A bacteriostatic medium for tissue culture of arundo donax and application thereof
By using specific antibacterial agents at different stages of Reed truncatum tissue culture, the problem of endophytic bacteria contamination during tissue culture was solved, improving seedling efficiency and tissue culture results, and providing a material basis for Reed truncatum biobreeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN RUNDO BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-02
AI Technical Summary
During the tissue culture process of Phragmites australis, endophytic bacteria contamination significantly reduced the induction, proliferation, and rooting abilities of the tissue culture seedlings, thus decreasing the seedling production efficiency.
Specific types and amounts of antibacterial agents were used to work synergistically in different stages of Arundo donax tissue culture, including induction medium, proliferation medium and rooting medium. Antibacterial agents such as chlorfenapyr, PPM and copper sulfate were added respectively. endophytic bacterial contamination was reduced through surface disinfection and specific culture conditions.
It significantly reduces endophytic bacteria contamination in Arundo donax tissue culture, improves the efficiency of Arundo donax tissue culture, enhances the induction, proliferation and rooting ability of tissue culture seedlings, ensures the preservation of valuable materials, and provides a material basis for bio-breeding.
Smart Images
Figure CN122123324A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to an antibacterial culture medium for Reed amurensis tissue culture and its application. Background Technology
[0002] Contamination in plant tissue culture is one of the biggest obstacles to tissue culture production, directly impacting production costs and the preservation of valuable research materials. Current technologies and industry understanding primarily focus on preventing contamination in open spaces, i.e., avoiding the invasion of exogenous microorganisms through purifying the operating environment, strengthening surface disinfection, and implementing aseptic procedures. However, conventional methods are completely ineffective against contamination caused by endophytic bacteria carried within the plant in aseptic spaces. This type of contamination mainly originates from within the plant, cannot be eradicated by surface disinfection, and after the endophytic bacteria recover and proliferate in the nutrient-rich environment of tissue culture, it leads to systemic and delayed contamination of the culture, causing incalculable research losses and production interruptions. Currently, the industry lacks efficient, specialized, and plant-friendly inhibition solutions for this type of endophytic bacterial contamination.
[0003] Arundo donax, a perennial plant belonging to the genus Arundo in the family Poaceae, has tall stems and a strong tillering ability, resulting in a large biomass. The harvested stems can be directly used to replace coal in thermal power generation, or processed downstream to produce clean energy sources such as hydrogen, natural gas, and biochar, thus potentially replacing fossil fuels and addressing the energy crisis. However, during tissue culture of Arundo donax, endophytic bacteria can cause contamination, significantly reducing the induction, proliferation, and rooting abilities of tissue-cultured seedlings, thereby lowering the efficiency of tissue culture seedling production.
[0004] In view of this, it is necessary to provide an antibacterial culture medium for Reed tissue culture to overcome the shortcomings of the prior art. Summary of the Invention
[0005] The purpose of this invention is to provide an antibacterial culture medium for Arundo donax tissue culture and its application. This addresses the problem that in existing Arundo donax tissue culture processes, endophytic bacteria can cause contamination, leading to a significant reduction in the induction, proliferation, and rooting abilities of tissue-cultured seedlings, thus reducing the efficiency of Arundo donax tissue culture seedling cultivation.
[0006] In a first aspect, the present invention provides an antibacterial culture medium for Arundo donax tissue culture, comprising a first antibacterial culture medium, a second antibacterial culture medium, and a third antibacterial culture medium; wherein the first antibacterial culture medium comprises an induction medium and 0.05-0.15% by weight of a bactericide; the second antibacterial culture medium comprises a proliferation medium, 0.03-0.07% by volume of PPM, and copper sulfate at a concentration of 5-15 mg / L; and the third antibacterial culture medium comprises a rooting medium and copper sulfate at a concentration of 3-7 mg / L.
[0007] In this invention, the inventors discovered that by adding specific types and amounts of antibacterial agents to the culture media used at different stages of Reed phlox tissue culture, the antibacterial agents in different culture media can significantly reduce the contamination of Reed phlox endophytic bacteria in Reed phlox tissue culture through synergistic effects, reduce the generation of contaminated seedlings during the Reed phlox tissue culture process, improve the efficiency of Reed phlox tissue culture, ensure the preservation of precious Reed phlox materials through tissue culture, and provide a material basis for the biological breeding of Reed phlox.
