Acer elegans tissue culture method

Abstract

The invention provides an acer elegans tissue culture method, and belongs to the technical field of tissue culture, and the acer elegans tissue culture method provided by the invention comprises the following steps: S1, explant selection; s2, disinfection; s3, salicylic acid treatment; s4, carrying out anti-oxidation treatment; s5, carrying out callus culture; s6, culturing cluster buds; s7, rooting culture; and S8, field planting. Through specific disinfection, treatment and culture steps, the technical problems of high pollution rate, serious browning and low survival rate in acer elegans tissue culture are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and more particularly, to a tissue culture method for Acer elegantulum Fang et P. L. Chiu Background Art

[0002] Acer elegantulum Fang et P. L. Chiu is a tree species with extremely high ornamental value. At present, the reproduction of Acer elegantulum Fang et P. L. Chiu mainly adopts traditional methods such as sowing, cutting and grafting. However, these methods have disadvantages such as low propagation coefficient, long cycle and strong seasonality, and it is difficult to meet the market demand for a large number of seedlings of excellent varieties. As a rapid propagation means, tissue culture technology can obtain a large number of plants with consistent genetic traits in a short time, and has become an important method for the reproduction of Acer elegantulum Fang et P. L. Chiu.

[0003] In the prior art, there have been reports on tissue culture methods for various maples. For the disinfection of explants in the tissue culture of maples, mercuric chloride is mainly used for disinfection at present. Mercuric chloride is highly toxic and harms both operators and the environment. Secondly, the explants of Acer elegantulum Fang et P. L. Chiu are prone to severe browning during the culture process, which is mainly caused by the oxidation of phenolic substances at the incision wound, seriously affecting the formation of callus and bud differentiation. Therefore, it is urgent to develop a new tissue culture method for Acer elegantulum Fang et P. L. Chiu to solve the problems of disinfectant toxicity and explant browning in the prior art. Summary of the Invention

[0004] The purpose of the present invention is to provide a tissue culture method for Acer elegantulum Fang et P. L. Chiu with low disinfection toxicity and not prone to browning.

[0005] To achieve the above object, the present invention provides a tissue culture method for Acer elegantulum Fang et P. L. Chiu, and the tissue culture method for Acer elegantulum Fang et P. L. Chiu includes the following steps: S1. Explant selection: Select non-lignified shoot tips with 1-2 axillary buds as explants in April every year, and rinse the explants with sterile water at least 2 times; S2. Disinfection: Immerse the explants in a silver disinfectant for 5-10 min, then rinse the explants with sterile water to remove the residual silver disinfectant. Then immerse the explants in 3% hydrogen peroxide for 5-10 min, and then rinse the explants with sterile water to remove hydrogen peroxide; S3. Salicylic acid treatment: Immerse the explants treated in step S2 in a salicylic acid solution for 12-24 h, and then rinse the explants with sterile water to remove the residual salicylic acid solution; S4. Antioxidant treatment: Immerse the explants treated in step S3 in an antioxidant buffer solution for 5-10 min, and then rinse the explants with sterile water to remove the residual antioxidant buffer solution; S5. Callus culture: Inoculate the explants treated in step S4 into a starting medium for starting culture to obtain callus; S6. Cluster bud culture: Inoculate the callus obtained in step S5 into a germination medium for germination culture to obtain cluster buds; S7. Rooting culture: Inoculate the cluster buds obtained in step S6 into a rooting medium for rooting culture to obtain cluster seedlings; S8. Transplanting and planting: Transplant the cluster seedlings obtained in step S7 into substrate soil for transplanting and planting.

[0006] In the tissue culture method of Acer elegantulum provided by the present invention, silver disinfectant and hydrogen peroxide are used in combination for disinfection to replace the traditional sodium hypochlorite or mercuric chloride disinfection. While ensuring the disinfection efficiency of the explants, it causes less harm to the explants and operators.

[0007] More preferably, in the technical solution of the present invention, the variety of Acer elegantulum is more specifically "Simingmeiwu".

[0008] Preferably, in step S2, the silver disinfectant is a 1% silver nitrate solution containing 0.1% Tween-20.

[0009] Preferably, in step S2, the silver disinfectant is a 200-450 ppm nano-silver solution containing 0.1% Tween-20.

