Tobacco sterile seedling cultivation method

Abstract

The application belongs to the field of plant culture, and specifically discloses a tobacco sterile seedling cultivation method, which comprises the following steps: placing dry compressed Jiffy-7 substrate blocks into transparent culture boxes, and adding newly prepared 1 / 2 modified LS nutrient solution into the culture boxes; transferring the prepared culture boxes into a transparent plastic incubator and sequentially placing the culture boxes; incompletely closing the cover of the incubator, and opening a circular hole with a diameter of 5-10 cm in the middle of the cover of the incubator, and sticking a filter membrane larger than the diameter of the circular hole on the cover, etc. Through the technical scheme of the application, the sterile tobacco seedling cultivation method is formed, and sterile tobacco seedlings are successfully cultivated, which can provide experimental conditions for revealing the mechanism of the interaction between tobacco and microbial communities in the research on the interaction between tobacco and microbial communities under greenhouse conditions.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation, and specifically relates to a method for cultivating aseptic tobacco seedlings. Background Technology

[0002] The complex structure and function of plant microbial communities are influenced by a variety of factors, including the environment, microbial interactions, and host factors. Similarly, the established microbial flora can affect many host phenotypes. While significant progress has been made in studying plant-microbial community interactions under open field or greenhouse conditions, little is known about the mechanisms underlying these interactions. Developing a usable sterile plant growth system could help elucidate the assembly rules of the microbiome, the functional interactions between the plant and microbiome, and isolate experimental variables. This would refine the understanding of the mechanisms of plant genotype-environment-microbiome-management interactions and advance plant microbiome research. Currently, there are no sterile growth systems for tobacco-related studies of tobacco-microbial interactions.

[0003] The main problems in the study of aseptic growth systems are as follows: contamination problems, mainly system contamination and seed contamination. Infected equipment can lead to contamination of the experimental system, and incomplete seed sterilization can also lead to contamination. Seed germination problems, such as incorrect seed sterilization time and method, can affect the germination rate. Improper nutrient solution formulation, due to the characteristics of some substrates, can cause nutrient deficiencies in plants, thus affecting plant growth and development. Summary of the Invention

[0004] The main objective of this invention is to provide a method for cultivating aseptic tobacco seedlings, thereby providing a research basis for understanding the mechanism of interaction between tobacco genotype, environment, microbiome, and management.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for cultivating aseptic tobacco seedlings includes the following steps:

[0007] S1: Place the dried and compressed substrate block into a transparent culture box and add 1 / 2 of the newly prepared modified LS nutrient solution to the culture box;

[0008] S2: Transfer the prepared culture boxes to a transparent plastic incubator and place them in order; do not close the lid of the incubator completely, and place them loosely on the incubator. Make a round hole with a diameter of 5-10cm in the middle of the lid of the incubator, and stick a filter membrane larger than the diameter of the round hole to the lid.

[0009] S3: Place the incubator into the autoclave bag, seal and fold the bag, perform the first sterilization, cool to about 50°C, remove and place in the sterile incubation room, without opening the autoclave bag during the process;

[0010] S4: Seed sterilization and disinfection shall be carried out the day after the first sterilization of the incubator;

[0011] S5: After the incubator has been placed for 36-48 hours, perform a second sterilization. After cooling to room temperature, close the lid of the incubator inside the sterilization bag in a sterile environment. Then reseal the sterilization bag and place it in a sterile room. Store for at least 24 hours before sowing.

[0012] S6: On a sterile operating table, remove the sterilization bag, open the incubator, take a small amount of substrate or culture box solution or solution in the incubator and spread it onto R2A and PDA culture media respectively. Incubate at 30°C for at least one week. Test the substrate for bacterial and fungal contamination. Irrigate each substrate block with sterilized modified LS nutrient solution from the top. After irrigation, the LS nutrient solution content in the culture box should not exceed 1 / 2 of the culture box volume.

[0013] S7: Use a sterile toothpick or disposable inoculation loop to transfer one or two seeds to the edge of the central substrate in each culture box. After closing the lid, place the incubator in a sterile culture room for incubation.

[0014] Furthermore, in step S1, the amount of 1 / 2 modified LS nutrient solution added is 10 times the mass of the substrate block.

