A potato virus-free breeding process

Abstract

The application discloses a potato virus-free breeding process and particularly relates to the field of agricultural biotechnology, and comprises the following steps: explant pretreatment: placing potato stem tip meristem in a pretreatment solution containing 0.5-1.0 mg / L salicylic acid and soaking in a dark condition at 4-6 DEG C; integrated virus-free culture: inoculating in MS integrated virus-free culture medium containing a mixture of forchlorfenuron, 6-benzylaminopurine, naphthaleneacetic acid, melatonin and silver nitrate to obtain test tube seedlings; virus detection: detecting five main potato viruses by using multiplex RT-PCR to screen for full-negative seedlings; rapid propagation and amplification: cutting and subculture, and the single generation propagation coefficient reaches 6-8 times; mist culture to induce microtubers. The virus-free rate of the application is not less than 95%, the breeding efficiency is high, the period is short, and the application is suitable for large-scale virus-free seedling production of multiple potato varieties.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically, to a virus-free propagation process for potatoes. Background Technology

[0002] Potatoes are an important crop used for both food and vegetables, but long-term asexual reproduction can easily lead to the accumulation of viruses, causing seed potato degeneration and seriously affecting yield and quality. Using virus-free breeding technology to obtain virus-free seed potatoes is a fundamental measure to prevent and control potato virus diseases and increase yield.

[0003] Currently, the main method for virus-free potato propagation is shoot tip tissue culture. The conventional process includes: selecting shoot tip meristems for virus-free culture, obtaining virus-free test-tube seedlings, transferring them to a proliferation medium for rapid propagation, screening qualified seedlings by virus detection, and finally producing mini-tubers in greenhouses or net houses. Although this method can effectively eliminate most viruses, it still has the following technical drawbacks: First, it is difficult to achieve both the virus elimination rate and the seedling rate. The smaller the shoot tip meristem is removed, the higher the virus elimination rate, but the lower the seedling rate. Existing culture medium formulas are difficult to obtain a high seedling rate while ensuring a high virus elimination rate. Secondly, the breeding cycle is relatively long. In conventional processes, steps such as virus-free culture, rapid propagation and amplification, and micro-tuber induction are relatively independent, and each stage requires multiple transfers. The overall cycle is usually more than 4 months, resulting in low production efficiency. Third, existing culture media mostly use conventional hormone combinations, such as 6-benzylaminopurine and naphthaleneacetic acid, which have limited effect on promoting shoot tip differentiation and are prone to browning, vitrification and other phenomena, affecting the detoxification effect.

[0004] Therefore, developing a potato virus-free propagation process with high virus-free rate, good seedling rate, short cycle, and suitability for large-scale production is of great practical significance. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution: a potato virus-free propagation process, comprising the following steps: S1. Explant pretreatment: Select potato shoot tip meristems and place them in a pretreatment solution containing 0.5-1.0 mg / L salicylic acid on MS liquid medium as the base, and soak them in the dark at 4-6℃ for 12-24 h. S2. Integrated Virus-Free Culture: Pretreated shoot tip meristems were inoculated into an integrated virus-free culture medium and cultured for 25-35 days at 22±1℃, light intensity of 2000-3000 Lux, and photoperiod of 16 h / d to obtain virus-free test-tube plantlets. Each liter of MS basal medium in the integrated virus-free culture medium contained: 0.3-0.6 mg of chlorpyrifos, 0.5-1.0 mg of 6-benzylaminopurine, and 0.1-0.2 mg of naphthaleneacetic acid. The culture medium consisted of 1.0-2.0 mg melatonin, 0.5-1.0 g activated charcoal, 2.0-5.0 mg silver nitrate, 25-30 g sucrose, and 6-8 g agar. The pH of the culture medium was 5.8-6.0. The illumination was provided by a 5:5 red-to-blue light LED light source, with the light intensity set in stages: 2000 Lux for the first 10 days of cultivation, 2500 Lux for days 11-25, and 3000 Lux from day 26 to the end of cultivation. S3. Virus Detection: Leaves of the virus-free test-tube seedlings obtained in step S2 were collected, and multiple reverse transcription polymerase chain reaction was used to detect potato virus X, virus Y, leafroll virus, virus A, and virus S. The virus-free rate was calculated, and qualified seedlings were screened. The virus-free rate was calculated as follows: number of test-tube seedlings with negative results for all five target viruses ÷ total number of test-tube seedlings tested × 100%. Test-tube seedlings with negative results for all five viruses were selected as qualified seedlings. S4. Rapid propagation and amplification: Cut the test-tube seedlings that have passed the test in step S3 into segments, each segment containing 1-2 nodes, and inoculate them into rapid propagation medium. Culture them at 23±1℃ and a photoperiod of 12h / d. Subculture them every 20-25 days. When the single-generation proliferation coefficient reaches 6-8 times, amplified seedlings are obtained. S5. Microtubule induction: Transplant the seedlings with a height of 3-5cm and well-developed root systems obtained in step S4 into the aeroponic system. Use intermittent nutrient solution spraying, control the relative humidity in the aeroponic system at 70%-85%, and the temperature at 20-25℃. Induce tuber formation for 30-40 days under dark conditions, and harvest microtubules.