[0008] In some embodiments, the induction medium in the first antimicrobial medium comprises the following components: MS + 1.5-2.5 mg / L 2,4-D + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0009] In some embodiments, the proliferation medium in the second antimicrobial medium comprises the following components: MS + 0.5-1.5 mg / L 6-BA + 0.1-0.3 mg / L NAA + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0010] In some embodiments, the rooting medium in the third antimicrobial medium comprises the following components: 1 / 2 MS + 0.3-0.7 mg / L IBA + 15-25 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0011] In a second aspect, the present invention provides the application of any of the above-mentioned antibacterial culture media in the tissue culture of Reed hyacinth.
[0012] In a third aspect, the present invention provides the use of any of the above-mentioned antibacterial culture media in inhibiting endophytic bacterial contamination in Arundo donax.
[0013] In a fourth aspect, the present invention provides a method for tissue culture of Reed hyacinth using any of the above-mentioned antibacterial culture media, comprising the following steps: taking the stem tip of Reed hyacinth, after surface disinfection, inoculating it in a first antibacterial culture medium for induction culture to obtain induced buds; inoculating the induced buds in a second antibacterial culture medium for proliferation culture to obtain clustered seedlings; and inoculating the clustered seedlings in a third antibacterial culture medium for rooting culture to obtain tissue culture seedlings.
[0014] In some implementation schemes, the step of taking the stem tips of *Arundinaria salsa*, disinfecting them, and then inoculating them into a first antibacterial culture medium for induction culture includes the following steps: first, rinsing with running water for 1-3 hours; then, immersing in 70-80% ethanol for 25-35 seconds under sterile conditions, followed by rinsing with sterile water 1-3 times, then immersing in 1-3% sodium hypochlorite solution for 10-20 minutes, and finally rinsing with sterile water 3-5 times; the induction culture includes culturing for 25-35 days under conditions of 23-27℃, 16h / 8h light intensity, and 1500-2500 lux light intensity.
[0015] In some implementations, the step of inoculating the induced buds into a second antibacterial medium for proliferation culture specifically includes: culturing for 40-50 days under conditions of 23-27°C, 16h / 8h light, and 1500-2500 lux light intensity; then transferring once and continuing the proliferation culture.
[0016] In some implementations, the rooting culture step of inoculating the clump seedlings into a third antibacterial medium specifically includes: culturing for 45-60 days under conditions of a temperature of 23-27°C, a light intensity of 16h / 8h, and a light intensity of 1500-2500 lux.
[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention adds specific types and amounts of antibacterial agents to the culture media used at different stages of Reed phlox tissue culture. Through synergistic effects, the antibacterial agents in different culture media can significantly reduce endophytic bacterial contamination in Reed phlox tissue culture, reduce the generation of contaminated seedlings during the Reed phlox tissue culture process, improve the efficiency of Reed phlox tissue culture, ensure the preservation of valuable Reed phlox materials, and provide a material basis for Reed phlox biobreeding. Therefore, the antibacterial culture medium provided by this invention has good application prospects in inhibiting endophytic bacterial contamination in Reed phlox and / or in Reed phlox tissue culture. Attached Figure Description
[0018] Figure 1 These are physical images of pollution (A) caused by the environment and operation and pollution (B) caused by plant endophytic bacteria in this invention; Figure 2 This is a diagram showing the growth of the clump-forming seedlings in groups PM-1 (A) and PM-5 (B) in Example 2 of the present invention. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.
[0021] Currently, during the tissue culture process of Arundo donax, endophytic bacteria can cause contamination, leading to a significant reduction in the induction, proliferation, and rooting abilities of tissue culture seedlings, and consequently reducing the efficiency of Arundo donax tissue culture seedling cultivation.
[0022] To address the problems of contamination by endophytic bacteria during existing tissue culture processes of Arundo donax, which significantly reduces the induction, proliferation, and rooting abilities of tissue culture seedlings and consequently lowers the efficiency of Arundo donax tissue culture seedling cultivation, this invention provides an antibacterial culture medium for Arundo donax tissue culture and its application.
