A rooting culture method for improving the survival rate of transplanted strawberry virus-free seedlings

Abstract

The application relates to a rooting culture method for improving the survival rate of strawberry virus-free seedlings, and belongs to the technical field of plant tissue culture. The rooting culture method comprises the steps of low-sugar culture pretreatment, staged rooting culture and seedling raising. The transplanted seedlings obtained by the rooting culture method are developed and robust in root systems, and the survival rate of the strawberry seedlings after transplanting can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology and relates to a rooting culture method for improving the survival rate of transplanted virus-free strawberry seedlings. Background Technology

[0002] Strawberry tissue culture is a core method for the rapid propagation of virus-free seedlings, but the low survival rate of transplanted tissue culture seedlings has always been a bottleneck restricting industrialization. Traditional rooting culture methods have the following drawbacks: Root structure defects: The roots induced in solid or liquid culture media in closed containers are mostly "aquatic roots" with few root hairs, thin cortex, and low mechanical strength, making it difficult for them to adapt to the soil environment after transplanting.

[0003] The acclimatization process is disconnected: rooting culture and transplanting acclimatization are carried out in separate steps, and seedlings need to be hardened off separately after being removed from the bottle, which increases the risk of root damage and the production cycle.

[0004] Weak environmental adaptability: Traditional tissue culture environment is characterized by high humidity, low light, and heterotrophic conditions, resulting in poor photosynthetic capacity and incomplete stomatal regulation function of seedlings, which easily lead to water loss and wilting after transplanting.

[0005] Although some studies have improved the rooting rate by optimizing hormone ratios (such as 1 / 2MS + 0.4-0.5 mg / L IBA) or enhanced seedling photosynthetic capacity through sugar-free tissue culture technology, none of them have fundamentally solved the adaptation problem of the transition from "aquatic roots" to "soil roots". Summary of the Invention

[0006] This invention aims to provide a method for strawberry tissue culture rooting, which induces the formation of a root system structure that combines aerial root characteristics and soil adaptability by simulating a gradual transition from an aquatic environment to an aerobic environment and then to a solid substrate during the rooting stage, thereby significantly improving the transplant survival rate.

[0007] The present invention employs the following technical solutions to achieve the above objectives: A rooting culture method for improving the survival rate of virus-free strawberry seedlings after transplanting mainly includes the following steps: Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low sugar medium, CO2 was added, and cultured for 10-12 days to obtain pretreated seedlings; Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I supplemented with indolebutyric acid and cultured in the dark at 25±2℃ for 5 to 7 days. Then, they were transferred to rooting medium II supplemented with indolebutyric acid and bisabolol and cultured for 10-15 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III containing indolebutyric acid, multi-walled carbon nanotubes, phloretin-β-D-glucan, and 1,8-cineole. They were cultured at 25±2℃, 2000 lx light intensity, and 16h / 8h photoperiod for 10-15 days to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle until it is completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with ventilation holes, cover it with a perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings.

[0008] Furthermore, In step one, the low-sugar culture medium contains the following components: 1 / 2 MS basal medium + 5~10 g / L + 6 g / L agar.

[0009] In step one, the CO2 concentration is 800–1000 ppm.

[0010] In step one, the light intensity is 3000-4000 lx.

[0011] In step two, the rooting medium I contains the following components: 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10~12 mg / L indolebutyric acid.

[0012] In step two, the rooting medium II contains the following components: 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 8~10 mg / L indolebutyric acid + 30~35 mg / L bisabolol.

[0013] In step two, the rooting medium III contains the following components: 1 / 2MS basal medium + 5 g / L sucrose + 6 g / L agar + 5~8 mg / L indolebutyric acid + 25~30 mg / L multi-walled carbon nanotubes + 50~55 mg / L phloretin-β-D-glucan + 20~25 mg / L 1,8-cineole.

[0014] In step three, the composite culture medium contains 50% culture medium and 50% MS basal culture medium.

[0015] Furthermore, the culture medium contains sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3.

