A tissue culture method for wild leeks

Abstract

This invention discloses a tissue culture method for Allium tuberosum, belonging to the field of tissue culture technology for Allium tuberosum. The method is as follows: (1) Aseptic treatment of explants: Select root tips of Allium tuberosum plants, disinfect them to obtain root tip explants; (2) Induction culture: Inoculate the root tip explants into the culture medium, culture them in the dark at 20-24℃ for 2-3 days, and then transfer them to the conditions of 20-24℃, light intensity 1500-2000 lx, and light time 10-12 h / day for 5-7 days to obtain root tip embryonic roots; (3) Proliferation culture: Inoculate the induced root tip embryonic roots into the proliferation culture medium, culture them at 20-24℃, light intensity 1500-2000 lx, and light time 10-12 h / day for 5-7 days to obtain root tip embryonic roots; (3) Cultivate for 15-20 days under the conditions of 500-2000 lx light intensity and 10-12 h / day; (4) Rooting culture: Transfer seedlings with a height of 2-3 cm to the rooting culture medium and cultivate for 8-10 days under the conditions of 20-24℃, 1500-2000 lx light intensity and 10-12 h / day. Then add the treatment agent and continue to cultivate for 7-10 days under the conditions of 24-26℃, 1800-2200 lx light intensity and 10-12 h / day. Then outdoor transplanting can be carried out, which solves the problem that mountain leek tissue culture seedlings are prone to vitrification and the tissue culture time is long.

Description

Technical Field

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

[0002] Wild leeks (Allium senescens L.) are perennial herbaceous plants belonging to the genus Allium in the family Liliaceae. Also known as mountain onion or rock onion, they are mainly distributed in Europe, Central Asia, Siberia, and northern China, growing in grasslands, meadows, or hillsides below 2000m in altitude. Wild leeks are rich in protein and vitamins; their tender leaves can be eaten as vegetables, and during their growing season, they serve as excellent forage for fattening livestock. Furthermore, wild leeks possess high medicinal value, capable of invigorating the spleen and stomach, and tonifying the kidneys and replenishing deficiencies.

[0003] The main propagation method for wild leeks is artificial cultivation. However, due to their weak natural tillering and low propagation coefficient, large-scale production is not possible in the short term. Therefore, tissue culture can be used to obtain new plants for field production. However, tissue culture of wild leeks is time-consuming and prone to vitrification. This is because the growth rate of tissue-cultured seedlings is too fast, exceeding the rate of dry matter accumulation, causing them to continuously absorb water to maintain cell volume, thus leading to vitrification. Currently, adding low concentrations of growth inhibitors such as paclobutrazol and chlormequat chloride is used to mitigate vitrification, but the effect is relatively poor, and vitrification still occurs. Furthermore, tissue-cultured seedlings often require a long hardening-off period to improve their adaptability and resistance before transplanting, further extending the culture time, which currently lacks effective methods to shorten.

[0004] Therefore, this invention provides a tissue culture method for Allium tuberosum, which shortens the tissue culture time and inhibits vitrification of tissue culture seedlings by improving the traditional tissue culture method. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a tissue culture method for *Allium chinense*, which solves the problems of vitrification in *Allium chinense* tissue culture seedlings and the long tissue culture time.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 1-2 hours, wash them with 75% ethanol solution for 1-2 minutes, then soak them in 0.1% mercuric chloride solution for 8-10 minutes, and rinse them with sterile water 4-6 times to obtain root tip explants.

[0008] (2) Induction culture: The root tip explants were inoculated in the culture medium and cultured in the dark at 20-24℃ for 2-3 days. Then, they were transferred to the conditions of 20-24℃, light intensity of 1500-2000 lx, and light time of 10-12 h / day for 5-7 days to obtain the root tip embryonic root.

[0009] (3) Proliferation culture: The induced root tip embryonic roots are inoculated into the proliferation culture medium and cultured for 15-20 days under the conditions of temperature 20-24℃, light intensity 1500-2000lx, and light time 10-12h / day.

[0010] (4) Rooting culture: Transfer seedlings with a height of 2-3cm to the rooting culture medium and culture them for 8-10 days at a temperature of 20-24℃, a light intensity of 1500-2000lx, and a light duration of 10-12h / day. Then add the treatment agent and continue to culture them for 7-10 days at a temperature of 24-26℃, a light intensity of 1800-2200lx, and a light duration of 10-12h / day. They can then be transplanted outdoors.

