Open tissue culture method for jujube trees
By adopting an open method in jujube tissue culture, using sand and wormwood culture medium, and using microcrystalline cellulose and chitosan coverings, the complexity and high cost of traditional jujube tissue culture are solved, and efficient and economical seedling breeding is achieved.
Patent Information
- Application Number
- CN202510501412.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-05-30
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Traditional jujube tree tissue culture requires a strict sterile environment, resulting in complex processes, high costs and low survival rate of seedlings.
Using an open tissue culture method, jujube tissue culture is achieved in a relatively open environment by adding sand and carbendazim to the culture medium and using a mixture of microcrystalline cellulose and chitosan as the surface covering.
The operation process is simplified, cost investment is reduced, and the survival rate of seedling refining and transplanting is improved.
Smart Images
Figure BDA0005368557380000091
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant tissue culture, and in particular to an open tissue culture method for jujube trees. Background Art
[0002] As an important economic forest tree, the jujube tree has high nutritional value and economic value. Its tissue culture technology is of great significance for seedling propagation, cultivation of excellent varieties, etc. Traditional jujube tree tissue culture usually requires a strict aseptic environment, including using high-pressure sterilization equipment to sterilize the culture medium and utensils, and performing inoculation and other operations in an aseptic operation space such as a laminar flow bench. However, such strict aseptic requirements make the tissue culture process complex, costly, and during the acclimatization and transplantation process, due to large environmental changes, the survival rate of seedlings is extremely low.
[0003] In recent years, the open tissue culture technology has gradually received attention. This technology enables plant tissue culture to be carried out in a relatively open and bacteria-containing environment by adding bacteriostatic agents to the culture medium, thereby reducing the dependence on an aseptic operation environment and reducing the cost investment in equipment, site, energy, etc. For example, some studies have applied this open culture technology to the tissue culture of ornamental plants such as chrysanthemums and orchids, as well as crops such as bananas and sugarcane, and achieved certain results. In the field of jujube tree tissue culture, traditional aseptic culture is still mainly used at present, and there is no systematic report on the research of open tissue culture methods. Therefore, developing an open tissue culture method suitable for jujube trees has important practical significance for simplifying the jujube tree tissue culture process, reducing costs, and improving the survival rate of seedling acclimatization and transplantation. Summary of the Invention
[0004] The present invention aims to simplify the complex process of traditional jujube tree tissue culture, reduce the strict requirements for an aseptic operation environment, thereby reducing cost investment, and at the same time improve the survival rate of seedling acclimatization and transplantation. This method successfully realizes jujube tree tissue culture in a relatively open and bacteria-containing environment by innovatively replacing agar with sand and adding bacteriostatic agents such as carbendazim to the culture medium. In addition, using a mixture of microcrystalline cellulose and chitosan as the covering on the surface of the culture medium not only effectively isolates the intrusion of external microorganisms, but also ensures the air permeability of the culture medium, providing sufficient oxygen for plant roots. By optimizing multiple key links such as the culture medium formula, selection of culture containers, treatment of plant shoot tips, and control of culture conditions, the present invention provides an efficient, economical and practical tissue culture technical solution for the rapid breeding and promotion of excellent jujube tree varieties, with remarkable innovation and broad application prospects.
[0005] An open tissue culture method for jujube trees, characterized by comprising the following steps:
[0006] (1) Prepare an open medium by mixing a basal medium: sand: sucrose: carbendazim in a mass ratio of 1000:4000:15 - 25:1 - 2 to form the open medium;
[0007] (2) Dispense the prepared open medium into culture containers, which can be culture flasks, Erlenmeyer flasks or other containers of appropriate size;
[0008] (3) Place the sterilized plant shoot tips into the culture containers filled with the medium;
[0009] (4) Prepare chitosan, and its steps include:
[0010] Raw material preparation: Select crushed fresh shrimp shells and crab shells as raw materials, ensuring that the raw materials are not spoiled, have a hard texture and uniform color;
[0011] Decalcification treatment: Soak the raw materials in 2.5% dilute hydrochloric acid to remove the calcium carbonate component in the shells;
[0012] Deproteinization and deacetylation reaction: Put the raw materials after decalcification treatment into a reaction kettle, and add 40% - 60% concentrated sodium hydroxide solution. Place the reaction kettle in a heating device, heat up to 90°C to 100°C, and maintain this temperature for 2 - 4 hours for the deacetylation reaction. During the reaction, use a mechanical stirrer to continuously stir at a speed of 100 revolutions per minute to ensure uniform reaction;
[0013] Terminate the reaction and wash: After the reaction is completed, pour the reaction product into distilled water to terminate the reaction, and then wash the solid part with distilled water and anhydrous ethanol respectively to remove the residual alkali and impurities;
[0014] Drying and pulverization: Dry the washed chitosan at 60°C and then perform re - pulverization to obtain chitosan powder;
[0015] (5) Prepare a surface covering. After mixing microcrystalline cellulose and chitosan powder evenly in a mass ratio of 30 - 40:1, cover it on the surface of the open medium and gently compact it, with a thickness of 2 - 4 mm. Microcrystalline cellulose can isolate the intrusion of external microorganisms, and at the same time its porous structure can ensure the air permeability of the open medium, providing sufficient oxygen for the plant roots and being beneficial to the respiration and growth of the roots.