[0016] The present invention has the following beneficial effects: This invention relates to a strawberry virus-free seedling cultivation process that constructs a rooting culture pathway encompassing induction, reinforcement, and acclimatization. This breaks away from traditional tissue culture seedling rooting methods, effectively completing the transformation of virus-free seedlings from aquatic roots to terrestrial roots, enhancing their adaptability, and improving the survival rate after transplanting. The main features are: Rootless tissue culture seedlings from proliferation culture were cultured using a low-sugar culture method, and the seedlings were induced to start photosynthesis by adding a small amount of exogenous sucrose. During the rooting culture stage, a three-stage progressive root cultivation method is adopted. The sucrose concentration is dynamically adjusted according to the cultivation process, and active ingredients are added. The unique nanotube structure of carbon nanotubes is used to stimulate the differentiation of root primordia, which greatly increases the number and density of adventitious roots. The addition of active ingredients such as bisabolol can help plant hormones induce the efficient differentiation of root primordia into the root hair zone, promote root development, and ensure efficient water absorption after transplanting. During the hardening-off period, traditional substrates were abandoned, and the culture medium was optimized. Clove essential oil was added to the culture medium to achieve a gradual adaptation of the root system from a sterile and humid environment to the natural environment, avoiding root damage caused by transplant shock. In addition, the antioxidant activity of clove essential oil can establish an antibacterial barrier in the rhizosphere microenvironment, significantly reducing the occurrence of root diseases during the hardening-off period. The hardening-off period also facilitated the transition between liquid culture medium and solid culture medium, effectively improving the transplant survival rate.

[0017] The root quality of virus-free strawberry seedlings obtained by the rooting culture method provided by this invention is significantly improved, and the root vitality is enhanced. The transition from liquid to solid phase is stable, the transplanting shock is small, and the root system collapse caused by sudden environmental changes is avoided. The transplanting survival rate is significantly improved, and the method has a wide range of applications, including major strawberry varieties such as 'Hongyan', 'Zhangji', 'Xiangye', and 'Fenyu'. Detailed Implementation

[0018] 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.

[0019] Example 1 Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low-sugar medium and transferred to sugar-free tissue culture boxes for low-sugar culture under the following conditions: Low-glucose medium: 1 / 2 MS basal medium + 5 g / L + 6 g / L agar; Introduce CO2 at a concentration of 1000 ppm; Light intensity: 4000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25±2℃ for 7 days. Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 8 mg / L indolebutyric acid + 35 mg / L bisabolol) and cultured for 15 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 5 mg / L indolebutyric acid + 30 mg / L multi-walled carbon nanotubes + 50 mg / L phloretin-β-D-glucan + 25 mg / L 1,8-cineole) and cultured for 15 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0020] Example 2 Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low-sugar medium and transferred to sugar-free tissue culture boxes for low-sugar culture under the following conditions: Low-glucose medium: 1 / 2 MS basal medium + 10 g / L + 6 g / L agar; CO2 was introduced at a concentration of 800 ppm; Light intensity: 3000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 12 mg / L indolebutyric acid) and cultured in the dark at 25 ± 2℃ for 5 days. Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid + 30 mg / L bisabolol) and cultured for 10 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 8 mg / L indolebutyric acid + 25 mg / L multi-walled carbon nanotubes + 55 mg / L phloretin-β-D-glucan + 20 mg / L 1,8-cineole) and cultured for 10 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0021] Example 3 Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low-sugar medium and transferred to sugar-free tissue culture boxes for low-sugar culture under the following conditions: Low-glucose medium: 1 / 2 MS basal medium + 8 g / L + 6 g / L agar; CO2 was introduced at a concentration of 900 ppm; Light intensity: 3000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25 ± 2℃ for 5 days. Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 9 mg / L indolebutyric acid + 33 mg / L bisabolol) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 6 mg / L indolebutyric acid + 28 mg / L multi-walled carbon nanotubes + 52 mg / L phloretin-β-D-glucan + 23 mg / L 1,8-cineole) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0022] Comparative Example 1 Step 1: Rooting Culture Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25±2℃ for 5 days; Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 9 mg / L indolebutyric acid + 33 mg / L bisabolol) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 6 mg / L indolebutyric acid + 28 mg / L multi-walled carbon nanotubes + 52 mg / L phloretin-β-D-glucan + 23 mg / L 1,8-cineole) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 2: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0023] Comparative Example 2 Step 1: Preprocessing In a low-sugar culture, rootless strawberry tissue culture seedlings after proliferation culture were inoculated into a low-sugar medium, transferred to a sugar-free tissue culture box, and cultured under low-sugar conditions. Culture medium: 1 / 2 MS basal medium + 8 g / L + 6 g / L agar; CO2 was introduced at a concentration of 900 ppm; Light intensity: 3000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25 ± 2℃ for 5 days. Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 9 mg / L indolebutyric acid) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 6 mg / L indolebutyric acid + 28 mg / L multi-walled carbon nanotubes) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0024] Comparative Example 3 Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low-sugar medium and transferred to sugar-free tissue culture boxes for low-sugar culture under the following conditions: Low-glucose medium: 1 / 2 MS basal medium + 8 g / L + 6 g / L agar; CO2 was introduced at a concentration of 900 ppm; Light intensity: 3000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 9 mg / L indolebutyric acid + 33 mg / L bisabolol + 28 mg / L multi-walled carbon nanotubes + 23 mg / L 1,8-cineole) and cultured for 20 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3) and 50% MS basal culture medium.

