo reeds, and relates to the technical field of tissue culture and rapid propagation of plants, the bacteriostatic agent is applied to the open type tissue culture and rapid propagation process of the polyploidy bamboo reeds, and the use method is that the bacteriostatic agent is added into a culture medium for use or sprayed or oscillated and flushed for use, and the bacteriostatic agent is applied to the open type tissue culture and rapid propagation process of the polyploidy bamboo reeds. The bacteriostatic agent is formed by mixing isothiazolinone and sodium dehydroacetate, the mass addition ratio of the isothiazolinone to the sodium dehydroacetate is (8-5): (2-5), and the method comprises the following steps: S1, primary culture of explants; s2, multiplication culture of seedlings; s3, rooting culture, wherein each liter of the culture medium contains 0.05-1g of the bacteriostatic agent in the above steps. The method has the beneficial effects that under the condition that the method does not need to be controlled in a sterile environment and does not depend on the quality of an inoculator, a large amount of propagation of the polyploidy bamboo reed tissue culture seedlings is realized by adopting an open tissue culture technical mode, the productivity speed of a tissue culture center is improved, and the construction threshold and cost of the tissue culture center are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture and rapid propagation, in particular to application of an antibacterial agent in open tissue culture and rapid propagation of polyploid Phragmites australis. Background Art

[0002] Arundo donax is a perennial herb of the genus Arundo donax in the Poaceae family and is a plant germplasm resource with great development value.

[0003] Polyploid Arundo donax is a novel energy plant developed using gene editing technology combined with traditional breeding methods. It is a superior Arundo donax variety. A perennial, tall, perennial herb resembling a reed or bamboo, it has short, sturdy, and heavily branched underground stems, broadly lanceolate leaves, and terminal, broom-shaped panicles. It flowers and bears fruit from September to December.

[0004] Polyploid Phragmites australis is highly resistant to stress, grows rapidly, and produces a large biomass. It can be cultivated in saline-alkali lands, tidal flats, swamps, sandy wastelands, barren land, and abandoned mining areas, with rainfall exceeding 500 mm, extreme temperatures exceeding -20°C, and salinity levels below 15‰. It can yield over 3 tons of dry matter per mu, potentially contributing to multiple trillion-yuan downstream industries. Its high content of cellulose, hemicellulose, and lignin, with a three-element content of ≥86%, and the high quality of its cellulose and hemicellulose make it a high-quality raw material for papermaking and textiles. Furthermore, its calorific value exceeds 4,000 kcal, similar to that of broad-leaved forests, making it the herbaceous plant closest to fossil fuels. When the plant reaches approximately 2.5 meters in height, its stems and leaves contain up to 16% crude protein, making it an excellent green fodder for poultry and livestock. In Tianjin, for example, it can be harvested three times a year, yielding 8-12 tons of fresh material per mu. Its bitter rhizome is cold in nature, clearing heat and promoting diuresis. It is used to treat fever-induced madness, consumptive bone steaming, stranguria, painful urination, and toothache caused by wind and fire. Furthermore, polyploid Arundo donax possesses greater stress resistance and a wider adaptability than other Arundo donax varieties. It can survive winters in northern regions at temperatures exceeding -20°C, is drought-resistant and infertile, and boasts a well-developed root system. Sand control experiments have shown that polyploid Arundo donax survives the winter normally in experimental plots. Three-year-old Arundo donax plants have dense and well-developed root systems, exceeding 150 cm in length, demonstrating significant sand-fixing properties. They can quickly re-establish vegetation in sandy areas, achieving wind and sand control. Furthermore, polyploid Arundo donax plants grow rapidly and tolerate flooding. Planted along rivers and lakes, their well-developed, fibrous root system minimizes soil disturbance on the opposite bank, effectively conserving water and soil. Therefore, polyploid reed bamboo can be widely used in windbreak and sand fixation, soil and water conservation, desertification control, forage production, edible fungus cultivation, saline-alkali land improvement, soil heavy metal remediation, and mine greening, with far-reaching implications for ecological management in northern my country. In recent years, as global carbon reduction targets have been implemented worldwide, reed bamboo, as the world's largest herbaceous plant by biomass, boasts high cellulose, hemicellulose, and lignin contents (≥86%), and high-quality cellulose and hemicellulose, making it a high-quality raw material for papermaking and textiles. Furthermore, polyploid reed bamboo boasts a calorific value exceeding 4,000 kcal, similar to that of broadleaf forests, making it the closest herbaceous plant to fossil energy. As an excellent energy crop, it has become a focus of industry attention and holds broad application prospects.

[0005] Our company has previously developed a highly efficient tissue culture rapid propagation method for Phragmites australis, with the publication number CN118000092A. This method and technology have revolutionized the tissue culture rapid propagation of Phragmites australis. By using this method, followed by multiplication and subsequent one-step seedling cultivation of the expanded Phragmites australis buds, the cultivation cycle of tissue cultured Phragmites australis seedlings can be shortened from 120-150 days to 60-90 days, significantly improving the quality of the tissue cultured seedlings by more than one grade. This method improves the growth potential of Phragmites australis seedlings, shortens their growth cycle, and significantly enhances their quality.

[0006] However, polyploid Phragmites australis cannot naturally produce fruit, making seed propagation impossible. Cutting propagation through seed nodes has an extremely low survival rate, significantly limiting its widespread application. Root propagation, however, is labor-intensive, requires significant soil damage, and is associated with high transportation costs over long distances. Currently, tissue culture propagation, a commonly used method, has addressed some of the challenges associated with these propagation methods. However, nationwide promotion has revealed that Phragmites australis differs from other plants in that current tissue culture propagation techniques require sterile environments and multiple disinfection steps prior to cultivation. Even under these conditions, contamination rates cannot be kept low. Currently, the contamination rate of axillary buds in Phragmites australis rarely remains below 0% in July and August, typically between 8 and 10%. Cultures in other months experience even higher rates. Therefore, tissue culture inoculation is challenging to implement and requires high-quality inoculators, hindering the rapid expansion of tissue culture centers. Consequently, the current supply of polyploid Phragmites australis tissue culture seedlings is limited by production capacity and will not be able to meet future market demand for high-quality polyploid Phragmites australis seedlings. Summary of the Invention

[0007] The invention aims to provide an application of an antibacterial agent in the open tissue culture rapid propagation of polyploid Arundo donax. By coordinating the antibacterial agent with a culture medium and culture conditions, the difficulty of open culture of Arundo donax is reduced, the survival rate is improved and the contamination rate is reduced.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] The invention discloses an application of an antibacterial agent in the open tissue culture rapid propagation of polyploid Arundo donax. The antibacterial agent is applied in the open tissue culture rapid propagation process of polyploid Arundo donax. The application method comprises the following steps: adding the antibacterial agent to a culture medium, spraying the agent, or vibrating and washing the agent. The antibacterial agent is prepared by mixing isothiazolinone and sodium dehydroacetate. The mass addition ratio of the isothiazolinone to sodium dehydroacetate is 8-5:2-5.

