Plant tissue culture method of edible chrysanthemum

Abstract

The invention discloses a plant tissue culture method of edible chrysanthemum, and relates to the technical field of tissue culture of edible chrysanthemum, and the plant tissue culture method comprises the following steps: selecting an edible chrysanthemum tender stem as an explant, washing with an SDS surfactant, flushing with running water, placing under an ultra-clean sterile condition, disinfecting for 10 minutes with a 0.1% mercury bichloride solution, shaking, then flushing with sterile water, and drying to obtain the plant tissue culture method of the edible chrysanthemum. Placing on sterile absorbent paper for dry absorption, cutting into segments with 1-2 leaves and length of 2-3cm, and vertically inserting the lower parts of the segments into a rooting culture medium; the method comprises the following steps: carrying out sterile rapid cultivation, starting tender stem culture, growing green protuberant differentiated tender stems on the fifth day, and enabling the plant height to be 3-5cm after about one month, and carrying out subculture proliferation, namely shearing the tender stems into small sections with one section, one bud and one leaf, inserting the base parts of the cut sections into a culture medium, forming axillary buds into new plants after 3-4 weeks, and repeatedly shearing and propagating. The edible chrysanthemum tissue culture method is high in propagation efficiency, rapid and large-scale propagation can be realized, excellent characters of female parents can be kept, the method is not limited by seasons, annual production can be realized, the survival rate and quality of seedlings are ensured, and the stress resistance is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of edible chrysanthemum tissue culture, in particular to a plant tissue culture method of edible chrysanthemum. Background Art

[0002] Edible chrysanthemum is a plant with both ornamental and edible value, with unique flavor and rich nutrients, showing broad application prospects in many fields such as food, health products and cosmetics. However, traditional edible chrysanthemum propagation methods mainly rely on seed propagation and division propagation, which have many limitations.

[0003] Seed propagation has the problems of slow propagation speed and easy separation of traits in offspring, which makes it difficult to stably inherit excellent traits, affecting the quality consistency and yield stability of edible chrysanthemum. Moreover, the cycle from sowing to harvesting is long, which is inefficient for large-scale commercial production and difficult to meet the growing market demand for edible chrysanthemum. Although ramet propagation can maintain the excellent traits of the mother plant, it has a low propagation coefficient, limited propagation materials, and requires the consumption of a large number of mother plants. In the long-term ramet propagation process, the plants are susceptible to diseases and insect pests, accumulate viruses, cause variety degeneration, and reduce the stress resistance and yield of edible chrysanthemum.

[0004] In addition, for some rare edible chrysanthemum varieties, it is difficult to achieve rapid and efficient propagation through traditional propagation methods, which limits the promotion and application of these varieties. As the market demand for edible chrysanthemums and their derivative products continues to expand, a technology that can efficiently, stably and large-scale propagate high-quality edible chrysanthemums is urgently needed.

[0005] Plant tissue culture technology provides a new approach to solving the above problems. Plant tissue culture is a technology that uses plant cells, tissues or organs as explants in a sterile and artificially controlled environment, and induces them to regenerate into complete plants by providing suitable nutrition and growth conditions. This technology has the advantages of fast reproduction, no seasonal restrictions, the ability to maintain the excellent traits of the mother plant, and the ability to achieve factory production. It has been widely used in the reproduction and seedling production of many plants.

[0006] However, the tissue culture technology for edible chrysanthemum is not yet fully mature and faces many problems in the actual operation process. For example, the disinfection of explants, the selection of culture medium formula, the optimization of the culture environment, and the management of rooting and seedling hardening all need further exploration and improvement. Different edible chrysanthemum varieties have different requirements for the external environment and nutrients. How to find a universal and efficient tissue culture method to achieve large-scale rapid reproduction while ensuring the survival rate and high-quality seedling rate is the key challenge in the development of edible chrysanthemum tissue culture technology.

[0007] In the prior art, although there are some plant tissue culture methods, the tissue culture methods for edible chrysanthemum are either cumbersome to operate or not detailed enough in key steps, resulting in low propagation efficiency and unstable seedling quality, which is difficult to meet the needs of large-scale commercial production and variety improvement. Therefore, it is necessary to develop a plant tissue culture method specifically for edible chrysanthemum to overcome the shortcomings of existing propagation technologies and provide strong technical support for the development of the edible chrysanthemum industry. Summary of the invention

[0008] 1. Technical issues to be solved

[0009] In view of the deficiencies in the prior art, the present invention provides a plant tissue culture method for edible chrysanthemum.

