Processing method of acer palmatum branch wood explant
Through the treatment of explants of the color leaf maple, including alcohol wipe, hydroponics, laundry detergent and benzolin soaking, and disinfection of ethanol and sodium hypochlorite solutions, the problems of low reproduction rate and incomplete explants of the maple family were solved, and efficient explants disinfection and high germination rate were achieved.
Patent Information
- Application Number
- CN202510287103.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-05-23
AI Technical Summary
The reproduction rate of the maple family plants is low and difficult to survive in traditional reproduction methods, and the existing explant treatment methods have the problem of excessive disinfection time leading to plant death or incomplete disinfection leading to contamination.
A treatment method for colored maple tree explant is adopted, including collecting branches of the axillary buds of the year, wiping and hydroponicing of alcohol, then cutting off the leaves and axillary buds, soaking the laundry powder and benzene, then disinfecting with ethanol and sodium hypochlorite solution, and finally inoculating it in the start-up medium.
Efficient explants disinfection was achieved, and the germination rates of stem tips and axillary buds reached 92.8% and 87.3% respectively. The disinfection effect was good, the toxic effect was small, and the operation was simple.
Smart Images
Figure CN120021555A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for processing colorful maple branch wood explants. Background Art
[0002] Plant tissue culture, also known as plant cloning, is a set of techniques and methods based on the theory of plant cell totipotency. It is to inoculate the plant's organs, tissues or cells - explants (root tips, stem segments, stem tips, young leaves, young embryos, anthers, etc.) in vitro on artificial culture media through aseptic operation, and to culture them in vitro under artificially controlled environmental conditions (temperature, humidity, light, etc.), and finally form complete plants. Plant tissue culture has attracted attention at home and abroad because of its fast reproduction speed, wide application, no seasonal restrictions, and the availability of non-toxic seedling systems. The treatment of plant explants is an indispensable and primary link in the field of plant tissue culture and the first factor in the success of plant tissue culture. However, different plant materials and different organs of the same plant have different treatment and disinfection methods. If the disinfection time is too long, the plant material will die. If the disinfection time is too short, the bacteria in the plant material cannot be killed, resulting in contamination of the plant material.
[0003] Aceraceae plants are mainly propagated by traditional seeds, cuttings or grafting, which are greatly affected by natural factors, and have low reproduction rates and difficulties in survival, which seriously affect the development of the industry. Plant tissue culture is an important way to expand plant propagation and obtain superior varieties. There have been reports on tissue culture technology for Aceraceae plants. The 2014 "Progress in Tissue Culture of Aceraceae in my country" in "Northern Horticulture" reported on the research on the time, location, and disinfection methods of explants of Aceraceae plants, but there are few reports on tissue culture research of colorful maples. Due to the different species of Aceraceae plants, complex genetic backgrounds, and obvious differences in biological characteristics, the explant treatment methods of different plants need to be continuously innovated. Summary of the invention
[0004] The present application provides a method for treating a colored maple branch wood explant, the method comprising the following steps:
[0005] (1) Collect the current year's branches of colored maple with axillary buds, remove the dirt on the surface, wipe with 65-75% alcohol, culture in water at 24-26°C for 10-20 days, spray with alcohol and sterile water 1-2 times in the morning and evening, and maintain the air humidity of the culture environment at 60-70%;
[0006] (2) After the hydroponic culture is completed, the leaves of the branches are cut off, the stem tips and axillary buds are removed, and each section of the stem tip is about 1-2 cm. The cut plant materials are first soaked in 0.15-0.25% detergent water for 10-20 min, soaked in 0.8-1.2 mg / L benomyl for 5-7 min, and then rinsed with tap water for 25-35 min;
[0007] (3) The stem tip and axillary buds were disinfected with 65-75% ethanol for 25-35 seconds, and then immersed in a mixture of 0.08-0.12% sodium hypochlorite solution and Tween 80 for 7-9 minutes, and then rinsed with sterile water after each disinfection; the mixture of 0.08-0.12% sodium hypochlorite solution and Tween 80 was prepared by adding 1 drop to 2 drops of Tween 80 to every 200 ml of 0.08-0.12% sodium hypochlorite solution.