[0010] The invention discloses an application of an antibacterial agent in the open tissue culture rapid propagation of polyploid Phragmites australis. The antibacterial agent is prepared by mixing isothiazolinone and sodium dehydroacetate. The mass addition ratio of the isothiazolinone to the sodium dehydroacetate is 8-5:2-5.

[0011] Furthermore, the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:3.

[0012] The application process of the antibacterial agent in the open tissue culture rapid propagation of polyploid Arundo donax is as follows:

[0013] S1: Primary explant culture

[0014] Insert the bud tip of the sterilized explant upward obliquely or vertically into the culture medium in the culture bottle. The inoculation depth should be 1-2 mm or even slightly below the surface of the culture medium at the node. After inoculation, cover the container with a lid and seal it tightly.

[0015] The culture medium used for primary culture is: 1 / 2MS as the basal medium, supplemented with 8-10 mg / L indolebutyric acid, 0.05 g / L-1 g / L antibacterial agent DY1, 15 g / L sucrose, and 7 g / L tissue culture type I or type K carrageenan;

[0016] Furthermore, the antibacterial agent DY1 is: a mixed solution of the isothiazolinone and sodium dehydroacetate, wherein the concentration of the antibacterial agent in the culture medium is 0.05 g / L-1 g / L, that is, 0.05 g-1 g of the antibacterial agent is contained per liter, and the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 8-5:2-5;

[0017] Preferably, the sum of the addition amounts of the isothiazolinone and sodium dehydroacetate is 0.1 g / L; the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:3;

[0018] The culture conditions were as follows: pH 6.5, culture room temperature 25 ± 2°C, photoperiod D / N = 12 h / 12 ​​h, and illumination 1500-2000 lx;

[0019] Furthermore, the pH value was adjusted to 6.5 using NaOH or HCl.

[0020] After 3-5 days, axillary buds are observed to sprout, and after 6-15 days, roots are sprouted from the base of leaf buds, thereby forming a complete polyploid Arundo donax seedling. The complete polyploid Arundo donax seedling is connected to a thicker stem segment, i.e., the original explant.

[0021] S2: Proliferation and culture of seedlings

[0022] The polyploid Arundo donax seedlings connected to the original explants are further cultured, and after induction culture, the buds are differentiated into polyploid Arundo donax seedling clusters with a plant height of more than 10 cm, and each of the clusters is divided to form a plurality of separate rootless buds with complete growth points;

[0023] Preferably, the polyploid Arundo donax seedlings connected to the original explants are further cultured for 15-40 days, and after induction culture, the buds are differentiated into polyploid Arundo donax seedling clusters with a plant height of more than 10 cm; each of the clusters is cut in a sterilized enclosed place to form 8-15 individual rootless buds with complete growth points;

[0024] The rootless buds are inoculated obliquely or vertically in a culture medium containing a pH of 6.5, wherein the culture medium is based on MS and supplemented with 8-10 mg / L indolebutyric acid, 0.05 g / L-1 g / L antibacterial agent DY1, 15 g / L sucrose, and 7 g / L tissue culture type I or type K carrageenan. The culture bottles are tightly capped and sealed, and subcultured according to the above-mentioned primary culture conditions, namely, the culture room temperature is 25±2° C., the photoperiod is D / N=12 h / 12 ​​h, and the illumination is 1500-2000 lx.

[0025] Furthermore, the antibacterial agent DY1 is: a mixed solution of the isothiazolinone and sodium dehydroacetate, wherein the concentration of the antibacterial agent in the culture medium is 0.05 g / L-1 g / L, that is, 0.05 g-1 g of the antibacterial agent is contained per liter, and the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 8-5:2-5;

[0026] Preferably, the sum of the addition amounts of the isothiazolinone and sodium dehydroacetate is 0.1 g / L; the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:3;

[0027] Furthermore, after 35-45 days of subculture and proliferation, the buds in the culture bottle can be differentiated again into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm, and the above proliferation culture is repeated, and proliferation is achieved through continuous subculture;

[0028] S3: Rooting Culture

[0029] After the S2 subculture induction culture, each of the clustered seedlings is cut into a number of individual rootless buds with complete growth points, and the buds are inoculated obliquely or vertically in a culture medium containing a pH of 6.5 for rooting culture, wherein the culture medium uses MS as a basal medium and is supplemented with 8-10 mg / L indolebutyric acid, 0.1 g / L antibacterial agent DY1, 15 g / L sucrose, and 7 g / L type I or type K carrageenan for tissue culture. The culture bottle is capped and sealed, and the antibacterial agent DY1 is the same as that in step S3. After 7-15 days, the inoculated buds in the culture bottle emit spiky root tissue.

[0030] Furthermore, the antibacterial agent DY1 is: a mixed solution of the isothiazolinone and sodium dehydroacetate, wherein the concentration of the antibacterial agent in the culture medium is 0.05 g / L-1 g / L, that is, 0.05 g-1 g of the antibacterial agent is contained per liter, and the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 8-5:2-5;

[0031] Preferably, the sum of the addition amounts of the isothiazolinone and sodium dehydroacetate is 0.1 g / L; the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:3;

[0032] After S3 subculture induction culture, the buds in the culture bottle differentiate into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm; each of the cluster seedlings is cut in a sterilized, enclosed space to form a plurality of individual rootless buds with intact growth points, generally forming a plurality of 8-15 individual rootless buds with intact growth points;

[0033] S4: Transplanting and hardening seedlings

[0034] Plants with a stem elongation of more than 10 cm were transplanted. The seedlings were removed from the culture medium, the culture medium remaining on the roots was washed with clean water, and then transplanted into a seedling container containing a seedling medium. The seedling medium used was a mixture of vermiculite, peat, perlite, and fine river sand. Before use, the medium was sprayed with 0.05 g / L to 1 g / L of the antibacterial agent DY1 for disinfection. The antibacterial agent DY1 was the same as that used in step S1.