[0010] In the tissue culture method of Acer elegantulum provided by the present invention, silver ions and nano-silver can cooperate with Tween-20 to improve the permeability of the disinfectant to miscellaneous bacteria, and its disinfection efficiency is much higher than that of existing disinfectants.

[0011] Preferably, in step S3, the concentration of the salicylic acid solution is 0.1-0.5 mg / L.

[0012] Preferably, in step S4, the antioxidant buffer solution includes 0.5-1 g / L of ascorbic acid and 0.1-0.5 g / L of glutathione.

[0013] In the tissue culture method of Acer elegantulum provided by the present invention, treating the explants with salicylic acid, ascorbic acid and glutathione can effectively inhibit the formation of phenolic substances, and at the same time can scavenge the free radicals generated by the explants to avoid the formation of brown substances.

[0014] Preferably, in step S5, the initiation medium is the QL medium supplemented with 0.5-2 mg / L of TDZ, 0.05-0.1 mg / L of NAA, and 0.05% by mass of activated carbon.

[0015] Preferably, in step S6, the germination medium is the QL medium supplemented with 1-2 mg / L of TDZ and 0.05% by mass of activated carbon.

[0016] Preferably, in step S7, the rooting medium is 1 / 2 MS medium supplemented with 0.1 - 0.5 mg / L of 6 - BA, 0.1 - 0.3 mg / L of IBA, and 0.05% by mass of activated carbon.

[0017] Preferably, in step S8, by mass, the substrate soil comprises 1 part of vermiculite, 1 part of perlite, and 1 - 3 parts of humus soil.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention constructs a mercury - free disinfection system for Acer elegantulum, uses silver - hydrogen peroxide synergistic disinfection, and through the broad - spectrum bactericidal effect of silver ions or silver nanoparticles in synergy with Tween - 20, realizes the deep inactivation of bacteria and fungi on the surface of explants, while reducing the contamination rate of explants and eliminating the harm of highly toxic mercury preparations to operators and the environment; 2. Through the synergistic effect of salicylic acid pretreatment and ascorbic acid - glutathione composite antioxidant solution, the present invention forms a dual protection mechanism of "enzyme activity inhibition - free radical scavenging", reduces the generation of brown substances in explants, and improves the success rate of tissue culture; 3. The present invention also optimizes the composition of the culture medium, improves the induction success rate of callus, and provides reliable technical support for the preservation of Acer elegantulum germplasm resources and industrialized seedling raising. Detailed implementation manners

[0019] To make the above - mentioned objects, features, and advantages of the present invention more obvious and understandable, the following detailed description is made of the specific embodiments of the present invention. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the protection scope of the claims of the present invention.

[0020] It should be noted that the endpoints and any values disclosed in this article are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0021] As described in the background art, the existing tissue culture method for Acer elegantulum has problems such as high toxicity of disinfectants and serious browning. In view of this, the detailed implementation manners of the present invention provide a tissue culture method for Acer elegantulum, which is specifically for the "Siming Meiwu" variety of Acer elegantulum, and includes the following steps: S1. Explant selection: Select non-lignified shoot tips with 1-2 axillary buds as explants in April every year, and rinse the explants with sterile water at least 2 times. S2. Disinfection: Immerse the explants in silver disinfectant for 5-10 minutes, then rinse the explants with sterile water to remove the residual silver disinfectant. Next, immerse the explants in 3% hydrogen peroxide for 5-10 minutes, and then rinse the explants with sterile water to remove hydrogen peroxide. S3. Salicylic acid treatment: Immerse the explants treated in step S2 in salicylic acid solution for 12-24 hours, and then rinse the explants with sterile water to remove the residual salicylic acid solution. S4. Antioxidant treatment: Immerse the explants treated in step S3 in antioxidant buffer for 5-10 minutes, and then rinse the explants with sterile water to remove the residual antioxidant buffer. S5. Callus culture: Inoculate the explants treated in step S4 into the initiation medium for initiation culture to obtain callus. S6. Multiple shoot culture: Inoculate the callus obtained in step S5 into the germination medium for germination culture to obtain multiple shoots. S7. Rooting culture: Inoculate the multiple shoots obtained in step S6 into the rooting medium for rooting culture to obtain multiple seedlings. S8. Transplanting: Transplant the multiple seedlings obtained in step S7 into the substrate soil for transplantation.

