A tissue culture method for strawberries

Abstract

The application discloses a tissue culture method of strawberry, and belongs to the technical field of strawberry tissue culture, and the specific method is as follows: a stolon or a leaf of a strawberry plant is selected as an explant, the explant is subjected to disinfection treatment, and then is subjected to primary culture, subculture and rooting and seedling training to obtain tissue culture seedlings; the rooting and seedling training medium and the method of rooting and seedling training are used for training the adaptability of the root system and the opening and closing ability of the leaf stomata at the same time, so that the obtained tissue culture seedlings can quickly adapt to the soil and air environment after being transplanted to a substrate, and can be directly grown in the open air, and the propagation cycle is shortened.

Description

Technical Field

[0001] This invention relates to the field of strawberry tissue culture technology, and more particularly to a method for strawberry tissue culture. Background Technology

[0002] Strawberry (Fragaria ananassa Duch.), a perennial herbaceous plant belonging to the genus Fragaria in the family Rosaceae, is also known as foreign strawberry, red strawberry, etc. It originated in South America and has a wide global distribution. In China, strawberries are cultivated throughout the country. Strawberries prefer warm, sunny growing environments. They are cold-hardy and shade-tolerant, but intolerant of drought, waterlogging, high temperatures, and severe cold. Strawberries are highly nutritious and are known as the "Queen of Fruits." They are rich in vitamins C, A, and E, which can alleviate night blindness, maintain the health of epithelial tissues, improve eyesight and liver function, and promote growth and development. Their abundant dietary fiber promotes gastrointestinal motility, food digestion, and relieves constipation, making them highly valuable for consumption. Traditionally, strawberries are mainly propagated by runners and division. Both methods have low propagation coefficients, and long-term asexual reproduction can easily lead to the accumulation of viruses in the strawberry plant, causing the strawberry variety to degenerate and yields to decrease year by year. Tissue culture is an effective way to solve these problems. By using strawberry leaves and stems for tissue culture, a large number of tissue culture seedlings with excellent traits can be obtained, enabling the rapid propagation of strawberries.

[0003] Strawberry tissue culture seedlings typically require two steps before transplanting: rooting and hardening-off. The latter primarily hardens the leaves and roots of the seedlings after rooting, improving their photosynthetic autotrophic capacity, enhancing stomatal regulation, and promoting root hair development to improve their environmental adaptability. Currently, in practice, strawberry tissue culture seedlings need to undergo rooting culture before being transplanted to a substrate. However, in the initial transplanting stage, the seedlings are prone to water loss and wilting, requiring a second hardening-off process involving artificial temperature and humidity management to allow them to gradually adapt to the external environment and resume growth. This process is cumbersome and time-consuming. Therefore, there is an urgent need to find a propagation method that shortens the cultivation time of strawberry tissue culture seedlings and improves the quality of rooting, hardening-off, and transplant survival rates. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a method for strawberry tissue culture that solves the problem that strawberry tissue culture seedlings need to be hardened off after rooting, and that the hardening-off process is time-consuming and cumbersome.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] (1) Explant acquisition: Select healthy strawberry plants that are growing well, free from pests and diseases and deformities. Take their stolons or leaves and cut them into 1-1.2 cm lengths. After rinsing, disinfect them with 3wt% sodium hypochlorite solution for 8-10 min. Then rinse them twice with sterile water and dry them to obtain sterile explants.

[0007] (2) Primary culture: Sterile explants were inoculated on primary culture medium and cultured for 13-15 days under the conditions of temperature 22±1℃, light intensity 1500-2000lx and light duration 12h / d to obtain adventitious shoots;

[0008] (3) Subculture: Adventitious buds were inoculated onto subculture medium and cultured for 22-25 days under the conditions of temperature 22±1℃, light intensity 1500-2000lx and light duration 12h / d to obtain clustered buds;

[0009] (4) Joint culture of rooting and hardening: Cut the clustered buds into individual plants and inoculate them into the rooting and hardening medium for rooting culture. After 12-15 days of constant temperature culture in the room under light conditions, transfer the culture bottle to the outdoors and continue culture for 5-7 days to obtain tissue culture seedlings.

[0010] (5) Transplanting: Remove the tissue culture seedlings from the culture medium, clean the roots of any remaining culture medium, and then transplant them to the substrate for outdoor shading culture.

[0011] Furthermore, the rooting and hardening culture medium comprises: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L.

[0012] Furthermore, the preparation method of the rooting and hardening-off culture medium is as follows:

[0013] After mixing MS stock solution, IAA, IBA, agar, sucrose and water evenly, the pH was adjusted to 5.8-6. After autoclaving, the mixture was poured into a culture flask and cooled to solidify to obtain the lower gel layer.

[0014] After mixing crown ether, IAA, IBA, agar, sucrose and water evenly, adjust the pH to 5.8-6, cool to 55-60℃, autoclave, pour into the surface of the lower gel layer, and cool and solidify to obtain the middle gel layer.

[0015] After thoroughly mixing MS stock solution, IAA, IBA, agar, sucrose and water, adjust the pH to 5.8-6, autoclave, cool to 55-60℃, pour onto the surface of the middle gel layer to cool and solidify, and obtain the upper gel layer, which is the rooting and seedling hardening medium.

[0016] Furthermore, the crown ether is benzo-15-crown-5.

[0017] Furthermore, the thickness of the lower gel layer of the rooting and seedling hardening culture medium is 2-2.5cm, the thickness of the middle gel layer is 1-1.5cm, and the thickness of the upper gel layer is 2-3.5cm.