[0035] Preferably, 0.7 g / L antibacterial agent DY1 is sprayed for disinfection before use, and the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:3;

[0036] Immediately after transplanting, water thoroughly with an atomizing nozzle, cover with a plastic film arch shed, and use a black sunshade net with a light transmittance of 60% to shade the soil to avoid direct sunlight. Three days after transplanting, the plastic film can be removed for proper ventilation during the period when sunlight is insufficient, and water can be added in an appropriate amount; after 7 days, the ventilation time can be gradually extended, and the film can be completely removed after 15 days. The sunshade net can be removed after 30 days as appropriate. During the entire transplanting and hardening period, pay attention to ensuring sufficient water supply. Depending on the facility conditions, choose an interval of 10-15 days to spray 0.05g / L-1g / L of antibacterial agent DY1. The antibacterial agent DY1 is the same as in step S1, and after the polyploid Phyllostachys australis seedlings begin to grow normally, choose to apply foliar fertilizers such as potassium dihydrogen phosphate.

[0037] Preferably, 0.7 g / L of antibacterial agent DY1 is sprayed at an interval of 10-15 days, and the mass addition ratio of isothiazolinone to sodium dehydroacetate is 7:3;

[0038] Before further primary explant culture, polyploid Phyllostachys australis plants are cut into stem segments with axillary buds as explants, cleaned, and sterilized. The specific steps are as follows:

[0039] The explant is placed in a sterilized sealed container, rinsed with a mixed solution of isothiazolinone and sodium dehydroacetate for 3-8 times by continuous shaking, then immersed in a 1.5‰ mercuric chloride solution for 7-9 minutes for sterilization by shaking, and rinsed with the mixed solution of isothiazolinone and sodium dehydroacetate for 3-4 times by continuous shaking, wherein the mass addition ratio of the isothiazolinone to sodium dehydroacetate in the above steps is (8-5):(2-5); the addition concentration of the isothiazolinone and sodium dehydroacetate is 0.05g / L-1g / L, and the remainder is water;

[0040] Preferably, in a confined place that has been disinfected, use 0.1g / L of isothiazolinone and sodium dehydroacetate antibacterial solution to continuously shake and rinse 3 times, and the addition ratio of isothiazolinone and sodium dehydroacetate is 6:4. Then, after pouring out the above solution, soak and shake disinfect with 1.5‰ mercuric chloride solution for 8 minutes, and continue to shake and rinse 3-4 times with 0.05g / L of isothiazolinone and sodium dehydroacetate sterile solution, and the mass addition ratio of isothiazolinone and sodium dehydroacetate is 7:3.

[0041] The beneficial effects of the present invention are:

[0042] This application does not require control in a sterile environment and does not rely on the quality of the vaccinator. It uses open tissue culture technology to achieve large-scale propagation of polyploid Phragmites australis tissue culture seedlings, improves the production capacity speed of the tissue culture center, and reduces the construction threshold and cost investment of the tissue culture center.

[0043] In the explant disinfection step, this method is used in combination with the cleaning and disinfecting agents in this application. The antibacterial effect of the mixed antibacterial agent of isothiazolinone + sodium dehydroacetate in a specific proportion used in this application is outstanding. It is combined with a 1.5‰ mercuric chloride solution. In the later culture process, this culture medium is used in combination with the antibacterial agent. Through comparative testing, it is found that it does not harm the explants and can achieve long-term antibacterial and disinfection effects. Moreover, when it lasts for 40 days, the contamination rate is maintained at 0%, and the survival rate is high. During the primary culture of Phragmites australis, the browning rate is ≤4.67 at 40 days, the contamination rate is 0%, and the survival rate is ≥94%.

[0044] The study showed that the application effects of different antibacterial agents in the subculture proliferation culture of polyploid Phragmites australis were such that the antibacterial effect of the mixed isothiazolinone + sodium dehydroacetate used in this step was outstanding. When the antibacterial effect lasted for 40 days, the browning rate was ≤4%, the contamination rate remained at 0%, the survival rate was ≥94.67%, and the proliferation multiples were ≥13.86, which was significantly better than other antibacterial agent treatments and the CK group.

[0045] The axillary buds of Phragmites australis in September were selected in this embodiment. In Tianjin, the applicant also tested other months and found that the contamination rate could reach 0% by this method. Therefore, the present application achieved good sterilization and disinfection effects, and the transplanting survival rate of the axillary buds of Phragmites australis in September reached 96.33%, which is the best material collection period. In actual operation, the present application can be operated normally without the need to specially set up high-pressure and high-temperature sterilization facilities, etc., so the cost is low and the operation is simple, which reduces the entry threshold of the industry. The present application provides an application of an antibacterial agent in the open tissue culture rapid propagation of polyploid Phragmites australis, which provides a direction for the improvement of saline-alkali land, tidal flats, swamps, sandy wastelands, barren land and abandoned mining areas in the later period of my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a photo of the roots of Phragmites australis in Example 1 after 15 days of rooting culture;

[0047] Figure 2 This is a frontal photograph of the Phragmites australis of Example 1 after 15 days of rooting culture;

[0048] Figure 3 This is a photo of the roots of Phragmites australis after 40 days of rooting culture in Example 1;

[0049] Figure 4 This is a frontal photograph of the Phragmites australis of Example 1 after 40 days of rooting culture; DETAILED DESCRIPTION

[0050] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0051] Note: The data in Tables 1 to 9 are all multi-processing averages. It should be noted that the data calculation formulas in Tables 1 to 9 are as follows:

[0052] Contamination rate = number of contaminations / number of inoculations × 100%;

[0053] Browning rate = browning number / inoculation number × 100%;

[0054] Survival rate = number of survivors / number of inoculated cells × 100%;

[0055] Rooting rate = number of rooted explants / number of surviving explants × 100%;

[0056] Multiplication multiple = number of multiplied seedlings / number of inoculated explants.