[0006] Preferably, the rapid propagation medium in step S4 contains the following per liter of MS basal medium: 0.2-0.5 mg of 6-benzylaminopurine, 0.05-0.1 mg of naphthaleneacetic acid, 0.5-1.0 mg of melatonin, 0.5 g of activated charcoal, 25-30 g of sucrose, and 6-7 g of agar, with a pH of 5.8-6.0.

[0007] Preferably, the nutrient solution in the aeroponic system in step S5 is a modified Hoagland nutrient solution, wherein the mass ratio of nitrogen, phosphorus and potassium is 1:0.5:1.5, and 0.05-0.1 mg / L of chlormequat chloride and 0.5-1.0 mg / L of melatonin are added. The pH value of the nutrient solution is adjusted to 5.8-6.2, and the conductivity is controlled at 1.5-2.0 mS / cm.

[0008] Preferably, the illumination for the integrated detoxification culture in step S2 uses a light-emitting diode light source with a red-to-blue light ratio of 5:5, and the light intensity is set in stages: 2000 Lux for the first 10 days of culture, 2500 Lux for days 11-25, and 3000 Lux from day 26 to the end of culture.

[0009] Preferably, the annealing temperature of the multiplex reverse transcription polymerase chain reaction in step S3 is 55-60℃, the number of cycles is 35-40, and the amplification product is confirmed by 1.5% agarose gel electrophoresis to identify the target band.

[0010] Preferably, in step S1, before peeling the shoot tip meristem, the potato tuber is placed at 25-28℃ and 80%-90% relative humidity to sprout for 10-15 days. When the sprouts grow to 2-3cm, the terminal bud or axillary bud is selected, and a shoot tip meristem with a length of 0.3-0.5mm is peeled off.

[0011] Preferably, during the rapid propagation process in step S4, the rapid propagation medium is replaced with fresh medium each time the culture is subcultured. During subculture, the callus tissue and aging leaves at the base are removed, and 1-2 young leaves at the top are retained.

[0012] Preferably, in step S5, the intermittent nutrient solution spraying is performed for 15 seconds every 30 minutes, the spraying pressure is controlled at 0.1-0.2 MPa, and the droplet size is 50-100 μm.

[0013] Preferably, after harvesting the mini-tubers in step S5, the mini-tubers are stored at low temperature for 30-45 days at 4-6℃ and 70%-80% relative humidity to break dormancy.

[0014] Preferably, in step S2, the mass ratio of silver nitrate to activated carbon in the integrated detoxification culture medium is 1:200-1:500, and the mass ratio of melatonin to chlorpyrifos is 2:1-5:1.

[0015] The technical effects and advantages of this invention are as follows: This invention employs an integrated detoxification culture medium, which combines chlorpyrifos, 6-benzylaminopurine, naphthaleneacetic acid, melatonin, and silver nitrate in a specific ratio. Combined with salicylic acid pretreatment and staged light culture, it can effectively inhibit shoot tip browning and vitrification, and promote rapid differentiation of meristematic tissue into seedlings. Verified by multiplex RT-PCR detection, the detoxification rate can reach over 95%, based on the percentage of test-tube seedlings with negative results for all five target viruses out of the total number of seedlings tested, which is significantly better than conventional detoxification processes. This invention integrates virus-free culture, virus detection, rapid propagation and amplification, and mini-tuber induction into a compact process. The virus-free culture stage achieves integrated seedling formation without the need for separate transfer and differentiation media, reducing transfer operations and contamination risks. The overall propagation cycle can be controlled within 90 days. Meanwhile, the rapid propagation stage involves subculturing every 20-25 days, with a single-generation proliferation coefficient of 6-8 times. Each mini-tuber plant can produce 11-13 tubers, enabling the production of a large number of high-quality virus-free seedlings in a short time, effectively reducing the production cost per seedling, and making it suitable for factory-scale production. The process parameters of this invention are reasonable and can achieve stable virus-free effects and breeding efficiency for different potato varieties such as Feverita, Shaboti, and Atlantic. It has good variety adaptability. At the same time, the process operation of this invention is standardized and the conditions are controllable, making it easy to promote and apply in factory seedling production, and providing reliable technical support for the large-scale production of virus-free mini-tubers. Attached Figure Description