[0025] Comparative Example 4 Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low-sugar medium and transferred to sugar-free tissue culture boxes for low-sugar culture under the following conditions: Low-glucose medium: 1 / 2 MS basal medium + 8 g / L + 6 g / L agar; CO2 was introduced at a concentration of 900 ppm; Light intensity: 3000 lx, 12h / d; Temperature: 25±2℃; The low-sugar culture period is 10-12 days, until white root primordia protrude from the base, and the pretreated seedlings are obtained. Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25 ± 2℃ for 5 days. Then, the culture medium was transferred to rooting medium II (containing 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 9 mg / L indolebutyric acid + 33 mg / L bisabolol) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 1500 lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III (containing 1 / 2 MS basal medium + 5 g / L sucrose + 6 g / L agar + 6 mg / L indolebutyric acid + 28 mg / L multi-walled carbon nanotubes + 52 mg / L phloretin-β-D-glucan + 23 mg / L 1,8-cineole) and cultured for 13 days at a temperature of 25±2℃, a light intensity of 2000 lx, and a photoperiod of 16h / 8h to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle within 24 hours to increase the air permeability gap until it is finally completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with air perforation, cover it with perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: 50% culture medium (containing sterilized vermiculite, coconut coir, and peat moss in a mass ratio of 2:1:1) and 50% MS basal culture medium.

[0026] Comparative Example 5 Step 1: Rooting Culture Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into rooting medium (containing 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10 mg / L indolebutyric acid) and cultured in the dark at 25±2℃ for 5 days; then the light intensity was gradually increased to 2000 lx and the photoperiod was 16h / 8h, and cultured for 25 days to obtain rooted seedlings. Step 2: Hardening off the seedlings Transplant the rooted seedlings into the culture medium, place them in a seedling box with ventilation holes, cover with a perforated plastic wrap to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings. The composite culture medium contains the following components: a mixture of peat moss, vermiculite, and perlite in a mass ratio of 2:1:1.

[0027] Effect evaluation I. The Influence of Different Culture Methods on Rooting in Strawberry Tissue Culture 1.1 Materials and Methods Tissue culture seedlings of the "Hongyan" strawberry variety, which had been subcultured five times, were selected. The seedlings were in a similar growth state, with a height of 2-3 cm and 3-4 leaves. They were rooted and cultured according to the tissue culture methods described in Examples 1-3 and Comparative Examples 1-5. The growth of the test-tube seedlings was recorded after 20 days.

[0028] Measure the longest root length, fresh root weight, dry root weight, and number of roots.

[0029] After the rooted test-tube seedlings were hardened off according to the methods described in Examples 1-3 and Comparative Examples 1-5, they were transplanted, and the transplant survival rate was counted.

[0030] 1.2 Results and Analysis As can be seen from the results in Table 1, During the tissue culture stage, different culture methods resulted in significant differences in root growth of tissue culture seedlings. In Examples 1-3, the rooting rate of tissue culture seedlings was high, the root development was high, and the number of roots was large. In Comparative Example 4, the tissue culture method was the same as that of the present invention, and the root development of its tissue culture seedlings was good. In Comparative Example 1, there was no pretreatment process. In Comparative Examples 2 and 3, the rooting culture methods were different. In Comparative Example 5, it was completely different from the present invention. The results showed that the root development of tissue culture seedlings in Comparative Example 5 was the worst. The root development of tissue culture seedlings in Comparative Example 1 was better than that in Comparative Examples 2 and 3, but overall it was worse than that in Examples 1-3 and Comparative Example 4. The survival rates of transplanted tissue culture seedlings varied significantly after being hardened off using different methods. The survival rates of transplanted seedlings in Examples 1-3 were relatively high. In Comparative Example 4, the hardening-off method was different, and although the seedlings already had a good root base from the tissue culture, the survival rate still decreased. The root development of tissue culture seedlings in Comparative Examples 1-3 and Comparative Example 5 was poor. Although Comparative Examples 1-3 used the same hardening-off method as the present invention, their root base was poor, resulting in a lower survival rate. Comparative Example 5 had the lowest survival rate.