[0011] Furthermore, the culture medium formula in step (2) is: MS + 6-BA 2mg / L + NAA 0.5mg / L + agar 6g / L + sucrose 20g / L.

[0012] Furthermore, the formulation of the proliferation medium in step (3) is: MS + 6-BA 0.5 mg / L + NAA 1 mg / L + agar 6 g / L + sucrose 20 g / L + inhibitor 2-3 g / L.

[0013] Furthermore, the rooting medium formula in step (4) is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L.

[0014] Furthermore, the inhibitor is characterized in that it comprises the following raw materials: glutathione, glutamic acid, sodium bisulfite, taurine, and activated carbon, wherein the mass ratio of glutathione, glutamic acid, sodium bisulfite, taurine, and activated carbon is (0.5-1):(1-1.5):(1-1.2):(0.8-1.2):(3-4).

[0015] Furthermore, the method for preparing the inhibitor is as follows:

[0016] Glutamic acid was added to a 30wt% ethanol solution and stirred at 300-350 rpm for 5-10 min to obtain a glutamic acid solution. Then, glutathione, sodium bisulfite, and taurine were added to the glutamic acid solution and stirred until completely dissolved. Activated carbon was then added and mixed evenly to obtain the inhibitor.

[0017] The inhibitor effectively promotes the accumulation of substances in tissue culture seedlings of *Allium tuberosum*, maintaining a balance between cell growth rate and substance accumulation rate, thereby inhibiting vitrification. The inhibitor contains a certain amount of glutathione and glutamic acid, which synergistically promote chlorophyll synthesis in the root tips of *Allium tuberosum*, enhance the seedlings' ability to capture light energy, and promote photosynthesis, thus increasing the rate of nutrient accumulation in the root tips. Simultaneously, the inhibitor also contains sodium bisulfite and taurine, which promote photosynthesis, thereby accelerating the conversion and accumulation of absorbed nutrients in the seedlings, further increasing the rate of dry matter accumulation, promoting cell wall thickening and strengthening, improving cell mechanical strength and protective capacity, reducing excessive cell expansion and abnormal water accumulation, and thus reducing vitrification. Furthermore, the activated carbon in the inhibitor effectively adsorbs excess cytokinins and auxins in the seedlings, slowing down their growth rate and preventing excessively rapid cell growth that could lead to vitrification. Activated carbon also aggregates the active ingredients in the inhibitor, promoting absorption by the seedlings. In addition, activated carbon can promote the proliferation and differentiation of tissue culture and increase the growth rate.

[0018] Furthermore, the treatment agent comprises raw materials in the following mass ratio: glyceryl monolaurate, sodium sulfate, soybean oil, and polysorbate, wherein the mass ratio of glyceryl monolaurate, sodium sulfate, potassium chloride, soybean oil, and Tween-80 is (1-1.2):(0.5-0.6):(0.8-1):(0.8-1.2):(0.04-0.06).

[0019] Furthermore, the preparation method of the treatment agent is as follows:

[0020] (1) Add Tween-80 to water and stir evenly to obtain Tween-80 solution. Heat the solution to 60-80℃ and add soybean oil. Stir at 300-350r / min for 15-20min and cool to room temperature to obtain soybean oil emulsion.

[0021] (2) Add glyceryl monolaurate to a 30wt% ethanol solution and stir for 10-15 min at 300-350 r / min to obtain a glyceryl monolaurate solution. Then add glyceryl monolaurate, sodium sulfate, potassium chloride and soybean oil emulsion and stir evenly to obtain the treatment agent.

[0022] Furthermore, the amount of the treatment agent added is 1 / 5 of the height of the culture medium.