[0023] In a first aspect, the present invention provides an antibacterial culture medium for *Arundinaria lobata* tissue culture, comprising a first antibacterial culture medium, a second antibacterial culture medium, and a third antibacterial culture medium; wherein the first antibacterial culture medium comprises an induction medium and a bactericide at a mass percentage of 0.05-0.15% (e.g., 0.05%, 0.07%, 0.1%, 0.13%, 0.15%, or other values within this range); the second antibacterial culture medium comprises a proliferation medium and a volume percentage of 0.03-0.07% (e.g., 0.03%, 0.07%, 0.1%, 0.13%, 0.15%, or other values within this range); The third antibacterial medium includes rooting medium and copper sulfate at a concentration of 3-7 mg / L (e.g., 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, or other values within the range);
[0024] The antibacterial culture medium provided by this invention, by adding specific types and amounts of antibacterial agents to the culture media used at different stages of Reed phlox tissue culture, can significantly reduce endophytic bacterial contamination in Reed phlox tissue culture through synergistic effects among the antibacterial agents in different culture media. This reduces the generation of contaminated seedlings during the Reed phlox tissue culture process, improves the efficiency of Reed phlox tissue culture, ensures the preservation of valuable Reed phlox materials, and provides a material basis for Reed phlox bio-breeding. Therefore, the antibacterial culture medium provided by this invention can enhance the induction, proliferation, and rooting ability of Reed phlox tissue culture seedlings, improve the efficiency of Reed phlox bio-breeding, and lay a solid foundation for Reed phlox to convert into new energy sources.
[0025] In some embodiments, the induction medium in the first antimicrobial medium comprises the following components: MS + 1.5-2.5 mg / L (e.g., 1.5 mg / L, 1.7 mg / L, 2 mg / L, 2.3 mg / L, 2.5 mg / L or other values within this range), 2,4-D + 25-35 g / L (e.g., 25 g / L, 27 g / L, 30 g / L, 33 g / L, 35 g / L or other values within this range), sucrose + 5-9 g / L (e.g., 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or other values within this range), and agar, with a pH of 5.6-6, e.g., 5.6, 5.7, 5.8, 5.9, 6 or other values within this range.
[0026] It is understood that the components in the induction medium can be routinely adjusted according to actual needs, as long as it can efficiently induce buds. For example, in this invention, the induction medium preferably includes the following components: MS + 1.5-2.5 mg / L 2,4-D + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0027] In some embodiments, the proliferation medium in the second antimicrobial medium comprises the following components: MS + 0.5-1.5 mg / L (e.g., 0.5 mg / L, 0.7 mg / L, 1 mg / L, 1.3 mg / L, 1.5 mg / L, or other values within this range) and 6-BA + 0.1-0.3 mg / L (e.g., 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, or other values within this range). Other values within this range) NAA + 25-35 g / L (e.g., 25 g / L, 27 g / L, 30 g / L, 33 g / L, 35 g / L or other values within this range) Sucrose + 5-9 g / L (e.g., 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or other values within this range) Agar, pH 5.6-6, e.g., 5.6, 5.7, 5.8, 5.9, 6 or other values within this range.
[0028] It is understood that the components in the proliferation medium can be routinely adjusted according to actual needs, as long as efficient proliferation of clump-forming seedlings can be achieved. For example, in this invention, the proliferation medium preferably includes the following components: MS + 0.5-1.5 mg / L 6-BA + 0.1-0.3 mg / L NAA + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0029] In some embodiments, the rooting medium in the third antimicrobial medium comprises the following components: 1 / 2 MS + 0.3-0.7 mg / L (e.g., 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L or other values within this range), IBA + 15-25 g / L (e.g., 15 g / L, 17 g / L, 20 g / L, 23 g / L, 25 g / L or other values within this range), sucrose + 5-9 g / L (e.g., 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or other values within this range), and agar, with a pH of 5.6-6, e.g., 5.6, 5.7, 5.8, 5.9, 6 or other values within this range.
[0030] It is understandable that the components in the rooting medium can be routinely adjusted according to actual needs, as long as efficient rooting and tissue culture seedlings can be obtained. For example, in this invention, the rooting medium preferably includes the following components: 1 / 2 MS + 0.3-0.7 mg / L IBA + 15-25 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
[0031] In a second aspect, the present invention provides the application of any of the above-mentioned antibacterial culture media in the tissue culture of Reed hyacinth.
[0032] In this invention, the use of antibacterial culture medium in Arundo donax tissue culture can significantly reduce endophytic bacterial contamination in Arundo donax tissue culture, enhance the induction, proliferation and rooting ability of Arundo donax tissue culture seedlings, and further improve the efficiency of Arundo donax biobreeding.