[0018] Furthermore, the specific operation of the rooting and hardening-off co-cultivation is as follows:

[0019] First, culture the culture in a closed bottle at a constant temperature of 25±1℃, light intensity of 2000-2500 lx, and light duration of 16 h / d for 10-12 days. Then, open the culture bottle and culture it for 2-3 days at a temperature of 25±1℃, humidity of 80%-85%, light intensity of 4000-5000 lx, and light duration of 14 h / d. After that, move the culture bottle outdoors and culture it in a natural environment with 50%-60% shade for 5-7 days, maintaining an air humidity of 70%-75%.

[0020] This invention achieves both direct rooting and hardening of strawberry tissue culture seedlings by using a rooting and hardening culture medium. This allows the strawberry tissue culture seedlings to improve their adaptability while rooting. After rooting and hardening, the seedlings can be quickly adapted to the environment and resume growth when transplanted into the substrate. They can be directly cultivated in the natural environment without artificial management, thus shortening the cultivation time of strawberry tissue culture seedlings.

[0021] After subculturing, the clump-forming shoots have already sprouted leaves. Therefore, this invention improves the adaptability of the clump-forming shoot leaves to changes in external temperature and humidity during the rooting and hardening process, promoting root formation while hardening off. Crown ethers are a class of macrocyclic polyether compounds. The ether bonds of crown ethers can interact with phospholipid molecules in the cell membrane, increasing the permeability of the cell membrane to potassium ions. By regulating the stomatal opening and closing of leaves through changes in potassium ion concentration, they regulate transpiration and gas exchange. However, crown ethers readily complex with alkali metals, and benzo-15-crown-5 crown ethers preferentially complex with potassium ions. Direct addition to the culture medium will reduce the potassium ion content, affecting the plant's absorption of potassium ions. Therefore, crown ethers need to act alone. However, if the clump-forming shoots are soaked in crown ether before rooting and hardening off, the crown ether will cause excessive absorption of potassium ions in the early stages of rooting culture, interfering with the plant's absorption of other nutrients and thus affecting rooting. Therefore, this invention sets the rooting and hardening-off culture medium as a three-layer structure. The top and bottom layers are gel layers containing conventional rooting culture components, and the middle layer is a salt-free gel layer containing crown ether. The bud clusters are first cultured in the upper gel layer in a closed bottle for rooting. The humidity in the culture bottle is high, the temperature is stable, and the stomatal opening and closing of the leaves is high, but water loss is not observed. When the roots of the bud clusters grow to the middle gel layer, the culture bottle is opened, and cultured under ventilation conditions. Leaf water evaporation accelerates. At this time, the roots come into contact with the crown ether, which enters the bud clusters and alters the permeability of the cell membrane to potassium ions. Since the middle gel layer does not contain potassium ions, the concentration difference causes potassium ions to leak out of the bud clusters. The potassium ion content in the guard cells of the leaves decreases, changing the osmotic pressure and causing the guard cells to lose water and shrink. This reduces the stomatal opening and closing of the leaves, thereby reducing... Transpiration prevents the bud clusters from wilting due to water loss in a well-ventilated environment, while also training the leaves' ability to control stomatal opening and closing. When the roots of the bud clusters continue to grow to the lower gel layer, the opened culture bottles are transferred to outdoor cultivation under 50-60% shade. At this time, the bud clusters have undergone crown ether treatment, which accelerates the absorption of potassium ions, increases the potassium ion content in the leaf guard cells, causes the cells to absorb water and swell, increases the degree of stomatal opening and closing, increases transpiration, lowers the leaf temperature to adapt to the environmental temperature, and accelerates gas exchange to promote photosynthesis, thereby improving the photosynthetic autotrophic capacity of the tissue culture seedlings and thus improving their adaptability.

[0022] Furthermore, this invention adds different amounts of agar to the multi-layered structure of the rooting and hardening culture medium, creating a gradient of hardness from top to bottom. This hinders ion movement between the culture medium layers and reduces the mutual consumption of crown ether and potassium ions at the contact surfaces. Simultaneously, the shoots root in the softer upper gel layer. The new roots are tender and grow more easily in the softer medium. When the root tips come into contact with the harder gel layer, root elongation slows down, promoting uniform root growth. At the same time, moderate hardness stimulation enhances root absorption and penetration, making them thicker and reducing the probability of root damage after transplanting, thus improving transplant survival rate.

[0023] Furthermore, the primary culture medium consists of MS + NAA 0.8-1.2 mg / L + 2-4D 0.3-0.5 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L; the secondary culture medium consists of MS + NAA 1-1.5 mg / L + 6-BA 1.5-2.0 mg / L + agar 7.5 g / L + sucrose 30 g / L.

[0024] Furthermore, the culture conditions during the primary and secondary cultures were a temperature of 22±1℃, a light intensity of 1500-2000 lx, and a light duration of 12 h / d.

[0025] Furthermore, the transplanting substrate is a mixture of peat moss, vermiculite, and perlite in a volume ratio of 3:1:1; the shading degree for outdoor cultivation after transplanting is 50%-60%.

[0026] Beneficial effects:

[0027] This invention discloses a method for strawberry tissue culture, which achieves rooting and hardening of seedlings through the use of a rooting and hardening-off medium. Crown ether and different concentrations of agar are added to the medium. During the culture process, the stomatal opening and closing of the leaves are controlled to improve the leaf's adaptability to changes in external temperature and humidity, thus hardening the leaves. The different concentrations of agar alter the hardening of the medium, promoting root development and improving root absorption capacity, thereby strengthening the roots and enhancing the adaptability of the tissue-cultured seedlings. After undergoing the rooting and hardening-off treatment of this invention, the tissue-cultured seedlings exhibit strong adaptability and a high survival rate after transplanting to the substrate. They can be directly placed outdoors for natural growth without artificial management, shortening the strawberry tissue culture seedling cultivation cycle. Attached Figure Description

[0028] Figure 1 : A diagram of the strawberry tissue culture process. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0030] Example 1:

[0031] Preparation of culture medium:

[0032] Primary culture medium:

[0033] The primary culture medium consists of MS + NAA 1.2 mg / L + 2-4D 0.3 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: Mix MS stock solution, NAA, 2-4D, activated carbon, agar, sucrose and 1 L of water evenly, adjust the pH to 6, autoclave, and cool to solidify to obtain the primary culture medium.