[0016] (6) Place the culture containers implanted with the open medium and jujube tree shoot tips in a culture environment with a temperature of 25 - 28°C, a light intensity of 1500 - 2000 lx, and a light duration of 12 - 14 hours per day.
[0017] Preferably, the formula of the basal medium in step (1) is: MS medium + 6 - BA 0.5 - 1.0 mg / L + IBA 0.1 - 0.3 mg / L.
[0018] Preferably, in step (1), the sand is screened river sand with a particle size of 0.5 - 2 mm and no impurities.
[0019] Preferably, in step (2), the culture container is made of transparent glass or plastic and does not require a lid.
[0020] Preferably, the plant shoot tip in step (3) is from a healthy jujube tree plant, free of pests and diseases, and has a length of 0.5 - 1.5 cm. Before being placed in the culture container, it is first disinfected with 1‰ mercuric chloride for 25 - 35 seconds, then disinfected with 75% alcohol for 10 - 20 seconds, and then rinsed with sterile water 3 - 5 times.
[0021] Preferably, in step (4), during the decalcification treatment, the mass ratio of dilute hydrochloric acid to the raw material is 10 - 15:1, the soaking time is 2 - 4 hours, and the temperature is at room temperature.
[0022] Preferably, in step (4), during the deproteinization and deacetylation reaction, the mass ratio of chitin powder to concentrated sodium hydroxide solution is 1:10 - 15.
[0023] Preferably, in step (4), microcrystalline cellulose and chitosan are mixed and stirred evenly to obtain a surface covering. And within 0.5 hour after the plant shoot tip is implanted into the open medium, the surface covering is evenly covered on the surface of the medium.
[0024] The beneficial effects of the present invention are as follows:
[0025] By replacing agar with sand and adding bacteriostatic agents such as carbendazim, the tissue culture of jujube trees can be carried out in a relatively open environment without a strictly aseptic operation environment, reducing the requirements for equipment, site, energy, etc., simplifying the operation process, and reducing costs.
[0026] Using the mixture of microcrystalline cellulose and chitosan as the surface covering can not only effectively isolate the intrusion of external microorganisms but also ensure the air permeability of the medium, providing sufficient oxygen for the plant roots.
[0027] The method of the present invention does not require a cumbersome seedling hardening process like traditional tissue culture, further simplifies the operation steps, improves the seedling propagation efficiency, and has important application value for the rapid breeding and promotion of excellent jujube tree varieties. Specific embodiments
[0029] The present invention discloses an open tissue culture method for jujube trees. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters for operation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The method and application of the present invention have passed relevant tests, and preferred embodiments have been selected for description. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.
[0030] Plant sample
[0031] The plant samples required for the experiments of the present invention were collected from the buds of the Ziziphus mauritiana Lam. rootstock (Ziziphus mauritiana Lam.) in the winter warm greenhouse of jujube trees in the Management Service Center of Shandong Binzhou National Agricultural Science and Technology Park. The lowest winter temperature in the winter warm greenhouse of jujube trees in the Management Service Center of Shandong Binzhou National Agricultural Science and Technology Park is 8°C, which can ensure the safe overwintering of Ziziphus mauritiana Lam. The Ziziphus mauritiana Lam. rootstock (Ziziphus mauritiana Lam.) starts to germinate at the end of January, and the experimental samples of this experiment were collected in the first ten days of February 2025. Each example / control example was set with 3 replicates, and 10 shoot tips were cultured in each replicate.
[0032] Example 1
[0033] Prepare an open culture medium:
[0034] Culture medium formula: MS medium + 6-BA 0.8 mg / L + IBA 0.2 mg / L + sucrose 25 g / L + sand (particle size 0.2 - 0.5 mm).