[0022] More specifically, in step S2 of the above embodiment, the silver disinfectant is a 1% silver nitrate solution containing 0.1% Tween-20.

[0023] More specifically, in step S2 of the above embodiment, the disinfectant is a 200-450 ppm silver nano solution containing 0.1% Tween-20.

[0024] More specifically, in step S3 of the above embodiment, the concentration of the salicylic acid solution is 0.1-0.5 mg / L.

[0025] More specifically, in step S4 of the above embodiment, the antioxidant buffer includes 0.5-1 g / L of ascorbic acid and 0.1-0.5 g / L of glutathione. More specifically, in step S5 of the above embodiment, the initiation medium is the QL medium supplemented with 0.5-2 mg / L of TDZ, 0.05-0.1 mg / L of NAA, and 0.05% by mass of activated carbon.

[0026] More specifically, in step S6 of the above embodiment, the germination medium is the QL medium supplemented with 1-2 mg / L of TDZ and 0.05% by mass of activated carbon.

[0027] More specifically, in step S7 of the above embodiment, the rooting medium is 1 / 2 MS medium supplemented with 0.1 - 0.5 mg / L of 6 - BA, 0.1 - 0.3 mg / L of IBA, and 0.05% by mass of activated carbon.

[0028] More specifically, in step S8 of the above embodiment, by mass, the substrate soil includes 1 part of vermiculite, 1 part of perlite, and 1 - 3 parts of humus soil.

[0029] The specific implementation mode of the present invention improves the success rate of tissue culture by optimizing the disinfection method and introducing anti - browning operations.

[0030] The technical solution of the present invention will be further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarification or easy reference, and such definitions herein should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited herein are generally well - understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments follows the protocols and parameters given by the manufacturers.

[0031] Example 1 A tissue culture method for Acer elegantulum, comprising the following steps: S1. Explant selection: In April 2023, select several non - lignified shoot tips of "Simingmeiwu" with 1 - 2 axillary buds as explants. The length of the explants is 3 - 5 cm, and rinse the explants twice with sterile water. S2. Disinfection: Immerse in a solution of 1% silver nitrate with 0.1% Tween - 20 for 8 min, gently shake to make the disinfectant fully contact with the explants. After disinfection, rinse the explants 5 times with sterile water, 2 min each time, to thoroughly remove the residual silver disinfectant. Subsequently, soak the explants in a 3% hydrogen peroxide solution for 7 min for secondary disinfection, and rinse again 5 times with sterile water, 2 min each time, to ensure that the hydrogen peroxide is completely removed. S3. Salicylic acid treatment: At 18°C, immerse the explants treated in step S2 in a 0.3 mg / L salicylic acid solution for 18 h. After treatment, rinse the explants 4 times with sterile water, 3 min each time, to ensure that the salicylic acid solution is completely removed. S4. Antioxidant treatment: After soaking the explants treated in step S3 in an antioxidant buffer containing 0.8 g / L ascorbic acid and 0.3 g / L glutathione for 8 min, rinse the explants 3 times with sterile water for 2 min each time to remove the residual antioxidant buffer; S5. Callus culture: Cut the explants treated in step S4 into small segments about 1 cm in length, with 1 axillary bud retained in each segment. Use forceps to vertically insert the explants into the initiation medium to a depth of about 0.5 cm. The initiation medium is QL medium supplemented with 1.5 mg / L TDZ, 0.08 mg / L NAA, and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture flask in a light incubator. The culture conditions are a temperature of 25 ± 1 °C, a light intensity of 2000 lux, and a light cycle of 16 h / d. After 2 weeks of culture, pale yellow callus begins to form at the base of the explants. Continue to culture for 1 week, and the callus gradually enlarges and shows a granular structure;

