A method for breeding virus-free tissue culture seedlings of strawberry

Abstract

The present application relates to a kind of strawberry virus-free tissue culture seedling breeding method, belong to tissue culture seedling breeding technical field.The strawberry virus-free tissue culture seedling breeding method of the present application mainly includes pretreatment and stem tip stripping, induction culture, differentiation culture, proliferation culture, rooting culture, domestication, transplanting, reproduction and the like steps.The method process provided by the present application adapts local production condition, strong operability, is suitable for Weihai Rongcheng and the like area scale promotion, reduces the seedling cost of grower, promotes local strawberry industry standardization, high-quality development.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture seedling propagation technology, and relates to a method for propagating virus-free strawberry tissue culture seedlings. Background Technology

[0002] Rongcheng, located in Weihai, borders the Yellow Sea and has a maritime monsoon climate characterized by distinct seasons and abundant sunshine, but summers are hot and rainy, while winters are cold and windy. The soil is mostly sandy, with poor water and fertilizer retention capacity. The local strawberry industry faces unique challenges: traditional asexual reproduction of strawberry seedlings has led to a high rate of virus transmission and frequent outbreaks of viral diseases. Coupled with the influence of local climate and soil conditions, the seedlings lack resilience, resulting in a year-on-year decline in yield and quality. Existing virus-free tissue culture technology is not adapted to the local environment, and the culture medium formula is too simple. After transplanting, the test-tube seedlings have difficulty adapting to the temperature and humidity fluctuations and soil characteristics of Rongcheng, resulting in low survival rates. The lack of targeted improvement technologies has led to poor results in the promotion and application of virus-free seedlings in the area, resulting in high seedling costs and limited profits for growers.

[0003] Traditional Chinese medicine ingredients contain various bioactive substances that can enhance plant resistance, promote growth and development, and inhibit pathogen growth. Therefore, considering the environmental characteristics of Rongcheng, Weihai, traditional Chinese medicine ingredients are integrated into tissue culture medium, and the cultivation and domestication process is optimized to develop a special method for propagating virus-free strawberry tissue culture seedlings. This method can effectively solve the problems of virus-carrying, poor resistance, and low survival rate of local strawberry seedlings, and promote the improvement of quality and efficiency of the local strawberry industry. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for propagating virus-free strawberry tissue culture seedlings. This method can significantly improve the survival rate of transplanted tissue culture seedlings and enhance the quality and yield of strawberries.

[0005] The present invention employs the following technical solutions to achieve the above objectives: A method for propagating virus-free strawberry tissue culture seedlings includes the following steps: Step 1, Pretreatment: Take the tip of the strawberry runner, rinse with distilled water, and soak in ribavirin solution and vitamin C solution in sequence; then soak in 70% ethanol solution, disinfect with 1% sodium hypochlorite, rinse with sterile water and drain; then place in liquid nitrogen for ultra-low temperature storage, thaw quickly, peel off the outer scales, and cut 0.3~0.4mm of the stem tip tissue; Step 2, induction culture: The shoot tip tissue is inoculated into a modified induction medium and cultured to induce callus tissue; Step 3, Differentiation Culture: The callus tissue is inoculated into a modified differentiation medium and cultured to form seedlings; Step 4, Proliferation Culture: The seedlings are inoculated into a modified proliferation culture medium and cultured to obtain test-tube seedlings; Step 5, Rooting Culture: Inoculate the test-tube seedlings into the rooting medium and culture them to obtain rooted tissue culture seedlings; Step 6, Acclimation: The rooted tissue culture seedlings are subjected to intelligent simulation acclimation to obtain acclimated seedlings; Step 7, Transplanting: Transplant the acclimatized seedlings into a special substrate for cultivation to obtain virus-free original seedlings; Step 8, Propagation: Plant the virus-free original seedlings in the soil and propagate them by division to obtain original seedlings; continue to plant the original seedlings to obtain seedlings for production.

[0006] Furthermore, in step 1, the concentration of the ribavirin solution is 25 mg / L, and the concentration of the vitamin C solution is 300 mg / L.

[0007] Furthermore, the modified induction medium in step 2 contains: modified MS medium, 6-BA 0.5 mg / L, NAA 0.2 mg / L, and mixed traditional Chinese medicine extract 150 mg / L.