[0010] (II) Technical solution

[0011] A plant tissue culture method for edible chrysanthemum, characterized in that it comprises the following steps:

[0012] Explant selection: Select the tender stems of edible chrysanthemum as explants;

[0013] Explant pretreatment: first wash the explant with SDS surfactant, then rinse it repeatedly with running water;

[0014] Disinfection: Place the pretreated explants under ultra-clean sterile conditions and disinfect them with 0.1% mercuric chloride solution for 10 minutes. Gently shake the explants during the disinfection process, then rinse them with sterile water five to six times and place them on sterile absorbent paper to absorb the moisture.

[0015] Explant segmentation: Cut the sterilized tender stem into segments with 1-2 leaves and a length of 2cm-3cm;

[0016] Inoculation: Insert the lower part of the explant stem segment vertically into the rooting medium;

[0017] Aseptic rapid cultivation: including starting tender stem culture and subculture proliferation. The culture medium used for starting tender stem culture is MS+IBA0.2mg / L+NAA0.1mg / L+carrageenan 5.5g / L+sucrose 30g / L. The pH of the culture medium is adjusted to 5.8. The culture is carried out under the conditions of a culture temperature of 25℃±1℃, a light intensity of 3000lx-4000lx, and a light duration of 8h / d. On the fifth day of culture, green protrusions appear on the stem segments and tender stems gradually differentiate. The plant height reaches 3cm-5cm in about one month. During subculture proliferation, the tender stems are cut into small segments with one node, one bud, and one leaf. The base of the cut segments is inserted into the culture medium of MS+IBA0.2mg / L+NAA0.1mg / L. The axillary buds are grown into new plantlets for 3-4 weeks. A large number of test tube seedlings are obtained by repeatedly cutting the stem segments.

[0018] Rooting and hardening of seedlings: When the sterile seedlings grow to 5cm-7cm in height, cut the rootless tender stems of the test tube seedlings of about 3cm and transfer them to the MS+NAA0.2mg / L culture medium. The rooting rate can reach 80% within 5-7 days and 100% within 10 days. After 15-20 days of cultivation, each test tube seedling will grow an average of 4-5 thick roots and the leaves will be dark green. Then the seedlings can be hardened. When hardening the seedlings, open the bottle mouth after 3-5 days and take out the test tube seedlings, wash the agar at the roots, transfer them to fine sand and fine mud media according to a certain row spacing, cover with an arch shed and cover with film to maintain humidity, and at the same time, carry out timely ventilation, regular fertilizer and pesticide spraying.

[0019] Furthermore, in the explant pretreatment step, the SDS surfactant is used at a concentration of 0.5%-1%, and the explant is washed at 20°C-30°C for 10-15 minutes.

[0020] Furthermore, in the disinfection step, the concentration of the mercuric chloride solution is in the range of 0.08%-0.12%, the disinfection time is 8-12 minutes, and during the disinfection process, the explant is gently shaken by an oscillating device with an oscillation frequency of 50-100 rpm.

[0021] Furthermore, in the rooting and seedling hardening step, when the test tube seedlings are transferred to the medium of fine sand and fine mud, the volume ratio of fine sand and fine mud is 1:1-2, and when the arch shed is covered with film, the relative humidity in the arch shed is maintained at 80%-90%, and ventilation is performed 2-3 times a day, each ventilation time is 15-30 minutes, and compound fertilizer containing N, P, and K elements is used when spraying fertilizer, and its concentration is 0.1%-0.2%, and carbendazim is used when spraying medicine, and the concentration is 0.05%-0.1% to prevent the occurrence of diseases and insect pests.

[0022] Furthermore, in the aseptic rapid cultivation step, when starting the tender stem culture, the light intensity is 2000lx-3000lx in the first 3 days of culture, and is increased to 3000lx-4000lx from the 4th day, and during subculture, the culture medium is replaced every 2-3 weeks to ensure an adequate supply of nutrients.

[0023] Furthermore, in the inoculation step, the explant stem segments are inserted into the rooting medium to a depth of 0.5 cm-1 cm, and 3-5 explant stem segments are inoculated in each culture container.

[0024] Furthermore, in the aseptic rapid cultivation step, for the subculture proliferation culture, the culture medium container is a triangular flask with a capacity of 100mL-250mL, 8-12 subcultured stem segments are inoculated in each triangular flask, and the triangular flask is sealed with a breathable sealing film to ensure gas exchange in the bottle and prevent contamination.

