Disinfection method of ginseng explant

Abstract

The invention provides a disinfection method of a ginseng explant, which comprises the following steps: soaking and disinfecting the ginseng explant by using a chemical disinfectant, soaking the soaked and disinfected explant into a first ethanol solution, taking out, igniting residual ethanol on the surface of the explant, burning, and cooling. According to the method disclosed by the invention, conventional chemical disinfection and high-concentration ethanol infiltration-flame burning are creatively combined, and the step-by-step and synergistic sterilization strategy is particularly suitable for explants with complicated structures, such as ginseng roots. Compared with a traditional disinfection method needing long-time soaking and multiple times of rinsing, the method has the advantages that the total disinfection time is greatly shortened and the explant preparation efficiency is remarkably improved by introducing an instantaneous firing step, and the explant preparation efficiency is greatly improved by reducing the total contact time of a chemical disinfectant, inactivating surface polyphenol oxidase by utilizing instantaneous high temperature and combining rapid cooling. The browning reaction is effectively inhibited, and the activity of the explant is ensured.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a method for disinfecting ginseng explants. Background Technology

[0002] Ginseng is a perennial herbaceous plant belonging to the Araliaceae family. Its roots are rich in active ingredients such as ginsenosides, possessing extremely high medicinal value and health benefits. However, wild ginseng resources are nearing depletion due to over-harvesting, and artificial cultivation is time-consuming and constrained by problems such as continuous cropping obstacles and soil-borne diseases. Plant tissue culture technology is an effective way to achieve rapid propagation of ginseng germplasm resources and large-scale production of active ingredients.

[0003] Explant disinfection is the first step in plant tissue culture. Ginseng root explants have unique characteristics: ① They grow in the soil for a long time and carry a large number of fungal spores and bacterial spores on their surface; ② The root surface is rough and wrinkled, with cracks of varying depths and residual fibrous root bases, making it difficult for conventional disinfectants to penetrate deep into the folds for thorough sterilization; ③ The cortex is rich in phenolic substances, and prolonged disinfection time or chemical stimulation can easily cause oxidative browning, leading to explant death.

[0004] Currently, commonly used methods for disinfecting plant explants are mainly divided into chemical disinfection and physical disinfection. For example, Chinese patent CN108094202A discloses a method for disinfecting explants of Magnolia rubrum, the core of which involves disinfecting pretreated explants with a 0.1-0.3% mercuric chloride (HgCl2) solution for 8-10 minutes. This method is a typical chemical disinfection method. Although the sterilization effect is good, mercuric chloride is highly toxic, posing a risk of residue, and the operation is cumbersome and unfriendly to personnel and the environment. Therefore, further improvement and development are needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies and solve the aforementioned problems, a method for disinfecting ginseng explants is proposed, and the following technical solution is provided: A method for disinfecting ginseng explants includes the following steps: soaking the ginseng explants in a chemical disinfectant, immersing the disinfected explants in a first ethanol solution, removing them, igniting the residual ethanol on the surface of the explants for burning treatment, and then cooling them.

[0006] Furthermore, the ginseng explants after soaking and disinfection are soaked in the first ethanol solution for 3-5 seconds, and the burning treatment is carried out for 5-10 seconds.

[0007] Furthermore, the volume fraction of the first ethanol solution is 95%.

[0008] Furthermore, the cooling process involves using deionized water at a temperature of 4-10°C for 10-15 seconds.

[0009] Furthermore, the chemical disinfectant includes a second ethanol solution, a sodium hypochlorite solution, and a potassium permanganate solution.

[0010] Furthermore, the process of disinfecting ginseng explants by soaking them in chemical disinfectants includes: immersing the ginseng explants in a second ethanol solution for shaking and rinsing; rinsing with deionized water and then soaking them in a sodium hypochlorite solution; rinsing again with deionized water and then soaking them in a potassium permanganate solution for disinfection.

[0011] Furthermore, the washing time in the second ethanol solution is 3-5 minutes, and the rotation speed is 80-100 rpm.

[0012] Furthermore, the soaking time in sodium hypochlorite solution is 15-20 minutes, with shaking for 30 seconds every 4 minutes during this period.

[0013] Furthermore, the soaking time in potassium permanganate solution is 15-20 minutes, with shaking for 30 seconds every 4 minutes during this period.

[0014] Furthermore, before soaking and disinfecting, the surface of the ginseng explant is cleaned.