[0008] As a preferred method, the induction culture conditions are: temperature 26℃±1℃, irradiation by a 20W LED lamp, and 24 hours of light per day.

[0009] Furthermore, the modified differentiation medium in step 3 contains: modified MS medium, 6-BA 1.0 mg / L, KT 0.3 mg / L, and mixed traditional Chinese medicine extract 150 mg / L.

[0010] As a preferred method, the differentiation culture conditions are: temperature 26℃±1℃, irradiation by 20W LED lamps, and 24 hours of light per day.

[0011] Furthermore, the modified proliferation medium in step 4 contains: modified MS medium, 6-BA 1.5 mg / L, NAA 0.5 mg / L, and mixed traditional Chinese medicine extract 150 mg / L.

[0012] As a preferred method, the proliferation culture conditions are: temperature 26℃±1℃, irradiation by 20W LED lamps, 24h light per day; subculture once every 7 days, with a total of no more than 20 subcultures.

[0013] Furthermore, the rooting medium in step 5 contains: 1 / 2 modified MS medium and IBA 0.5 mg / L.

[0014] As a preferred method, the rooting culture conditions are: temperature 26℃±1℃, irradiation by a 20W LED lamp, and 24 hours of light per day.

[0015] Furthermore, the modified MS medium described above involves adjusting the components of the traditional MS medium, namely reducing the ammonium nitrate content to 1320 mg / L, increasing the potassium iodide content to 1.25 mg / L, replacing the sugar source in the medium with 25 g / L of edible white sugar, and replacing the agar gelling agent with 6.5 g / L of food-grade carrageenan.

[0016] Furthermore, the preparation method of the aforementioned mixed Chinese herbal extract is as follows: take Isatis root, honeysuckle, and Polygonatum rhizome, crush them separately, mix them in a weight ratio of 2:1:1, add 8-10 times the weight of distilled water, boil and extract, filter, concentrate and dry the extract to obtain the mixed Chinese herbal extract.

[0017] As a preferred method, the acclimatization process in step 6 is as follows: the rooted tissue culture seedlings are first subjected to 7 days of intelligent simulation acclimatization in a tissue culture sterile room: the light duration is set to 10-14 hours and the light intensity is changed in a gradient of 'weak-strong-weak'; then they are transferred to a dedicated hardening-off room, initially set with a shading rate of 75%, a temperature of 26℃±1℃, and an air relative humidity of 70%±5%, and hardened off for 7 days; then the shading rate is gradually reduced to 50%, the day-night temperature difference is set to 30℃±1℃ during the day and 15℃±1℃ at night, and the air relative humidity is maintained at 70%±5%, and hardening off continues for another 7 days.

[0018] Furthermore, the special substrate in step 7 contains: peat moss, vermiculite, and perlite in a weight ratio of 1:1:1, and 1.0 kg / m³ of nitrogen, phosphorus, and potassium compound fertilizer is added, wherein the nitrogen, phosphorus, and potassium content in the compound fertilizer is 1:1:1.

[0019] As a preferred method, step 7, the transplanting and cultivation process, is as follows: the acclimatized seedlings are transplanted into plug trays containing the above-mentioned special substrate. A small arched shed is built above the plug trays, and a breathable plastic film is covered. The humidity is adjusted in a gradient of 'closed for 2 days (maintaining humidity of 80%±5%), loosened for 3 days (maintaining humidity of 70%±5%), and uncovered for 5 days (maintaining humidity of 60%±5%)'. Shading is provided at midday. Ventilation is carried out for 1 hour per day in the early stage of transplanting, and the ventilation time is gradually increased to 8 hours in the later stage.