[0033] In a third aspect, the present invention provides the use of any of the above-mentioned antibacterial culture media in inhibiting endophytic bacterial contamination in Arundo donax.
[0034] In this invention, the antibacterial culture medium can significantly reduce the contamination of endophytic bacteria in Arundo donax tissue culture, reduce the generation of contaminated seedlings during the Arundo donax tissue culture process, and further improve the efficiency of Arundo donax tissue culture.
[0035] In a fourth aspect, the present invention provides a method for tissue culture of Reed hyacinth using any of the above-mentioned antibacterial culture media, comprising the following steps: taking the stem tip of Reed hyacinth, after surface disinfection, inoculating it in a first antibacterial culture medium for induction culture to obtain induced buds; inoculating the induced buds in a second antibacterial culture medium for proliferation culture to obtain clustered seedlings; and inoculating the clustered seedlings in a third antibacterial culture medium for rooting culture to obtain tissue culture seedlings.
[0036] The method for tissue culture of Reed hyacinth provided by this invention is simple, uses inexpensive and readily available raw materials, and through the synergistic effect between different antibacterial agents in the culture medium, it can significantly reduce the contamination of Reed hyacinth endophytic bacteria in tissue culture. Therefore, it has good application prospects.
[0037] In some implementations, in the step of taking *Arundinaria salsa* stem tips, surface disinfecting them, and then inoculating them into a first antibacterial culture medium for induction culture, the surface disinfection treatment specifically includes: first rinsing with running water for 1-3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or other values within this range; then, under sterile conditions, immersing in 70-80% (for example, 70%, 72%, 75%, 78%, 80%, or other values within this range) ethanol for 25-35 seconds, for example, 25 seconds, 27 seconds, 30 seconds, 33 seconds, 35 seconds, or other values within this range; then rinsing with sterile water 1-3 times, for example, once, twice, three times, or other values within this range; and finally, rinsing with 1-3% (for example, 1%, 1.5%, 2%, 2.5%, 3%, or other values within this range) hypochlorite solution. Soak in sodium sulfate solution for 10-20 minutes, for example, 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes or other values within this range; finally rinse with sterile water 3-5 times, for example, 3 times, 4 times, 5 times or other values within this range; induction culture specifically includes: culturing for 25-35 days under the following conditions: temperature of 23-27℃ (for example, 23℃, 24℃, 25℃, 26℃, 27℃ or other values within this range), light intensity of 16h / 8h, and light intensity of 1500-2500 lux (for example, 1500 lux, 1700 lux, 2000 lux, 2300 lux, 2500 lux or other values within this range).
[0038] In this invention, by using a first antibacterial culture medium during the induction culture process, the contamination of endophytic bacteria in Arundo donax can be significantly reduced, thereby increasing the formation rate of induced buds.
[0039] In some embodiments, the step of inoculating the induced buds into a second antibacterial medium for proliferation culture specifically includes: culturing for 40-50 days, for example, at a temperature of 23-27°C (e.g., 23°C, 24°C, 25°C, 26°C, 27°C, or other values within this range), a light intensity of 16h / 8h, and a light intensity of 1500-2500 lux (e.g., 1500 lux, 1700 lux, 2000 lux, 2300 lux, 2500 lux, or other values within this range); then subculturing once and continuing the proliferation culture.
[0040] In this invention, by using a second antibacterial culture medium during the proliferation culture process, the contamination of endophytic bacteria in Arundo donax can be significantly reduced, thereby increasing the proliferation coefficient of clump seedlings.
[0041] In some implementations, the rooting culture step of inoculating the clump seedlings into a third antibacterial medium specifically includes: culturing for 45-60 days under conditions of a temperature of 23-27°C (e.g., 23°C, 24°C, 25°C, 26°C, 27°C or other values within this range), a light intensity of 16h / 8h, and a light intensity of 1500-2500 lux (e.g., 1500 lux, 1700 lux, 2000 lux, 2300 lux, 2500 lux or other values within this range).
[0042] In this invention, by using a third antibacterial culture medium during the rooting culture process, the contamination of endophytic bacteria in Phyllostachys amurensis can be significantly reduced, thereby increasing the rooting rate of tissue culture seedlings.
[0043] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0044] Please see Figure 1 The images show physical examples of pollution caused by the environment and operations (A) and pollution caused by plant endophytes (B), in which plant endophyte contamination produces transparent filamentous substances and affects the normal growth of Reed.