[0034] Subculture medium:

[0035] The subculture medium consists of MS + NAA 1.5 mg / L + 6-BA 2.0 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: Mix MS stock solution, NAA, 6-BA, agar, sucrose and 1 L of water evenly, adjust the pH to 6, autoclave, and cool to solidify to obtain the subculture medium.

[0036] Rooting and hardening-off culture medium:

[0037] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.05 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L.

[0038] The specific preparation method of the rooting and hardening-off culture medium is as follows:

[0039] After thoroughly mixing MS stock solution, IAA, IBA, agar, sucrose, and water, the pH was adjusted to 6. The mixture was then autoclaved, poured into culture flasks, and allowed to cool and solidify to obtain the lower gel layer, which was 2.5 cm thick. Next, crown ether, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 6. This mixture was then autoclaved, cooled to 55°C, poured onto the surface of the lower gel layer, and allowed to cool and solidify to obtain the middle gel layer, which was 1.5 cm thick. Finally, MS stock solution, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 6. This mixture was then autoclaved, cooled to 55°C, poured onto the surface of the middle gel layer, and allowed to cool and solidify to obtain the upper gel layer, which was 3.5 cm thick. This final rooting and hardening culture medium was obtained.

[0040] Tissue culture methods for strawberries:

[0041] (1) Explant acquisition: Select healthy strawberry plants that are growing well, free from pests and diseases and deformities. Cut their stolons into 1-1.2 cm lengths, rinse with clean water, disinfect with 3wt% sodium hypochlorite solution for 8 min, rinse twice with sterile water, and then dry to obtain sterile explants.

[0042] (2) Primary culture: Sterile explants were inoculated on primary culture medium and cultured under the following conditions: temperature 22±1℃, light intensity 2000lx, light duration 12h / d. Adventitious shoots were obtained after 13 days of culture.

[0043] (3) Subculture: Adventitious buds were inoculated onto subculture medium and cultured under the following conditions: temperature 22±1℃, light intensity 2000lx, light duration 12h / d. After 25 days of culture, clustered buds were obtained.

[0044] (4) Co-culture of rooting and hardening: The clustered buds were cut into single plants and inoculated into the rooting and hardening medium. They were first cultured for 10 days at a temperature of 25±1℃, a light intensity of 2000lx, and a light duration of 16h / d. Then the culture bottle was opened and cultured for 3 days at a temperature of 25±1℃, a humidity of 80%-85%, a light intensity of 4000lx, and a light duration of 14h / d. After that, the culture bottle was moved outdoors and cultured for 7 days in a natural environment with a shade degree of 50% and an air humidity of 70%-75% to obtain tissue culture seedlings.

[0045] (5) Transplanting: Remove the tissue culture seedlings from the culture medium, clean the roots of any remaining culture medium, and then transplant them into a substrate with a volume ratio of peat moss, vermiculite, and perlite of 3:1:1. Place them outdoors in shade with a shade level of 50%.

[0046] Example 2:

[0047] Preparation of culture medium:

[0048] Primary culture medium:

[0049] The primary culture medium consisted of MS + NAA 0.8 mg / L + 2-4D 0.5 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: the MS stock solution, NAA, 2-4D, activated carbon, agar, sucrose and 1 L of water were mixed evenly and the pH was adjusted to 5.8. The mixture was then autoclaved and cooled to solidify to obtain the primary culture medium.

[0050] Subculture medium:

[0051] The subculture medium consists of MS + NAA 1 mg / L + 6-BA 1.5 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: Mix MS stock solution, NAA, 6-BA, agar, sucrose and 1 L of water evenly, adjust the pH to 5.8, autoclave, and cool to solidify to obtain the subculture medium.

[0052] Rooting and hardening-off culture medium:

[0053] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L.

[0054] The specific preparation method of the rooting and hardening-off culture medium is as follows:

[0055] After thoroughly mixing MS stock solution, IAA, IBA, agar, sucrose, and water, the pH was adjusted to 5.8. The mixture was then autoclaved, poured into culture flasks, and cooled to solidify, resulting in a lower gel layer with a thickness of 2 cm. Next, crown ether, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 5.8. The mixture was then autoclaved, cooled to 55°C, poured onto the surface of the lower gel layer, and cooled to solidify, resulting in an intermediate gel layer with a thickness of 1 cm. Finally, MS stock solution, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 5.8. The mixture was then autoclaved, cooled to 55°C, poured onto the surface of the intermediate gel layer, and cooled to solidify, resulting in an upper gel layer with a thickness of 2 cm. This final rooting and hardening culture medium was obtained.

[0056] Tissue culture methods for strawberries:

[0057] (1) Explant acquisition: Select healthy strawberry plants that are growing well, free from pests and diseases and deformities. Cut their leaves into 1-1.2 cm lengths, rinse with clean water, disinfect with 3wt% sodium hypochlorite solution for 10 min, rinse twice with sterile water, and then dry to obtain sterile explants.

[0058] (2) Primary culture: Sterile explants were inoculated on primary culture medium and cultured under the following conditions: temperature 22±1℃, light intensity 1500lx, light duration 12h / d. Adventitious shoots were obtained after 15 days of culture.

[0059] (3) Subculture: Adventitious buds were inoculated onto subculture medium and cultured under the following conditions: temperature 22±1℃, light intensity 1500 lx, light duration 12h / d. After 22 days of culture, clustered buds were obtained.