[0020] The present invention has the following beneficial effects: This invention is designed to optimize the strawberry breeding process in response to the marine monsoon climate and sandy soil characteristics of Rongcheng, Weihai. The seedlings are highly adaptable and can withstand the local climate conditions of hot and rainy summers and cold and windy winters, as well as the poor water and fertilizer retention characteristics of sandy soil. The environmentally friendly detoxification pretreatment technology uses a combination of non-toxic and harmless reagents such as ribavirin and vitamin C, along with ultra-low temperature storage, to replace traditional toxic chemical detoxification reagents such as mercuric chloride, thus avoiding safety risks to staff and environmental pollution. The ultra-low temperature can effectively destroy the apical cells containing the virus, with a detoxification efficiency of up to 98.7%, which is more than 12% higher than traditional detoxification technology, and the seedlings have no chemical residues. The culture medium formula was optimized, using a modified MS basal medium adapted to local soil conditions. Food-grade carrageenan replaced agar, and white sugar replaced sucrose. Combined with precise proportions, the cost per liter of culture medium was reduced by 52.9%, solving the problem of high cost (35% higher than traditional seedlings) in the promotion of traditional virus-free tissue culture seedlings, while ensuring seedling vigor. An innovative modified culture medium using traditional Chinese medicine ingredients was also developed. The bioactive substances contained in extracts of traditional Chinese medicinal herbs such as Isatis root, honeysuckle, and Polygonatum sibiricum, improved the culture medium for virus-free strawberry seedlings, resulting in superior fruit quality. Compared to traditional strawberry fruits, the soluble solids content, vitamin C content, and antioxidant enzyme activity and quantity were significantly better, solving the problem of insufficient seedling vigor in local areas. Through an intelligent and precise acclimatization system of 'tissue culture pre-adaptation, seedling hardening gradient acclimatization, and transplanting in a specialized substrate', the final survival rate reached 97.5%, solving the industry pain point of low survival rate of virus-free seedling transplantation. The optimized localized seedling hardening process, simulating the diurnal temperature range and humidity changes in Rongcheng, significantly improved the survival rate of transplanted test-tube seedlings, increasing it by more than 80% compared to traditional techniques. This invention combines indicator plant detection methods with isolated propagation to strictly control the virus-carrying rate of seedlings. Comparative planting trials were conducted in the same cultivation environment and management conditions in Rongcheng, Weihai. The results showed that seedlings propagated using this invention yielded more than 30% more than ordinary seedlings, and the fruit quality was significantly improved. The method and process provided by this invention are adapted to local production conditions, are highly operable, and are suitable for large-scale promotion in Rongcheng, Weihai, reducing seedling costs for growers and promoting the standardized and high-quality development of the local strawberry industry. Attached Figure Description

[0021] Figure 1 Comparison of strawberry fruit quality index test results; Figure 2 Comparison of antioxidant enzyme assay results in strawberry fruits; Figure 3 Comparison of strawberry fruits; Figure 4 Comparison of individual strawberry fruits. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0023] Example 1 Raw material preparation: Modified MS medium: ammonium nitrate 1320 mg / L, potassium iodide 1.25 mg / L, granulated sugar 25 g / L, agar 6.5 g / L, the rest are the same as the traditional MS medium.

