Open culture method of arundo donax

Abstract

The application belongs to the technical field of plant propagation, and provides an open type tissue culture method of bamboo reed, which comprises the following steps: step S1, explant treatment; step S2, growth aid solution preparation and use step; S3, explant disinfection step; S4, culture medium preparation step; S5, inoculation step; and S6, culture. The method is characterized in that 0.1 g / L indole butyric acid is used, the explant is soaked for 12-36 h before disinfection, and the mixed bacteriostatic agent of 0.075 g / L of chlorothalonil and 0.075 g / L of streptomycin is added to the culture medium, so that the cost of bamboo reed propagation is significantly reduced, the advantages of tissue culture are retained, the seedlings are uniform, the propagation coefficient is large, the propagation speed is fast, and a new idea is provided for the open type tissue culture of bamboo reed.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation technology, and specifically relates to an open tissue culture method for Reed sphagnum. Background Technology

[0002] Arundo donax L., a perennial tall herbaceous plant belonging to the genus Arundo of the subfamily Arundoideae in the family Poaceae, is widely used in artificial wetlands, landscaping, medicine, weaving, and papermaking due to its strong adaptability, resilience, ornamental value, and high economic value. Current research generally recognizes Arundo donax as a polyploid plant, as its megasporocytes cannot divide normally, leading to seed abortion. Therefore, it can only reproduce asexually, with common propagation methods including division, cuttings, and tissue culture. However, each of these methods has its advantages and disadvantages, and none are sufficient for rapid propagation to meet the seedling needs of Arundo donax.

[0003] Propagation by tillering involves cultivating suckers that emerge from adventitious buds at the base of the root or stem, separating them from the parent plant to become independent plants. This method is one of the most traditional asexual propagation methods, characterized by high survival rates, rapid seedling growth, low propagation coefficient, and inconsistent seedling size. However, due to the low propagation coefficient and inconsistent plant size and growth, it is difficult to meet the demand for large-scale seedling production.

[0004] Propagation by cuttings is a common method of plant propagation. It involves taking a section of the plant's vegetative organs and inserting it into loose, moist soil or fine sand. The cuttings then utilize the plant's regenerative ability to root and sprout new shoots. Its advantages include simplicity and low cost. However, it also has disadvantages such as a low propagation coefficient, inconsistent seedling size, and a lower survival rate than division propagation.

[0005] Tissue culture technology refers to the technique of isolating tissues, organs, cells, protoplasts, etc. from plants and inoculating them under sterile conditions on a culture medium containing various nutrients and plant hormones to obtain regenerated complete plants or produce other products with economic value. It has the advantages of high propagation coefficient, uniform seedling emergence, and fast propagation speed. However, this seedling system must be carried out under sterile conditions throughout the process, so the operation is complicated, and the labor and time costs are high. At the same time, it is difficult to avoid contamination in the later stage, resulting in significant economic losses. Summary of the Invention

[0006] To address the challenges of aseptic operation and high costs associated with establishing a sterile environment in traditional tissue culture techniques, this method utilizes antibacterial agents and explant disinfection to prevent significant contamination while retaining the advantages of tissue culture, such as a high propagation coefficient, uniform emergence, and rapid propagation speed.

[0007] This invention is implemented as follows:

[0008] An open tissue culture method for Reed przewalskii includes the following steps:

[0009] Step S1, Explant treatment: Select healthy lateral branches of Reed, prune them into sections with one bud each, wash and soak them in a laundry detergent solution for 15-30 minutes, then rinse them with running water for later use.

[0010] Step S2, preparation and use of growth-promoting solution: Prepare an indolebutyric acid solution of a certain concentration, take the explants from step S1, soak them for 12-36 hours and then wash them for later use.

[0011] Step S3, Explant Disinfection: Take the explant from step S2, soak it in 70% alcohol for 30 seconds, then transfer it to 2% sodium hypochlorite solution and soak it for 15 minutes, then rinse it with sterile water for later use.