[0031] Table 1. Effects of different culture methods on rooting during tissue culture. II. The Influence of Different Culture Methods on Root Growth and Transplanting during the Hardening-off Period 2.1 Materials and Methods Tissue culture was performed as described in Example 1. Rooted tissue culture seedlings with similar growth were selected and hardened off as described in Examples 1-3 and Comparative Examples 1-5, respectively. At the end of the hardening-off period, 5 seedlings were randomly selected to observe their root growth. They were then transplanted, and the survival rate was recorded 20 days after transplanting.

[0032] As can be seen from the results in Table 2, the survival rate after transplanting varies significantly depending on the seedling hardening method used. Examples 1-3 and Comparative Examples 1-3 used the same seedling hardening method, and the tissue culture seedlings had a good root system, with a survival rate of 100%. Comparative Examples 4 and 5 used different seedling hardening methods than the present invention. Although the tissue culture seedlings had the same root system, the root development during the hardening period was different, resulting in uneven root systems and a lower survival rate after hardening.

[0033] Table 2. Effects of different culture methods on rooting during hardening-off and transplanting stages.

Claims

1. A rooting culture method for improving the survival rate of virus-free strawberry seedlings after transplanting, characterized in that, Includes the following steps: Step 1: Preprocessing Strawberry rootless tissue culture seedlings after proliferation culture were inoculated into low sugar medium, CO2 was added, and cultured for 10-12 days to obtain pretreated seedlings; Step 2: Rooting Culture The pretreated seedlings were inoculated into rooting medium I supplemented with indolebutyric acid and cultured in the dark at 25±2℃ for 5 to 7 days. Then, they were transferred to rooting medium II supplemented with indolebutyric acid and bisabolol and cultured for 10-15 days at a temperature of 25±2℃, a light intensity of 1500lx, and a photoperiod of 16h / 8h. The seedlings were then transferred to rooting medium III containing indolebutyric acid, multi-walled carbon nanotubes, phloretin-β-D-glucan, and 1,8-cineole. They were cultured at 25±2℃, 2000 lx light intensity, and 16h / 8h photoperiod for 10-15 days to obtain rooted seedlings. Step 3: Hardening off the seedlings Gradually open the sealing film of the above-mentioned rooted seedling culture bottle until it is completely exposed to the room temperature environment; then transfer the rooted seedling to the composite culture medium, put it into the seedling box with ventilation holes, cover it with a perforated plastic film to maintain 60-70% humidity, and culture for 14 days to obtain transplanted seedlings.

2. The rooting culture method as described in claim 1, characterized in that, In step one, the low-sugar culture medium contains the following components: 1 / 2 MS basal medium + 5~10 g / L + 6 g / L agar.

3. The rooting culture method as described in claim 1, characterized in that, In step one, the CO2 concentration is 800-1000 ppm.

4. The rooting culture method as described in claim 1, characterized in that, In step one, the light intensity is 3000-4000 lx.

5. The rooting culture method as described in claim 1, characterized in that, In step two, the rooting medium I contains the following components: 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 10~12 mg / L indolebutyric acid.

6. The rooting culture method as described in claim 1, characterized in that, In step two, the rooting medium II contains the following components: 1 / 2 MS basal medium + 15 g / L sucrose + 6 g / L agar + 8~10 mg / L indolebutyric acid + 30~35 mg / L bisabolol.

7. The rooting culture method as described in claim 1, characterized in that, In step two, the rooting medium III contains the following components: 1 / 2MS basal medium + 5 g / L sucrose + 6 g / L agar + 5~8 mg / L indolebutyric acid + 25~30 mg / L multi-walled carbon nanotubes + 50~55 mg / L phloretin-β-D-glucan + 20~25 mg / L 1,8-cineole.

8. The rooting culture method as described in claim 1, characterized in that, In step three, the composite culture medium contains 50% culture medium and 50% MS basal culture medium.

9. The rooting culture method as described in claim 8, characterized in that, The culture medium contains sterilized vermiculite, wheat bran, coconut coir, and clove oil in a mass ratio of 2:1:1:0.3.

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