[0057] Exploration Example 1-9:

[0058] Exploration Examples 1-9 were conducted simultaneously. The operation steps were the same, but the antibacterial agents were different. The antibacterial agents in Exploration Examples 1-9 were used for experiments and the experimental effects were observed. To verify the effect of different antibacterial agents in culture, we used a concentration of 0.1 g / L for the study. A total of 9 treatment groups were set up, and a blank control group CK without antibacterial agent was also set up. An open tissue culture experiment was performed according to the following experimental steps. 50 explants were inoculated in each treatment and 3 groups were repeated. The antibacterial agents selected in Exploration Examples 1-9 included potassium permanganate, sodium hypochlorite, mancozeb, chlorothalonil, carbendazim, streptomycin, isothiazolinone, and sodium dehydroacetate. The steps are as follows:

[0059] Step 1: Primary cultivation of Arundo donax

[0060] The main purpose of this study was to study the effects of different antimicrobial agents on primary culture of Phragmites australis. An open tissue culture experiment was conducted according to the following experimental steps. 50 explants were inoculated in each treatment, and three replicates were performed:

[0061] 1. Explant Preparation

[0062] Around mid-September in Tianjin, on a sunny day with an outside temperature of 23°C-30°C and a relative humidity of 35%-55%, healthy, pest-free polyploid Phragmites australis plants were selected. The middle and upper stem segments were cut as explants, and tissues and organs such as leaves and leaf sheaths were removed. The stem segments were then cut into approximately 2 cm long segments with a node (retaining the axillary bud), ensuring a distance of 0.5 cm below the node and 1.5 cm above the node. The resulting stem segments with nodes and axillary buds contained nutrients that promoted the rapid growth of the axillary buds.

[0063] 2. Explant Sterilization

[0064] In a closed place that has been disinfected, use 0.1g / L antibacterial solution with continuous shaking and flushing for 3 times. The above solution contains 0.1g antibacterial solution per 1L of water. Then pour out the above solution, soak and disinfect with 1.5‰ mercuric chloride solution for 8 minutes, and then use 0.05g / L antibacterial solution to continuously shake and flush 4 times;

[0065] The antibacterial solutions are all the antibacterial agents listed in Table 1. Experiments were conducted on 9 antibacterial agents respectively and the experimental effects were observed respectively.

[0066] 3. Culture medium preparation

[0067] Fill a heating medium filling machine with distilled water and heat to 95°C before entering the heat preservation stage. Then, add all the necessary chemicals to dissolve and adjust the volume. While stirring continuously, adjust the pH to 6.5 using sodium hydroxide (NaOH) or hydrochloric acid (HCl). Quickly dispense the prepared medium solution into 350ml culture bottles and seal them tightly with a cap. Place the newly filled bottles in a clean, enclosed environment to cool and avoid exposure to the outside environment. The medium should be prepared and used immediately. It can be stored in a clean, enclosed, low-temperature environment for a short period of time, but the storage period should not exceed 30 days.

[0068] 4. Primary Induction Culture of Explants

[0069] The sterilized explant stem segment with nodes and axillary buds is inserted obliquely or vertically into the culture medium in the culture bottle with the bud tip facing upward. The inoculation depth should be 1-2 mm slightly below the surface of the culture medium or flush with the node. After inoculation, the bottle cap is quickly closed and sealed. The culture medium used for primary culture is: 1 / 2MS basal medium, 8-10 mg / L indolebutyric acid, 0.1 g / L antibacterial agent, 15 g / L sucrose, and 7 g / L type I or type K carrageenan for tissue culture. The antibacterial agents described in this step are the antibacterial agents listed in Table 1. Nine antibacterial agents were tested separately and the experimental effects were observed. During this process, the pH value was adjusted to 6.5 using NaOH or HCl. The culture conditions were as follows: the culture room temperature was 25±2°C, the photoperiod was D / N=12h / 12h, and the illumination was 1500-2000lx. Axillary buds were observed to sprout after 3-5 days, and roots sprouted from the base of the leaf buds after 6-15 days, forming complete polyploid Phragmites australis seedlings connected to the thicker stem segments, i.e., the original explants. Statistics were taken at 15 and 40 days of culture to determine the browning rate, contamination rate, and survival rate after 40 days. The results are shown in Table 1.

[0070] Table 1 Statistics of the results of different single antibacterial agents in the primary induction culture process of open tissue culture of Phragmites australis

[0071]

[0072] As shown in Table 1, we selected a blank control group (CK) without antibacterial agents. Since no antibacterial agents were added, the primary culture of Phragmites australis was minimally damaged, resulting in a low browning rate, but a high contamination rate. By selecting several different types of fungicides, including the oxidizing fungicides sodium hypochlorite and potassium permanganate, the protective fungicide mancozeb, the potent, non-systemic fungicides carbendazim and chlorothalonil, the antibiotic fungicide streptomycin, and the broad-spectrum, non-oxidizing fungicides isothiazolinone and sodium dehydroacetate, we found that potent, non-systemic, protective fungicides were less effective in the primary induction culture of Phragmites australis in open tissue culture. Potassium permanganate, streptomycin, isothiazolinone, and sodium dehydroacetate resulted in lower browning and contamination rates at 15 and 40 days, and a higher survival rate than the other antibacterial agents.

[0073] Step 2: Study on the application effect of polyploid Arundo donax in subculture proliferation culture

[0074] Based on the primary culture of Phragmites australis in Exploration Example 1-9, surviving shoots were selected for further cultivation. The results are as follows:

[0075] 1. Culture medium preparation

[0076] Fill a heating medium filling machine with distilled water and heat to 95°C before entering the heat preservation stage. Then, add all the necessary chemicals to dissolve and adjust the volume. While stirring continuously, adjust the pH to 6.5 using sodium hydroxide (NaOH) or hydrochloric acid (HCl). Quickly dispense the prepared medium solution into 350ml culture bottles and seal them tightly with a cap. Place the newly filled bottles in a clean, enclosed environment to cool and avoid exposure to the outside environment. The medium should be prepared and used immediately. It can be stored in a clean, enclosed, low-temperature environment for a short period of time, but the storage period should not exceed 30 days.