[0060] (4) Co-culture of rooting and hardening: The clustered buds were cut into single plants and inoculated into the rooting and hardening medium. They were first cultured for 12 days at a temperature of 25±1℃, a light intensity of 2500lx, and a light duration of 16h / d. Then the culture bottle was opened and cultured for 2 days at a temperature of 25±1℃, a humidity of 80%-85%, a light intensity of 5000lx, and a light duration of 14h / d. After that, the culture bottle was moved outdoors and cultured for 5 days in a natural environment with a shade degree of 50% and an air humidity of 70%-75% to obtain tissue culture seedlings.

[0061] (5) Transplanting: Remove the tissue culture seedlings from the culture medium, clean the roots of any remaining culture medium, and then transplant them into a substrate with a volume ratio of peat moss, vermiculite, and perlite of 3:1:1. Place them outdoors in shade with a shade level of 60%.

[0062] Example 3:

[0063] Preparation of culture medium:

[0064] Primary culture medium:

[0065] The primary culture medium consisted of MS + NAA 0.8 mg / L + 2-4D 0.5 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: the MS stock solution, NAA, 2-4D, activated carbon, agar, sucrose and 1 L of water were mixed evenly and the pH was adjusted to 5.8. The mixture was then autoclaved and cooled to solidify to obtain the primary culture medium.

[0066] Subculture medium:

[0067] The subculture medium consists of MS + NAA 1 mg / L + 6-BA 2 mg / L + agar 7.5 g / L + sucrose 30 g / L. The specific preparation method is as follows: Mix MS stock solution, NAA, 6-BA, agar, sucrose and 1 L of water evenly, adjust the pH to 5.8, autoclave, and cool to solidify to obtain the subculture medium.

[0068] Rooting and hardening-off culture medium:

[0069] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L.

[0070] The specific preparation method of the rooting and hardening-off culture medium is as follows:

[0071] After thoroughly mixing MS stock solution, IAA, IBA, agar, sucrose, and water, the pH was adjusted to 6. The mixture was then autoclaved, poured into culture flasks, and cooled to solidify, resulting in a lower gel layer with a thickness of 2.5 cm. Next, crown ether, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 6. The mixture was then autoclaved, cooled to 60°C, poured onto the surface of the lower gel layer, and cooled to solidify, resulting in an intermediate gel layer with a thickness of 1 cm. Finally, MS stock solution, IAA, IBA, agar, sucrose, and water were thoroughly mixed, and the pH was adjusted to 6. The mixture was then autoclaved, cooled to 60°C, poured onto the surface of the intermediate gel layer, and cooled to solidify, resulting in an upper gel layer with a thickness of 3 cm. This final rooting and hardening culture medium was obtained.

[0072] Tissue culture methods for strawberries:

[0073] (1) Explant acquisition: Select healthy strawberry plants that are growing well, free from pests and diseases and deformities. Cut their stolons into 1-1.2 cm lengths, rinse with clean water, disinfect with 3wt% sodium hypochlorite solution for 10 min, rinse twice with sterile water, and then dry to obtain sterile explants.

[0074] (2) Primary culture: Sterile explants were inoculated on primary culture medium and cultured under the following conditions: temperature 22±1℃, light intensity 1500lx, light duration 12h / d. Adventitious shoots were obtained after 15 days of culture.

[0075] (3) Subculture: Adventitious buds were inoculated onto subculture medium and cultured under the following conditions: temperature 22±1℃, light intensity 2000lx, light duration 12h / d. After 23 days of culture, clustered buds were obtained.

[0076] (4) Co-culture of rooting and hardening: The clustered buds were cut into single plants and inoculated into the rooting and hardening medium. They were first cultured for 12 days at a temperature of 25±1℃, a light intensity of 2000lx, and a light duration of 16h / d. Then the culture bottle was opened and cultured for 3 days at a temperature of 25±1℃, a humidity of 80%-85%, a light intensity of 4000lx, and a light duration of 14h / d. After that, the culture bottle was moved outdoors and cultured for 6 days in a natural environment with a shade degree of 60% and an air humidity of 70%-75% to obtain tissue culture seedlings.

[0077] (5) Transplanting: Remove the tissue culture seedlings from the culture medium, clean the roots of any remaining culture medium, and then transplant them into a substrate with a volume ratio of peat moss, vermiculite, and perlite of 3:1:1. Place them outdoors in shade with a shade level of 60%.

[0078] Comparative Example 1:

[0079] Compared with Example 1, the only difference is the formulation of the rooting and hardening-off medium. Specifically, the middle gel layer of the rooting and hardening-off medium is a conventional rooting culture component without crown ether. The specific formulation of the rooting and hardening-off medium is as follows:

[0080] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L. The preparation method of the medium is the same as in Example 1.

[0081] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0082] Comparative Example 2:

[0083] Compared with Example 1, the only difference lies in the formulation and preparation method of the rooting and hardening-off culture medium. Specifically, crown ether is directly added to the conventional rooting culture components to obtain the rooting and hardening-off culture medium. The specific formulation and preparation method of the rooting and hardening-off culture medium are as follows:

[0084] The rooting and hardening culture medium consisted of MS + crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7.5 g / L + sucrose 30 g / L;

[0085] Preparation of rooting and hardening medium: MS stock solution, crown ether, IAA, IBA, agar, sucrose and water were mixed evenly and the pH was adjusted to 6. The mixture was then autoclaved, poured into culture flasks, and cooled to solidify to obtain the rooting and hardening medium.

[0086] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0087] Comparative Example 3:

[0088] Compared with Example 1, the only difference is the formulation of the rooting and hardening-off medium. The only difference is that the agar content in each layer of the rooting and hardening-off medium is 6 g / L. The specific formulation of the rooting and hardening-off medium is as follows:

[0089] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L. The preparation method of the medium is the same as in Example 1.