[0025] Furthermore, in the rooting and hardening step, after the test tube seedlings are transferred to the fine sand and fine mud medium, they are shaded at a shading rate of 30%-50% for 1-2 weeks to promote the adaptation and growth of the test tube seedlings.

[0026] (III) Beneficial technical effects

[0027] First, in terms of reproduction efficiency, this tissue culture method can achieve rapid reproduction of edible chrysanthemum. Using tender stems as explants, after optimized explant treatment, aseptic rapid cultivation and rooting and seedling hardening processes, a large number of high-quality test tube seedlings can be obtained in a short period of time, greatly shortening the reproduction cycle, improving the reproduction coefficient, overcoming the problem of slow traditional seed reproduction and division reproduction, and meeting the needs of large-scale commercial production.

[0028] Secondly, this method can maintain the excellent traits of the mother plant. Through strict aseptic operation and appropriate culture conditions, the separation of offspring traits and variety degeneration are avoided, the quality consistency of edible chrysanthemums is ensured, and the quality and yield stability of the products are guaranteed.

[0029] Furthermore, the tissue culture method can achieve year-round production. It is not restricted by seasons and can produce seedlings at any time according to market demand, thereby improving the supply capacity of edible chrysanthemums and providing a stable source of seedlings for the sustainable development of related industries.

[0030] In addition, through meticulous management during the rooting and seedling hardening stage, such as reasonable medium ratio, ventilation, fertilizer and pesticide spraying, the adaptability and stress resistance of test tube seedlings can be enhanced, the survival rate of seedlings can be improved, and they can better adapt to the natural environment after transplanting, reducing the occurrence of diseases and pests and reducing planting risks.

[0031] Finally, the tissue culture method of the present invention is helpful for the preservation and promotion of rare edible chrysanthemum varieties. For some rare varieties with outstanding excellent traits, a large number of reproduction can be carried out through this technology, which promotes the development and utilization of edible chrysanthemum variety resources and lays a foundation for the wide application of edible chrysanthemum in industries such as food, health products and cosmetics.

[0032] In summary, the present invention provides an efficient, stable and high-quality tissue culture method for the reproduction of edible chrysanthemums, promotes the development of the edible chrysanthemum industry, and has important economic and social benefits. DETAILED DESCRIPTION

[0033] Embodiment 1:

[0034] Explant selection and pretreatment:

[0035] The tender stems of the edible chrysanthemum variety "Hangbaiju" were selected as explants. The explants were washed with 0.8% SDS surfactant at 25°C for 12 minutes, and then rinsed with running water for 30 minutes to ensure that the dirt and impurities on the surface of the explants were completely removed.

[0036] Disinfection:

[0037] The pretreated explants were placed in a clean bench and disinfected with 0.1% mercuric chloride solution for 10 minutes. During the disinfection process, the explants were gently shaken with an oscillating device at an oscillation frequency of 80 rpm to make the disinfection more uniform. After the disinfection was completed, the explants were rinsed six times with sterile water and placed on sterile absorbent paper to absorb the water.

[0038] Explant segmentation:

[0039] The sterilized tender stems were cut into segments with one leaf and a length of 2.5 cm.

[0040] Vaccination:

[0041] Insert the lower part of the explant stem segment vertically into the rooting medium with a depth of 0.8 cm, and inoculate 4 explant stem segments in each culture container (culture dish). The rooting medium is MS+IBA 0.2 mg / L+NAA 0.1 mg / L+carrageenan 5.5 g / L+sucrose 30 g / L, and the pH of the medium is adjusted to 5.8.

[0042] Aseptic rapid cultivation:

[0043] Start the tender stem culture: Cultivate at a temperature of 25°C, a light intensity of 3000 lx, and a light duration of 8 h / d. On the fifth day of cultivation, green protrusions begin to appear on the stem segments, and then tender stems gradually differentiate. After one month, the plant height reaches about 4 cm.

[0044] Subculture proliferation: Cut the tender stem into small segments with one node, one bud and one leaf, and insert the base of the segment into the culture medium of MS+IBA0.2mg / L+NAA0.1mg / L. Use a 150mL triangular flask as the culture container, inoculate 10 stem segments in each triangular flask, and seal it with a breathable sealing film. After 3.5 weeks of culture, the axillary buds grow into new plantlets. Continue to cut the stem segments repeatedly for culture to obtain a large number of test tube seedlings.