[0077] 2. Proliferation and Culture of Seedlings

[0078] After about 40 days of primary induction culture, the buds in the culture bottles differentiated into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm; each of the above cluster seedlings was cut in a closed place that had been disinfected to form 12-20 separate rootless buds with complete growth points; the above rootless buds were obliquely or vertically inoculated into culture bottles containing pH = 6.5, MS basal culture medium, 8-10 mg / L indolebutyric acid, 0.1 g / L antibacterial agent, 15 g / L sucrose, and 7 g K-type carrageenan for tissue culture, and the bottles were quickly covered and sealed, wherein the antibacterial agents were the antibacterial agents listed in Table 2, and experiments were conducted on 9 antibacterial agents, and the experimental effects were observed respectively. The amount of the antibacterial agent added was 0.1 g / L, where 0.1 g / L was the concentration of the antibacterial agent in the culture medium solution; subculture proliferation culture was performed according to the above-mentioned primary culture conditions; after about 40 days of subculture proliferation culture, the buds in the culture bottle could be differentiated again into polyploid Phragmites australis clustered seedlings with a plant height of more than 10 cm, and the above-mentioned proliferation culture was repeated, and proliferation was achieved through continuous subculture; statistics were performed at 15 days and 40 days of culture to determine the browning rate, contamination rate, and survival rate and value-added rate after 40 days, and the results are shown in Table 2.

[0079] Table 2 Statistics of the results of single antibacterial agent in the open tissue culture subculture process of Arundo donax

[0080]

[0081] Through research, it is shown that the application effects of different antibacterial agents in the subculture proliferation culture of polyploid Phragmites australis were found. The antibacterial effects of potassium permanganate, streptomycin, isothiazolinone and sodium dehydroacetate used in this step on Phragmites australis were outstanding. When the antibacterial effects lasted for 40 days, the browning rate was ≤15%, the contamination rate was ≤10%, the survival rate was ≥69%, and the proliferation multiples were ≥5%, which were significantly better than those of other antibacterial agent treatment groups.

[0082] Step 3: Study on the application effect of polyploid Phragmites australis rooting culture

[0083] Based on the research on the application effect of polyploid Phragmites australis in subculture proliferation culture in the above-mentioned Exploration Examples 1-9, the process is that after about 40 days of subculture induction culture, the buds in the culture bottle differentiate into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm; each of the above-mentioned cluster seedlings is cut in a closed place that has been disinfected as above to form 12-20 separate rootless buds with complete growth points, and they are obliquely inserted or vertically inoculated in a culture bottle filled with pH = 6.5, MS basal culture medium + 8-10 mg / L indolebutyric acid + 0.1 g / L antibacterial agent + 15 g sucrose + 7 g type I carrageenan for tissue culture. The antibacterial agent in the above steps is the antibacterial agent in Exploration Examples 1-9 in the following table. The 9 antibacterial agents were experimented with separately and the experimental effects were observed separately. Rooting culture was carried out according to the above culture conditions. After about 7-15 days, the inoculated buds in the culture bottle began to emit thorn-like root tissue. Statistics were taken at 15 days and 40 days of culture to determine the browning rate, contamination rate, rooting rate, and survival rate after 40 days, number of roots, root length, etc. The results are shown in Table 3 below.

[0084] Table 3 Statistics of rooting results of single antibacterial agent in open tissue culture of Phragmites australis

[0085]

[0086] Studies have shown that in the open tissue culture rooting process of Phragmites australis, the use of potassium permanganate, streptomycin, isothiazolinone, sodium dehydroacetate and sodium hypochlorite has an outstanding antibacterial effect on Phragmites australis, with high rooting rate and survival rate.

[0087] Step 4: Study on the application effect in transplanting and hardening seedlings

[0088] Based on the research on the application effect of polyploid Phragmites australis rooting culture in Exploratory Examples 1-9 above, after 30-60 days of rooting culture, the buds in the culture bottles grow into plants over 10 cm in diameter with 3-5 stem nodes and complete root systems, and then enter the seedling hardening stage. The plants are removed from the culture bottles, the culture medium remaining on the roots is washed with clean water, and then transplanted into a seedling container filled with seedling medium. The seedling medium used is a combination of vermiculite, peat, perlite, and fine river sand, and is sprayed with 0.7 g / L of antibacterial agent for disinfection before use. The antibacterial agents are each listed in Table 4. Each of the nine antibacterial agents was tested and the experimental effects were observed. Immediately after transplanting, water is poured thoroughly with an atomizing nozzle, and then a plastic film arch is added to the shed. A black shade net with a light transmittance of 60% is used to shade the plants from direct sunlight. Three days after transplanting, the plastic film can be removed during the period when sunlight is insufficient to allow proper ventilation and add water in moderation; after 7 days, the ventilation time can be gradually lengthened, and the film can be completely removed after 15 days. The shade net can be removed after 30 days as appropriate. During the entire transplanting and hardening period, pay attention to ensuring adequate water supply. Depending on the conditions of the facilities, spray 0.7 ml / L of antibacterial agent at intervals of 10-15 days, and apply foliar fertilizers such as potassium dihydrogen phosphate after the polyploid Phragmites australis seedlings begin to grow normally; record the contamination status, plant height and survival rate of the test Phragmites australis seedlings at 30 days and 60 days, as shown in Table 4. The following is the application effect of different antibacterial agents in the rooting culture of polyploid Phragmites australis:

[0089] Table 4 Data on the application effect of a single antibacterial agent in transplanting and hardening seedlings

[0090]

[0091] Experiments have shown that different antibacterial agents have little effect on the survival rate and plant height of Phragmites australis seedlings during the transplanting and hardening stage. The survival rate of carbendazim, thiophanate-methyl, streptomycin and isothiazolinone is higher than 92%.

[0092] It should be noted that the survival rate of plants using only streptomycin during the transplanting and hardening process was significantly lower than that of chlorothalonil. Analysis showed that the use of streptomycin in the first three steps may have made the plants in this step resistant to the drug. Therefore, streptomycin can be used for short-term disinfection of Phragmites australis.