[0012] Step S4: Prepare the culture medium: Use 1 / 8 to 1 / 2 MS as the basal medium, add 20 to 30 g / L sucrose, 4 to 8 g / L agar, and 0.5 to 2 g / L activated carbon. Select a certain concentration of streptomycin solution and chlorothalonil solution as antibacterial agents and add them to the culture medium.

[0013] Step S5, Inoculation: Inoculate the explants from step S3 onto the culture medium from step S4, and then seal and culture them.

[0014] Step S6, Cultivation: Place the inoculation bottle in an environment of 24-32℃, with a light intensity of 800-1200 lux, a light duration of 12h / d, and cultivate for 28-60 days.

[0015] Furthermore, in step S2, the concentration of the indolebutyric acid solution is 0.05–0.2 mg / L.

[0016] Furthermore, in step S1, the concentration of the indolebutyric acid solution is 0.1 mg / L.

[0017] Furthermore, in step S4, the concentrations of both streptomycin solution and chlorothalonil solution are 0.05–0.2 g / L.

[0018] Furthermore, in step S4, the concentrations of both streptomycin solution and chlorothalonil solution are 0.075 g / L.

[0019] Furthermore, the order in which steps 2 and 3 are performed can be interchanged.

[0020] The beneficial effects of this invention are:

[0021] This invention promotes the development and rooting of axillary buds in stem nodes by soaking the plant in 0.1 mg / L indolebutyric acid for 12–36 hours. A mixed antibacterial agent of chlorothalonil and streptomycin, both at a concentration of 0.075 g / L, is added to the culture medium to inhibit fungal and bacterial infections, reducing the sterilization steps and costs associated with tissue culture. This significantly lowers the cost of Arundo donax propagation while retaining the advantages of tissue culture, such as uniform emergence, high propagation coefficient, and rapid propagation speed, providing a new approach for open tissue culture of Arundo donax.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0027] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0028] Example 1

[0029] This study investigated the browning, contamination, and survival of *Arundo donax* after treatment with different antibacterial agents.

[0030] Common antibacterial agents, including chlorothalonil, carbendazim, mancozeb, streptomycin, cefotaxime sodium, kanamycin, sodium hypochlorite, and potassium permanganate, were selected and studied at a concentration of 0.1 g / L. The antibacterial agents described in this application were prepared as a mixture of chlorothalonil solution and streptomycin solution, each at a concentration of 0.075 g / L. Nine treatment groups and one control group without antibacterial agents were established. Open tissue culture was performed according to the following steps, with 30 explants inoculated for each treatment, and three replicates:

[0031] Step 1, Explant treatment: Select healthy lateral branches of Reed, prune them into sections with one bud each, wash and soak them in a laundry detergent solution for 15-30 minutes, then rinse them with running water for later use.

[0032] Step 2, preparation and use of growth-promoting solution: Prepare a 0.05-0.2 mg / L indolebutyric acid solution. Take the explants from Step 1, soak them for 12-36 hours, and then rinse them for later use.

[0033] Step 3, Explant disinfection: Take the explant from step 2, soak it in 70% alcohol for 30 seconds, then transfer it to a 2% sodium hypochlorite solution and soak it for 15 minutes. Rinse it with sterile water and set aside.

[0034] Step 4: Prepare the culture medium: Use 1 / 8 to 1 / 2 MS as the basal medium, and add 20 to 30 g / L sucrose, 4 to 8 g / L agar, and 0.5 to 2 g / L activated carbon;

[0035] Step 5, Inoculation: Inoculate the explants from Step 3 onto the culture medium from Step 4, and then seal and culture.

[0036] Step 6, Cultivation: Place the inoculation bottle in an environment of 24-32℃, with a light intensity of 800-1200 lux, a light duration of 12h / d, and cultivate for 28-60 days.

