A method for raising seedlings of Castanopsis chinensis cell suspension culture

Abstract

The invention relates to the field of forestry seedling production, and discloses a method for raising seedlings by cell suspension culture of tung oil tree, comprising the following steps: disinfecting the surface of tung oil tree tender stems with a 1% sodium hypochlorite solution, and then soaking the tender stems in 70% ethanol alcohol; chopping the tung oil tree tender stems treated in step 2 into small pieces with a blade; placing them in a 1.2% Cellulase R-10 enzyme solution, and incubating them in a constant temperature incubator; filtering the treated sample with a cell pore size filter paper to remove residual tung oil tree cell wall fragments, washing them multiple times with a clean buffer solution, so as to make the single cell protoplasts clean, etc. In the invention, the separation and culture of tung oil tree cells are effectively supported, the problems of long breeding time and low survival rate in traditional tung oil tree breeding methods are improved, cell division and growth are promoted, and ultraviolet light sterilization ensures the sterility of the culture environment and reduces exogenous pollution.

Description

Technical Field

[0001] The invention relates to the field of forestry seedling production, and in particular to a method for raising seedlings through cell suspension culture of Castanopsis chinensis. Background Art

[0002] Ailanthus altissima is a tree with economic and environmental value, and is widely used in landscaping and medicinal purposes. Traditional propagation methods of Ailanthus altissima often have problems such as long propagation time and low survival rate, so a more efficient and reliable propagation method is needed.

[0003] After searching, the publication number CN107258452 discloses a method for raising seedlings of Castanopsis truncatum, which uses smoldering high temperature to harmlessly treat the soil in the seedbed, kill weed seeds, bacteria, nematodes, viruses and eggs, and uses tea dregs to prevent diseases and insects, which comprehensively solves the problems of diseases and insect pests in the production of Castanopsis truncatum seedlings, and solves the problem of weeds in the seedbed. However, the seed culture used in the above application has problems such as long breeding time and low survival rate. Summary of the invention

[0004] In order to make up for the above shortcomings, the present invention provides a method for raising seedlings of Castanopsis schrenkiana cell suspension culture, aiming to improve the problems of long breeding time and low survival rate in traditional methods of breeding Castanopsis schrenkiana.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for raising seedlings of Castanopsis schrenkiana cell suspension culture, comprising the following steps:

[0006] Step 1, take the tender stems of Castanopsis jasminoides, and prepare the required culture medium and sterilizing equipment;

[0007] Step 2: The surface of the tender stem of the tung tree fruit is disinfected with a 1% sodium hypochlorite solution, and then the tender stem is immersed in 70% ethanol;

[0008] Step 3: Use a blade to chop the tender stems of the tung tree processed in step 2 into small pieces;

[0009] Step 4, placing the small pieces processed in step 3 in a 1.2% Cellulase R-10 enzyme solution and incubating in a constant temperature incubator;

[0010] Step 5, placing the tissue of Castanopsis serrata in an enzymatic hydrolysate containing 1.2% Pectolyase Y-23, placing the container containing the enzymatic hydrolysate and the tissue in a constant temperature incubator for incubation, and then adding a digestive enzyme inhibitor and placing it in a water bath again to heat for a period of time;

[0011] Step 6: Filter the treated sample using cell pore filter paper to remove the residual cell wall fragments of Castanopsis schrenkiana, and wash the sample several times using a clean buffer to make the single-cell protoplasts clean;

[0012] Step 7, after enzymatic incubation, observe the changes in the tissue, separate the tissue and release the single cells, then transfer the separated single cells to a new MS+0.2 mg / L IAA liquid culture medium to culture the Castanopsis sylvestris cell fluid;

[0013] Step 8, under sterile conditions, placing the Castanopsis schrenkiana cell culture medium obtained in the previous step on a shaker, maintaining a certain rotation speed for cultivation, and culturing under light for a period of time every day;