[0023] During rooting culture, the treatment agent is poured into the culture medium, gradually softening its hardness. This alteration of the medium's morphology creates environmental stress, thus hardening the seedlings. Combining this stress with rooting culture stimulates seedling growth, improving the adaptability and resistance of *Allium tuberosum* tissue culture seedlings. Hardening occurs simultaneously with rooting culture, shortening the tissue culture time. The culture medium contains a three-dimensional network structure formed by the cross-linking of gelatin molecules. Soybean oil in the treatment agent disrupts these bonds, promoting gelatin dispersion and loosening the gel's three-dimensional network structure, thus reducing the gelling properties of the culture medium. Sodium sulfate and glyceryl monolaurate in the treatment agent synergistically disrupt the hydrogen bonds between gelatin molecules, reducing cross-linking and further decreasing the medium's coagulation and hardness. Furthermore, potassium chloride is added to the treatment agent, which further disrupts the spatial structure of gelatin, further reducing its gelling properties. The environmental stress caused by the gradual decrease in the hardness of the culture medium can harden the tissue culture seedlings without the need for additional hardening, thus reducing the tissue culture time of wild leeks.

[0024] Beneficial effects:

[0025] This invention improves traditional tissue culture methods by inhibiting the formation of glassy seedlings during the tissue culture process, thereby increasing the effective proliferation coefficient and improving the quality of tissue culture seedlings. Simultaneously, this invention combines rooting culture with hardening-off, which not only stimulates the rooting rate of tissue culture seedlings but also shortens the hardening-off time. The resulting tissue culture seedlings exhibit strong adaptability and high stress resistance, effectively meeting market demand for wild chives. Furthermore, this invention is simple to operate, easy to implement, and worthy of widespread promotion. Attached Figure Description

[0026] Figure 1 : Growth of *Leek tissue culture* seedlings after transplanting into planting pots. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments:

[0028] Example 1: Tissue culture of Allium tuberosum

[0029] Preparation of inhibitors:

[0030] Add 1g of glutamic acid to 100mL of 30wt% ethanol solution and stir at 300r / min for 5min to obtain a glutamic acid solution. Then add 0.5g of glutathione, 1g of sodium bisulfite and 0.8g of taurine to the glutamic acid solution and continue stirring until completely dissolved. Finally, add 3g of activated charcoal and mix well to obtain the inhibitor.

[0031] Preparation of the treatment agent:

[0032] (1) Add 0.04g of Tween-80 to 80mL of water and stir until homogeneous to obtain a Tween-80 solution. Heat the solution to 60℃ and add 0.8g of soybean oil. Stir at 300r / min for 15min and cool to room temperature to obtain a soybean oil emulsion.

[0033] (2) Add 1g of lauric acid monoglyceride to 100mL of 30wt% ethanol solution and stir at 300r / min for 10min to obtain lauric acid monoglyceride solution. Then add 0.5g of sodium sulfate, 0.8g of potassium chloride and soybean oil emulsion and stir evenly to obtain treatment agent.

[0034] Tissue culture methods for wild leeks:

[0035] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 1 hour, soak them in 75% ethanol solution for 1 minute, then soak them in 0.1% mercuric chloride solution for 8 minutes, rinse them with sterile water 4 times to obtain root tip explants.

[0036] (2) Induction culture: Root tip explants were inoculated in a culture medium containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L + agar 6 g / L + sucrose 20 g / L. After being cultured in the dark at 20℃ for 2 days, they were transferred to a culture medium with a temperature of 20℃, a light intensity of 1500 lx, and a light duration of 10 h / day for 5 days to obtain root tip embryonic roots.

[0037] (3) Proliferation culture: The induced root tip embryonic roots were inoculated into the proliferation culture medium, which was: MS + 6-BA 0.5mg / L + NAA 1mg / L + agar 6g / L + sucrose 20g / L + inhibitor 2g / L. The culture was carried out for 15 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day.

[0038] (4) Rooting culture: Transfer seedlings with a height of 2cm to the rooting culture medium, which is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L. After culturing for 8 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day, add the treatment agent, the amount of which is 1 / 5 of the height of the culture medium. Continue culturing for 7 days at a temperature of 24℃, a light intensity of 1800lx, and a light duration of 10h / day before outdoor transplanting.

[0039] Example 2: Tissue culture of Allium tuberosum

[0040] Preparation of inhibitors:

[0041] 1.25 g of glutamic acid was added to 125 mL of 30 wt% ethanol solution and stirred at 330 r / min for 5 min to obtain a glutamic acid solution. Then, 0.75 g of glutathione, 1.1 g of sodium bisulfite, and 1 g of taurine were added to the glutamic acid solution and stirred until completely dissolved. Finally, 3.5 g of activated charcoal was added and mixed evenly to obtain the inhibitor.