[0024] Preparation of mixed Chinese herbal extract: Take Isatis root, honeysuckle, and Polygonatum rhizome separately, crush them, mix them in a weight ratio of 2:1:1, add 8-10 times the weight of distilled water, boil and extract, filter, concentrate and dry the extract to prepare mixed Chinese herbal extract; Prepare a modified induction medium: modified MS medium, 6-BA 0.5 mg / L, NAA 0.2 mg / L, mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave and cool for later use; Prepare a modified differentiation medium: Modified MS medium, 6-BA 1.0 mg / L, KT 0.3 mg / L, and mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave, and cool for later use; Prepare the modified proliferation medium: modified MS medium, 6-BA 1.5 mg / L, NAA 0.5 mg / L, mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave and cool for later use; The rooting medium formula is as follows: 1 / 2 modified MS medium, IBA 0.5 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture bottles, autoclave and cool for later use. Step 1: Pretreatment and stem tip peeling High-quality Xiangye strawberry varieties grown locally in Rongcheng, Weihai, were selected. Healthy, disease-free plants were chosen, and stem tips approximately 0.5cm in length were cut from the top of newly sprouted runners, removing surface leaves and impurities. The stem tips were rinsed with distilled water for 30 minutes to remove surface dust and some pathogens. They were then pre-treated for detoxification by immersing them sequentially in a 25mg / L ribavirin solution for 8 minutes and a 300mg / L LVc solution for 5 minutes. Next, they were immersed in a 70% ethanol solution for 30 seconds, quickly shaken, and then disinfected in a 1% sodium hypochlorite solution for 15 minutes with continuous stirring. Finally, they were rinsed 6 times with sterile water for 1 minute each time and drained. The strawberries were then temporarily stored in liquid nitrogen at ultra-low temperature for 30 minutes, rapidly thawed for 2 minutes, and then placed on a sterile operating table. Using a dissecting microscope and a sterile dissecting needle, the outer scales were peeled off, and 0.3-0.4mm sections were precisely cut. The shoot tip tissue (precisely preserving the growth point and leaf primordia) was immediately placed in a sterile petri dish for later use; Step 2: Induction Culture Shoot tip culture: The excised shoot tip tissue was inoculated into a modified induction medium, with 8 shoot tips inoculated into each culture bottle; the culture bottles were placed in a culture room and the culture conditions were set as follows: temperature 26℃±1℃, 20W LED lamp irradiation, 24h light per day, and after 30 days of culture, callus tissue was induced. Step 3: Differentiation Culture When the callus tissue grows to about 2 cm, it is transferred to a modified differentiation medium. The inoculated culture bottles are placed in a culture room at a temperature of 26℃±1℃ and under 20W LED light for 24 hours a day. After 30 days of culture, aseptic buds differentiate to form dense seedling clusters. Three healthy leaves are randomly cut from each seedling cluster and tested for strawberry mottle virus, strawberry wrinkle virus, strawberry vein virus, and strawberry mild yellow edge virus using indicator plant detection methods. Seedling clusters with positive test results are discarded, and only virus-free seedling clusters are retained for further culture. Step 4: Proliferation Culture Virus-free seedlings were divided into small clumps using sterile scissors and inoculated into a modified proliferation medium. The culture conditions were 26℃±1℃, 20W LED light, and 24h light per day. Subculture was performed every 7 days, with a total of no more than 20 subcultures to avoid seedling degeneration and to rapidly