[0014] Step 9: Prepare a solid culture medium of MS+agar+1.5mg / L IAA+0.1mg / L IBA, adjust the pH value of the culture medium to acidic, pour the prepared culture medium into the culture dish, immediately cover the culture dish lid, wait for the culture medium in the culture dish to solidify, and then place it in a clean bench and sterilize it with ultraviolet light;

[0015] Step 10, evenly apply the cell solution of the Mandelieu sempervirens cultured in step 7 to the prepared culture dish in the clean bench, and culture under light;

[0016] Step 11. Cover the sugar-free tissue culture box with vermiculite in advance and irrigate it with rooting solution. Then transplant the seedlings cultured in the culture dish into the sugar-free culture box, cover the box, and culture it under light every day. The box must be forced to ventilate and the carbon dioxide concentration should be maintained at 1000-1500pmol / L. After a period of time, it will grow into Castanopsis affine tissue culture seedlings.

[0017] As a further description of the above technical solution, in step 2, the disinfection treatment is performed for 10 minutes and the immersion in ethanol alcohol is performed for 10 minutes.

[0018] As a further description of the above technical solution, in step 4, when incubating in a constant temperature incubator, the incubation is carried out at a temperature of 28°C for 45 minutes.

[0019] As a further description of the above technical solution, in step 5, when incubating in a constant temperature incubator, incubate at 30°C for 45 minutes, then add a digestive enzyme inhibitor and place it in a 50°C water bath for 5 minutes.

[0020] As a further description of the above technical scheme, step 8 specifically includes the following steps: under sterile conditions, the Castanopsis schrenkiana cell culture container obtained in the previous step is placed on a shaker at 25°C, cultured at a speed of 10 r / min, with an illumination of 3000 lux at 460 nm, a lighting time of 10 h per day, and cultured for 14 days.

[0021] As a further description of the above technical solution, in step 9, the pH value of the culture medium is adjusted to 5.8, and the ultraviolet lamp sterilization time is 30 minutes.

[0022] As a further description of the above technical solution, in step 10, the conditions for culturing under light are 4500 lux at 460nm+630nm, 10 hours of light per day, and a temperature of 26°C. After 21 days, young shoots grow and the culture is continued for 35 days.

[0023] As a further description of the above technical solution, in step 11, the conditions for daily illumination culture are 10 hours of illumination per day, full spectrum illumination, 15,000 lux, and a culture temperature of 28°C.

[0024] The present invention has the following beneficial effects:

[0025] The present invention not only effectively supports the separation and culture of Castanopsis japonici cells, but also promotes their growth and development through optimized environmental conditions, thereby improving the problems of long breeding time and low survival rate in traditional Castanopsis japonici methods, and provides healthy plant materials with growth potential for further research or application.

[0026] In the present invention, enzymatic hydrolyzing agents are used to treat the tender stems of Castanopsis schrenkiana, and the cells are successfully separated and transferred to a suitable culture medium, thereby ensuring the survival and continued growth of single cells. Light culture on a shaker provides cells with suitable photosynthesis conditions, promoting cell division and growth. Ultraviolet light sterilization ensures the sterility of the culture environment and reduces exogenous pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention provides a flow chart of a method for raising seedlings through cell suspension culture of Castanopsis affine. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Reference Figure 1 , an embodiment provided by the present invention: a method for raising seedlings through cell suspension culture of Castanopsis affine, comprising the following steps:

[0030] S1. Preparation: First, take the tender stems of Castanopsis jasminoides, because the cells in the tender stems are easier to separate and the cells are more active.

[0031] S2. Preparation: Prepare the required culture medium and sterilizing equipment at the same time.

[0032] S3. Sampling: Take out the tender stems of Castanopsis affine, and try to avoid taking in too much accompanying tissue.

[0033] S4. Sample treatment: Disinfect the surface of the tender stems of Castanopsis truncatula with 1% sodium hypochlorite solution for 10 minutes to remove possible bacteria and fungi. Then soak the tender stems in 70% ethanol for 10 minutes.

[0034] S5. Cut tissue: Use a blade to chop the sterilized tender stems of Castanopsis schrenkiana into small pieces.