[0042] Preparation of the treatment agent:

[0043] (1) Add 0.05g Tween-80 to 100mL of water and stir until homogeneous to obtain Tween-80 solution. Heat the solution to 70℃ and add 1g of soybean oil. Stir at 330r / min for 18min and cool to room temperature to obtain soybean oil emulsion.

[0044] (2) Add 1.1g of lauric acid monoglyceride to 110mL of 30wt% ethanol solution and stir at 330r / min for 13min to obtain lauric acid monoglyceride solution. Then add 0.55g of sodium sulfate, 0.9g of potassium chloride and soybean oil emulsion and stir evenly to obtain treatment agent.

[0045] Tissue culture methods for wild leeks:

[0046] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 2 hours, soak them in 75% ethanol solution for 2 minutes, then soak them in 0.1% mercuric chloride solution for 10 minutes, rinse them with sterile water 6 times to obtain root tip explants.

[0047] (2) Induction culture: Root tip explants were inoculated in a culture medium containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L + agar 6 g / L + sucrose 20 g / L. After being cultured in the dark at 22℃ for 2 days, the culture was transferred to a temperature of 22℃, a light intensity of 1800 lx, and a light duration of 11 h / day for 6 days to obtain root tip embryonic roots.

[0048] (3) Proliferation culture: The induced root tip embryonic roots were inoculated into the proliferation culture medium, which was: MS + 6-BA 0.5mg / L + NAA 1mg / L + agar 6g / L + sucrose 20g / L + inhibitor 2.5g / L. The culture was carried out for 18 days at a temperature of 22℃, a light intensity of 1800lx, and a light duration of 11h / day.

[0049] (4) Rooting culture: Transfer seedlings with a height of 2cm to the rooting culture medium, which is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L. After culturing for 9 days at a temperature of 22℃, a light intensity of 1800lx, and a light duration of 11h / day, add the treatment agent, the amount of which is 1 / 5 of the height of the culture medium. Continue culturing for 8 days at a temperature of 25℃, a light intensity of 2000lx, and a light duration of 11h / day before outdoor transplanting.

[0050] Example 3: Tissue culture of Allium tuberosum

[0051] Preparation of inhibitors:

[0052] Add 1.5g of glutamic acid to 150mL of 20wt% ethanol solution and stir at 350r / min for 10min to obtain a glutamic acid solution. Then add 1g of glutathione, 1.2g of sodium bisulfite and 1.2g of taurine to the glutamic acid solution and continue stirring until completely dissolved. Then add 4g of activated charcoal and mix well to obtain the inhibitor.

[0053] Preparation of the treatment agent:

[0054] (1) Add 0.06g of Tween-80 to 120mL of water and stir until homogeneous to obtain a Tween-80 solution. Heat the solution to 80℃ and add 1.2g of soybean oil. Stir at 350r / min for 20min and cool to room temperature to obtain a soybean oil emulsion.

[0055] (2) Add 1.2g of lauric acid monoglyceride to 120mL of 30wt% ethanol solution and stir for 15min at 350r / min to obtain lauric acid monoglyceride solution. Then add 0.6g of sodium sulfate, 1g of potassium chloride and soybean oil emulsion and stir evenly to obtain treatment agent.

[0056] Tissue culture methods for wild leeks:

[0057] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 2 hours, soak them in 75% ethanol solution for 2 minutes, then soak them in 0.1% mercuric chloride solution for 10 minutes, rinse them with sterile water 6 times to obtain root tip explants.

[0058] (2) Induction culture: Root tip explants were inoculated in a culture medium containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L + agar 6 g / L + sucrose 20 g / L. After being cultured in the dark at 24℃ for 3 days, the culture was transferred to a temperature of 24℃, a light intensity of 2000 lx, and a light duration of 12 h / day for 10 days to obtain root tip embryonic roots.

[0059] (3) Proliferation culture: The induced root tip embryonic roots were inoculated into the proliferation culture medium, which was: MS + 6-BA 0.5mg / L + NAA 1mg / L + agar 6g / L + sucrose 20g / L + inhibitor 3g / L. The culture was carried out for 20 days at a temperature of 24℃, a light intensity of 2000lx, and a light duration of 12h / day.