[0037] Statistical analysis was conducted at 15 and 30 days of culture to determine the browning rate and the rates of fungal and bacterial contamination. The survival rate after 30 days was also calculated. The results are shown in Table 1 below:

[0038] Table 1

[0039] antibacterial agent 15d browning 15-day contamination fungi 15-day contamination bacteria 30-day browning 30-day contamination fungi 30-day contamination bacteria Survival Chlorothalonil 1.11% 0.00% 12.22% 5.56% 1.11% 16.67% 76.67% Carbendazim 4.44% 3.33% 11.11% 6.67% 5.56% 20.00% 67.78% Manganese zinc 15.56% 4.44% 15.56% 25.56% 11.11% 25.56% 37.78% Streptomycin 1.11% 4.44% 1.11% 4.44% 5.56% 2.22% 87.78% Cefotaxime sodium 2.22% 2.22% 3.33% 11.11% 4.44% 4.44% 80.00% Kanamycin 3.33% 3.33% 2.22% 8.89% 5.56% 6.67% 78.89% Sodium hypochlorite 5.56% 1.11% 2.22% 12.22% 5.56% 10.00% 72.22% potassium permanganate 5.56% 1.11% 2.22% 12.22% 4.44% 7.78% 75.56% Chlorothalonil + Streptomycin 1.11% 0.00% 1.11% 5.56% 1.11% 2.22% 91.11% CK 1.11% 3.33% 4.44% 2.22% 5.56% 14.44% 77.78%

[0040] The results showed that the antibacterial effect of the mixed antibacterial agent of chlorothalonil and streptomycin used in this application was excellent, significantly better than other antibacterial agents, while the browning rate was also low and the overall survival rate was extremely high.

[0041] Example 2:

[0042] The effects of growth-promoting solutions on the timing of application were studied.

[0043] Indolebutyric acid (IBA) solution was used as a rooting inducer for Reed (Prunus armeniaca) at the following times: ① 0.1 mg / L IBA, used before sterilization; ② 0.1 mg / L IBA, used after sterilization and before inoculation; ③ 0.1 mg / L IBA, added to the culture medium; ④ 0.05 mg / L IBA, added to the culture medium; ⑤ IBA was not used.

[0044] Open tissue culture was performed according to the following steps, with 30 explants inoculated per treatment, and 3 replicates:

[0045] Step 1, Explant treatment: Select healthy lateral branches of Reed, prune them into sections with one bud each, wash and soak them in a laundry detergent solution for 15-30 minutes, then rinse them with running water for later use.

[0046] Step 2, Explant disinfection: Take the explant from Step 2, soak it in 70% alcohol for 30 seconds, then transfer it to 2% sodium hypochlorite solution for 15 minutes, and rinse it with sterile water for later use.

[0047] Step 3: Prepare the culture medium: Use 1 / 8 to 1 / 2 MS as the basal medium, add 20 to 30 g / L sucrose, 4 to 8 g / L agar, and 0.5 to 2 g / L activated carbon. Use streptomycin (0.05 to 0.2 g / L) and chlorothalonil (0.05 to 0.2 g / L) together as antibacterial agents.

[0048] Step 4, Inoculation: Inoculate the explants from Step 3 onto the culture medium from Step 4, and then seal and culture.

[0049] Step 5, Cultivation: Place the inoculation bottle in an environment of 24-32℃, with a light intensity of 800-1200 lux, a light duration of 12h / d, and cultivate for 28-60 days.

[0050] Rooting was recorded at 7 days and 30 days of cultivation, and root length and number were also recorded at 30 days. The results are shown in Table 2.