[0045] Example 1 Screening of antibacterial agents In this embodiment, the aim is to screen antibacterial agents that have a good inhibitory effect on the endophytic bacteria (Ailanthus albus) of Arundo donax.
[0046] Specifically, it includes the following steps: 1) Take the tender stems of healthy Reed japonica plants from the field and rinse them under running water for 2 hours. In a clean bench, soak them in 75% ethanol for 30 seconds, rinse once with sterile water, then soak them in 2% sodium hypochlorite solution (with 1-2 drops of Tween-80) for 15 minutes, shaking constantly. Finally, rinse 3-5 times with sterile water. Cut the surface-sterilized stem segments into 0.5cm pieces, grind them in a sterile mortar, and prepare a tissue homogenate with sterile physiological saline. Spread 100μL of the homogenate onto NA and PDA plates and incubate at 28℃ for 3-7 days. Select and purify dominant colonies of different morphologies. Perform 16S rDNA sequencing on a frequently occurring, fast-growing dominant bacterial strain RD-B1, which was identified as *Bacillus pallida* (…). Ochrobactrum sp .).
[0047] 2) Using *Bacillus oryzae* RD-B1 from step 1) as the target, the antibacterial effects of PPM, phytosin, chlorfenapyr, uniconazole, and copper sulfate were tested. Specifically, the liquid dilution method was used: each antibacterial agent was prepared into a stock solution with sterile water and added to sterilized NA liquid medium cooled to 50-60℃. The final concentrations were: PPM 0.1% (volume percentage), phytosin 1.2% (volume percentage), chlorfenapyr 0.15% (mass percentage), uniconazole 20 mg / L, and copper sulfate 20 mg / L. An equal volume of RD-B1 bacterial suspension was inoculated into each tube, with an RD-B1 bacterial suspension without antibacterial agents serving as a blank control. After incubation at 28℃ and 180 rpm for 24 h with shaking, the OD was measured using a microplate reader. 600nm The results are shown in Table 1 below.
[0048] Table 1. Screening results of antibacterial agents
[0049] As shown in Table 1, 0.1% PPM, 20 mg / L copper sulfate, and 0.15% chlorhexidine all showed inhibition rates of more than 95% against Aristolochic Acid RD-B1. Therefore, these three antibacterial agents were selected for subsequent research on Arundo donax tissue culture.
[0050] Example 2: Screening and preparation of antibacterial culture medium for Reed Arundo donax tissue culture and application test of the antibacterial culture medium In this embodiment, an antibacterial culture medium for Arundo donax tissue culture is first screened and prepared, and then a method for Arundo donax tissue culture is developed based on the antibacterial culture medium.
[0051] Specifically, the first step is to screen the types of antibacterial agents added to the induction culture medium, including the following steps: 1) Prepare induction medium according to the composition of the induction medium (MS + 2 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.8). After autoclaving the induction medium, cool it to about 50°C in a laminar flow hood. Add the filtered sterilized antibacterial stock solution under aseptic conditions and perform the following treatments: IM-1: induction medium (control), IM-2: induction medium + 0.1% PPM, IM-3: induction medium + 15 mg / L copper sulfate, IM-4: induction medium + 0.1% chlorhexidine. 2) The stem segments that were surface disinfected in Example 1 were cut into 1-1.5cm pieces and inoculated into the culture medium of the above treatment. Each treatment had 30 explants, and the results were repeated 3 times. The plants were cultured for 30 days under the conditions of 25℃, 16h / 8h light (16h light, 8h darkness) and 2000 lux light intensity. The contamination of endophytic fungi in each treatment was observed, and the induced bud germination rate was counted. The results are shown in Table 2 below.
[0052] Table 2. Screening results of antibacterial agents in induction culture medium
[0053] As shown in Table 2, adding 0.1% of Chlorfenapyr to the induction medium significantly inhibits contamination by endophytic fungi in Arundo donax, thereby improving the bud germination rate. Therefore, Chlorfenapyr is the optimal antibacterial agent in the induction medium.