[0060] (4) Rooting culture: Transfer seedlings with a height of 5cm to the rooting culture medium, which is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L. After culturing for 10 days at a temperature of 24℃, a light intensity of 2000lx, and a light duration of 12h / day, add the treatment agent, the amount of which is 1 / 5 of the height of the culture medium. Continue culturing for 10 days at a temperature of 26℃, a light intensity of 2200lx, and a light duration of 12h / day before outdoor transplanting.

[0061] Comparative Example 1:

[0062] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the inhibitor; specifically, glutathione is not added. The specific preparation process is as follows:

[0063] Add 1g of glutamic acid to 100mL of 30wt% ethanol solution and stir for 5min at 300r / min to obtain a glutamic acid solution. Then add 1g of sodium bisulfite and 0.8g of taurine to the glutamic acid solution and continue stirring until completely dissolved. Finally, add 3g of activated carbon and mix well to obtain the inhibitor.

[0064] The preparation of the treatment agent and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0065] Comparative Example 2:

[0066] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the inhibitor; specifically, taurine is not added. The specific preparation process is as follows:

[0067] 1g of glutamic acid was added to 100mL of 30wt% ethanol solution and stirred at 300r / min for 5min to obtain a glutamic acid solution. Then, 0.5g of glutathione and 1g of sodium bisulfite were added to the glutamic acid solution and stirred until completely dissolved. Finally, 3g of activated charcoal was added and mixed evenly to obtain the inhibitor.

[0068] The preparation of the treatment agent and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0069] Comparative Example 3:

[0070] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the inhibitor; specifically, activated carbon is not added. The specific preparation process is as follows:

[0071] 1g of glutamic acid was added to 100mL of 30wt% ethanol solution and stirred at 300r / min for 5min to obtain a glutamic acid solution. Then, 0.5g of glutathione, 1g of sodium bisulfite, and 0.8g of taurine were added to the glutamic acid solution and stirred until completely dissolved to obtain the inhibitor.

[0072] The preparation of the treatment agent and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0073] Comparative Example 4:

[0074] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the treatment agent; specifically, soybean oil and Tween-80 are not added. The specific preparation process is as follows:

[0075] (2) Add 1g of lauric acid monoglyceride to 100mL of 30wt% ethanol solution and stir at 300r / min for 10min to obtain lauric acid monoglyceride solution. Then add 0.5g of sodium sulfate and 0.8g of potassium chloride and stir evenly to obtain the treatment agent.

[0076] The preparation of the inhibitor and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0077] Comparative Example 5:

[0078] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the treatment agent. Specifically, glyceryl monolaurate and sodium sulfate are not added. The specific preparation process is as follows:

[0079] (1) Add 0.04g of Tween-80 to 80mL of water and stir until homogeneous to obtain a Tween-80 solution. Heat the solution to 60℃ and add 0.8g of soybean oil. Stir at 300r / min for 15min and cool to room temperature to obtain a soybean oil emulsion.

[0080] (2) Mix 0.5g of sodium sulfate with 100mL of water to obtain a sodium sulfate solution. Add soybean oil emulsion to the sodium sulfate solution and stir evenly to obtain the treatment agent.

[0081] The preparation of the inhibitor and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0082] Comparative Example 6:

[0083] This comparative example is compared with Example 1, the only difference being the raw materials used to prepare the treatment agent; specifically, potassium chloride is not added. The specific preparation process is as follows:

[0084] (1) Add 0.04g of Tween-80 to 80mL of water and stir until homogeneous to obtain a Tween-80 solution. Heat the solution to 60℃ and add 0.8g of soybean oil. Stir at 300r / min for 15min and cool to room temperature to obtain a soybean oil emulsion.

[0085] (2) Add 1g of lauric acid monoglyceride to 100mL of 30wt% ethanol solution and stir for 10min at 300r / min to obtain lauric acid monoglyceride solution. Then add 0.5g of sodium sulfate and soybean oil emulsion and stir evenly to obtain treatment agent.

[0086] The preparation of the inhibitor and the tissue culture method of *Allium tuberosum* were the same as in Example 1.

[0087] Comparative Example 7:

[0088] This comparative example is compared with Example 1, the only difference being that no inhibitors were added during the proliferation culture process. The specific method is as follows:

[0089] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 1 hour, soak them in 75% ethanol solution for 1 minute, then soak them in 0.1% mercuric chloride solution for 8 minutes, rinse them with sterile water 4 times to obtain root tip explants.