[0035] S6. Enzymatic hydrolysis: Place the cut tissue in a 1.2% Cellulase R-10 enzyme solution at an appropriate concentration, place it in a constant temperature incubator, and incubate it at 28°C for 45 minutes to degrade the colloid substance between cells and promote cell separation.

[0036] S7. Place the tissue of the mountain ash in a solution containing 1.2% Pectolyase Y-23. Make sure that the tissue is completely soaked in the solution and that the amount of liquid is sufficient to cover the tissue sample. Place the container containing the solution and tissue in a constant temperature incubator and incubate at 30°C for 45 minutes. Then add the digestive enzyme inhibitor and place it in a 50°C water bath for 5 minutes.

[0037] S8, filtration and washing: Use cell pore filter paper to filter the treated sample to remove the residual cell wall fragments of Castanopsis schrenkiana, and use clean buffer to wash several times to make the single-cell protoplasts relatively clean;

[0038] S9. Separation of cells: After enzymatic incubation, observe the changes in the tissue. It is expected that the tissue will be separated and single cells will be released. Use a pipette, tweezers and other tools to carefully transfer the separated single cells into a new MS + 0.2 mg / L IAA liquid culture medium to culture the Castanopsis sylvestris cell fluid.

[0039] S10. Illumination culture: Under sterile conditions, the cell culture container obtained in the previous step was placed on a shaker at 25°C, cultured at a speed of 10 r / min, with an illumination of 3000 lux at 460 nm and a daily illumination time of 10 h for 14 days.

[0040] S11. Prepare a solid culture medium of MS + agar + 1.5 mg / L IAA + 0.1 mg / L IBA, adjust the pH value of the culture medium to 5.8, pour the prepared culture medium into the culture dish, immediately cover the culture dish lid, and after the culture medium in the culture dish solidifies, place it in the clean bench and sterilize it with ultraviolet light for 30 minutes.

[0041] S12. Evenly apply the cell fluid of Castanopsis schrenkiana cultured in step 9 to the prepared culture dish in the clean bench, and culture it under a light of 4500 lux at 460nm+630nm, 10 hours of light per day, and a temperature of 26°C. After 21 days, it can be observed that very small buds have grown, and the culture is continued for 35 days.

[0042] S13. Transplantation: Cover the sugar-free tissue culture box with vermiculite in advance and irrigate it with rooting solution. Then transplant the seedlings cultured in the culture dish into the sugar-free culture box. Transplant 100 plants in each box. Cover the box and expose it to sunlight for 10 hours a day. Use full-spectrum light, 15,000 lux, and the culture temperature is 28°C. The box must be forced to ventilate and the carbon dioxide concentration should be maintained at 1000-1500pmol / L. After 21 days, it will grow into a Tung oil plant with a rich root system.

[0043] In the above embodiments, each step has an important impact on the final culture result: Cell separation and culture solution preparation: The tender stems of Castanopsis schrenkiana were treated with an enzymatic agent to successfully separate the cells and transfer them to a suitable culture medium (MS+0.2mg / LIAA). This ensures the survival and continued growth of single cells. Light and temperature control: The light culture (25°C, 460nm, 3000 lux, 10 hours a day) carried out on a shaker provides cells with suitable photosynthesis conditions, promoting cell division and growth. Preparation of solid culture medium and ultraviolet light sterilization: The prepared solid culture medium (MS+agar+1.5mg / L IAA+0.1mg / L IBA, pH 5.8) provides nutrition and support, and ultraviolet light sterilization ensures the sterility of the culture environment and reduces exogenous contamination. Observation of bud growth and continuation of culture: Under the control of light (460nm+630nm, 4500 lux, 10 hours a day) and temperature (26°C), the buds can grow healthily in the culture dish. After 21 days, small shoots were observed to grow, and the culture continued for 35 days for further development and growth. Transplantation and rooting: The cultured seedlings were transplanted into sugar-free tissue culture boxes and the root system was promoted by watering with rooting solution. The lighting and ventilation conditions (full spectrum lighting, 15,000 lux, 28°C, CO2 concentration maintained at 1000-1500 pmol / L) maintained the ideal environmental conditions required for plant growth. Through this series of steps, the Tung tree tissue culture seedlings with rich root systems can be obtained, which can continue to grow and propagate in subsequent laboratory studies or field transplants.