proliferate a large number of virus-free test-tube seedlings. Step 5: Rooting Culture Select robust test-tube seedlings with a height of 3cm or more and transfer them to a modified rooting medium. Place the culture bottles in a culture room and maintain the temperature at 26℃±1℃. Illuminate them with a 20W LED lamp for 24 hours a day. After 14 days of culture, the test-tube seedlings will grow robust roots and form rooted tissue culture seedlings. Step Six: Domestication The rooted tissue culture seedlings were first subjected to 7 days of intelligent simulation acclimatization in a sterile tissue culture room, with a light duration of 10-14 hours and a light intensity gradient of 'weak-strong-weak', and temperature and humidity adjusted according to the season. Then, they were transferred to a special hardening-off room that simulated the general environment of Rongcheng, Weihai, initially with a shading rate of 75%, a temperature of 26℃±1℃, and a relative humidity of 70%±5%, for 7 days. After that, the shading rate was gradually reduced to 50%, simulating the diurnal temperature difference of Rongcheng (30℃±1℃ during the day and 15℃±1℃ at night), and the relative humidity was maintained at 70%±5%, for another 7 days of hardening-off. After the hardening-off was completed, the seedlings were processed according to the process of "seedling selection → removal of culture medium → rinsing with sterile water → repairing seedlings → sterilization" and then prepared for use. Step 7: Transplanting in seed trays and acclimatization during the seedling stage Preparation of nutrient substrate suitable for sandy soil in Rongcheng, Weihai: Peat moss, vermiculite and perlite were mixed in a weight ratio of 1:1:1, and then 1.0 kg / m³ of nitrogen, phosphorus and potassium (1-1-1) compound fertilizer was added. The mixture was sterilized at 121℃ for 0.5 h and then put into use. Fill the sterilized nutrient substrate into the seedling trays and gently compact it. Transplant the acclimatized seedlings (after washing off the culture medium) into the holes of the seedling trays, following the principle of "shallow transplanting without exposing the roots and deep transplanting without burying the crown." Transplant one seedling per hole. Water thoroughly immediately after transplanting to ensure close contact between the roots and the substrate. Construct a small arched shed 1.5m high above the seedling trays and cover it with a breathable plastic film. Adjust the humidity in a gradient: cover for 2 days (maintaining 80%±5% humidity), loosen the cover for 3 days (maintaining 70%±5% humidity), and open the cover for 5 days (maintaining 60%±5% humidity). Provide shade during midday. Ventilate for 1 hour daily in the early stages of transplanting, gradually increasing the ventilation time to 8 hours later. Regularly observe the plant growth and promptly remove diseased and weak leaves. Three weeks after transplanting, the test-tube seedlings will grow vigorously and form virus-free original seedlings. Step 8: Propagation of original seedlings and production seedlings In the Rongcheng area of ​​Weihai, select plots free of soil nematodes and strawberry pathogens to build insect-proof isolation zones. Plant virus-free original seedlings in the isolation zones and propagate them by division. The first generation of plants obtained from this propagation are the original seedlings. Continue to plant the original seedlings in the isolation zones and propagate them by division. The offspring are the seedlings for production and can be directly used for local strawberry production. During the propagation process of original seedlings and production seedlings, virus testing is carried out regularly. The virus infection rate does not exceed 0.5%, and the seedling quality is good.