[0090] (2) Induction culture: Root tip explants were inoculated in a culture medium containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L + agar 6 g / L + sucrose 20 g / L. After being cultured in the dark at 20℃ for 2 days, they were transferred to a culture medium with a temperature of 20℃, a light intensity of 1500 lx, and a light duration of 10 h / day for 5 days to obtain root tip embryonic roots.

[0091] (3) Proliferation culture: The induced root tip embryonic roots were inoculated into the proliferation culture medium, which was: MS + 6-BA 0.5mg / L + NAA 1mg / L + agar 6g / L + sucrose 20g / L. The culture was carried out for 15 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day.

[0092] (4) Rooting culture: Transfer seedlings with a height of 2cm to the rooting culture medium, which is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L. After culturing for 8 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day, add the treatment agent, the amount of which is 1 / 5 of the height of the culture medium. Continue culturing for 7 days at a temperature of 24℃, a light intensity of 1800lx, and a light duration of 10h / day before outdoor transplanting.

[0093] The preparation of the inhibitor and the treatment agent were the same as in Example 1.

[0094] Comparative Example 8:

[0095] This comparative example is compared with Example 1, specifically in that no treatment agent is added during the rooting culture and hardening-off process. The specific method is as follows:

[0096] (1) Aseptic treatment of explants: Take the root tips of healthy, disease-free ivy plants, rinse them with running water for 1 hour, soak them in 75% ethanol solution for 1 minute, then soak them in 0.1% mercuric chloride solution for 8 minutes, rinse them with sterile water 4 times to obtain root tip explants.

[0097] (2) Induction culture: Root tip explants were inoculated in a culture medium containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L + agar 6 g / L + sucrose 20 g / L. After being cultured in the dark at 20℃ for 2 days, they were transferred to a culture medium with a temperature of 20℃, a light intensity of 1500 lx, and a light duration of 10 h / day for 5 days to obtain root tip embryonic roots.

[0098] (3) Proliferation culture: The induced root tip embryonic roots were inoculated into the proliferation culture medium, which was: MS + 6-BA 0.5mg / L + NAA 1mg / L + agar 6g / L + sucrose 20g / L + inhibitor 2g / L. The culture was carried out for 15 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day.

[0099] (4) Rooting culture: Transfer seedlings with a height of 2cm to the rooting culture medium, which is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L. Culture for 15 days at a temperature of 20℃, a light intensity of 1500lx, and a light duration of 10h / day before outdoor transplanting.

[0100] The preparation of the inhibitor and the treatment agent were the same as in Example 1.

[0101] Experiment 1:

[0102] Root tips from healthy, disease-free *Allium tuberosum* plants were collected, rinsed with running water for 1 hour, soaked in 75% ethanol solution for 1 minute, then soaked in 0.1% mercuric chloride solution for 8 minutes, and rinsed four times with sterile water to obtain root tip explants. *Allium tuberosum* tissue culture was performed according to the methods in Example 1 and Comparative Examples 1-8. Thirty explants were cultured in each experimental group, and each experimental group underwent three parallel experiments. The number of adventitious bud differentiation and vitrified seedlings in each experimental group were statistically analyzed, and the adventitious bud differentiation rate and vitrification rate were calculated. The results are shown in Table 1.

[0103] Table 1

[0104] Glass transition rate (%) Adventitious bud differentiation rate (%) Example 1 3.33 94.44 Comparative Example 1 22.22 70.00 Comparative Example 2 20.00 74.44 Comparative Example 3 18.89 73.33 Comparative Example 4 5.56 92.22 Comparative Example 5 4.44 93.33 Comparative Example 6 4.44 93.33 Comparative Example 7 38.89 54.44 Comparative Example 8 4.44 92.22

[0105] Analysis of Table 1 shows that:

[0106] 1. Compared with Example 1, Comparative Example 1 did not add glutathione, resulting in lower chlorophyll synthesis in the root tips of the tissue culture seedlings and a slower photosynthetic rate. Consequently, the accumulation and transformation of nutrients in the tissue culture of *Allium tuberosum* was slower, the rate of cell enlargement was greater than the rate of material accumulation in the tissue culture seedlings, and the cells continuously absorbed water, leading to vitrification of the tissue culture seedlings, physiological pathological changes, and a reduced differentiation rate of adventitious buds.