[0015] Due to the adoption of the above technical solutions, the beneficial technical effects of the present invention are as follows: 1. This invention creatively combines "conventional chemical disinfection" with "high-concentration ethanol immersion-flame burning". Conventional chemical disinfection first kills most of the surface bacteria, while the subsequent "ethanol immersion-flame burning" step utilizes the strong penetrability of high-concentration ethanol to immerse the root surface micro-cracks that are difficult for chemical disinfectants to reach. Then, the high temperature generated by instantaneous combustion completely kills the remaining fungal spores and bacterial spores. This step-by-step and synergistic sterilization strategy is especially suitable for explants with complex structures such as ginseng roots. The contamination rate can be controlled below 5%, which is significantly better than traditional methods. 2. Compared with the traditional mercuric chloride disinfection method, which requires long soaking and multiple rinsing, this invention significantly shortens the total disinfection time by introducing an instantaneous burning step, thus significantly improving the efficiency of explant preparation. 3. Prolonged chemical soaking is one of the main causes of browning in ginseng roots. This method effectively inhibits the browning reaction and ensures the viability of explants by reducing the total contact time with chemical disinfectants, using instantaneous high temperature to inactivate surface polyphenol oxidase, and combining it with rapid cooling. Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Based on the embodiments in this application, other similar embodiments obtained by those skilled in the art without creative effort should all fall within the scope of protection of this application.

[0017] A method for disinfecting ginseng explants includes the following steps: soaking the ginseng explants in a chemical disinfectant, immersing the disinfected explants in a first ethanol solution, removing them, igniting the residual ethanol on the surface of the explants for burning treatment, and then cooling them. This invention creatively combines conventional chemical disinfection with high-concentration ethanol immersion-flame burning. Conventional chemical disinfection first kills most surface bacteria, while the subsequent ethanol immersion-flame burning step utilizes the strong penetrability of high-concentration ethanol to penetrate the microscopic fissures on the root surface that are difficult for chemical disinfectants to reach. Then, the high temperature generated by instantaneous combustion thoroughly kills any remaining fungal spores and bacterial spores. This step-by-step, synergistic sterilization strategy is particularly suitable for explants with complex structures, such as ginseng roots, and the contamination rate can be controlled to below 5%, significantly superior to traditional methods. Compared to the traditional mercuric chloride disinfection method, which requires long soaking and multiple rinsings, this invention significantly shortens the total disinfection time by introducing an instantaneous burning step, significantly improving the efficiency of explant preparation. This method effectively inhibits browning reaction and ensures the viability of explants by reducing the total contact time of chemical disinfectants and using instantaneous high temperature to inactivate surface polyphenol oxidase, combined with rapid cooling.

[0018] Example 1 A method for disinfecting root explants in ginseng tissue culture, the specific steps of which are as follows: (1) Surface cleaning pretreatment: Take fresh ginseng roots that have grown for 3 years and are healthy and free from disease, cut off the small fibrous roots and rhizomes, and retain the main root. Soak the ginseng roots under running tap water for 5 minutes to soften the mud and sand on the surface. Use a soft polyester fiber brush, dip it in an appropriate amount of detergent solution, and gently brush the surface wrinkles and the base of the remaining fibrous roots along the longitudinal axis of the root in one direction. The brushing force should be such that it does not scratch the periderm. After brushing, rinse under running tap water for 15 minutes to thoroughly remove detergent residue.

[0019] (2) Initial chemical disinfection: Transfer the root explants treated in step (1) to a glass bottle, add 75% ethanol solution until the liquid surface completely covers the material, place it on a shaker and shake and wash at 100 rpm for 3 min; discard the ethanol solution, add sterile deionized water and shake and wash for 1 min, discard the waste liquid; repeat the sterile water washing operation twice.

[0020] (3) Secondary chemical disinfection: Transfer the root explants treated in step (2) to a clean bench, add a sodium hypochlorite solution with a mass-volume concentration of 1.0%, soak for 15 minutes, and shake the container for 30 seconds every 4 minutes during this period; discard the sodium hypochlorite solution, add sterile deionized water and shake to clean for 1 minute, and repeat the cleaning twice.

[0021] (4) Three chemical disinfection: Add 0.1% potassium permanganate solution to the root explants treated in step (3) and soak for 15 min, shaking the container for 30 s every 4 min during the process; discard the potassium permanganate solution, add sterile deionized water and shake to clean for 1 min, and repeat the cleaning twice.

[0022] (5) Physical-chemical synergistic disinfection: Use sterile tweezers to pick up the root explants treated in step (4), drain off the excess water on the surface, and immerse them in a 95% ethanol solution for 3 seconds. Quickly remove the explants, tilt them at 45° and let them stand for 2 seconds to allow the excess ethanol droplets on the surface to drip down. Immediately move the explants to the outer flame of an alcohol lamp, ignite the residual ethanol on the surface, and let the flame burn naturally for 8 seconds. After the flame goes out on its own, quickly immerse the explants completely in pre-cooled sterile deionized water (4°C) for 10 seconds.