[0015] Furthermore, the modified LS nutrient solution comprises: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, anhydrous calcium chloride 332.2 mg / L, anhydrous magnesium sulfate 180.7 mg / L, potassium dihydrogen phosphate 170 mg / L, disodium ethylenediaminetetraacetate 37.26 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, boric acid 6.2 mg / L, manganese sulfate monohydrate 16.9 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, vitamin B1 0.4 mg / L, inositol 180 mg / L, glycine 1 mg / L, sucrose 30000 mg / L, with the remainder being water.

[0016] Furthermore, in step S4, the method for disinfecting tobacco seeds is as follows: initial disinfection with 70% ethanol solution for 30 seconds, the solution is aspirated and discarded using a 1000ml pipette tip, then 10% H2O2 solution is added for sterilization for 10 minutes, the solution is aspirated and discarded, and the seeds in the centrifuge tube are rinsed 5 times with sterile water.

[0017] Furthermore, in step S7, the culture conditions are: temperature 26℃, humidity 70%–80%, dark culture for 4 days, followed by light for 12 hours, and alternating dark and dark periods for 25–40 days, with a light intensity of 2000–3000 lx.

[0018] Furthermore, in step S2, the pore size of the filter membrane is 0.22 μm.

[0019] The technical effects achieved by this invention are as follows:

[0020] The sterile tobacco seedling cultivation method developed through the technical solution of this invention has successfully cultivated sterile tobacco seedlings, which can provide experimental conditions for revealing the mechanism of interaction between tobacco and microbial communities in later research on the interaction between tobacco and microbial communities under greenhouse conditions.

[0021] In the tobacco seedling cultivation process of this invention, the seed disinfection method of this invention is a disinfection method obtained through several experiments. After R2A and PDA culture medium coating tests, no bacterial or fungal infection occurred, and the seed germination rate after sowing reached 98%, indicating that the disinfection method did not damage the germplasm and the disinfection effect was good.

[0022] Under the operating steps and methods of this invention, various components in the incubator were sampled and tested using R2A and PDA culture media. No bacterial or fungal infections were found, indicating that the culture method of this invention is free from system contamination.

[0023] This invention improves the composition of the original LS nutrient solution by increasing the inositol content and adding glycine. Considering the differences between seed germination and plant tissue culture, this invention improves the LS nutrient solution. Tobacco seedlings grown with the improved LS nutrient solution are relatively robust and do not exhibit post-germination decline, yellowing leaves, or rotting due to nutrient deficiencies. Attached Figure Description

[0024] Figure 1 Figure 1: Water absorption and swelling of Jiffy-7 substrate block (A: Substrate block before water absorption, B: Substrate block after water absorption);

[0025] Figure 2 A photo of an incubator containing culture boxes.

[0026] Figure 3 Diagram of seed surface disinfection process; (A: Add 70% ethanol solution; B: Add 10% H2O2 solution; C: Rinse with sterile water; D: Blot dry with sterile filter paper)

[0027] Figure 4 Streaking patterns of sterilized seed R2A and PDA culture media plates;

[0028] Figure 5 Photos of the incubator after two sterilization cycles;

[0029] Figure 6 Plate spread diagrams of R2A and PDA culture media contents after sterilization of the incubator;

[0030] Figure 7 Photo of sterilized seeds being inoculated onto the substrate;

[0031] Figure 8 Photos of the incubator after inoculation;

[0032] Figure 9 A picture showing the condition of tobacco seedlings after 15 days of cultivation.

[0033] Figure 10 A picture showing the condition of tobacco seedlings after 25 days of cultivation. Detailed Implementation

[0034] Unless otherwise specified, the reagents and consumables involved in this invention are all commercially available. Unless otherwise specified, the experimental conditions are generally performed under conventional conditions or as recommended by the reagent company.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments and accompanying drawings, but they should not be construed as limiting the scope of protection of the present invention.

[0036] The R2A culture medium in this invention consists of: 0.5 g / L yeast extract, 0.5 g / L peptone, 0.5 g / L casein hydrolysate, 0.5 g / L glucose, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.024 g / L anhydrous magnesium sulfate, 0.3 g / L sodium pyruvate, 15.0 g / L agar, with the balance being water, and a pH of 7.2 ± 0.2.