[0090] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0091] Comparative Example 4:

[0092] Compared with Example 1, the only difference is the formulation of the rooting and hardening-off medium. The only difference is that the agar content in each layer of the rooting and hardening-off medium is 7 g / L. The specific formulation of the rooting and hardening-off medium is as follows:

[0093] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L. The preparation method of the medium is the same as in Example 1.

[0094] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0095] Comparative Example 5:

[0096] Compared with Example 1, the only difference is the formulation of the rooting and hardening-off medium. The only difference is that the agar content in each layer of the rooting and hardening-off medium is 8 g / L. The specific formulation of the rooting and hardening-off medium is as follows:

[0097] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L; an intermediate gel layer composed of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L. The preparation method of the medium is the same as in Example 1.

[0098] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0099] Comparative Example 6:

[0100] Compared with Example 1, the only difference is the thickness of the middle gel layer in the rooting and hardening-off medium. Specifically, the thickness of the upper and middle gel layers in the rooting and hardening-off medium is 3.5 cm. The specific preparation method of the rooting and hardening-off medium is as follows:

[0101] The culture medium formulation is the same as in Example 1;

[0102] After thoroughly mixing MS stock solution, IAA, IBA, agar, sucrose and water, the pH was adjusted to 6, autoclaved, poured into culture flasks, and cooled to solidify to obtain the lower gel layer, which was 2.5 cm thick.

[0103] After mixing crown ether, IAA, IBA, agar, sucrose and water evenly, the pH was adjusted to 6, autoclaved, cooled to 55°C, poured onto the surface of the lower gel layer, and cooled and solidified to obtain the middle gel layer with a thickness of 3.5 cm.

[0104] After mixing MS stock solution, IAA, IBA, agar, sucrose and water evenly, the pH was adjusted to 6, autoclaved, cooled to 55°C, poured onto the surface of the middle gel layer, and cooled and solidified to obtain the lower gel layer with a thickness of 3.5 cm. Finally, the rooting and seedling hardening medium was obtained.

[0105] The remaining procedures for explant acquisition, primary culture, subculture, rooting and hardening-off co-culture, and transplanting are the same as in Example 1.

[0106] Comparative Example 7:

[0107] Compared with Example 1, the only difference is the culture conditions for the joint rooting and hardening-off culture. Specifically, the entire rooting and hardening-off culture is conducted indoors. The specific joint rooting and hardening-off culture conditions are as follows:

[0108] The tissue culture seedlings were first cultured at a temperature of 25±1℃, a light intensity of 2000 lx, and a light duration of 16 h / d for 10 days. Then, the culture bottles were opened and cultured at a temperature of 25±1℃, a humidity of 80%-85%, a light intensity of 4000 lx, and a light duration of 14 h / d for another 10 days to obtain tissue culture seedlings. The rooting and hardening culture medium used was the same as that used in Example 1.

[0109] The remaining procedures for explant acquisition, primary culture, subculture, and transplantation are the same as in Example 1.

[0110] Comparative Example 8:

[0111] Compared with Example 1, the only difference is the culture conditions for the joint rooting and hardening-off culture. Specifically, after indoor culture, the culture was directly opened and cultured outdoors. The specific rooting and hardening-off culture conditions are as follows:

[0112] The tissue culture seedlings were first cultured at a temperature of 25±1℃, a light intensity of 2000lx, and a light duration of 16h / d for 10 days. Then, the culture bottles were opened and transferred outdoors to be cultured in a natural environment with 50% shade and 70%-75% humidity for 10 days to obtain tissue culture seedlings. The rooting and hardening culture medium used was the same as that in Example 1.

[0113] The other procedures for explant acquisition, primary culture, subculture, and transplantation are the same as in Example 1.

[0114] Comparative Example 9:

[0115] Compared with Example 1, the only difference is the timing of crown ether application. Specifically, the base of the clustered buds is first soaked in crown ether solution before inoculation into a rooting and hardening-off medium without crown ether. The specific operation is as follows:

[0116] Co-culture of rooting and hardening: The clustered buds were cut into individual plants, and the base of the clustered buds was soaked in 0.05wt% crown ether solution for 4 hours before being inoculated into the rooting and hardening medium. The plants were first cultured for 10 days at a temperature of 25±1℃, a light intensity of 2000 lx, and a light duration of 16 h / d. Then the culture bottles were opened and cultured for 3 days at a temperature of 25±1℃, a humidity of 80%-85%, a light intensity of 4000 lx, and a light duration of 14 h / d. The culture bottles were then moved outdoors and cultured for 7 days in a natural environment with 50% shade and 70%-75% air humidity to obtain tissue culture seedlings.

[0117] The rooting and hardening-off medium consists of three gel layers: an upper gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer composed of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L. The preparation method of the medium is the same as in Example 1.

[0118] The remaining procedures for explant acquisition, primary culture, subculture, and transplantation are the same as in Example 1.

[0119] Comparative Example 10:

[0120] Compared with Example 1, the only difference is that the rooting and hardening-off processes are handled separately. The clustered buds are first rooted and cultured before being hardened off again. The specific culture operation is as follows:

[0121] Preparation of rooting medium:

[0122] The rooting medium consists of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7.5 g / L + sucrose 30 g / L. After mixing MS stock solution, IAA, IBA, agar and sucrose evenly, the pH is adjusted to 6 and then autoclaved to obtain the rooting medium.

[0123] Rooting culture: The clustered buds were cut into individual plants and inoculated into the rooting medium for rooting culture. After 20 days of culture under the conditions of temperature 25±1℃, light intensity 2000lx and light duration 16h / d, rooted seedlings were obtained.