[0051] Table 2

[0052] deal with Rooting in 7 days Rooting in 30 days 30 days per strand 30d root length / cm ① 75.56% 98.89% 13.33 8.43 ② 66.67% 86.67% 11.67 7.27 ③ 47.78% 65.56% 5.67 2.77 ④ 52.22% 81.11% 12.00 4.40 ⑤ 3.33% 25.56% 2.67 1.87

[0053] The results show that the concentration and timing of indolebutyric acid application used in this application are very effective in promoting the growth of Arundo donax, and far exceed other application schemes in terms of rooting rate, root length and root number.

[0054] This invention significantly reduces the cost of Arundinaria propagation by soaking explants in 0.1 mg / L indolebutyric acid for 12–36 h before disinfection, and by adding a mixed antibacterial agent of chlorothalonil and streptomycin at a concentration of 0.075 g / L to the culture medium, while retaining the advantages of tissue culture, such as uniform emergence, high propagation coefficient, and fast propagation speed. This provides a new approach for open tissue culture of Arundinaria.

[0055] It should be understood that the specific order or hierarchy of steps in the disclosed process is an example of an exemplary method. Based on design preferences, it should be understood that the specific order or hierarchy of steps in the process may be rearranged without departing from the scope of this disclosure. The appended method claims provide elements of various steps in an exemplary order and are not intended to limit the scope to a specific order or hierarchy.

[0056] In the detailed description above, various features are combined together in a single embodiment to simplify this disclosure. This approach to disclosure should not be construed as reflecting an intention that embodiments of the claimed subject matter require more features than are explicitly stated in each claim. Rather, as reflected in the appended claims, the invention is presented with fewer features than all of the features in a single disclosed embodiment. Therefore, the appended claims are hereby explicitly incorporated into the detailed description, with each claim representing a separate preferred embodiment of the invention.

[0057] The foregoing description includes examples of one or more embodiments. It is certainly impossible to describe all possible combinations of components or methods in order to describe the above embodiments, but those skilled in the art will recognize that further combinations and arrangements of the various embodiments are possible. Therefore, the embodiments described herein are intended to cover all such changes, modifications, and variations that fall within the scope of the appended claims. Furthermore, the term "comprising" as used in the specification or claims is interpreted in a manner similar to the term "including," as interpreted when used as a conjunction in the claims. Additionally, the use of any term "or" in the specification of the claims is intended to mean "non-exclusive or."

Claims

1. An open type tissue culture method of Arundo donax, characterized by, The method comprises the following steps: Step S1, explant treatment: select healthy lateral branches of Arundo donax, trim each section to one bud, clean and soak in laundry detergent solution for 15-30 min, then rinse with running water for standby; Step S2, preparation and use of growth promoting solution: prepare 0.05-0.2 mg / L indole butyric acid solution, take the explants in step S1, soak for 12-36 h, then clean and standby; Step S3, explant disinfection: take the explants in step S2, soak in 70% alcohol for 30 s, then transfer to 2% sodium hypochlorite solution for 15 min, then clean with sterile water for standby; Step S4, preparation of culture medium: use 1 / 8-1 / 2 MS as the basic culture medium, add 20-30 g / L sucrose, 4-8 g / L agar, 0.5-2 g / L activated carbon, and add 0.05-0.2 g / L streptomycin solution and 0.05-0.2 g / L chlorothionol solution as bacteriostatic agents; Step S5, inoculation: inoculate the explants in step S3 into the culture medium in step S4, and seal the film for culture; Step S6, culture: place the inoculation bottle in an environment of 24-32°C for culture, the light intensity is 800-1200 lux, the light duration is 12 h / d, and the culture time is 28-60 d.

2. The open type tissue culture method of Arundo donax according to claim 1, characterized in that, In step S2, the concentration of the indole butyric acid solution is 0.1 mg / L.

3. The open type tissue culture method of Arundo donax according to claim 1, characterized in that, In step S4, the concentrations of the streptomycin solution and the chlorothionol solution are both 0.075 g / L.

4. The open type tissue culture method of Arundo donax according to claim 1, characterized in that, The order of steps S2 and S3 is exchanged.

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