[0054] Next, the types of antibacterial agents added to the proliferation medium were screened, including the following steps: 1) Prepare the proliferation medium according to the composition of the proliferation medium (MS + 1 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8). After autoclaving the proliferation medium, cool it to about 50°C in a clean bench, and add the filtered sterilized antibacterial stock solution under aseptic conditions. Perform the following treatments respectively: PM-1: proliferation medium (control), PM-2: proliferation medium + 0.1% PPM, PM-3: proliferation medium + 15 mg / L copper sulfate, PM-4: proliferation medium + 0.1% chlorhexidine, PM-5: proliferation medium + 0.05% PPM + 10 mg / L copper sulfate; 2) The induced shoots from the IM-4 treatment group were inoculated into the culture medium of the above treatment, and repeated 3 times. They were cultured for 45 days under the conditions of 25℃, 16h / 8h light (16h light, 8h darkness) and 2000 lux light intensity. They were then transferred once and cultured for another 45 days under the above conditions. The contamination of endophytic fungi in each treatment was observed and the proliferation coefficient was calculated. The results are shown in Table 3 below.
[0055] The growth of the clump-forming seedlings in the PM-1 and PM-5 groups is as follows: Figure 2 As shown.
[0056] Table 3. Screening results of antibacterial agents in proliferation culture medium
[0057] From Table 3 and Figure 2 The results show that adding 0.05% PPM + 10 mg / L copper sulfate to the proliferation medium can significantly inhibit the contamination of endophytic fungi in Arundo donax, thereby increasing the proliferation coefficient of clump-forming seedlings. Therefore, the optimal antibacterial agent in the proliferation medium is the combination of PPM and copper sulfate.
[0058] Finally, the types of antibacterial agents added to the rooting medium were screened, including the following steps: 1) Prepare rooting medium according to the components of the rooting medium (1 / 2 MS + 0.5 mg / L IBA + 20 g / L sucrose + 7 g / L agar, pH 5.8). After autoclaving the rooting medium, cool it to about 50°C in a clean bench, and add the filtered sterilized antibacterial stock solution under aseptic conditions. Perform the following treatments respectively: RM-1: rooting medium (control), RM-2: rooting medium + 0.1% PPM, RM-3: rooting medium + 5 mg / L copper sulfate, RM-4: rooting medium + 0.1% chlorhexidine, RM-5: rooting medium + 0.05% PPM + 10 mg / L copper sulfate; 2) The clump-forming seedlings in the PM-5 treatment group were inoculated into the culture medium of the above treatment, and repeated 3 times; and cultured for 55 days under the conditions of temperature of 25℃, light of 16h / 8h (16h light, 8h darkness) and light intensity of 2000 lux; the endophytic fungal contamination of Arundo donax in each treatment was observed, and the rooting rate and average number of roots were counted. The results are shown in Table 4 below.
[0059] Table 4. Screening results of antibacterial agents in rooting culture medium
[0060] As shown in Table 4, adding 5 mg / L copper sulfate to the rooting medium can significantly inhibit the contamination of endophytic fungi in Arundo donax, thereby improving the rooting rate and average number of roots in tissue culture seedlings. Therefore, copper sulfate is the optimal antibacterial agent in the rooting medium.
[0061] Based on the above results, the antibacterial culture medium for Arundo donax tissue culture determined by the present invention includes a first antibacterial culture medium, a second antibacterial culture medium, and a third antibacterial culture medium. The first antibacterial culture medium included induction medium (MS + 2 mg / L 2,4-D + 30 g / L sucrose + 7 g / L agar, pH 5.8) and 0.1% of chlorfenapyr. The second antibacterial culture medium includes proliferation medium (MS + 1 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8), 0.05% PPM by volume, and 10 mg / L copper sulfate by mass. The third antibacterial culture medium includes rooting medium (1 / 2 MS + 0.5 mg / L IBA + 20 g / L sucrose + 7 g / L agar, pH 5.8) and copper sulfate with a mass concentration of 5 mg / L.
[0062] Based on the above-mentioned antibacterial culture medium, the present invention provides a method for tissue culture of *Arundinaria lobata* using the above-mentioned antibacterial culture medium, comprising the following steps: S1. Take the stem tips of Reed Bamboo, rinse with running water for 2 hours, soak in 75% ethanol for 30 seconds in a clean bench, rinse once with sterile water, then soak in 2% sodium hypochlorite solution (with 1-2 drops of Tween-80) for 15 minutes, shaking continuously during the process. Finally, rinse with sterile water 3-5 times, cut the stem segments into 1-1.5cm pieces, and inoculate them into the first antibacterial medium. Under the conditions of 25℃, 16h / 8h light (16h light, 8h darkness), and 2000 lux light intensity, culture for 30 days to obtain induced buds. S2. The induced buds obtained in step S1 are inoculated into the second antibacterial medium and cultured for 45 days under the conditions of 25℃, 16h / 8h light (16h light, 8h darkness) and 2000 lux light intensity. The buds are then transferred once and cultured for another 45 days under the above conditions to obtain clump seedlings. S3. The clump-forming seedlings were inoculated into the third antibacterial medium and cultured for 55 days at a temperature of 25℃, a light intensity of 16h / 8h (16h light, 8h darkness) and a light intensity of 2000 lux; tissue culture seedlings were obtained.