[0032] S6. Multiple shoot culture: Inoculate the callus obtained in step S5 into the germination medium. The germination medium is QL medium supplemented with 1.5 mg / L TDZ and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture flask in a light incubator. The culture conditions are a temperature of 25 ± 1 °C, a light intensity of 3000 lux, and a light cycle of 16 h / d. After 1 week of culture, green protrusions begin to appear on the surface of the callus. Continue to culture for 1 week, and the green protrusions develop into obvious bud points, forming multiple shoots. Subculture once every 1 week for 2 consecutive subcultures to obtain a large number of robust multiple shoots; S7. Rooting culture: Select the well-grown multiple shoots with a height of about 2 - 3 cm in step S6, separate them from the callus with a sterile scalpel, and transfer them to the rooting medium. The rooting medium is 1 / 2 MS medium supplemented with 0.3 mg / L 6 - BA, 0.2 mg / L IBA, and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture dish in a light incubator. The culture conditions are a temperature of 22 ± 1 °C, a light intensity of 2500 lux, and a light cycle of 14 h / d. After 2 weeks of culture, white root primordia begin to appear at the base of the multiple shoots. Continue to culture for 2 weeks, and the roots gradually elongate and branch to form complete multiple plantlets; S8. Planting: Transplant the cluster seedlings obtained in step S7 into the substrate soil for planting. The substrate soil consists of 1 part of vermiculite, 1 part of perlite, and 2 parts of humus soil. After planting, place the cluster seedlings in the greenhouse for hardening. Control the temperature at 20 - 25°C, maintain the relative humidity above 80%, use a sunshade net to adjust the light intensity, keep spraying throughout the day for the first 7 days, then gradually reduce the spraying frequency, and carry out normal management after 15 days. After 1 month of hardening, the survival rate reaches 94%, the plants grow vigorously, and then transfer them to field cultivation.

[0033] Example 2 A tissue culture method for Acer elegantulum, comprising the following steps: S1. Explant selection: In April 2023, select several non-lignified shoot tips of "Simingmeiwu" with 1 - 2 axillary buds as explants. The length of the explants is 3 - 5 cm, and rinse the explants twice with sterile water. S2. Disinfection: Immerse the explants in a 300 ppm silver nanoparticle solution containing 0.1% Tween - 20 for 7 minutes, gently shake to make the disinfectant fully contact with the explants. After disinfection, rinse the explants 4 times with sterile water, 3 minutes each time, to thoroughly remove the residual silver disinfectant. Subsequently, soak the explants in a 3% hydrogen peroxide solution for 6 minutes for secondary disinfection, and rinse 4 times again with sterile water, 3 minutes each time, to ensure that the hydrogen peroxide is completely removed. S3. Salicylic acid treatment: Immerse the explants treated in step S2 in a 0.2 mg / L salicylic acid solution at 18°C for 15 hours. After treatment, rinse the explants 3 times with sterile water, 4 minutes each time, to ensure that the salicylic acid solution is completely removed. S4. Antioxidant treatment: Immerse the explants treated in step S3 in an antioxidant buffer solution containing 0.6 g / L of ascorbic acid and 0.2 g / L of glutathione for 6 minutes, and then rinse the explants 3 times with sterile water, 2 minutes each time to remove the residual antioxidant buffer solution. S5. Callus culture: Cut the explants treated in step S4 into small segments about 1 cm in length, with 1 axillary bud retained in each segment. Use forceps to vertically insert the explants into the initiation medium, with the insertion depth about 0.5 cm. The initiation medium is QL medium supplemented with 2.0 mg / L of TDZ, 0.06 mg / L of NAA, and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture bottle in a light incubator. The culture conditions are a temperature of 25 ± 1°C, a light intensity of 2000 lux, and a light cycle of 16 h / d. After 2 weeks of culture, pale yellow callus begins to form at the base of the explants. Continue to culture for 1 week, and the callus gradually enlarges and shows a granular structure.