[0008] Preferably, in step (1), 70% alcohol is used for wiping.
[0009] More preferably, the hydroponic temperature in step (1) is 25°C.
[0010] More preferably, during the hydroponic period in step (1), the water is changed once every 2 days.
[0011] More preferably, the cut plant material in step (2) is first soaked in 0.2% washing powder water for 15 minutes, and then soaked in 1 mg / L benomyl for 6 minutes.
[0012] More preferably, in step (3), the stem tip and axillary buds are sterilized with 70% ethanol for 30 seconds respectively.
[0013] More preferably, in step (3), the mixture is then immersed in a mixture of 0.1% sodium hypochlorite solution and Tween 80 for 8 minutes for disinfection.
[0014] More preferably, after disinfection with sodium hypochlorite solution in step (3), rinse with sterile water 5-7 times, each time for no less than 3 minutes.
[0015] The invention also provides application of the treatment method in disinfecting explants of colorful maple branches.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The invention has good disinfection effect, small toxicity and simple operation, and can provide a basis for the treatment of ornamental tree explants. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the germination status of the shoot tip in Example 1 3 weeks after inoculation on the priming medium.
[0019] Figure 2 , 3 The germination conditions of different axillary buds in Example 1 3 weeks after inoculation into the priming medium. DETAILED DESCRIPTION
[0020] Example 1
[0021] 1. Preparation of sterilized vessels:
[0022] Select a 250ml plastic tissue culture bottle, clean it, and sterilize it with high-pressure steam for later use.
[0023] 2. Preparation of disinfectant:
[0024] Sterile water sterilized by high temperature and high pressure (pressure 110 kpa, temperature 121°C, 26 min), 1 mg / L benomyl suspension, 0.1% sodium hypochlorite solution.
[0025] 3. Preparation of equipment:
[0026] Sterilize filter paper, scalpel, scissors, and tweezers.
[0027] Collect the branches of the colored maple with axillary buds from mid-April to the end of May, use clean water to remove the surface dirt, and then pre-treat and wipe with 70% alcohol, and hydroponic for 10-20 days at 25℃ in a closed environment covered with indoor film. During the hydroponic period, change the water once every 2 days, spray alcohol and sterile water once in the morning and evening, and keep the air humidity of the culture environment at 60-70%. After the hydroponic culture is completed, cut off the leaves of the branches, take the stem tips and peel off the axillary buds, and each section of the stem tips is about 1.5cm. The cut plant materials are first soaked in 0.2% laundry detergent water for 15min, 1mg / L benomyl for 6min, and then rinsed under running water for 30min. The rinsed plant materials are transferred to the clean bench for infiltration and disinfection. The stem tip was disinfected with 70% ethanol for 30 seconds, 0.1% sodium hypochlorite solution + Tween 80 (one drop of Tween 80 was added to every 200 ml of sodium hypochlorite solution, and Tween 80 was sucked with a pipette). The main function of the surfactant Tween 80 was to destroy the water film formed on the surface of the explant material during the soaking process, so that the sterilization effect of the explant surface and cortex was more sufficient. Soak and stir for disinfection for 4 minutes; the axillary buds were disinfected with 70% ethanol for 30 seconds, 0.1% sodium hypochlorite disinfectant + Tween 80 (the amount of Tween 80 added was the same as above), and soaked and stirred for disinfection for 8 minutes. After each disinfection, it was necessary to rinse with sterile water. The sodium hypochlorite residue was difficult to remove, so it should be rinsed with sterile water 5-7 times, each time for no less than 3 minutes.
[0028] The wounds of the disinfected stem tips and axillary buds were cut off from the contact parts with the disinfectant and inoculated into the start-up medium, which was the basic medium for woody plants (Med medium) Med+6-BA 1.5mg / L+IBA 0.01mg / L, and incubated at 25℃ for 13h·d. -1 The sterilization rate of the disinfection method of the present invention is 100%. After 3 weeks of inoculation of the stem tips and axillary buds, some of the stem tips and axillary buds begin to sprout and grow, and the sprouting rates of the stem tips and axillary buds reach 92.8% and 87.3% respectively.