[0124] Hardening off and transplanting: Open the culture bottles of rooted seedlings and harden them indoors for 3 days under the conditions of temperature 25±1℃, humidity 80%-85%, light intensity 4000lx, and light duration 16h / d. After that, take the tissue culture seedlings out of the culture medium, clean the roots of any remaining culture medium, and transplant them into a substrate of peat moss, vermiculite, and perlite in a volume ratio of 3:1:1. Harden them off outdoors in a natural environment with 50% shade and 70%-75% humidity for 7 days. After that, no more humidity management is required.

[0125] The remaining procedures for explant acquisition, primary culture, and subculture are the same as in Example 1.

[0126] Comparative Example 11:

[0127] Compared with Example 1, the only difference is that rooting and hardening are treated separately. The clustered buds are first rooted and cultured, and then hardened off twice. The specific culture operation is as follows:

[0128] Preparation of rooting medium:

[0129] The rooting medium consists of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 7.5 g / L + sucrose 30 g / L. After mixing MS stock solution, IAA, IBA, agar and sucrose evenly, the pH is adjusted to 6 and then autoclaved to obtain the rooting medium.

[0130] Rooting culture: The clustered buds were cut into individual plants and inoculated into the rooting medium for rooting culture. After 20 days of culture at a temperature of 25±1℃, a light intensity of 2000lx, and a light duration of 16h / d, rooted seedlings were obtained.

[0131] Hardening off and transplanting: Open the culture bottles of rooted seedlings and harden them off for 3 days under the conditions of temperature 25±1℃, humidity 80%-85%, light intensity 4000lx, and light duration 16h / d. After that, remove them from the culture medium, clean the roots of any remaining culture medium, and transplant them into a substrate of peat moss, vermiculite, and perlite in a volume ratio of 3:1:1. Cover the tissue culture seedlings with a transparent plastic cover and culture them indoors for 3 days. Then remove the plastic cover and transfer them outdoors for 5 days of hardening off in a natural environment with 50% shade and 70%-75% humidity. After that, no more humidity management is required.

[0132] experiment:

[0133] (1) Explant acquisition: Using “Hongyan” strawberry as the experimental variety, healthy strawberry plants that were cultivated from the same batch of seeds, grew well, were free from pests and diseases, and were free from deformities were selected for the experiment. The stolons of the strawberry plants were cut into 1.2cm lengths, rinsed with clean water, disinfected with 3wt% sodium hypochlorite solution for 8 minutes, rinsed twice with sterile water, and then dried to obtain sterile explants.

[0134] (2) Primary culture: Sterile explants were inoculated on a primary culture medium prepared with MS + NAA 0.8 mg / L + 2-4D 0.5 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L for primary culture. After 13 days of culture at a temperature of 22±1℃, a light intensity of 2000 lx and a light duration of 12 h / d, clustered shoots were obtained.

[0135] (3) Subculture: Adventitious shoots were inoculated onto subculture medium and cultured on a subculture medium prepared with MS + NAA 1 mg / L + 6-BA 1.5 mg / L + agar 7.5 g / L + sucrose 30 g / L. The shoots were cultured for 25 days at a temperature of 22±1℃, a light intensity of 2000 lx and a light duration of 12 h / d to obtain clustered shoots.

[0136] (4) Rooting culture: Cut the clustered buds into individual plants, and randomly select 100 plants from each group to be inoculated into the rooting and hardening medium for rooting culture to obtain rooted tissue culture seedlings. Repeat three times, with one clustered bud inoculated into one culture bottle.

[0137] (5) Transplanting: Select 50 tissue culture seedlings from each group, clean the culture medium at the base, and transplant them into a substrate with a volume ratio of peat moss, vermiculite and perlite of 3:1:1 and place them outdoors for natural growth.

[0138] The following experiments were conducted following the tissue culture process described above:

[0139] 1. Experimental group 1 used the rooting and hardening culture medium of Example 1 and the co-culture method of rooting and hardening culture of Example 1;

[0140] 2. Comparative groups 1-6 were cultured using the rooting and hardening-off culture media prepared in comparative examples 1-6 and the rooting and hardening-off co-culture method of Example 1, respectively;

[0141] 3. Comparative groups 7-8 used the rooting and hardening culture medium prepared in Example 1 and the rooting and hardening culture method of comparative groups 7-8.

[0142] 4. The control group 9-11 used the rooting culture medium prepared by the control group 9-11 and the rooting and hardening culture method of the control group 9-11.

[0143] On the 20th day of rooting culture, the survival rate of each group was counted. At the same time, 10 tissue culture seedlings were randomly selected from each group to count the average root-to-shoot ratio and average leaf width.

[0144] After transplanting tissue culture seedlings to the substrate and moving them outdoors, the propagation cycle was recorded as long as new shoots sprouted outdoors; the survival rate was recorded on day 30 after transplanting. The results are shown in Table 1.

[0145] Survival rate (%) = (Number of survivors / Number of doses administered) × 100%

[0146] Root-to-shoot ratio = Fresh weight of roots / Fresh weight of above-ground parts;

[0147] Transplant survival rate (%) = (Number of surviving transplanted plants / Number of transplanted plants) × 100%.