[0035] Add carbendazim 800-fold solution to the culture medium.
[0036] Dispense the culture medium:
[0037] Dispense the culture medium into transparent plastic culture bottles without lids.
[0038] Put in plant shoot tips:
[0039] Select plant shoot tips with a length of 1.0 cm, disinfect them with 1‰ mercuric chloride for 25 seconds first, then disinfect them with 75% alcohol for 10 seconds, and then rinse them with sterile water 3 times., and then put them into the culture bottle.
[0040] Prepare chitosan:
[0041] Raw material preparation: Select fresh shrimp shells that have been crushed.
[0042] Decalcification treatment: Soak the raw materials in 2.5% dilute hydrochloric acid at room temperature, the mass ratio of dilute hydrochloric acid to raw materials is 12:1, and the soaking time is 3 hours.
[0043] Deproteinization and deacetylation reaction: Put the decalcified raw materials into a reaction kettle, add 50% concentrated sodium hydroxide solution, and the mass ratio of chitin powder to concentrated sodium hydroxide solution is 1:12. Place the reaction kettle in a heating device, heat up to 95°C, and maintain this temperature for 2 hours for the deacetylation reaction. During the reaction, use a mechanical stirrer to continuously stir at a speed of 100 revolutions per minute to ensure uniform reaction.
[0044] Termination of reaction and washing: After the reaction is completed, pour the reactants into distilled water to terminate the reaction, and wash with distilled water and absolute ethanol respectively.
[0045] Drying and pulverization: Dry at 60°C and then pulverize to obtain chitosan powder.
[0046] Preparation of surface cover:
[0047] Mix microcrystalline cellulose and chitosan evenly at a mass ratio of 350:1, cover the surface of the culture medium and gently compact it, with a thickness of 3 mm.
[0048] Cultivation conditions:
[0049] Temperature: 26°C.
[0050] Light intensity: 1800 lx.
[0051] Light duration: 13 hours per day.
[0052] Example 2
[0053] Preparation of open culture medium:
[0054] Culture medium formula: MS medium + 6-BA 1.0 mg / L + IBA 0.1 mg / L + sucrose 20 g / L + sand (particle size 0.5 - 0.8 mm).
[0055] Add 500-fold solution of carbendazim to the culture medium.
[0056] Sub-packaging of culture medium:
[0057] Sub-package the culture medium into transparent glass culture bottles without lids.
[0058] Put in plant shoot tips:
[0059] Select plant shoot tips with a length of 0.8 cm, first disinfect with 1‰ mercuric chloride for 35 seconds, then disinfect with 75% alcohol for 20 seconds, and then rinse with sterile water 5 times. Then put them into the culture bottles.
[0060] Preparation of chitosan:
[0061] Raw material preparation: Select crushed fresh crab shells.
[0062] Decalcification treatment: Soak the raw materials in 2.5% dilute hydrochloric acid at room temperature. The mass ratio of dilute hydrochloric acid to raw materials is 10:1, and the soaking time is 4 hours.
[0063] Deproteinization and deacetylation reaction: Put the raw materials after decalcification treatment into a reaction kettle, and add 40% concentrated sodium hydroxide solution. The mass ratio of chitin powder to concentrated sodium hydroxide solution is 1:10. Place the reaction kettle in a heating device, heat up to 90 °C, and maintain this temperature for 3 hours for the deacetylation reaction. During the reaction process, use a mechanical stirrer to continuously stir at a speed of 100 revolutions per minute to ensure uniform reaction.
[0064] Terminate the reaction and wash: After the reaction is completed, pour the reactants into distilled water to terminate the reaction, and wash with distilled water and absolute ethanol respectively.
[0065] Drying and pulverizing: Dry at 60 °C and then pulverize to obtain chitosan powder.
[0066] Make the surface covering:
[0067] Mix microcrystalline cellulose and chitosan evenly according to the mass ratio of 400:1, cover the surface of the culture medium and gently compact it, with a thickness of 2 mm.
[0068] Cultivation conditions:
[0069] Temperature: 27 °C.
[0070] Light intensity: 1500 lx.
[0071] Light time: 12 hours / day.
[0072] Example 3
[0073] Make an open culture medium:
[0074] Culture medium formula: MS medium + 6-BA 1.0 mg / L + IBA 0.1 mg / L + sucrose 20 g / L + sand (particle size 0.5 - 0.8 mm).
[0075] Add carbendazim 1500-fold solution to the culture medium.