[0107] 2. Compared with Example 1, Comparative Example 2 did not add taurine. The enzyme activity in the tissue culture seedlings was lower, which led to a decrease in the material conversion rate in the tissue culture seedlings. The rate of dry matter accumulation in the tissue culture seedlings was less than the rate of cell enlargement, which led to vitrification of the tissue culture seedlings and further reduced the differentiation rate of adventitious buds.

[0108] 3. Compared to Example 1, without activated carbon, Comparative Example 3 showed a higher vitrification rate and a lower differentiation rate of adventitious buds in the tissue culture seedlings. This is because activated carbon effectively adsorbs excess cytokinins and auxins at the tissue culture seedling site, reducing the cell growth rate and balancing the dry matter accumulation rate and cell proliferation rate, thereby lowering the vitrification rate. Furthermore, activated carbon can promote proliferation and differentiation in tissue culture, increasing the differentiation rate of adventitious buds. Therefore, the tissue culture seedlings in Comparative Example 3 without activated carbon showed a higher vitrification rate and a lower differentiation rate.

[0109] 5. Compared with Example 1, Comparative Example 7 did not add any inhibitors. During the tissue culture process, the cells of the seedlings continuously increased in size, while the accumulation of dry matter was slower. This resulted in the cells continuously absorbing water without maintaining their volume, leading to severe vitrification of the seedlings and a decrease in the differentiation rate of adventitious buds. This demonstrates that the inhibitor prepared in Example 1 can effectively reduce the vitrification rate of tissue culture seedlings and promote their normal growth.

[0110] Experiment 2:

[0111] Rooting medium was prepared as follows: MS + NAA 1 mg / L + agar 3 g / L + sucrose 20 g / L. Treatment agents prepared in Examples 1 and 4-6 were added to each medium, with the added amount being 1 / 5 of the medium height. No treatment agent was added to Comparative Example 8. The hardness of the medium was tested before adding the treatment agent; the hardness was 250 g / cm³. 2The hardness of the culture medium was tested on days 3, 5, and 7 after the addition of the treatment agent. After rooting culture, the plants were directly transplanted into planting pots (the transplanting substrate in the planting pots consisted of topsoil and well-rotted organic fertilizer in a mass ratio of 8:2). Twenty plants were transplanted per group, with three replicates. The plants were then moved outdoors for growth. The survival rate was recorded after 30 days of outdoor growth. The results are shown in Table 2.

[0112] Table 2

[0113] <![CDATA[Hardness 3d (g / cm 2 )]]> <![CDATA[Hardness 5d (g / cm 2 )]]> <![CDATA[Hardness 7d (g / cm 2 )]]> Transplant survival rate (%) Example 1 220 172 136 93.33 Comparative Example 4 238 208 186 81.67 Comparative Example 5 236 207 185 83.33 Comparative Example 6 235 206 183 85.00 Comparative Example 8 242 238 234 68.33

[0114] Analysis of Table 2 shows that:

[0115] 1. Compared with Example 1, Comparative Example 4 did not add soybean oil to the treatment agent, and the hardness of the culture medium changed less. This is because the three-dimensional structure of gelatin in the culture medium is stable, resulting in higher gelling properties of the culture medium in Comparative Example 1. Due to the small change in the culture medium, the time required for hardening the seedlings was longer. Therefore, under the same culture and hardening time as Example 1, the tissue culture seedlings in Comparative Example 4 had poor adaptability after hardening, resulting in a low transplant survival rate.

[0116] 2. Compared with Example 1, Comparative Example 5 did not add glyceryl monolaurate or sodium sulfate to the treatment agent. The gelatin molecules in the culture medium had strong intermolecular forces and were difficult to decompose. Therefore, the gelation properties of the culture medium in Comparative Example 5 were better, resulting in a smaller change in the hardness of the culture medium after the treatment agent was added. The stress effect on the tissue culture seedlings was insufficient, resulting in a less effective hardening effect than in Example 1. The adaptability of the tissue culture seedlings was poor, leading to a lower transplant survival rate.