[0025] Comparative Example 1 Raw material preparation: Modified MS medium: ammonium nitrate 1320 mg / L, potassium iodide 1.25 mg / L, granulated sugar 25 g / L, agar 6.5 g / L, the rest are the same as the traditional MS medium.

[0026] Preparation of mixed Chinese herbal extract: Take Isatis root and honeysuckle, pulverize them separately, mix them in a weight ratio of 2:1, add 8 to 10 times the weight of distilled water, boil and extract, filter, concentrate and dry the extract to prepare mixed Chinese herbal extract. Prepare a modified induction medium: modified MS medium, 6-BA 0.5 mg / L, NAA 0.2 mg / L, mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave and cool for later use; Prepare a modified differentiation medium: Modified MS medium, 6-BA 1.0 mg / L, KT 0.3 mg / L, and mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave, and cool for later use; Prepare the modified proliferation medium: modified MS medium, 6-BA 1.5 mg / L, NAA 0.5 mg / L, mixed Chinese herbal extract 150 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture flasks, autoclave and cool for later use; The rooting medium formula is as follows: 1 / 2 modified MS medium, IBA 0.5 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into culture bottles, autoclave and cool for later use. Step 1: Pretreatment and stem tip peeling High-quality Xiangye strawberry varieties grown locally in Rongcheng, Weihai, were selected. Healthy, disease-free plants were chosen, and stem tips approximately 0.5cm in length were cut from the top of newly sprouted runners, removing surface leaves and impurities. The stem tips were rinsed with distilled water for 30 minutes to remove surface dust and some pathogens. They were then pre-treated for detoxification by immersing them sequentially in a 25mg / L ribavirin solution for 8 minutes and a 300mg / L LVc solution for 5 minutes. Next, they were immersed in a 70% ethanol solution for 30 seconds, quickly shaken, and then disinfected in a 1% sodium hypochlorite solution for 15 minutes with continuous stirring. Finally, they were rinsed 6 times with sterile water for 1 minute each time and drained. The strawberries were then temporarily stored in liquid nitrogen at ultra-low temperature for 30 minutes, rapidly thawed for 2 minutes, and then placed on a sterile operating table. Using a dissecting microscope and a sterile dissecting needle, the outer scales were peeled off, and 0.3-0.4mm sections were precisely cut. The shoot tip tissue (precisely preserving the growth point and leaf primordia) was immediately placed in a sterile petri dish for later use; Step 2: Induction Culture Shoot tip culture: The excised shoot tip tissue was inoculated into a modified induction medium, with 8 shoot tips inoculated into each culture bottle; the culture bottles were placed in a culture room and the culture conditions were set as follows: temperature 26℃±1℃, 20W LED lamp irradiation, 24h light per day, and after 30 days of culture, callus tissue was induced. Step 3: Differentiation Culture When the callus tissue grows to about 2 cm, it is transferred to a modified differentiation medium. The inoculated culture bottles are placed in a culture room at a temperature of 26℃±1℃ and under 20W LED light for 24 hours a day. After 30 days of culture, aseptic buds differentiate to form dense seedling clusters. Three healthy leaves are randomly cut from each seedling cluster and tested for strawberry mottle virus, strawberry wrinkle virus, strawberry vein virus, and strawberry mild yellow edge virus using indicator plant detection methods. Seedling clusters with positive test results are discarded, and only virus-free seedling clusters are retained for further culture. Step 4: Proliferation Culture Virus-free seedlings were divided into small clumps using sterile scissors and inoculated into a modified proliferation medium. The culture conditions were 26℃±1℃, 20W LED light, and 24h light per day. Subculture was performed every 7 days, with a total of no more than 20 subcultures to avoid seedling degeneration and to rapidly proliferate a large number of virus-free test-tube seedlings. Step 5: Rooting Culture Select robust test-tube seedlings with a height of 3cm or more and transfer them to a modified rooting medium. Place the culture bottles in a culture room and maintain the temperature at 26℃±1℃. Illuminate them with a 20W LED lamp for 24 hours a day. After 14 days of culture, the test-tube seedlings will grow robust roots and form rooted tissue culture seedlings. Step Six: Transplanting in Seedling Trays and Seedling Acclimatization Preparation of nutrient substrate suitable for sandy soil in Rongcheng, Weihai: Peat moss, vermiculite and perlite were mixed in a weight ratio of 1:1:1, and then 1.0 kg / m³ of nitrogen, phosphorus and potassium (1-1-1) compound fertilizer was added. The mixture was sterilized at 121℃ for 0.5 h and then put into use. Fill the seedling trays with the sterilized nutrient substrate and gently compact it. After washing away the culture medium, transplant the rooted tissue culture seedlings into the holes of the seedling trays, following the principle of "shallow transplanting without exposing the roots and deep transplanting without burying the crown." Transplant one seedling per hole. Water thoroughly immediately after transplanting to ensure close contact between the roots and the substrate. Construct a small arched shed with a height of 1.5m above the seedling trays and cover it with a breathable plastic film. Adjust the humidity in a gradient: cover for 2 days (maintain humidity 80%±5%), loosen the cover for 3 days (maintain humidity 70%±5%), and open the cover for 5 days (maintain humidity 60%±5%). Provide shade during midday. Ventilate for 1 hour daily in the early stages of transplanting, gradually increasing the ventilation time to 8 hours later. Regularly observe the plant growth and promptly remove diseased and weak leaves. Three weeks after transplanting, the test-tube seedlings will grow vigorously and form virus-free original seedlings. Step 7: Propagation of original seedlings and production seedlings In the Rongcheng area of ​​Weihai, select plots free of soil nematodes and strawberry pathogens, and build insect-proof isolation zones; plant virus-free original seedlings in the isolation zones and propagate them by division. The first generation of plants obtained by division are the original seedlings; continue to plant the original seedlings in the isolation zones, and propagate them by division to produce seedlings for production.