[0045] Rooting and refining seedlings:

[0046] When the sterile seedlings grow to 6cm in height, cut the rootless stems of the test tube seedlings about 3cm long and transfer them to the medium of MS+NAA0.2mg / L. Within 6 days, the rooting rate reaches 80%, and within 9 days, the rooting rate reaches 100%. After 18 days of cultivation, each test tube seedling grows 4 strong roots on average and the leaves are dark green.

[0047] After hardening the seedlings, open the bottle mouth and take out the test tube seedlings after 3 days, wash the agar at the roots, transfer them to the medium of fine sand and fine mud with a volume ratio of 1:1.5 at a row spacing of 5 cm, cover the arch shed with film, keep the relative humidity in the arch shed at 85%, ventilate twice a day, and ventilate for 20 minutes each time. When spraying fertilizer, use compound fertilizer containing N, P, and K elements at a concentration of 0.15%, and when spraying medicine, use carbendazim at a concentration of 0.08%. After transferring the test tube seedlings to the medium, shade them with a shading rate of 40% for 1.5 weeks.

[0048] Embodiment 2:

[0049] Explant selection and pretreatment:

[0050] The tender stems of the edible chrysanthemum variety "Jinsi Huangju" were selected as explants. The explants were washed with 1% SDS surfactant at 22°C for 15 minutes and then rinsed with running water for 25 minutes.

[0051] Disinfection:

[0052] Place it in a clean bench and disinfect it with 0.09% mercuric chloride solution for 11 minutes. During disinfection, use an oscillating device with an oscillation frequency of 70 rpm to gently shake it. After disinfection, rinse the explant five times with sterile water and place the explant on sterile absorbent paper to dry it.

[0053] Explant segmentation:

[0054] The sterilized tender stems were cut into segments with two leaves and a length of 3 cm.

[0055] Vaccination:

[0056] Insert the lower part of the explant stem segment vertically into the rooting medium with a depth of 0.7 cm, and inoculate 5 explant stem segments in each culture container. The rooting medium is MS+IBA 0.2 mg / L+NAA 0.1 mg / L+carrageenan 5.5 g / L+sucrose 30 g / L, and the pH of the medium is adjusted to 5.8.

[0057] Aseptic rapid cultivation:

[0058] Start tender stem culture: Cultivate at a temperature of 24°C, a light intensity of 3500 lx, and a light duration of 8 h / d. On the fifth day of cultivation, green protrusions appear on the stem segments and tender stems begin to differentiate. One month later, the plant height reaches 4.5 cm.

[0059] Subculture: Cut the tender stem into small segments with one bud and one leaf, insert the base of the segment into the culture medium of MS+IBA0.2mg / L+NAA0.1mg / L, use a 200mL triangular flask as the culture container, inoculate 12 stem segments in each triangular flask, and seal it with a breathable sealing film. After 4 weeks of culture, the axillary buds grow into new plantlets, and the stem segments are repeatedly cut for propagation to obtain a large number of test tube seedlings.

[0060] Rooting and refining seedlings:

[0061] When the sterile seedlings grow to 7cm in height, cut the rootless stems of the test tube seedlings about 3cm long and transfer them to the medium of MS+NAA0.2mg / L. Within 7 days, the rooting rate reaches 80%, and within 10 days, the rooting rate reaches 100%. After 20 days of cultivation, each test tube seedling grows 5 strong roots on average and the leaves are dark green.

[0062] After hardening the seedlings, open the bottle mouth and take out the test tube seedlings after 4 days, wash the agar at the roots, transfer them to the medium of fine sand and fine mud with a volume ratio of 1:2 at a row spacing of 6 cm, cover the arch shed with film, keep the relative humidity in the arch shed at 88%, ventilate 3 times a day, and ventilate for 25 minutes each time. When spraying fertilizer, use compound fertilizer containing N, P, and K elements at a concentration of 0.2%, and when spraying pesticides, use carbendazim at a concentration of 0.1%. After the test tube seedlings are transferred to the medium, they are shaded with a shading rate of 50% for 2 weeks.

[0063] Embodiment 3:

[0064] Explant selection and pretreatment:

[0065] The tender stems of the edible chrysanthemum variety "Chuju" were selected as explants. The explants were washed with 0.5% SDS surfactant at 30°C for 10 minutes, and then rinsed with running water for 35 minutes.

[0066] Disinfection:

[0067] In a clean bench, disinfect with 0.12% mercuric chloride solution for 8 minutes. During the disinfection process, gently shake with an oscillating device with an oscillation frequency of 100 rpm. After disinfection, rinse the explant six times with sterile water and place the explant on sterile absorbent paper to dry.