[0016] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0018] Example 1

[0019] This embodiment provides a virus-free potato propagation process, which specifically includes the following steps: S1, Explant Pretreatment Healthy tubers of the potato variety Fiorentina were selected and sprouted for 12 days at 25°C and 85% relative humidity. When the sprouts reached 2.5 cm in length, the terminal buds were cut off as explants. In a clean bench, the explants were disinfected with 75% ethanol for 30 seconds, then disinfected with 0.1% mercuric chloride solution for 10 minutes, and rinsed 5 times with sterile water. After disinfection, 0.4 mm long shoot tip meristems were peeled off under a dissecting microscope and placed in a pretreatment solution containing 0.8 mg / L salicylic acid on MS liquid medium as a base and soaked in the dark at 5°C for 18 hours. S2, Integrated Detoxification Culture Pretreated shoot tip meristems were inoculated into an integrated virus-free culture medium. This step employs an integrated culture mode, meaning that the shoot tip meristems simultaneously complete virus removal, meristem differentiation, and seedling growth in the same culture medium, eliminating the need for transfer to a differentiation medium. Each liter of MS basal medium in the integrated virus-free medium contains: 0.5 mg chlorpyrifos, 0.8 mg 6-benzylaminopurine, 0.15 mg naphthaleneacetic acid, 1.5 mg melatonin, 0.8 g activated charcoal, 3.5 mg silver nitrate, 28 g sucrose, and 7 g agar. The pH of the culture medium was adjusted. Up to 5.9, the cultivation conditions were as follows: temperature 22±1℃, light source using LED light with a red-to-blue light ratio of 5:5, and light intensity set in stages: 2000 Lux for the first 10 days of cultivation, 2500 Lux for days 11-25, and 3000 Lux for days 26-30, with a photoperiod of 16 h / d. After 30 days of cultivation, virus-free test-tube seedlings were obtained. According to statistics, the shoot tip seedling rate in this embodiment was 86.2%. Virus detection showed that the virus-free test-tube seedlings had a virus-free rate of 96.5% against potato virus X, virus Y, leafroll virus, virus A, and virus S.

[0020] S3, Virus Detection Leaves from the virus-free test-tube seedlings obtained in step S2 were collected, and multiplex reverse transcription polymerase chain reaction (RT-PCR) was used to detect potato virus X, Y, leafroll virus, A, and S. RNA extraction was performed using the Trizol method, with total RNA extracted from the leaves according to standard molecular biology procedures. cDNA was synthesized using a commercially available reverse transcription kit. Specific primers for the five viruses were the commonly used potato virus detection primer sequences. The PCR reaction was conducted in a 25 μL system: 12.5 μL of 2×Taq PCR Mix, 0.5 μL each of forward and reverse primers, 2 μL of cDNA template, and sterile water to a final volume of 25 μL. The reaction conditions were: annealing temperature 58℃, 38 cycles. The amplified products were confirmed by 1.5% agarose gel electrophoresis to identify the target bands. Statistically, in this embodiment, 96.5% of the test-tube seedlings tested negative for all five target viruses. These seedlings were selected for the next step. S4, Rapid Propagation Cut the qualified test-tube seedlings from step S3 into segments, each segment containing one node, and inoculate them into a rapid propagation medium. The rapid propagation medium contains the following per liter of MS basal medium: 0.35 mg of 6-benzylaminopurine, 0.08 mg of naphthaleneacetic acid, 0.8 mg of melatonin, 0.5 g of activated carbon, 28 g of sucrose, and 6.5 g of agar. Adjust the pH of the medium to 5.9. The culture conditions are: temperature 23±1℃, photoperiod 12 h / d, and subculture every 22 days. During subculture, remove the basal callus and aging leaves, retain one young leaf at the top, and replace with fresh rapid propagation medium. The single-generation proliferation coefficient of this process can be stably up to 7 times, and sufficient amplified seedlings for transplanting can be obtained after 2 subcultures. S5, Micro-potato Induction The seedlings obtained in step S4, with a height of 3-5 cm, well-developed root systems, and 3-4 functional leaves, were transplanted into an aeroponic system. The nutrient solution in the aeroponic system was a modified Hogland nutrient solution, with a nitrogen, phosphorus, and potassium mass ratio of 1:0.5:1.5, and 0.08 mg / L of chlormequat chloride and 0.8 mg / L of melatonin were added. The pH of the nutrient solution was adjusted to 6.0, and the conductivity was controlled at 1.8 mS / cm. The intermittent nutrient solution spraying parameters were: spraying for 15 seconds every 30 minutes, spraying pressure controlled at 0.15 MPa, droplet size of 75 μm, relative humidity controlled at 78%, and temperature controlled at 23℃. Tuber formation was induced for 35 days under dark conditions, and mini-tubers were harvested. The harvested mini-tubers were then stored at 5℃ and 75% relative humidity for 38 days to break dormancy.