[0027] Comparative Example 2 Prepare induction medium: conventional MS medium, 6-BA 0.5 mg / L, NAA 0.2 mg / L, adjust pH to 5.8~6.0; dispense the prepared medium, autoclave, and cool for later use. Prepare differentiation medium: conventional MS medium, 6-BA 1.0 mg / L, KT 0.3 mg / L, adjust pH to 5.8-6.0; dispense the prepared medium, autoclave, and cool for later use. Prepare the proliferation medium: conventional MS medium, 6-BA 1.5 mg / L, NAA 0.5 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium into containers, autoclave and cool for later use. The rooting medium formula is as follows: 1 / 2 conventional MS medium, IBA 0.5 mg / L, adjust the pH to 5.8~6.0; dispense the prepared medium, autoclave and cool for later use. Step 1: Pretreatment and stem tip peeling High-quality Xiangye strawberry varieties grown locally in Rongcheng, Weihai, were selected. Healthy plants free from pests and diseases were chosen, and stem tips approximately 0.5cm long were cut from the top of newly sprouted runners. Surface leaves and impurities were removed. The stem tips were then immersed in a 70% ethanol solution for 30 seconds, quickly shaken, and then placed in a 1% sodium hypochlorite solution for 15 minutes for sterilization, stirring constantly. Finally, they were rinsed 6 times with sterile water for 1 minute each time and drained. They were then temporarily stored in liquid nitrogen at ultra-low temperature for 30 minutes, rapidly thawed for 2 minutes, and placed on a sterile operating table. Using a dissecting microscope and a sterile dissecting needle, the outer scales were peeled off, and 0.3-0.4mm stem tip tissue was precisely cut (precisely preserving the growth point and leaf primordia) and immediately placed in a sterile petri dish for later use. Step 2: Induction Culture Shoot tip culture: The excised shoot tip tissue was inoculated into the induction medium, with 8 shoot tips inoculated into each culture bottle; the culture bottles were placed in the culture room and the culture conditions were set as follows: temperature 26℃±1℃, 20W LED lamp irradiation, 24h light per day, and after 30 days of culture, callus tissue was induced. Step 3: Differentiation Culture When the callus tissue grows to about 2 cm, it is transferred to differentiation medium. The inoculated culture bottles are placed in a culture room and kept at a temperature of 26℃±1℃ under 20W LED light for 24 hours a day. After 30 days of culture, aseptic buds differentiate to form dense seedling clusters. Three healthy leaves are randomly cut from each seedling cluster and tested for strawberry mottle virus, strawberry wrinkle virus, strawberry vein virus, and strawberry mild yellow edge virus using indicator plant detection methods. Seedling clusters with positive test results are discarded, and only virus-free seedling clusters are retained for further culture. Step 4: Proliferation Culture Virus-free seedlings were divided into small clumps using sterile scissors and inoculated into proliferation medium. The culture conditions were 26℃±1℃, 20W LED light, and 24h light per day. Subculture was performed every 7 days, with a total of no more than 20 subcultures to avoid seedling degeneration and to rapidly proliferate and obtain a large number of virus-free test-tube seedlings. Step 5: Rooting Culture Select robust test-tube seedlings with a height of 3cm or more and transfer them to rooting medium. Place the culture bottles in a culture room and maintain the temperature at 26℃±1℃. Illuminate them with a 20W LED lamp for 24 hours a day. After 14 days of culture, the test-tube seedlings will grow robust roots and form rooted tissue culture seedlings. Step Six: Transplanting in Seedling Trays and Seedling Acclimatization Preparation of nutrient substrate suitable for sandy soil in Rongcheng, Weihai: Peat moss, vermiculite and perlite were mixed in a weight ratio of 1:1:1, and then 1.0 kg / m³ of nitrogen, phosphorus and potassium (1-1-1) compound fertilizer was added. The mixture was sterilized at 121℃ for 0.5 h and then put into use. Fill the seedling trays with the sterilized nutrient substrate and gently compact it. After washing away the culture medium, transplant the rooted tissue culture seedlings into the holes of the seedling trays, following the principle of "shallow transplanting without exposing the roots and deep transplanting without burying the crown." Transplant one seedling per hole. Water thoroughly immediately after transplanting to ensure close contact between the roots and the substrate. Construct a small arched shed with a height of 1.5m above the seedling trays and cover it with a breathable plastic film. Adjust the humidity in a gradient: cover for 2 days (maintain humidity 80%±5%), loosen the cover for 3 days (maintain humidity 70%±5%), and open the cover for 5 days (maintain humidity 60%±5%). Provide shade during midday. Ventilate for 1 hour daily in the early stages of transplanting, gradually increasing the ventilation time to 8 hours later. Regularly observe the plant growth and promptly remove diseased and weak leaves. Three weeks after transplanting, the test-tube seedlings will grow vigorously and form virus-free original seedlings. Step 7: Propagation of original seedlings and production seedlings In the Rongcheng area of ​​Weihai, select plots free of soil nematodes and strawberry pathogens, and build insect-proof isolation zones; plant virus-free original seedlings in the isolation zones and propagate them by division. The first generation of plants obtained by division are the original seedlings; continue to plant the original seedlings in the isolation zones, and propagate them by division to produce seedlings for production.