[0093] Exploration Example 10-15

[0094] Step 1: Primary cultivation of Arundo donax

[0095] In order to further reduce the contamination rate and browning rate and improve the survival rate of Phragmites australis in the open tissue culture process, we selected four fungicides with better effects and mixed them in pairs, namely streptomycin, isothiazolinone, potassium permanganate, and sodium dehydroacetate, and observed the experimental effects respectively. The steps and addition amounts were the same as those in Exploration Examples 1-9. The addition ratio of the two antibacterial agents in each group was 1:1, and the sum of the addition amounts of the two antibacterial agents in each group was 0.1g / L. The following are the experimental results after the primary culture of Phragmites australis:

[0096] Table 5 Statistics of the results of the combination of two antibacterial agents in the primary induction culture process of Arundo donax open tissue culture

[0097]

[0098]

[0099] Through exploration of Examples 10-15, it can be seen that by compounding potassium permanganate with isothiazolinone and sodium dehydroacetate, the browning rate and contamination rate are reduced, and the overall survival rate is increased. By compounding streptomycin with potassium permanganate, isothiazolinone and sodium dehydroacetate, the effect is not as good as that of using streptomycin. Only the survival rate is high, and the browning rate and contamination rate are increased. Analysis may be that potassium permanganate is a strong oxidant and streptomycin is an antibiotic. In the compound system, potassium permanganate will undergo redox reaction with streptomycin. The chemical structure of streptomycin may be destroyed under oxidation, causing the key active groups in its molecular structure to change, resulting in its inability to normally bind to bacteria or interfere with bacteria, thereby reducing the bactericidal ability of streptomycin. Streptomycin kills bacteria mainly by inhibiting bacterial protein synthesis and interfering with bacterial cell wall synthesis. Potassium permanganate kills bacteria by oxidizing bacterial cellular components and destroying bacterial structure and function. Isothiazolinone works by destroying bacterial cell membranes. When they are combined, the different pathways of action may interfere with each other, resulting in poor browning and contamination rates. By using isothiazolinone and sodium dehydroacetate in a 1:1 addition ratio, the browning and contamination rates are controlled, reaching 3.25% and 2.36% respectively, and the survival rate reaches 86.36%.

[0100] Exploration Example 16-19

[0101] Step 1: Primary cultivation of Arundo donax

[0102] In order to further improve the survival rate, we selected the best and conducted in-depth research using isothiazolinone and sodium dehydroacetate. By adjusting the addition amount and the addition ratio of the two antibacterial agents, the experimental results were observed. The following Table 6 shows the results obtained by compounding the two fungicides. Among them, in Exploration Example 16 and Exploration Example 17, the sum of the addition amounts of the two antibacterial agents was 0.1 g / L, but the mass addition ratio of isothiazolinone to sodium dehydroacetate in Exploration Example 16 was 7:3, and the mass addition ratio of isothiazolinone to sodium dehydroacetate in Exploration Example 17 was 8:2.

[0103] By using the isothiazolinone and sodium dehydroacetate addition ratio of 1:1, the browning rate and the contamination rate were controlled, reaching 3.25% and 2.36% respectively, and the survival rate reached 86.36%. Since isothiazolinone has a better effect in controlling the browning rate and the contamination rate, the addition ratio was adjusted to be more isothiazolinone and less sodium dehydroacetate. When the isothiazolinone and dehydroacetic acid addition ratio was 7:3, as shown in FIG. Figure 1 、 Figure 2 As shown, the survival rate is ≥94%. By further adjusting the ratio of the two, the effect is better. Therefore, when the addition ratio of isothiazolinone to dehydroacetic acid is 8:2, the survival rate does not increase but decreases to 88.25%. It is speculated that excessive addition of isothiazolinone has damaged the cultivation process of Phragmites australis. Therefore, it is concluded that the effect is best when the addition ratio of isothiazolinone to dehydroacetic acid is 7:3.

[0104] Then we changed the addition amount. In Exploration Examples 18 and 19, the mass addition ratio of isothiazolinone and sodium dehydroacetate was 7:3. In Exploration Example 17, the sum of the addition amounts of the two antibacterial agents, isothiazolinone and sodium dehydroacetate, was 0.03 g / L. In Exploration Example 17, the sum of the addition amounts of the two antibacterial agents, isothiazolinone and sodium dehydroacetate, was 0.5 g / L.

[0105] Table 6 Statistics of the results of the combination of isothiazolinone and sodium dehydroacetate in the primary induction culture process of Arundo donax open tissue culture

[0106]

[0107] We tried to achieve better results by increasing the proportion of isothiazolinone and sodium dehydroacetate in the culture medium, but the experimental results did not meet expectations. It is estimated that the excessive amount of antibacterial agents inhibited the growth of some plants. We tried to achieve better results by reducing the proportion of isothiazolinone and sodium dehydroacetate in the culture medium, but the experimental results did not meet expectations. The contamination rate was not well controlled, resulting in the survival rate not meeting expectations.

[0108] Therefore, it was concluded that the antibacterial effect of a mixed antibacterial agent using isothiazolinone + sodium dehydroacetate in a specific proportion and specific addition amount is outstanding, will not inhibit the growth of explants, will not harm the explants, and can achieve long-term antibacterial and disinfection effects. Moreover, when the contamination rate lasts for 40 days, the survival rate is high, and the browning rate is also low, and the survival seedling rate is good. By adopting the method of Exploration Example 16, the browning rate is ≤4.67% at 40 days during the primary culture of Phyllostachys australis, the contamination rate is 0%, and the survival rate is ≥94%.

[0109] Example 1

[0110] Step 2: Study on the application effect of polyploid Arundo donax in subculture proliferation culture

[0111] 1. Culture medium preparation

[0112] Fill a heated culture medium filling machine with distilled water and heat to 95°C before entering the heat preservation stage. Then, add all the necessary chemicals to the culture medium and dissolve them to volume. While stirring continuously, adjust the pH to 6.5 with NaOH or HCl. Quickly dispense the prepared culture medium solution into 350ml culture bottles and seal them tightly with a cap. Place the newly filled culture bottles in a clean, enclosed environment to cool and avoid exposure to the outside world. Culture medium should be prepared and used immediately. It can be stored in a clean, enclosed, low-temperature environment for short periods of time, but the storage period should not exceed 30 days.