[0021] Tests showed that the number of tubers per mini-tuber plant obtained in this embodiment was 12, with an average weight of 1.8g per tuber, and a germination rate of 92% after storage. The overall propagation cycle from shoot tip inoculation to harvest of mini-tuber was about 85 days, which could be controlled within 90 days.

[0022] Example 2

[0023] This embodiment is basically the same as Embodiment 1, except that the potato variety is Shaboti, and some parameters are adjusted as follows: In step S1, the germination temperature is 27℃, the germination time is 10 days, the length of the stem tip meristem is 0.3mm, the salicylic acid concentration is 0.5mg / L, and the soaking time is 12h. In step S2, the integrated virus-free culture medium consisted of: 0.3 mg chlorpyrifos, 0.5 mg 6-benzylaminopurine, 0.1 mg naphthaleneacetic acid, 1.0 mg melatonin, 0.5 g activated charcoal, 2.0 mg silver nitrate, 25 g sucrose, and 6 g agar, with a pH of 5.8 and a culture time of 25 days. The light intensity was set in stages: 2000 Lux for the first 10 days of culture, and 2500 Lux for days 11-25. Statistically, the shoot tip seedling rate in this embodiment was 82.7%. According to virus detection statistics, in this embodiment, the number of test tube seedlings that tested negative for all five target viruses accounted for 95.2% of the total number of test tube seedlings tested, i.e., the virus-free rate was 95.2%. In step S4, the rapid propagation culture medium consists of: 0.2 mg of 6-benzylaminopurine, 0.05 mg of naphthaleneacetic acid, 0.5 mg of melatonin, 0.5 g of activated carbon, 25 g of sucrose, and 6 g of agar, with a pH of 5.8, a subculture cycle of 20 days, and a single-generation proliferation coefficient of 6 times. In step S5, the amount of chlormequat chloride added was 0.05 mg / L, the amount of melatonin added was 0.5 mg / L, the pH of the nutrient solution was 5.8, the conductivity was 1.5 mS / cm, the spraying pressure was 0.1 MPa, the droplet size was 50 μm, the relative humidity was 70%, and tuber formation was induced for 30 days. The number of tubers per plant was 11, and the average weight of a single tuber was 1.6 g. The overall breeding cycle from shoot tip inoculation to harvesting mini-tubers was approximately 78 days.