[0028] Performance testing 1. Effects of different treatments on the survival rate of strawberry seedlings Eighty-one rooted tissue culture seedlings obtained from Example 1, Comparative Example 1, and Comparative Example 2 were used for transplanting experiments. The transplant survival rate was calculated, and the results are shown in Table 1.

[0029] Table 1 Comparison of transplant survival rates As shown in Table 1, the survival rate of rooted tissue culture seedlings obtained by the method described in this invention through direct transplantation is over 95%, which is significantly better than that of Comparative Example 1 and the conventional method (Comparative Example 2). Compared with the conventional method, the survival rate is increased by 80.2%, demonstrating a significant effect.

[0030] 2. The impact of different treatments on strawberry yield and quality 1000 seedlings from each of Examples 1, 1 Comparative Example 1, and 2 were planted, and the final strawberry yield was calculated. The results are shown in Table 2.

[0031] Ten strawberry fruits from each group were selected for quality testing, including soluble solids content, vitamin C content, antioxidant enzymes, and fruit size. Results are shown below. Figure 1-4 .

[0032] Table 2 Comparison of Strawberry Yields From Table 2, Figure 1-4 The results show that the strawberry seedlings obtained by the method described in this invention have a higher strawberry yield after planting, with a yield increase of more than 30% compared with seedlings propagated by traditional methods, and the fruit quality is significantly improved.

Claims

1. A method for propagating virus-free strawberry tissue culture seedlings, characterized in that, Includes the following steps: Step 1, Pretreatment: Soak strawberry stem tips in ribavirin solution and vitamin C solution; then soak in ethanol solution, disinfect with sodium hypochlorite, rinse with sterile water and drain; then place in liquid nitrogen for ultra-low temperature storage, thaw quickly, peel off the outer scales and cut the stem tip tissue; Step 2, induction culture: The shoot tip tissue was inoculated into a modified induction medium containing modified MS medium, 0.5 mg / L 6-BA, 0.2 mg / L NAA, and 150 mg / L mixed Chinese herbal extract to induce callus tissue; Step 3, Differentiation Culture: The callus tissue was inoculated into a modified differentiation medium containing modified MS medium, 6-BA 1.0 mg / L, KT 0.3 mg / L, and mixed Chinese herbal extract 150 mg / L and cultured to form seedlings; Step 4, Proliferation Culture: The seedlings were inoculated into a modified proliferation medium containing modified MS medium, 6-BA 1.5 mg / L, NAA 0.5 mg / L, and mixed Chinese herbal extract 150 mg / L to obtain test-tube seedlings; Step 5, rooting culture: Inoculate the test-tube seedlings into rooting medium containing 1 / 2 modified MS medium and 0.5 mg / L IBA to obtain rooted tissue culture seedlings; Step Six, Acclimation: The rooted tissue culture seedlings are subjected to intelligent simulation acclimation to obtain acclimated seedlings. The intelligent simulation acclimation process is as follows: the light duration is set to 10-14 hours, and the light intensity is changed in a gradient of 'weak-strong-weak'. Then, the seedlings are transferred to a special hardening-off room. Initially, the shading rate is set to 75%, the temperature to 26℃±1℃, and the relative humidity to 70%±5%, and the seedlings are hardened off for 7 days. After that, the shading rate is gradually reduced to 50%, the day-night temperature difference is set to 30℃±1℃ during the day and 15℃±1℃ at night, and the relative humidity is maintained at 70%±5%, and the seedlings are hardened off for another 7 days. Step 7, Transplanting: Transplant the acclimatized seedlings into a special substrate for cultivation to obtain virus-free original seedlings; Step 8, Propagation: Plant the virus-free original seedlings in the soil and propagate them by division to obtain original seedlings; The original seedlings are then transplanted to obtain seedlings for production. in, The mixed Chinese herbal extract is prepared from Isatis root, honeysuckle, and Polygonatum. The preparation method is as follows: take Isatis root, honeysuckle, and Polygonatum separately, crush them, mix them in a weight ratio of 2:1:1, add 8 to 10 times the weight of water for extraction, filter, concentrate and dry the extract to prepare the mixed Chinese herbal extract. The modified MS medium is as follows: the ammonium nitrate content is reduced to 1320 mg / L, the potassium iodide content is increased to 1.25 mg / L, white sugar is used instead of sucrose, and carrageenan is used instead of agar in the traditional MS culture; and the content of white sugar is 25 g / L and the content of carrageenan is 6.5 g / L.

2. The breeding method as described in claim 1, characterized in that, In step 1, the concentration of ribavirin solution is 25 mg / L, and the concentration of vitamin C solution is 300 mg / L.

3. The breeding method as described in claim 1, characterized in that, The special substrate in step 7 contains peat moss, vermiculite, and perlite in a weight ratio of 1:1:1, and 1.0 kg / m³ of nitrogen, phosphorus, and potassium compound fertilizer is added, wherein the nitrogen, phosphorus, and potassium content in the compound fertilizer is 1:1:1.

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