[0034] S6. Clustered bud culture: Inoculate the callus obtained in step S5 into the germination medium, which is the QL medium supplemented with 1.0 mg / L of TDZ and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture flask in a light incubator. The culture conditions are a temperature of 25 ± 1°C, a light intensity of 3000 lux, and a light cycle of 16 h / d. After 1 week of culture, green protrusions begin to appear on the surface of the callus. Continue to culture for 1 week, and the green protrusions develop into obvious bud points, forming clustered buds. Conduct subculture once every 1 week and subculture continuously for 2 times to obtain a large number of robust clustered buds; S7. Rooting culture: Select the well-grown clustered buds with a height of about 2 - 3 cm in step S6, separate them from the callus with a sterile scalpel, and transfer them to the rooting medium, which is the 1 / 2 MS medium supplemented with 0.5 mg / L of 6-BA, 0.3 mg / L of IBA, and 0.05% by mass of activated carbon. Adjust the pH value of the medium to 5.8. After inoculation, place the culture dish in a light incubator. The culture conditions are a temperature of 22 ± 1°C, a light intensity of 2500 lux, and a light cycle of 14 h / d. After 2 weeks of culture, white root primordia begin to appear at the base of the clustered buds. Continue to culture for 2 weeks, and the roots gradually elongate and branch to form complete clustered seedlings; S8. Transplanting: Transplant the clustered seedlings obtained in step S7 into the substrate soil for transplantation. The substrate soil includes 1 part of vermiculite, 1 part of perlite, and 2 parts of humus soil. Place the transplanted clustered seedlings in the greenhouse for hardening. Control the temperature at 20 - 25°C, keep the relative humidity above 80%, adjust the light intensity with a sunshade net, keep spraying all day for the first 7 days, then gradually reduce the spraying frequency, and conduct normal management after 15 days. After 1 month of hardening, the survival rate reaches 97%, the plants grow robustly, and then transfer them to field cultivation.

[0035] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention.

Claims

1. A tissue culture method for Acer elegantulum, characterized in that, It includes the following steps: S1. Explant selection: Select non-lignified shoot tips with 1-2 axillary buds as explants in April every year, and rinse the explants with sterile water at least 2 times. S2. Disinfection: Immerse the explants in silver disinfectant for 5-10 min, then rinse the explants with sterile water to remove the residual silver disinfectant. Then immerse the explants in 3% hydrogen peroxide for 5-10 min, and rinse the explants with sterile water to remove hydrogen peroxide. S3. Salicylic acid treatment: Immerse the explants treated in step S2 in salicylic acid solution for 12-24 h, and then rinse the explants with sterile water to remove the residual salicylic acid solution. S4. Antioxidant treatment: Immerse the explants treated in step S3 in antioxidant buffer for 5-10 min, and then rinse the explants with sterile water to remove the residual antioxidant buffer. S5. Callus culture: Inoculate the explants treated in step S4 into the initiation medium for initiation culture to obtain callus. S6. Multiple shoot culture: Inoculate the callus obtained in step S5 into the germination medium for germination culture to obtain multiple shoots. S7. Rooting culture: Inoculate the multiple shoots obtained in step S6 into the rooting medium for rooting culture to obtain multiple plantlets. S8. Transplanting: Transplant the multiple plantlets obtained in step S7 into the substrate soil for transplantation.

2. The tissue culture method of Acer elegantulum as described in claim 1, characterized in that, In step S2, the silver disinfectant is a 1% silver nitrate solution containing 0.1% Tween-20.

3. The tissue culture method of Acer elegantulum as described in claim 1, characterized in that, In step S2, the silver disinfectant is a 200-450 ppm silver nanosol solution containing 0.1% Tween-20.

4. The tissue culture method of Acer elegantulum as claimed in claim 1, characterized in that, In step S3, the concentration of the salicylic acid solution is 0.1-0.5 mg / L.

5. The tissue culture method of Acer elegantulum as claimed in claim 1, characterized in that, In step S4, the antioxidant buffer includes 0.5-1 g / L of ascorbic acid and 0.1-0.5 g / L of glutathione.

6. The tissue culture method of Acer elegantulum as claimed in claim 1, wherein, In step S5, the initiation medium is the QL medium supplemented with 0.5-2 mg / L of TDZ, 0.05-0.1 mg / L of NAA, and 0.05% by mass of activated carbon.

7. The tissue culture method of Acer elegantulum as claimed in claim 1, characterized in that, In step S6, the germination medium is the QL medium supplemented with 1-2 mg / L of TDZ and 0.05% by mass of activated carbon.

8. The tissue culture method of Acer elegantulum as described in claim 1, characterized in that, In step S7, the rooting medium is the 1 / 2 MS medium supplemented with 0.1-0.5 mg / L of 6-BA, 0.1-0.3 mg / L of IBA, and 0.05% by mass of activated carbon.

9. The tissue culture method of Acer elegantulum as described in claim 1, characterized in that, In step S8, by mass, the substrate soil includes 1 part of vermiculite, 1 part of perlite, and 1-3 parts of humus soil.