[0024] Example 3

[0025] This embodiment is basically the same as Embodiment 1, except that the potato variety is Atlantic and some parameters are adjusted as follows: In step S1, the germination temperature is 28℃, the germination time is 15 days, the length of the stem tip meristem is 0.5mm, the salicylic acid concentration is 1.0mg / L, and the soaking time is 24h. In step S2, the integrated virus-free culture medium consisted of: 0.6 mg chlorpyrifos, 1.0 mg 6-benzylaminopurine, 0.2 mg naphthaleneacetic acid, 2.0 mg melatonin, 1.0 g activated carbon, 5.0 mg silver nitrate, 30 g sucrose, and 8 g agar, with a pH of 6.0 and a culture time of 35 days. The light intensity was set in stages: 2000 Lux for the first 10 days of culture, 2500 Lux for days 11-25, and 3000 Lux for days 26-35. Statistical analysis showed that the shoot tip seedling rate in this embodiment was 89.4%. According to virus detection statistics, in this embodiment, the number of test tube seedlings that tested negative for all five target viruses accounted for 97.8% of the total number of test tube seedlings tested, i.e., the virus-free rate was 97.8%. In step S4, the rapid propagation culture medium consists of: 0.5 mg of 6-benzylaminopurine, 0.1 mg of naphthaleneacetic acid, 1.0 mg of melatonin, 0.5 g of activated carbon, 30 g of sucrose, and 7 g of agar, with a pH of 6.0, a subculture cycle of 25 days, and a single-generation proliferation coefficient of 8 times. In step S5, the amount of chlormequat chloride added was 0.1 mg / L, the amount of melatonin added was 1.0 mg / L, the pH of the nutrient solution was 6.2, the conductivity was 2.0 mS / cm, the spraying pressure was 0.2 MPa, the droplet size was 100 μm, the relative humidity was 85%, and tuber formation was induced for 40 days. The number of tubers per plant was 13, and the average weight of a single tuber was 2.0 g. The overall breeding cycle from shoot tip inoculation to harvesting mini-tubers was about 90 days.

[0026] Comparative Example 1 This comparative example uses a conventional potato virus-free propagation process. The culture medium used in step S2 is MS basal medium supplemented with 0.8 mg / L 6-benzylaminopurine and 0.15 mg / L naphthaleneacetic acid, without the addition of chlorpyrifos, melatonin, and silver nitrate. The remaining steps are the same as in Example 1.

[0027] The results showed that the virus detoxification rate of Comparative Example 1 was 78.3%, the seedling survival rate was 65%, and the single-generation proliferation coefficient was 4 times. Compared with Example 1, the virus detoxification rate decreased by 18.2 percentage points, the seedling survival rate decreased by about 20 percentage points, and the proliferation coefficient decreased by about 43%.

[0028] Comparative Example 2 The difference between this comparative example and Example 1 is that silver nitrate is not added to the integrated detoxification culture medium, while the other components and parameters are the same as in Example 1.

[0029] The results showed that the browning rate of the shoot tips in Comparative Example 2 was as high as 42%, the seedling rate was only 58%, and the detoxification rate was 81.5%. Compared with Example 1, the browning rate was significantly increased and the seedling rate was significantly reduced, indicating that silver nitrate plays a key role in resisting browning and promoting seedling growth in the integrated detoxification culture medium of the present invention.

[0030] Comparative Example 3 The difference between this comparative example and Example 1 is that melatonin is not added to the integrated detoxification culture medium, while the other components and parameters are the same as in Example 1.

[0031] The results showed that the virus elimination rate of Comparative Example 3 was 82.1%, and the seedling survival rate was 71%. The test-tube seedlings showed obvious yellowing and slow growth during the rapid propagation stage. Compared with Example 1, the virus elimination rate and seedling survival rate were significantly reduced, indicating that melatonin has a synergistic effect with hormones such as chlorpyrifos and 6-benzylaminopurine, which can significantly improve the virus elimination effect and plant vitality.

[0032] The comparative results of Examples 1-3 and Comparative Examples 1-3 above demonstrate that the synergistic combination of multiple components such as chlorpyrifos, 6-benzylaminopurine, naphthaleneacetic acid, melatonin, and silver nitrate in the integrated detoxification culture medium provided by the present invention can significantly improve the detoxification rate and seedling rate of potato shoot tips, while obtaining a high proliferation coefficient and mini-tubule yield. The process of the present invention is applicable to multiple potato varieties such as Fiurita, Shaboti, and Atlantic, and has universality and industrial application prospects.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for the virus-free multiplication of potato, characterized in that: Includes the following steps: S1. Explant pretreatment: Select potato shoot tip meristems and place them in a pretreatment solution containing 0.5-1.0 mg / L salicylic acid on MS liquid medium as the base, and soak them in the dark at 4-6℃ for 12-24 h. S2. Integrated Virus-Free Culture: Pretreated shoot tip meristems were inoculated into an integrated virus-free culture medium and cultured for 25-35 days at 22±1℃, light intensity of 2000-3000 Lux, and photoperiod of 16 h / d to obtain virus-free test-tube plantlets. Each liter of MS basal medium in the integrated virus-free culture medium contained: 0.3-0.6 mg of chlorpyrifos, 0.5-1.0 mg of 6-benzylaminopurine, and 0.1-0.2 mg of naphthaleneacetic acid. The culture medium consisted of 1.0-2.0 mg melatonin, 0.5-1.0 g activated charcoal, 2.0-5.0 mg silver nitrate, 25-30 g sucrose, and 6-8 g agar. The pH of the culture medium was 5.8-6.