[0044] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for raising seedlings of Castanopsis affine cell suspension culture, characterized in that: The following steps are involved: Step 1, take the tender stems of Castanopsis jasminoides, and prepare the required culture medium and sterilizing equipment; Step 2: The surface of the tender stem of the tung tree fruit is disinfected with a 1% sodium hypochlorite solution, and then the tender stem is immersed in 70% ethanol; Step 3: Use a blade to chop the tender stems of the tung tree processed in step 2 into small pieces; Step 4, placing the small pieces processed in step 3 in a 1.2% Cellulase R-10 enzyme solution and incubating in a constant temperature incubator; Step 5, placing the tissue of Castanopsis serrata in an enzymatic hydrolysate containing 1.2% Pectolyase Y-23, placing the container containing the enzymatic hydrolysate and the tissue in a constant temperature incubator for incubation, and then adding a digestive enzyme inhibitor and placing it in a water bath again to heat for a period of time; Step 6: Filter the treated sample using cell pore filter paper to remove the residual cell wall fragments of Castanopsis schrenkiana, and wash the sample several times using a clean buffer to make the single-cell protoplasts clean; Step 7, after enzymatic incubation, observe the changes in the tissue, separate the tissue and release the single cells, then transfer the separated single cells to a new MS+0.2 mg / L IAA liquid culture medium to culture the Castanopsis sylvestris cell fluid; Step 8, under sterile conditions, placing the Castanopsis schrenkiana cell culture medium obtained in the previous step on a shaker, maintaining a certain rotation speed for cultivation, and culturing under light for a period of time every day; Step 9: Prepare a solid culture medium of MS+agar+1.5mg / L IAA+0.1mg / L IBA, adjust the pH value of the culture medium to acidic, pour the prepared culture medium into the culture dish, immediately cover the culture dish lid, wait for the culture medium in the culture dish to solidify, and then place it in a clean bench and sterilize it with ultraviolet light; Step 10, evenly apply the cell solution of the Mandelieu sempervirens cultured in step 7 to the prepared culture dish in the clean bench, and culture under light; Step 11. Cover the sugar-free tissue culture box with vermiculite in advance and irrigate it with rooting solution. Then transplant the seedlings cultured in the culture dish into the sugar-free culture box, cover the box, and culture it under light every day. The box must be forced to ventilate and the carbon dioxide concentration should be maintained at 1000-1500pmol / L. After a period of time, it will grow into Castanopsis affine tissue culture seedlings.

2. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In step 2, the disinfection treatment is performed for 10 minutes and the sample is immersed in ethanol for 10 minutes.

3. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In step 4, the incubation is carried out at a temperature of 28° C. for 45 minutes in a constant temperature incubator.

4. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In step 5, the cells are placed in a constant temperature incubator for incubation at 30°C for 45 minutes, and then a digestive enzyme inhibitor is added and the cells are placed in a 50°C water bath for 5 minutes.

5. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: The step 8 specifically includes the following steps: under sterile conditions, placing the Castanopsis schrenkiana cell culture container obtained in the previous step on a shaker at 25°C, culturing at a speed of 10 r / min, irradiating with 3000 lux at 460 nm, 10 h of illumination per day, and culturing for 14 days.

6. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In step 9, the pH value of the culture medium is adjusted to 5.8, and the ultraviolet lamp sterilization time is 30 minutes.

7. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In the step 10, the conditions for culturing under light are culturing at 4500 lux of 460nm+630nm, 10 hours of light per day, and a temperature of 26°C. After 21 days, young shoots grow and the culture is continued for 35 days.

8. The method for raising seedlings of Castanopsis affine cell suspension culture according to claim 1, characterized in that: In step 11, the conditions for daily illumination culture are 10 hours of illumination per day, full spectrum illumination, 15,000 lux, and a culture temperature of 28°C.

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