[0076] Dispense the culture medium:
[0077] Dispense the culture medium into transparent glass culture bottles without lids.
[0078] Put in the plant shoot tip:
[0079] Select a plant shoot tip with a length of 0.8 cm, disinfect it with 1‰ mercuric chloride for 35 seconds first, then disinfect it with 75% alcohol for 20 seconds, and then rinse it with sterile water 5 times. Then put it into the culture bottle.
[0080] Make chitosan:
[0081] Raw material preparation: Select fresh crushed crab shells.
[0082] Decalcification treatment: Soak the raw materials in 2.5% dilute hydrochloric acid at room temperature. The mass ratio of dilute hydrochloric acid to raw materials is 10:1, and the soaking time is 4 hours.
[0083] Deproteinization and deacetylation reaction: Put the raw materials after decalcification treatment into a reaction kettle, and add 40% concentrated sodium hydroxide solution. The mass ratio of chitin powder to concentrated sodium hydroxide solution is 1:10. Place the reaction kettle in a heating device, heat up to 90 °C, and maintain this temperature for 3 hours for the deacetylation reaction. During the reaction process, use a mechanical stirrer to continuously stir at a speed of 100 revolutions per minute to ensure uniform reaction.
[0084] Terminate the reaction and wash: After the reaction is completed, pour the reaction product into distilled water to terminate the reaction, and wash with distilled water and absolute ethanol respectively.
[0085] Drying and pulverization: Dry at 60 °C and then pulverize to obtain chitosan powder.
[0086] Make the surface covering:
[0087] Mix microcrystalline cellulose and chitosan evenly at a mass ratio of 400:1, cover the surface of the culture medium and gently press it, with a thickness of 1 mm.
[0088] Cultivation conditions:
[0089] Temperature: 27 °C.
[0090] Light intensity: 1500 lx.
[0091] Light duration: 12 hours / day.
[0092] Control Example 1
[0093] Make a traditional culture medium:
[0094] Culture medium formula: MS medium + 6-BA 0.8 mg / L + IBA 0.2 mg / L + sucrose 25 g / L + agar 7 g / L.
[0095] Sterilize the culture medium and culture bottles at 120 °C for 20 minutes.
[0096] Dispense the culture medium:
[0097] Under sterile conditions, dispense the culture medium into transparent plastic culture bottles with lids and ventilation holes on the lids.
[0098] Put in the plant shoot tip:
[0099] Select a plant shoot tip with a length of 1.0 cm, first disinfect it with 1‰ mercuric chloride for 35 seconds, then disinfect it with 75% alcohol for 20 seconds, and then rinse it 4 times with sterile water. Place the treated plant shoot tip in a culture bottle in a laminar flow hood.
[0100] Culture conditions:
[0101] Temperature: 26 °C.
[0102] Light intensity: 1800 lx.
[0103] Light duration: 13 hours / day.
[0104] Comparative Example 2
[0105] Prepare an open medium:
[0106] Medium formula: MS medium + 6-BA 0.8 mg / L + IBA 0.2 mg / L + sucrose 25 g / L + sand (particle size 0.3 mm).
[0107] Add a 500-fold solution of carbendazim to the medium.
[0108] Dispense the medium:
[0109] Dispense the medium into transparent glass culture bottles without lids.
[0110] Place the plant shoot tip:
[0111] Select a plant shoot tip with a length of 1.0 cm, first disinfect it with 1‰ mercuric chloride for 35 seconds, then disinfect it with 75% alcohol for 20 seconds, then rinse it 5 times with sterile water, and then place it in a culture bottle.
[0112] Culture conditions:
[0113] Temperature: 26 °C.
[0114] Light intensity: 1800 lx.
[0115] Light duration: 13 hours / day.
[0116] Observe the contamination rate of the medium and the survival rate of acclimatized transplanting in Examples 1-3 and Comparative Examples 1-2.
[0117] Statistical table of medium contamination rate and survival rate of acclimatized transplanting (%)
[0118]
[0119] Note: Different lowercase letters in the table indicate significant differences in the same column of numbers at the 5% significance level.