[0029] The composition of the plant basic medium (Med medium) used in this experiment is as follows:
[0030]
[0031]
[0032] Comparative Example 1
[0033] The experimental operation was the same as in Example 1, except that the following operations were omitted: the shoot tip was disinfected with 70% ethanol for 30 s, 0.1% sodium hypochlorite solution + Tween 80 (one drop of Tween 80 was added to every 200 ml of sodium hypochlorite solution, and Tween 80 was sucked with a pipette), and the immersion was stirred for disinfection for 4 min; the axillary buds were disinfected with 70% ethanol for 30 s, 0.1% sodium hypochlorite disinfectant + Tween 80 (one drop of Tween 80 was added to every 200 ml of sodium hypochlorite solution, and Tween 80 was sucked with a pipette), and the immersion was stirred for disinfection for 8 min.
[0034] Comparative Example 2
[0035] The experimental operation was the same as in Example 1, except that the operation before "disinfecting the shoot tip with 70% ethanol for 30 s, 0.1% sodium hypochlorite solution + Tween 80 (adding one drop of Tween 80 to every 200 ml of sodium hypochlorite solution, and taking Tween 80 with a pipette), soaking and stirring for disinfection for 4 min; disinfecting the axillary buds with 70% ethanol for 30 s, 0.1% sodium hypochlorite disinfectant + Tween 80 (adding one drop of Tween 80 to every 200 ml of sodium hypochlorite solution, and taking Tween 80 with a pipette), soaking and stirring for disinfection for 8 min" was omitted.
[0036] The percentage (%) of sterile complete plants obtained by culturing axillary buds in Example 1 and Comparative Example 1-2 is as follows:
[0037] Table 1
[0038] explant Example 1 Comparative Example 1 Comparative Example 2 Shoot tip 92.8％ 0 18.7％ axillary bud 87.3％ 5.6％ 19.3％
[0039] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for treating a colored maple branch explant, characterized in that: The processing method comprises the following steps: (1) Collect the current year's branches of colored maple with axillary buds, remove the dirt on the surface, wipe with 65-75% alcohol, culture in water at 24-26°C for 10-20 days, spray with alcohol and sterile water 1-2 times in the morning and evening, and maintain the air humidity of the culture environment at 60-70%; (2) After the hydroponic culture is completed, the leaves of the branches are cut off, the stem tips and axillary buds are removed, and each section of the stem tip is about 1-2 cm. The cut plant materials are first soaked in 0.15-0.25% detergent water for 10-20 min, soaked in 0.8-1.2 mg / L benomyl for 5-7 min, and then rinsed with tap water for 25-35 min; (3) The stem tip and axillary buds were disinfected with 65-75% ethanol for 25-35 seconds, and then immersed in a mixture of 0.08-0.12% sodium hypochlorite solution and Tween 80 for 7-9 minutes, and then rinsed with sterile water after each disinfection; the mixture of 0.08-0.12% sodium hypochlorite solution and Tween 80 was prepared by adding 1 drop to 2 drops of Tween 80 to every 200 ml of 0.08-0.12% sodium hypochlorite solution.
2. The processing method according to claim 1, characterized in that: In step (1), 70% alcohol is used for wiping.
3. The processing method according to claim 2, characterized in that: The hydroponic temperature in step (1) is 25°C.
4. The processing method according to claim 3, characterized in that: During the hydroponic culture in step (1), the water is changed once every 2 days.
5. The processing method according to claim 4, characterized in that: The cut plant material in step (2) is first soaked in 0.2% washing powder water for 15 minutes, and then soaked in 1 mg / L benomyl for 6 minutes.
6. The processing method according to claim 5, characterized in that: In the step (3), the stem tip and axillary buds are disinfected with 70% ethanol for 30 seconds respectively.
7. The processing method according to claim 6, characterized in that: In step (3), the mixture is then immersed in a mixture of 0.1% sodium hypochlorite solution and Tween 80 for 8 minutes for stirring and disinfection.
8. The processing method according to claim 7, characterized in that: After disinfection with sodium hypochlorite solution in step (3), rinse with sterile water 5-7 times, each time for no less than 3 minutes.
9. Use of the treatment method according to any one of claims 1 to 8 in disinfecting colored maple branch explants.