[0148] Table 1

[0149]

[0150] Analysis of the data in Table 1 shows that:

[0151] 1. Compared with experimental group 1, control group 1 did not add crown ether to its rooting and hardening medium. The resulting tissue culture seedlings had a high root-to-shoot ratio, narrow leaves, low rooting survival rate, and low transplanting survival rate. This is because in control group 1, leaf water evaporation was too rapid during the open culture process, causing the leaves of the clustered buds to wilt due to water loss, leading to a low survival rate during the rooting stage, poor quality of the tissue culture seedlings, and a low transplanting survival rate. Experimental group 1 added crown ether to the middle gel layer of its rooting and hardening medium, and the crown ether modified the culture medium during the open culture process. The altered permeability of cell membranes to potassium ions causes potassium ion leakage from clustered shoots, changes the osmotic pressure of leaf guard cells, leads to cell dehydration, reduces stomatal opening and closing, and decreases leaf water evaporation, thereby improving the survival rate during the rooting stage, the quality of tissue culture seedlings, and the transplant survival rate. In contrast, control group 2, where crown ether was directly added to conventional rooting culture components to prepare a one-layer rooting and seedling hardening medium, resulted in tissue culture seedlings with a high root-to-shoot ratio, narrow leaves, low survival rate during the rooting stage, and low transplant survival rate. This is because the crown ether was directly added to the conventional... After rooting and hardening culture medium is used, it forms a stable complex with potassium ions in MS stock solution, reducing the potassium ion concentration in the culture medium. This inhibits the rooting and growth of clustered shoots. At the same time, the crown ether cannot play its role in regulating stomatal opening and closing of leaves. When cultured in open bottles, leaves wilt due to water loss, resulting in a lower survival rate, poor quality of tissue culture seedlings, and low transplant survival rate. Experiment 1 prepared the rooting and hardening culture medium into a multi-layered structure. The middle layer was a salt-free culture medium containing crown ether. The three gel layers had different hardness, which hindered the movement and complexation of crown ether and potassium ions, ensuring sufficient free potassium ions in the culture medium, promoting the rooting and growth of clustered shoots. At the same time, the crown ether can change the permeability of cell membranes to potassium ions. When cultured in open bottles indoors, it regulates the stomatal opening and closing of leaves to reduce transpiration. When cultured outdoors, it promotes potassium ion absorption, increases stomatal opening and closing, and transpiration helps leaves adapt to environmental temperature. Accelerated gas exchange promotes photosynthesis and wax layer synthesis, improving the adaptability of tissue culture seedlings to environmental changes, thereby improving the rooting survival rate and transplant survival rate.

[0152] 2. Compared with experimental group 1, the rooting and hardening-off media in control groups 3-5 showed no change in hardness. The rooting and hardening-off media in control group 3 was a soft, semi-solid state, resulting in low survival rates during the rooting stage, high root-to-shoot ratios, narrow leaves, and low transplant survival rates. The rooting and hardening-off media in control group 4 was a relatively soft, solidified state, resulting in high survival rates during the rooting stage, moderate root-to-shoot ratios, and wider leaves, but still low transplant survival rates. This is because the roots in control groups 3 and 4 were cultured in a relatively soft medium, leading to insufficient root penetration and absorption capacity, making it difficult for them to quickly adapt to the soil environment. This resulted in insufficient nutrient supply after transplanting, reducing transplant survival rates. Simultaneously, the semi-solid medium in control group 3 accelerated the movement of crown ether and potassium ions between layers, leading to greater mutual consumption, reduced free potassium ion content, and inability to regulate stomatal opening and closing, resulting in narrow leaves, weak roots, and low survival rates in control group 3. The rooting and hardening-off media in control group 5 was relatively soft, solidified. In a hard, solidified medium, the survival rate during the rooting stage is low, the root-to-shoot ratio is low, and the transplant survival rate is low. This is because the high-hardness medium inhibits root elongation and growth, resulting in slow root growth of the bud clusters. During culture and after transplanting, the short roots cannot supply enough nutrients to the plant, leading to a decrease in rooting and transplant survival rates. In experimental group 1, different amounts of agar were added to the multi-layered gel layer of the rooting and hardening medium, gradually increasing the hardness from top to bottom. This can hinder the complexation of crown ether and potassium ions, allowing the crown ether to function and training the leaves' ability to regulate stomatal opening and closing. At the same time, the bud clusters root and then elongate in the softer medium. When the roots come into contact with the harder medium, the elongation slows down, but the rhizomes thicken, enhancing the root system's absorption and penetration capabilities. After transplanting, the roots can quickly penetrate the soil to absorb sufficient nutrients to supply the plant's growth, thus improving the transplant survival rate.

[0153] 3. Compared with experimental group 1, the thickness of the middle gel layer in the rooting and hardening medium of control group 6 was increased, resulting in a lower root survival rate, a lower root-to-shoot ratio, and a lower transplant survival rate. This is because the middle gel layer does not contain salts and lacks essential elements for plant growth. The increased thickness of the middle gel layer means that the roots have to grow in a salt-free environment for a longer period of time. The lack of metal elements over a long period of time hinders the growth of clustered shoots, reduces the root survival rate, results in poor quality tissue culture seedlings, and reduces the transplant survival rate.

[0154] 4. Compared with experimental group 1, the rooting and hardening-off culture conditions of control groups 7 and 8 were different. Control group 7 was cultured indoors throughout the rooting and hardening-off stage, resulting in a high survival rate during the rooting stage but a low transplant survival rate. This is because the tissue culture seedlings were always cultured in a stable indoor environment, and after transplanting, the leaves had difficulty adapting quickly to changes in natural light and temperature and humidity, resulting in weak photosynthetic autotrophic capacity and a low transplant survival rate. Control group 8 was placed outdoors directly after the initial rooting and hardening-off culture, resulting in a low survival rate, narrow leaves, and a low transplant survival rate. This is because after opening the bottle, the direct outdoor culture caused multiple stimuli from changes in natural light, temperature, and humidity, leading to leaf dehydration and wilting, reducing the survival rate, resulting in poor quality tissue culture seedlings and a low transplant survival rate. Experimental group 1 was cultured in a stable indoor environment during the early rooting stage, and after opening the bottle, it was first transitioned indoors for 3 days before being transferred outdoors, allowing the tissue culture seedlings to gradually adapt to environmental changes, thus improving the survival rate and transplant survival rate.