[0113] 2. Proliferation and Culture of Seedlings

[0114] After about 40 days of primary induction culture, the buds in the culture bottle differentiate into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm; each of the cluster seedlings is cut in a closed place that has been disinfected to form 12-20 separate rootless buds with complete growth points; the rootless buds are obliquely or vertically inoculated in a culture bottle containing pH=6.5, MS basal medium, 8-10 mg / L indolebutyric acid, 0.1 g / L antibacterial agent DY1, 15 g / L sucrose, and 7 g K-type carrageenan for tissue culture, and the bottle is quickly covered and sealed, wherein the antibacterial agent DY1 is an isocyanate. A mixed solution of isothiazolinone and sodium dehydroacetate, wherein the mass addition ratio of the isothiazolinone to sodium dehydroacetate solution is 7:3, and the concentration of the antibacterial agent in the culture medium solution is 0.1 g / L; subculture proliferation culture is performed according to the above-mentioned primary culture conditions; after about 40 days of subculture proliferation culture, the buds in the culture bottle can be differentiated again into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm, and the above-mentioned proliferation culture is repeated, and proliferation is achieved through continuous subculture; statistics are performed on the 15th and 40th days of culture to determine the browning rate, contamination rate, and survival rate and value-added rate after 40 days, and the results are shown in Table 7.

[0115] Table 7 Statistics of experimental results of isothiazolinone and sodium dehydroacetate in open tissue culture subculture proliferation of Arundo donax

[0116]

[0117] Research has shown that the antibacterial effect of the mixed antibacterial agent of isothiazolinone + sodium dehydroacetate used in this step on Phragmites australis is outstanding. When it lasts for 40 days, the browning rate is ≤4%, the contamination rate remains at 0%, the survival rate is ≥94.67%, and the proliferation multiples are ≥13.86%.

[0118] Step 3: Study on the application effect of polyploid Phragmites australis rooting culture

[0119] After about 40 days of subculture induction culture, the buds in the culture bottle differentiated into polyploid Phragmites australis cluster seedlings with a plant height of more than 10 cm; in a closed place after the above disinfection treatment, each cluster seedling was cut to form 12-20 separate rootless buds with complete growth points, and they were inserted obliquely or vertically in a medium containing pH = 6.5, MS basic medium + 8-10 mg / L indolebutyric acid + 0.1 g / L antibacterial agent DY1 + 15 g sucrose + 7 g type I card for tissue culture. In the culture bottle of the glue pulling, the antibacterial agent DY1 in the above step is a mixed solution of isothiazolinone and sodium dehydroacetate, and the addition ratio of the isothiazolinone to sodium dehydroacetate solution is 7:3; rooting culture is carried out according to the above culture conditions. After about 7-15 days, the inoculated buds in the culture bottle begin to emit thorn-like root tissue; statistics are taken at 15 days and 40 days of culture to determine the browning rate, contamination rate, rooting rate, and survival rate, number of roots, and root length after 40 days. The results are shown in Table 8. Figure 3 、 Figure 4 shown.

[0120] Table 8 Statistics of the results of the combination of isothiazolinone and sodium dehydroacetate in the rooting of Arundo donax open tissue culture

[0121]

[0122]

[0123] Studies have shown that in the open tissue culture rooting process of Arundo donax, the antibacterial effect of a mixed solution of isothiazolinone and sodium dehydroacetate on Arundo donax is outstanding. Combined with the culture medium of this application, the effect is good. When it lasts for 40 days, the rooting rate reaches 93.33%, the contamination rate remains at 0%, and the survival rate is ≥93.33%.

[0124] Step 4: Study on the application effect in transplanting and hardening seedlings

[0125] After 30-60 days of rooting culture, the buds in the culture bottles can grow into plants over 10 cm in length with 3-5 stem nodes and complete root systems, and then enter the seedling hardening stage; the above plants are removed from the culture bottles, and the culture medium remaining on the roots is washed with clean water, and then transplanted into a seedling container filled with seedling medium. The seedling medium used is a compound of vermiculite, peat, perlite and fine river sand, and is sprayed with 0.7g / L of antibacterial agent DY1 for disinfection before use. The antibacterial agent DY1 is a mixed solution of isothiazolinone and sodium dehydroacetate, and the isothiazolinone to sodium dehydroacetate solution is added in a ratio of 7:3. Immediately after transplanting, water thoroughly with an atomizing nozzle, then cover with a plastic film arch shed, and shade with a black sunshade net with a light transmittance of 60% to avoid direct sunlight. Three days after transplanting, the plastic film can be removed to allow adequate ventilation during periods of insufficient sunlight, and water can be added as needed. After seven days, the ventilation period can be gradually increased, and the film can be completely removed after 15 days. After 30 days, the shade net can be removed as appropriate. Ensure adequate water supply throughout the transplanting and hardening period. Depending on the facility, spray 0.7 ml / L of DY1, a mixed solution of isothiazolinone and sodium dehydroacetate, at a ratio of 7:3, every 10-15 days. Foliar fertilizers such as potassium dihydrogen phosphate should be applied after the polyploid Phragmites australis seedlings begin to grow normally. Contamination status, plant height, and survival rate of the tested Phragmites australis seedlings were recorded at 30 and 60 days, as shown in Table 9.

[0126] Table 9 Application effect data of isothiazolinone and sodium dehydroacetate in transplanting and hardening seedlings

[0127]

[0128] The experiment showed that, in the transplanting and hardening stage, the application effect of the antibacterial agent in Example 1 in the transplanting and hardening was that the survival rate reached 96.33%, the plant height was 27.35 cm, and the contamination rate was 0%.

[0129] The culture solution of the present invention is compounded with a suitable plant growth regulator and a novel antibacterial agent to form an open tissue culture medium formula. The tissue culture rapid propagation medium of the present invention is used, and axillary bud stem segments of superior polyploid Phragmites australis plants are used as explants. The entire culture process does not require high-temperature and high-pressure sterilization or strict sterile environment control, and the culture process is simple and easy. The subculture proliferation culture stage adopts a bud culture method, which is free of mutation. During the rooting culture stage, no callus tissue is formed at the base, resulting in good rooting effect and a high transplant survival rate.