[0037] The PDA culture medium in this invention consists of: 5.0 g / L potato starch, 20.0 g / L glucose, 14.0 g / L agar, with the remainder being water, and a pH of 5.6 ± 0.2.

[0038] The modified LS nutrient solution in this invention consists of: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, anhydrous calcium chloride 332.2 mg / L, anhydrous magnesium sulfate 180.7 mg / L, potassium dihydrogen phosphate 170 mg / L, disodium ethylenediaminetetraacetate 37.26 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, boric acid 6.2 mg / L, manganese sulfate monohydrate 16.9 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, vitamin B1 0.4 mg / L, inositol 180 mg / L, glycine 1 mg / L, sucrose 30000 mg / L, with the remainder being water, and the pH being natural.

[0039] Example 1

[0040] 1. Place the dried and compressed Jiffy-7 substrate block (50mm in size, approximately 100g) into a transparent culture box. Add 1L of freshly prepared 1 / 2 modified LS nutrient solution to the culture box (pour from the top of the substrate) to completely hydrate the substrate block. Immerse the substrate block completely in the nutrient solution and wait 30-40 minutes. The substrate block will absorb water and expand to approximately 5 times its original height. Figure 1 .

[0041] 2. Transfer the prepared culture boxes (including substrate blocks and remaining nutrient solution) to a transparent rectangular plastic incubator and arrange them sequentially. The bottom of the culture boxes is hollowed out, and the lid of the incubator is equipped with a silicone sealing ring. Loosely place the culture boxes on the incubator with the lid partially closed. Make a circular hole with a diameter of 5-10 cm in the center of the lid. Attach a filter membrane larger than the diameter of the hole to the lid, ideally with filter membranes attached to both sides of the lid. The filter membrane pore size should be 0.22 μm. See details below. Figure 2 As shown, the culture box and incubator are both conventional food preservation containers that are resistant to high temperatures.

[0042] 3. Place the sealed incubator into an autoclave bag (Xingu S6386 autoclave bag), seal and fold the autoclave bag, and then place the assembled incubator into an autoclave for the first sterilization. After autoclaving at 121℃ for 45 minutes, cool it to about 50℃, take it out and place it in a sterile incubation room. Do not open the autoclave bag during the process.

[0043] 4. The day after the first sterilization of the incubator, perform seed sterilization. Add an appropriate amount of tobacco seeds (Bunsen) to a 2ml centrifuge tube, add 70% ethanol solution for initial sterilization for 30 seconds, remove and discard the solution using a 1000ml pipette tip, then add 10% H2O2 solution for sterilization for 10 minutes, remove and discard the solution, and rinse the seeds in the centrifuge tube 5 times with sterile water. The process is as follows. Figure 3 Using a disposable inoculation loop, apply the solution from the final seed rinse to R2A and PDA media, respectively. Incubate at 30°C for at least one week, then test the seeds for bacterial and fungal contamination. Figure 4 The technical solution of this invention is pollution-free, and the seed germination rate reaches over 98% in the later cultivation process.

[0044] 5. After placing the incubator for 36-48 hours, perform a second sterilization by autoclaving at 121℃ for 45 minutes. Then, cool to room temperature for 3-4 hours. Remove the incubator and operate on a sterile workbench. Close the incubator lid inside the sterilization bag to ensure a seal between the lid and the incubator body. Reseal the sterilization bag and place it in a sterile room. Figure 5 Place the incubator in a sterilized bag and store it for at least 24 hours before sowing.

[0045] 6. Wipe the sterile workbench in the laboratory with 75% alcohol. Spray the internal environment of the sterile workbench and disinfect all items to be used with alcohol and UV lamp. After disinfection for at least 15 minutes, remove the incubator from the sterilization bag and remove the bag from the sterile workbench.

[0046] 7. Open the incubator. Use a disposable inoculation loop to take a small amount of substrate, culture box solution, or solution from the incubator and spread it onto R2A and PDA media respectively. Incubate at 30°C for at least one week. Test the substrate for bacterial and fungal contamination. Figure 6 After testing, no bacterial or fungal contamination was found under the operating method of this invention. Then, 15-20 mL of sterilized modified LS nutrient solution was added to each culture box from the top of each substrate block. After irrigation, the LS nutrient solution content did not exceed 1 / 2 of the culture box volume, so that the water content of the substrate block returned to saturation.