[0120] By comparing the contamination rate and acclimatized survival rate of the examples and comparative examples, the following conclusions can be drawn:
[0121] The contamination rates of Example 1 and Example 2 are very low, and the survival rates of seedling hardening are relatively high, indicating that the method of the present invention has significant advantages in reducing the contamination rate and increasing the survival rate of seedling hardening. Although the contamination rate of Comparative Example 1 is relatively low, the survival rate of seedling hardening is only 13.33%, indicating that there are obvious defects in the traditional aseptic culture method during the seedling transplanting stage. The culture medium of Comparative Example 2 is completely contaminated, indicating that chitosan, microcrystalline cellulose and carbendazim play important roles in reducing the contamination rate and increasing the survival rate of seedling hardening. The open tissue culture method of the present invention can effectively carry out jujube tree tissue culture in a relatively open environment, simplify the operation process, reduce costs, and at the same time increase the survival rate of seedlings, with significant innovation and broad application prospects.
Claims
1. A method for open tissue culture of jujube trees, characterized in that: The steps include: (1) preparing an open culture medium by mixing a basic culture medium, sand, sucrose, and carbendazim in a mass ratio of 1000:4000:15-25:1-2 to prepare an open culture medium; (2) dispensing the prepared open culture medium into a culture container, which may be a culture bottle, a triangular flask, or other container of suitable size; (3) placing the sterilized plant stem tip into a culture container filled with culture medium; (4) making chitosan, the steps include: Raw material preparation: Choose crushed fresh shrimp shells and crab shells as raw materials, and ensure that the raw materials are not rotten or deteriorated, have a hard texture and uniform color; Decalcification: Soak the raw material in 2.5% dilute hydrochloric acid to remove the calcium carbonate component in the shell; Deproteinization and deacetylation reaction: Place the decalcified raw materials into a reactor and add 40%-60% concentrated sodium hydroxide solution. Place the reactor in a heating device and heat it to 90°C to 100°C. Maintain this temperature for 2-4 hours for deacetylation reaction. During the reaction, use a mechanical stirrer to stir continuously at a speed of 100 rpm to ensure uniform reaction. Termination of reaction and washing: After the reaction is completed, pour the reactants into distilled water to terminate the reaction, and then wash the solid part with distilled water and anhydrous ethanol respectively to remove residual alkali and impurities; Drying and crushing: drying the washed chitosan at 60°C, and then crushing it again to obtain chitosan powder; (5) Make a surface covering: mix microcrystalline cellulose and chitosan powder at a mass ratio of 30-40:1, cover the surface of the open culture medium and gently compact it to a thickness of 2-4 mm. Microcrystalline cellulose can isolate the invasion of external microorganisms, and its porous structure can ensure the air permeability of the open culture medium, providing sufficient oxygen for the plant roots, which is beneficial to the respiration and growth of the roots.
2. The open tissue culture method of jujube tree according to claim 1, characterized in that: The formula of the basic culture medium is: MS culture medium + 6-BA 0.5-1.0 mg / L + IBA 0.1-0.3 mg / L.
3. The open tissue culture method of jujube tree according to claim 1, characterized in that: The culture container is made of transparent glass or plastic and does not need a cover.
4. The open tissue culture method of jujube tree according to claim 1, characterized in that: The length of the plant stem tip is 0.5-1.5 cm, and before being placed in a culture container, it is first disinfected with 1‰ mercuric liter for 25-35 seconds, then disinfected with 75% alcohol for 10-20 seconds, and then rinsed with sterile water for 3-5 times.
5. The open tissue culture method of jujube tree according to claim 1, characterized in that: In step 4, during the decalcification treatment, the mass ratio of dilute hydrochloric acid to raw material is 10-15:1, and the mixture is soaked at room temperature for 2-4 hours.
6. The open tissue culture method of jujube tree according to claim 1, characterized in that: In step 4, during the deproteinization and deacetylation reaction, the mass ratio of chitosan powder to concentrated sodium hydroxide solution is 1:10-15.
7. The open tissue culture method of jujube tree according to claim 1, characterized in that: The culture container is placed in a culture environment with a temperature of 25-28° C., a light intensity of 1500-2000 lx, and a light duration of 12-14 hours / day.
8. The open tissue culture method of jujube tree according to claim 1, characterized in that: The sand is screened river sand with a particle size of 0.5-2 mm and no impurities.
9. The open tissue culture method of jujube tree according to claim 1, characterized in that: The plant stem tip is derived from a healthy Yunnan jujube tree free of diseases and insect pests.
10. The open tissue culture method of jujube tree according to claim 1, characterized in that: The microcrystalline cellulose and chitosan are mixed and stirred evenly to obtain a surface covering. The surface covering is evenly covered on the surface of the culture medium within 0.5 hours after the plant stem tip is implanted into the open culture medium, with a thickness of 2-4 mm.