[0068] Explant segmentation:

[0069] The sterilized tender stems were cut into segments with one leaf and a length of 2 cm.

[0070] Vaccination:

[0071] The lower part of the explant stem segment was vertically inserted into the rooting medium with a depth of 0.5 cm, and 3 explant stem segments were inoculated in each culture container. The rooting medium was MS + IBA 0.2 mg / L + NAA 0.1 mg / L + carrageenan 5.5 g / L + sucrose 30 g / L, and the pH of the medium was adjusted to 5.8.

[0072] Aseptic rapid cultivation:

[0073] Start tender stem culture: Cultivate at a temperature of 26°C, a light intensity of 3200 lx, and a light duration of 8 h / d. On the fifth day of cultivation, green protrusions appear on the stem segments, followed by differentiation of tender stems. One month later, the plant height reaches 3.5 cm.

[0074] Subculture proliferation: Cut the tender stem into small segments with one bud and one leaf, and insert the base of the segment into the culture medium of MS+IBA0.2mg / L+NAA0.1mg / L. Use a 100mL triangular flask as the culture container, inoculate 8 stem segments in each triangular flask, and seal it with a breathable sealing film. After 3 weeks of cultivation, the axillary buds grow into new plantlets. By cutting the stem segments multiple times, a large number of test tube seedlings can be cultivated.

[0075] Rooting and refining seedlings:

[0076] When the sterile seedlings grow to 5cm in height, cut the rootless stems of the test tube seedlings about 3cm long and transfer them to the medium of MS+NAA0.2mg / L. Within 5 days, the rooting rate reaches 80%, and within 10 days, the rooting rate reaches 100%. After 15 days of cultivation, each test tube seedling grows 4 strong roots on average and the leaves are dark green.

[0077] After hardening the seedlings, open the bottle mouth and take out the test tube seedlings after 5 days, wash the agar at the roots, transfer them to the medium of fine sand and fine mud with a volume ratio of 1:1 at a row spacing of 4 cm, cover the arch shed with film, keep the relative humidity in the arch shed at 80%, ventilate twice a day, and ventilate for 15 minutes each time. When spraying fertilizer, use compound fertilizer containing N, P, and K elements at a concentration of 0.1%, and when spraying medicine, use carbendazim at a concentration of 0.05%. After transferring the test tube seedlings to the medium, shade them with a shading rate of 30% for 1 week.

[0078] Comparative Example:

[0079] The edible chrysanthemum variety "Hangbaiju" is propagated by the traditional division propagation method:

[0080] Division operation:

[0081] Select healthy side shoots from the mother plant and carefully separate them from the mother plant, making sure they have a certain root system, stems and leaves.

[0082] The divided plants are transplanted directly into the field soil, which has been plowed and fertilized in advance.

[0083] Post-management:

[0084] Water, fertilize and weed regularly, but no special pest control and other treatments are carried out.

[0085] During the growth process, it was found that the growth rate of the plant propagated by division was relatively slow, the propagation coefficient was low, and within the same growth cycle, the number of new plants obtained was far less than the number of plants obtained by the tissue culture method of the present invention. Moreover, after several propagation cycles, the plant propagated by division showed a phenomenon of variety degeneration, which was manifested as weakened plant growth potential, decreased resistance to pests and diseases, and reduced quality and yield of flowers. At the same time, this propagation method is restricted by seasons and can generally only be carried out in spring and autumn, and cannot meet the production needs throughout the year. The tissue culture method of the present invention can overcome these problems, reflecting the significant advantages of the tissue culture method in the propagation of edible chrysanthemums.

[0086]

[0087]

[0088] This table compares three embodiments and one comparative example from four aspects: reproduction cycle, reproduction coefficient, seedling quality and seasonality of production. In terms of reproduction cycle, the tissue culture method of the present invention can complete the cultivation from explant to transplantable seedling in a relatively short time, while the traditional ramet propagation of the comparative example takes a long time. In terms of reproduction coefficient, the tissue culture method can achieve a large number of propagations, while the traditional ramet propagation is limited by the number of mother plants and has a low reproduction coefficient. As for seedling quality, the seedlings obtained by the tissue culture method have stable traits and strong resistance, while the seedlings propagated by traditional ramet are prone to degeneration and weakened resistance after several reproduction cycles. Finally, the tissue culture method is not restricted by seasons and can be produced throughout the year, while the traditional ramet propagation can only be carried out in spring and autumn. This table clearly shows the advantages of the tissue culture method of the present invention over traditional ramet propagation.