[0155] 5. Compared with experimental group 1, control group 9, where the clustered shoots were first soaked in crown ether solution before being inoculated into the rooting and hardening medium, showed lower survival rates, narrower leaves, and lower transplant survival rates. This is because crown ether alters the permeability of cell membranes to potassium ions. After contact with the medium, the clustered shoots absorb large amounts of potassium ions for an extended period, affecting the absorption of other nutrients and causing osmotic imbalance, resulting in low survival rates, weak tissue culture seedlings, and low transplant survival rates.

[0156] 6. Compared with experimental group 1, control groups 10-11 separated the rooting and hardening-off processes. Control group 10 underwent conventional rooting culture followed by hardening-off in the bottle, and then transplanted to the substrate for outdoor culture. The survival rate during the rooting stage was high, the transplant survival rate was high, and the propagation cycle was long. This is because tissue culture seedlings undergoing rooting and hardening-off indoors have weak stomatal regulation ability, weak photosynthetic autotrophic ability, and insufficient root absorption and penetration. After transplanting, they cannot quickly adapt to the environment, resulting in a low transplant survival rate and requiring a longer time to recover growth, thus leading to a long propagation cycle. Control group 11 underwent conventional rooting followed by hardening-off in the bottle, then transplanted to the substrate for a second hardening-off indoors before being transplanted outdoors. The survival rate during the rooting stage was high, and the transplant survival rate was high, but the time required for artificial management was long, resulting in a longer propagation cycle.

[0157] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for tissue culture of strawberries, characterized in that, The method is as follows: (1) Explant acquisition: Select healthy strawberry plants that are growing well, free from pests and diseases and deformities. Take their stolons or leaves and cut them into 1-1.2 cm lengths. After rinsing, disinfect them with 3wt% sodium hypochlorite solution for 8-10 min. Then rinse them twice with sterile water and dry them to obtain sterile explants. (2) Primary culture: Sterile explants were inoculated on primary culture medium and cultured for 13-15 days to obtain adventitious shoots; (3) Subculture: Adventitious buds are inoculated onto subculture medium and cultured for 22-25 days to obtain clustered buds; (4) Joint culture of rooting and hardening: Cut the clustered buds into individual plants and inoculate them into the rooting and hardening medium for rooting culture. After 12-15 days of constant temperature culture in the room under light conditions, transfer the culture bottle to the outdoors and continue culture for 5-7 days to obtain tissue culture seedlings. (5) Transplanting: Remove the tissue culture seedlings from the culture medium, clean the roots of any remaining culture medium, and then transplant them to the substrate for outdoor shading culture.

2. The method for tissue culture of strawberry according to claim 1, characterized in that, The rooting and hardening culture medium comprises: an upper gel layer consisting of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L; an intermediate gel layer consisting of crown ether 0.03 g / L + IAA 1 mg / L + IBA 0.5 mg / L + agar 7 g / L + sucrose 30 g / L; and a lower gel layer consisting of MS + IAA 1 mg / L + IBA 0.5 mg / L + agar 8 g / L + sucrose 30 g / L.

3. The method for tissue culture of strawberry according to claim 2, characterized in that, The method for preparing the rooting and seedling hardening culture medium is as follows: After mixing MS stock solution, IAA, IBA, agar, sucrose and water evenly, the pH was adjusted to 5.8-6. After autoclaving, the mixture was poured into a culture flask and cooled to solidify to obtain the lower gel layer. After mixing crown ether, IAA, IBA, agar, sucrose and water evenly, adjust the pH to 5.8-6, cool to 55-60℃, autoclave, pour into the surface of the lower gel layer, and cool and solidify to obtain the middle gel layer. After mixing MS stock solution, IAA, IBA, agar, sucrose and water evenly, the pH was adjusted to 5.8-6. The mixture was then autoclaved, cooled to 55-60℃, and poured onto the surface of the middle gel layer to cool and solidify, thus obtaining the upper gel layer. Finally, the rooting and seedling hardening medium was obtained.

4. The method for tissue culture of strawberry according to claim 3, characterized in that, The crown ether is benzo-15-crown-5.

5. The method for tissue culture of strawberry according to claim 4, characterized in that, The thickness of the lower gel layer of the rooting and seedling hardening culture medium is 2-2.5cm, the thickness of the middle gel layer is 1-1.5cm, and the thickness of the upper gel layer is 2-3.5cm.

6. The method for tissue culture of strawberry according to claim 5, characterized in that, The specific procedures for the combined rooting and seedling hardening-off cultivation are as follows: First, culture the culture in a closed bottle at a constant temperature of 25±1℃, light intensity of 2000-2500 lx, and light duration of 16 h / d for 10-12 days. Then, open the culture bottle and culture it for 2-3 days at a temperature of 25±1℃, humidity of 80%-85%, light intensity of 4000-5000 lx, and light duration of 14 h / d. After that, move the culture bottle outdoors and culture it in a natural environment with 50%-60% shade for 5-7 days, maintaining an air humidity of 70%-75%.

7. The method for tissue culture of strawberry according to claim 6, characterized in that, The primary culture medium consisted of MS + NAA 0.8-1.2 mg / L + 2-4D 0.3-0.5 mg / L + activated carbon 1 mg / L + agar 7.5 g / L + sucrose 30 g / L.

8. The method for tissue culture of strawberry according to claim 7, characterized in that, The subculture medium consisted of MS + NAA 1-1.5 mg / L + 6-BA 1.5-2.0 mg / L + agar 7.5 g / L + sucrose 30 g / L.

9. A method for tissue culture of strawberry according to claim 8, characterized in that, The culture conditions for both the primary and secondary cultures were a temperature of 22±1℃, a light intensity of 1500-2000 lx, and a light duration of 12 h / d.

10. A method for tissue culture of strawberry according to claim 9, characterized in that, The transplanting substrate is a mixture of peat moss, vermiculite, and perlite in a volume ratio of 3:1:1.

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