[0117] 3. Compared with Example 1, Comparative Example 6 did not add potassium chloride, and the change in the hardness of the culture medium was smaller. This is because potassium chloride can destroy the spatial structure of gelatin in the culture medium, thereby reducing the gelling properties of the culture medium and promoting hardening of the tissue culture seedlings due to the stress caused by changes in the culture medium during rooting. Therefore, the tissue culture seedlings of Comparative Example 6 without added potassium chloride had a poor hardening effect and a lower transplant survival rate.

[0118] 4. Compared with Example 1, Comparative Example 8 did not use any treatment agent, and the culture medium underwent less change during the rooting culture process. The hardness of the culture medium did not change significantly, which made it impossible to harden the tissue culture seedlings at the same time during the rooting culture period. As a result, the transplanted tissue culture seedlings were difficult to survive.

[0119] 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 Allium tuberosum, characterized in that, The method includes the following steps: (1) Aseptic treatment of explants: Root tips of wild leek plants were selected and disinfected to obtain root tip explants; (2) Induction culture: The root tip explants were inoculated in the culture medium and cultured in the dark at 20-24℃ for 2-3 days. Then, they were cultured in the environment at 20-24℃, light intensity of 1500-2000 lx, and light intensity of 10-12 h / day for 5-7 days to obtain the root tip embryonic root. (3) Proliferation culture: The induced root tip embryonic roots are inoculated into the proliferation culture medium and cultured for 15-20 days under the conditions of temperature 20-24℃, light intensity 1500-2000lx, and light time 10-12h / day. (4) Rooting culture: Transfer seedlings with a height of 2-3cm to rooting culture medium and culture them for 8-10 days at a temperature of 20-24℃, a light intensity of 1500-2000lx, and a light duration of 10-12h / day. Then add treatment agent and continue to culture them for 7-10 days at a temperature of 24-26℃, a light intensity of 1800-2200lx, and a light duration of 10-12h / day. They can then be transplanted outdoors. The formulation of the proliferation medium in step (3) is: MS + 6-BA 0.5 mg / L + NAA 1 mg / L + agar 6 g / L + sucrose 20 g / L + inhibitor 2-3 g / L; The inhibitor is composed of the following raw materials: glutathione, glutamic acid, sodium bisulfite, taurine, and activated carbon, wherein the mass ratio of glutathione, glutamic acid, sodium bisulfite, taurine, and activated carbon is (0.5-1):(1-1.5):(1-1.2):(0.8-1.2):(3-4). The treatment agent is a raw material in the following mass ratio: glyceryl monolaurate, sodium sulfate, soybean oil, and potassium chloride, wherein the mass ratio of glyceryl monolaurate, sodium sulfate, potassium chloride, soybean oil, and Tween-80 is (1-1.2):(0.5-0.6):(0.8-1):(0.8-1.2):(0.04-0.06).

2. The method for tissue culture of Allium tuberosum according to claim 1, characterized in that, The culture medium formula in step (2) is: MS + 6-BA 2mg / L + NAA 0.5mg / L + agar 6g / L + sucrose 20g / L.

3. The method for tissue culture of Allium tuberosum according to claim 2, characterized in that, The formula for the rooting medium in step (4) is: MS + NAA 1mg / L + agar 3g / L + agar 3g / L + sucrose 20g / L.

4. The method for tissue culture of Allium tuberosum according to claim 3, characterized in that, The method for preparing the inhibitor is as follows: Glutamic acid was added to a 30 wt% ethanol solution and stirred at 300-350 r / min for 5-10 min to obtain a glutamic acid solution. Then, glutathione, sodium bisulfite, and taurine were added to the glutamic acid solution and stirred until completely dissolved. Activated carbon was then added and mixed evenly to obtain the inhibitor.

5. The method for tissue culture of Allium tuberosum according to claim 4, characterized in that, The preparation method of the treatment agent is as follows: (1) Add Tween-80 to water and stir evenly to obtain Tween-80 solution. Heat the solution to 60-80℃ and add soybean oil. Stir at 300-350r / min for 15-20min and cool to room temperature to obtain soybean oil emulsion. (2) Add glyceryl monolaurate to a 30wt% ethanol solution and stir for 10-15 min at 300-350 r / min to obtain a glyceryl monolaurate solution. Then add sodium sulfate, potassium chloride and soybean oil emulsion and stir evenly to obtain the treatment agent.

6. The method for tissue culture of Allium tuberosum according to claim 5, characterized in that, The amount of the treatment agent used is 1 / 5 of the height of the culture medium.

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