[0063] In summary, this invention, by adding specific types and amounts of antibacterial agents to the culture media used at different stages of Arundo donax tissue culture, can significantly reduce endophytic bacterial contamination in Arundo donax tissue culture through the synergistic effect of antibacterial agents in different culture media, thereby reducing the generation of contaminated seedlings during the Arundo donax tissue culture process and improving the efficiency of Arundo donax tissue culture.
[0064] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.
[0065] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. An antibacterial culture medium for Reed amurensis tissue culture, characterized in that, Including the first antibacterial culture medium, the second antibacterial culture medium, and the third antibacterial culture medium; The first antibacterial culture medium includes an induction culture medium and 0.05-0.15% (by weight) of a bactericide. The second antibacterial culture medium includes a proliferation medium, 0.03-0.07% PPM by volume, and copper sulfate with a mass concentration of 5-15 mg / L; The third antibacterial culture medium includes a rooting medium and copper sulfate with a mass concentration of 3-7 mg / L.
2. The antibacterial culture medium according to claim 1, characterized in that, In the first antibacterial culture medium, the induction culture medium comprises the following components: MS + 1.5-2.5 mg / L 2,4-D + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
3. The antibacterial culture medium according to claim 1, characterized in that, In the second antibacterial culture medium, the proliferation medium comprises the following components: MS + 0.5-1.5 mg / L 6-BA + 0.1-0.3 mg / L NAA + 25-35 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
4. The antibacterial culture medium according to claim 1, characterized in that, In the third antibacterial medium, the rooting medium comprises the following components: 1 / 2 MS + 0.3-0.7 mg / L IBA + 15-25 g / L sucrose + 5-9 g / L agar, with a pH of 5.6-6.
5. The application of the antibacterial culture medium as described in any one of claims 1-4 in the tissue culture of Reed truncata.
6. The use of the antibacterial culture medium as described in any one of claims 1-4 in inhibiting endophytic bacteria contamination in Reed ferns.
7. A method for tissue culture of *Arundo donax* using the antibacterial culture medium according to any one of claims 1-4, characterized in that, Includes the following steps: The tips of Reed shoots were taken, and after surface disinfection, they were inoculated into the first antibacterial culture medium for induction culture to obtain induced buds; The induced buds were inoculated into a second antibacterial medium for proliferation culture to obtain clustered seedlings; The clump-forming seedlings were inoculated into a third antibacterial medium for rooting culture to obtain tissue culture seedlings.
8. The method according to claim 7, characterized in that, In the step of taking the stem tips of Reed hyacinth, after surface disinfection, and inoculating them into the first antibacterial culture medium for induction culture, the surface disinfection treatment specifically includes: first rinsing with running water for 1-3 hours; then soaking in 70-80% ethanol for 25-35 seconds under sterile conditions, followed by rinsing with sterile water 1-3 times, then soaking in 1-3% sodium hypochlorite solution for 10-20 minutes, and finally rinsing with sterile water 3-5 times. The induction culture specifically includes: culturing for 25-35 days under conditions of temperature of 23-27℃, light intensity of 16h / 8h, and light intensity of 1500-2500 lux.
9. The method according to claim 7, characterized in that, In the step of inoculating the induced buds into the second antibacterial medium for proliferation culture, the proliferation culture specifically includes: culturing for 40-50 days under the conditions of temperature of 23-27℃, light intensity of 16h / 8h, and light intensity of 1500-2500 lux; then transferring once and continuing the proliferation culture.
10. The method according to claim 7, characterized in that, In the step of inoculating the clump seedlings into the third antibacterial medium for rooting culture, the rooting culture specifically includes: culturing for 45-60 days under the conditions of a temperature of 23-27℃, a light intensity of 16h / 8h, and a light intensity of 1500-2500 lux.