0. The illumination was provided by a 5:5 red-to-blue light LED light source, with the light intensity set in stages: 2000 Lux for the first 10 days of cultivation, 2500 Lux for days 11-25, and 3000 Lux from day 26 to the end of cultivation. S3. Virus Detection: Leaves of the virus-free test-tube seedlings obtained in step S2 were collected, and multiple reverse transcription polymerase chain reaction was used to detect potato virus X, virus Y, leafroll virus, virus A, and virus S. The virus-free rate was calculated, and qualified seedlings were screened. The virus-free rate was calculated as follows: number of test-tube seedlings with negative results for all five target viruses ÷ total number of test-tube seedlings tested × 100%. Test-tube seedlings with negative results for all five viruses were selected as qualified seedlings. S4. Rapid propagation and amplification: Cut the test-tube seedlings that have passed the test in step S3 into segments, each segment containing 1-2 nodes, and inoculate them into rapid propagation medium. Culture them at 23±1℃ and a photoperiod of 12h / d. Subculture them every 20-25 days. When the single-generation proliferation coefficient reaches 6-8 times, amplified seedlings are obtained. S5. Microtubule induction: Transplant the seedlings with a height of 3-5cm and well-developed root systems obtained in step S4 into the aeroponic system. Use intermittent nutrient solution spraying, control the relative humidity in the aeroponic system at 70%-85%, and the temperature at 20-25℃. Induce tuber formation for 30-40 days under dark conditions, and harvest microtubules.

2. Potato virus-free multiplication process according to claim 1, characterized in that: Step S4: The rapid propagation medium contains the following per liter of MS basal medium: 0.2-0.5 mg of 6-benzylaminopurine, 0.05-0.1 mg of naphthaleneacetic acid, 0.5-1.0 mg of melatonin, 0.5 g of activated charcoal, 25-30 g of sucrose, and 6-7 g of agar. The pH of the medium is 5.8-6.

0.

3. Potato virus-free multiplication process according to claim 1, characterized in that: In step S5, the nutrient solution in the aeroponic system is a modified Hoagland nutrient solution, in which the mass ratio of nitrogen, phosphorus and potassium is 1:0.5:1.5, and 0.05-0.1 mg / L of chlormequat chloride and 0.5-1.0 mg / L of melatonin are added. The pH of the nutrient solution is adjusted to 5.8-6.2, and the conductivity is controlled at 1.5-2.0 mS / cm.

4. The potato virus-free propagation process according to claim 1, characterized in that: In step S3, the annealing temperature of the multiplex reverse transcription polymerase chain reaction is 55-60℃, the number of cycles is 35-40, and the amplification product is confirmed by 1.5% agarose gel electrophoresis to identify the target band.

5. The potato virus-free propagation process according to claim 1, characterized in that: Before peeling the shoot tip meristem in step S1, the potato tubers are placed at 25-28℃ and 80%-90% relative humidity to sprout for 10-15 days. When the sprouts grow to 2-3cm, the terminal bud or axillary bud is selected and the shoot tip meristem with a length of 0.3-0.5mm is peeled off.

6. The potato virus-free propagation process according to claim 1, characterized in that: During the rapid propagation process in step S4, the medium should be replaced with fresh medium each time the culture is subcultured. During subculture, callus tissue and aging leaves at the base should be removed, and 1-2 young leaves at the top should be retained.

7. The potato virus-free propagation process according to claim 1, characterized in that: In step S5, the intermittent nutrient solution spraying is carried out for 15 seconds every 30 minutes, with the spraying pressure controlled at 0.1-0.2 MPa and the droplet size at 50-100 μm.

8. The potato virus-free propagation process according to claim 1, characterized in that: After harvesting the mini-tubers in step S5, the mini-tubers are stored at low temperature for 30-45 days at 4-6℃ and 70%-80% relative humidity to break dormancy.

9. The potato virus-free propagation process according to claim 1, characterized in that: In step S2, the mass ratio of silver nitrate to activated carbon in the integrated detoxification culture medium is 1:200-1:500, and the mass ratio of melatonin to chlorpyrifos is 2:1-5:1.

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