[0047] 8. Using a sterile toothpick or disposable inoculation loop, transfer one or two seeds to the edge of the substrate in the center of each culture box, such as... Figure 7 After closing the lid, place the incubator in a sterile culture room for incubation. Figure 8 Culture conditions: temperature 26℃, humidity 70%–80%, dark culture for 4 days, followed by alternating periods of 12 hours of light and 12 hours of darkness for 25–40 days, light intensity 2000–3000 lx. The resulting plants showed… Figure 9 , 10 As shown, the plants can grow normally. On days 15 and 25, small amounts of substrate, culture box solution, incubator solution, or tobacco leaves were respectively applied to R2A and PDA culture media and incubated at 30°C for at least one week to detect bacterial and fungal contamination. The tests showed that, under the operating method of this invention, there was no bacterial or fungal contamination.

[0048] In the above-mentioned aseptic system, the microorganisms to be studied can be inoculated at any stage after seed sowing (the soil, nutrient solution, or tobacco seedling tissue, etc., are streak-cultured and tested to be free of contamination before inoculation). After inoculation, the corresponding follow-up studies can be carried out (the whole process is aseptic). The filter membrane can filter out microorganisms in the air and provide gas exchange for tobacco seedling growth. Aseptic tobacco seedlings are obtained by cultivation. Then, a single microorganism or community of microorganisms of interest can be introduced to observe, study, and detect the phenotypic changes and functional interactions of the tobacco seedlings.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cultivating aseptic tobacco seedlings, characterized in that, Includes the following steps: S1: Place the dried and compressed substrate block into a transparent culture box, and add 1 / 2 of the prepared modified LS nutrient solution to the culture box. The amount of 1 / 2 modified LS nutrient solution added is 10 times the mass of the substrate block. S2: Transfer the prepared culture boxes to a transparent plastic incubator and place them in order; do not close the lid of the incubator completely, and place them loosely on the incubator. Make a circular hole with a diameter of 5-10cm in the middle of the lid of the incubator, and stick a filter membrane with a pore size of 0.22μm larger than the diameter of the circular hole to the lid. S3: Place the incubator into the autoclave bag, seal and fold the bag, perform the first sterilization, cool to 50°C, remove and place in a sterile incubation room, without opening the autoclave bag during the process; S4: The seeds are sterilized the day after the first sterilization of the incubator. The sterilization method for tobacco seeds is as follows: the seeds are initially sterilized with 70% ethanol solution for 30 seconds, the solution is aspirated and discarded with a 1000 ml pipette tip, then 10% H2O2 solution is added for sterilization for 10 minutes, the solution is aspirated and discarded, and the seeds in the centrifuge tube are rinsed 5 times with sterile water. S5: After the incubator has been placed for 36-48 hours, perform a second sterilization. After cooling to room temperature, close the lid of the incubator inside the sterilization bag under sterile conditions. Then reseal the sterilization bag and place it in a sterile room. Store for at least 24 hours before sowing. S6: On a sterile operating table, open the incubator and take a small amount of substrate or culture box solution or solution in the incubator and spread it onto R2A and PDA culture media respectively. Incubate at 30°C for at least one week and test the substrate for bacterial and fungal contamination. Irrigate each substrate block with sterilized modified LS nutrient solution from the top. After irrigation, the LS nutrient solution content in the culture box should not exceed 1 / 2 of the culture box volume. S7: Use a sterile toothpick or disposable inoculation loop to transfer one or two seeds to the edge of the central substrate in each culture box. After closing the lid, place the incubator in a sterile culture room for culture. The culture conditions are: temperature 26℃, humidity 70%~80%, dark culture for 4 days, then light for 12 hours and dark for 12 hours, alternating for 25~40 days, with a light intensity of 2000-3000lx. The modified LS nutrient solution consists of: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, anhydrous calcium chloride 332.2 mg / L, anhydrous magnesium sulfate 180.7 mg / L, potassium dihydrogen phosphate 170 mg / L, disodium ethylenediaminetetraacetate 37.26 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, boric acid 6.2 mg / L, manganese sulfate monohydrate 16.9 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, vitamin B1 0.4 mg / L, inositol 180 mg / L, glycine 1 mg / L, sucrose 30000 mg / L, with the remainder being water.

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