[0023] (6) Inoculation and culture: After disinfection, the ginseng root explants were placed on sterile filter paper to absorb surface moisture. Root cortex tissue blocks (each block was of uniform thickness, approximately 4 mm) were cut using a sterile scalpel and inoculated into MS solid medium (containing 1.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose, 6.5 g / L agar, pH 5.8). Five blocks were inoculated per bottle, and each treatment was repeated 10 times. The explants were incubated in the dark at (25±2)℃ for 7 days, and the contamination rate and browning rate were recorded.

[0024] Example 2 The difference between this embodiment and Embodiment 1 is that the flame burning time in step (5) is 5 seconds. The remaining steps and parameters are exactly the same as in Embodiment 1.

[0025] Example 3 The difference between this embodiment and Embodiment 1 is that the flame burning time in step (5) is 10 seconds. The remaining steps and parameters are exactly the same as in Embodiment 1.

[0026] Comparative Example 1 Using traditional disinfection methods, the ginseng root explants, after surface cleaning pretreatment, were immersed in a 0.1% mercuric chloride solution for 8 minutes for disinfection, followed by repeated rinsing with sterile water. Comparative Example 2 The ginseng root explants, which have undergone surface cleaning pretreatment, were soaked in 95% ethanol for 3 seconds and then directly scorched for 8 seconds before being cooled for later use.

[0027] The explants treated with the disinfection methods of Examples 1-3 and Comparative Examples 1-2 were subjected to tissue culture experiments. The sample size of each treatment group was 50 explants. The contamination rate, browning rate and survival rate after 7 days of culture were statistically analyzed. The results are shown in Table 1.

[0028] Table 1. Comparison of the effects of different disinfection methods on ginseng root explants As shown in Table 1, the disinfection methods of Examples 1-3 of this invention have significantly lower contamination and browning rates than traditional disinfection methods, and a much higher survival rate than Comparative Example 1. Although Comparative Example 2 has an extremely low browning rate, its contamination rate is extremely high, indicating that ethanol soaking and burning alone cannot effectively kill microorganisms deep within the folds of ginseng roots, and its survival rate is also the lowest. This fully demonstrates that combining "conventional chemical disinfection" with "physical-chemical synergistic disinfection" is essential for treating ginseng, a root explant with a complex structure, achieving a synergistic and enhanced technical effect.

[0029] Data from Examples 1-3 show that good disinfection effects can be achieved within the parameter range defined by the present invention, with the parameter combination in Example 1 achieving the best balance between contamination rate, browning rate, and survival rate.

[0030] In summary, the disinfection method for root explants in ginseng tissue culture provided in this application solves the problems of difficult sterilization and high risk of mercuric chloride residue in the deep folds of ginseng root explants through a synergistic physical-chemical sterilization technique of "ethanol immersion-flame burning". This method has outstanding advantages such as thorough sterilization, rapid operation, no chemical residue, and strong versatility, providing a reliable explant preparation scheme for the tissue culture of ginseng and other rhizomatous medicinal plants, and has good potential for widespread application.

[0031] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for disinfecting ginseng explants, characterized in that, Includes the following steps: Ginseng explants were disinfected by soaking in a chemical disinfectant. After soaking, the explants were immersed in a first ethanol solution. After removal, the residual ethanol on the surface of the explants was ignited for burning treatment, followed by cooling.

2. The method for disinfecting ginseng explants according to claim 1, characterized in that, The ginseng explants after soaking and disinfection were soaked in the first ethanol solution for 3-5 seconds, and the burning treatment was carried out for 5-10 seconds.

3. The method for disinfecting ginseng explants according to claim 1, characterized in that, The volume fraction of the first ethanol solution is 95%.

4. The method for disinfecting ginseng explants according to claim 1, characterized in that, The cooling process involves using deionized water at a temperature of 4-10°C for 10-15 seconds.

5. The method for disinfecting ginseng explants according to claim 1, characterized in that, The chemical disinfectant includes a second ethanol solution, a sodium hypochlorite solution, and a potassium permanganate solution.

6. The method for disinfecting ginseng explants according to claim 4, characterized in that, The process of disinfecting ginseng explants by soaking them in chemical disinfectants includes: immersing the ginseng explants in a second ethanol solution for shaking and rinsing; rinsing with deionized water and then soaking them in a sodium hypochlorite solution; rinsing again with deionized water and then soaking them in a potassium permanganate solution for disinfection.

7. The method for disinfecting ginseng explants according to claim 5, characterized in that, The washing time in the second ethanol solution is 3-5 minutes with shaking at a speed of 80-100 rpm.

8. A method for disinfecting ginseng explants according to claim 5, characterized in that, Soak in sodium hypochlorite solution for 15-20 minutes, shaking for 30 seconds every 4 minutes.

9. A method for disinfecting ginseng explants according to claim 5, characterized in that, Soak in potassium permanganate solution for 15-20 minutes, shaking for 30 seconds every 4 minutes.

10. A method for disinfecting ginseng explants according to claim 1, characterized in that, Before soaking and disinfecting, the surface of the ginseng explant is cleaned.

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