[0089] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A plant tissue culture method for edible chrysanthemum, characterized in that: The following steps are involved: Explant selection: Select the tender stems of edible chrysanthemum as explants; Explant pretreatment: first wash the explant with SDS surfactant, then rinse it repeatedly with running water; Disinfection: Place the pretreated explants under ultra-clean sterile conditions and disinfect them with 0.1% mercuric chloride solution for 10 minutes. Gently shake the explants during the disinfection process, then rinse them with sterile water five to six times and place them on sterile absorbent paper to absorb the moisture. Explant segmentation: Cut the sterilized tender stem into segments with 1-2 leaves and a length of 2cm-3cm; Inoculation: Insert the lower part of the explant stem segment vertically into the rooting medium; Aseptic rapid cultivation: including starting tender stem culture and subculture proliferation. The culture medium used for starting tender stem culture is a mixture of MS, IBA0.2mg / L, NAA0.1mg / L, carrageenan 5.5g / L and sucrose 30g / L. The pH of the culture medium is adjusted to 5.

8. The culture is carried out under the conditions of a culture temperature of 25℃±1℃, a light intensity of 3000lx-4000lx and a light duration of 8h / d. On the fifth day of culture, green protrusions appear on the stem segments and tender stems gradually differentiate. The plant height reaches 3cm-5cm in one month. During subculture proliferation, the tender stems are cut into small segments with one node, one bud and one leaf. The base of the cut segments is inserted into a culture medium mixed with MS, IBA0.2mg / L and NAA0.1mg / L. The axillary buds are grown into new plantlets after 3-4 weeks of culture. A large number of test tube seedlings are obtained by repeatedly cutting the stem segments. Rooting and hardening of seedlings: When the sterile seedlings grow to 5cm-7cm in height, cut the rootless tender stems of the test tube seedlings of about 3cm and transfer them to the MS+NAA0.2mg / L culture medium. The rooting rate can reach 80% within 5-7 days and 100% within 10 days. After 15-20 days of cultivation, each test tube seedling will grow an average of 4-5 thick roots and the leaves will be dark green. Then the seedlings can be hardened. When hardening the seedlings, open the bottle mouth after 3-5 days and take out the test tube seedlings, wash the agar at the roots, transfer them to fine sand and fine mud media, cover with arch shed and cover with film, and ventilate, spray fertilizer and spray pesticides at the same time.

2. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the explant pretreatment step, the concentration of SDS surfactant is 0.5%-1%, and the explant is washed at 20°C-30°C for 10-15 minutes.

3. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the disinfection step, the concentration of the mercuric chloride solution ranges from 0.08% to 0.12%, the disinfection time is 8 to 12 minutes, and during the disinfection process, an oscillating device with an oscillation frequency of 50 to 100 rpm is used to gently shake the explant.

4. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the rooting and seedling hardening step, when the test tube seedlings are transferred to the fine sand and fine mud medium, the volume ratio of the fine sand and the fine mud is 1:1-2, and when the arch shed is covered with film, the relative humidity in the arch shed is maintained at 80%-90%, ventilation is performed 2-3 times a day, and each ventilation time is 15-30 minutes. When spraying fertilizer, a compound fertilizer containing N, P, and K elements is used, and the concentration thereof is 0.1%-0.2%, and when spraying medicine, carbendazim is used, and the concentration thereof is 0.05%-0.1%.

5. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the aseptic rapid cultivation step, when starting the tender stem culture, the light intensity is 2000lx-3000lx in the first 3 days of culture, and is increased to 3000lx-4000lx from the 4th day, and the culture medium is replaced every 2-3 weeks during subculture proliferation.

6. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the inoculation step, the explant stem segments are inserted into the rooting medium to a depth of 0.5 cm-1 cm, and 3-5 explant stem segments are inoculated in each culture container.

7. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the aseptic rapid cultivation step, for subculture proliferation culture, the medium container is a triangular flask with a capacity of 100mL-250mL, 8-12 subcultured stem segments are inoculated in each triangular flask, and the triangular flask is sealed with a breathable sealing film.

8. The plant tissue culture method of edible chrysanthemum according to claim 1, characterized in that: In the rooting and seedling hardening step, after the test tube seedlings are transferred to the fine sand and fine mud medium, they are shaded with a shading rate of 30%-50% for 1-2 weeks.

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