[0130] At present, there are no reports in the field of polyploid reed tissue culture seedlings. Conventional tissue culture seedling methods are divided into two steps: subculture and rooting induction, and the seedling cycle is long. In addition, the inoculation technology is difficult to implement and has certain requirements on the quality of the inoculator, which is not convenient for improving the production capacity and speed of the tissue culture center. The required high-pressure and high-temperature sterilization facilities consume huge electricity and have high requirements on the sterility level of the facilities and the environment, and the construction and maintenance investment are huge. The present invention adopts a new type of bactericidal and antibacterial agent to carry out open tissue culture and expansion. The open tissue culture and rapid propagation method of polyploid reed of the present invention does not require high-temperature and high-pressure sterilization in the entire culture process, does not require strict sterile environment control, and reduces the quality requirements of the inoculators for tissue culture production, thereby reducing the construction threshold and cost investment of the tissue culture center. The present invention achieves cost reduction and efficiency improvement in the tissue culture and expansion production of polyploid reed, and the comprehensive cost savings of the entire culture process are 15%-30%, shortening the tissue culture production cycle, which is conducive to the expansion of the production capacity of polyploid reed seedlings and the promotion of the polyploid reed industry.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of an antibacterial agent in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: The antibacterial agent is applied in the open tissue culture rapid propagation process of polyploid Phragmites australis, and is used by adding it to a culture medium, spraying it, or washing it with vibration. The antibacterial agent is prepared by mixing isothiazolinone and sodium dehydroacetate, and the mass addition ratio of the isothiazolinone to the sodium dehydroacetate is 8-5:2-5.

2. The use of a bacteriostatic agent according to claim 1 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: The mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:

3.

3. The use of a bacteriostatic agent according to claim 2 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that The application process is as follows: S1: Primary explant culture Insert the sterilized explants into the culture medium for cultivation; S2: Proliferation and culture of seedlings The polyploid Phragmites australis seedlings cultured in step S1 are connected to the stem segments of the original explants and then cultured. After induction culture, each clustered seedling is cut into a plurality of rootless buds with complete growth points; inoculating the rootless buds into a culture medium and continuing to culture; S3: Rooting Culture After S2 subculture induction culture, each of the clustered seedlings is cut into a number of individual rootless buds with complete growth points, and the buds are inoculated into a culture medium for rooting culture; In steps S1, S2, and S3, the antibacterial agent added to the culture medium is a mixed solution of isothiazolinone and sodium dehydroacetate, and the culture medium contains 0.05 g-1 g / L of the antibacterial agent.

4. The use of a bacteriostatic agent according to claim 3 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: The sum of the added amounts of the isothiazolinone and sodium dehydroacetate is 0.1 g / L; the mass addition ratio of the isothiazolinone to sodium dehydroacetate is 7:

3.

5. The use of a bacteriostatic agent according to claim 4 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: In step S1, the culture medium used for primary culture is: 1 / 2MS as the basal medium, supplemented with 8-10 mg / L indolebutyric acid, 0.05 g / L-1 g / L antibacterial agent, 15 g / L sucrose, and 7 g / L tissue culture type I or type K carrageenan; The culture conditions were as follows: pH 6.5, culture room temperature 25 ± 2°C, photoperiod D / N = 12 h / 12 ​​h, and illumination 1500-2000 lx; Step S2: inoculating the rootless buds into a medium containing a pH of 6.5, using MS as a basal medium, supplemented with 8-10 mg / L indolebutyric acid, 0.05 g / L-1 g / L antibacterial agent, 15 g / L sucrose, and 7 g / L tissue culture type I or type K carrageenan, tightly capping the culture bottle and sealing it. The culture conditions are: a culture room temperature of 25±2°C, a photoperiod of D / N=12h / 12h, and an illumination of 1500-2000lx. In step S3, after the subculture induction culture in S2, each of the clustered seedlings is cut into several separate rootless buds with complete growth points, and inoculated into a culture medium with pH = 6.5 for rooting culture. MS is used as the basic culture medium, and 8-10 mg / L indolebutyric acid, 0.05 g-1 g / L antibacterial agent, 15 g / L sucrose, and 7 g / L type I or type K carrageenan for tissue culture are added. The culture bottle is covered with a cap and sealed.

6. The use of a bacteriostatic agent according to claim 5 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: After culturing in step S3, the buds in the culture bottles differentiate into polyploid Phragmites australis clustered seedlings with a plant height of more than 10 cm; each of the clustered seedlings is cut in a sterilized, enclosed space to form a plurality of individual rootless buds with complete growth points; Plants with stems extending to more than 10 cm are transplanted. When transplanting, the seedlings are removed from the culture medium, the culture medium remaining on the roots is washed with clean water, and then transplanted into a seedling container filled with a seedling medium; After transplanting, water thoroughly and cover with plastic film shed and shade net; 3 days after transplanting, remove the plastic film to allow air to flow and water to the plants; After 7 days, extend the ventilation time, remove the film after 15 days, and remove the sunshade net after 30 days.

7. The use of a bacteriostatic agent according to claim 6 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: The seedling culture medium is compounded by vermiculite, peat, perlite and fine river sand, and is sprayed with 0.05g / L-1g / L antibacterial agent for disinfection before use. The antibacterial agent is the same as that in step S1; During the entire transplanting and hardening period, 0.05g / L-1g / L antibacterial agent is sprayed at intervals of 10-15 days. The antibacterial agent is the same as that in step S1, and foliar fertilizers such as potassium dihydrogen phosphate are applied after the polyploid Phragmites australis seedlings begin to grow normally.

8. The use of a bacteriostatic agent according to claim 7 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: Spray 0.7g / L antibacterial agent for disinfection before use, and the mass addition ratio of isothiazolinone to sodium dehydroacetate is 7:3; The 0.7 g / L antibacterial agent was sprayed at an interval of 10-15 days, and the mass addition ratio of the isothiazolinone to sodium dehydroacetate was 7:

3.

9. The use of a bacteriostatic agent according to any one of claims 1 to 8 in the open tissue culture rapid propagation of polyploid Phragmites australis, characterized in that: Before primary explant culture, polyploid Phragmites australis plants are cut into stem segments with axillary buds as explants, which are then cleaned and disinfected. The specific steps are as follows: The explant is placed in a sterilized sealed container, and is continuously shaken and rinsed 3-8 times with a mixed solution of isothiazolinone and sodium dehydroacetate, then immersed in a 1.5‰ mercuric chloride solution for 7-9 minutes for sterilization, and is continuously shaken and rinsed 3-4 times with the mixed solution of isothiazolinone and sodium dehydroacetate. The mass addition ratio of the isothiazolinone to sodium dehydroacetate in the above steps is (8-5):(2-5); the added concentration of the isothiazolinone and sodium dehydroacetate is 0.05g / L-1g / L.

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