A tissue culture medium and a tissue culture method for regenerating lycoris

Abstract

This invention belongs to the field of plant propagation and cultivation technology, specifically relating to a tissue culture medium and method for the regeneration of Lycoris radiata. This invention provides a tissue culture medium for Lycoris radiata regeneration, comprising a callus induction medium, a callus proliferation medium, and a callus differentiation medium. In the tissue culture medium and method provided by this invention, single scales are selected as explants, and modified MS medium and B5 medium are used as basal media, with appropriate concentrations of 6-BA, NAA, carbon nanotubes, and acid-hydrolyzed casein added. Combined with suitable temperature and light, this facilitates the induction of Lycoris radiata callus proliferation and differentiation, increases the propagation coefficient of Lycoris radiata, and yields a large number of uniform Lycoris radiata tissue culture seedlings.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation and cultivation technology, specifically relating to a tissue culture medium and a regeneration tissue culture method for Lycoris radiata. Background Technology

[0002] Lycoris radiata, also known as cockroach flower, one-arrow arrow, crow garlic, dragon claw flower, and wild daffodil, is a plant belonging to the genus Lycoris in the family Amaryllidaceae. Lycoris radiata includes varieties such as Chinese Lycoris, Lycoris floribunda, Lycoris floribunda, Lycoris rosea, and Lycoris longituba. Among them, Lycoris longituba (Y.Hsu & G.J.Fan) is a perennial herb of the genus Lycoris in the family Amaryllidaceae. Its leaves are strap-shaped and basal, gradually narrowing at the apex, rounded, green, with a distinct pale central band; the umbel-shaped inflorescence has 5-7 flowers; the buds are pale purple, the flowers are white, the perianth lobes have slightly pale red stripes on the ventral side, are oblong, slightly revolute at the apex, and the edges are not wrinkled; the corolla tube is 4-6 cm long; the stamens are slightly shorter than the perianth. Lycoris longituba flowers from July to August, produces capsules, and the seeds are black. Lycoris chinensis Traub prefers to grow in moist environments, such as shady and damp hillsides, under rocks and cliffs, but it also grows well in sunny locations. It is also tolerant of partial shade and drought, somewhat cold-hardy, and quite resilient. It is not particular about soil type and is naturally distributed only in Jiangsu Province. Lycoris chinensis Traub is a perennial herb with an ovoid bulb about 4 cm in diameter. Leaves emerge in spring; the leaves are strap-shaped, about 35 cm long and 2 cm wide, with a rounded apex, green in color, and a distinct pale band in the middle. The flowering stem is about 60 cm tall. There are two oblanceolate bracts. The umbel has 5-6 yellow flowers. The perianth lobes have a pale yellow midrib on the back, are oblanceolate, about 6 cm long and 1 cm wide, and exhibit strong recurving and wrinkling. The perianth tube is about 1.7-2.5 cm long. The stamens are nearly equal in length to or slightly protrude beyond the perianth, with yellow filaments. The upper part of the style is rose-red. Chinese Lycoris is distributed in Henan, Jiangsu, Zhejiang, Shaanxi and other places, and mostly grows in shady and damp places on hillsides.

[0003] The bulbs of *Lycoris radiata* and *Lycoris radiata* contain alkaloids such as lycorine, lycoramine, galantamine, and lycoramine, which can be used as raw materials for pharmaceuticals. Among these, galantamine and lycoramine are present in higher concentrations and have detoxifying and nodule-dispersing effects, treating sore throat, boils, carbuncles, and scrofula. *Lycoris radiata* bulbs are typically harvested in spring and autumn after 2-3 years, washed, and dried, or sliced ​​and dried. For external use, they are crushed and applied to the affected area. In landscaping, *Lycoris radiata* can be used as ground cover, planted in flower borders, or naturally planted among rocks. Both *Lycoris radiata* and *Lycoris radiata* have robust flower stalks and tall flower stems, making them ideal cut flower materials.

[0004] However, *Lycoris radiata* is a species with a narrow distribution, found only on shady, damp mountain slopes in Jiangsu Province, and its population is relatively small. While *Lycoris chinensis* has a wider distribution than *Lycoris radiata*, its reserves are only slightly larger in Jiangsu and Zhejiang provinces. Both species typically reproduce by seeds and bulb division, but both suffer from low seed yields, long germination times, and short vegetative growth periods. From sowing to flowering takes 5-6 years, resulting in a long bulb formation cycle and high cultivation costs. For bulb division, a single flowering bulb can only naturally produce 1-2 offsets per year, leading to a very low propagation coefficient. Therefore, there is an urgent need to improve the regeneration tissue culture medium for *Lycoris radiata* to increase its propagation coefficient. Summary of the Invention

[0005] The purpose of this invention is to provide a tissue culture medium and a method for regenerating Lycoris radiata. By using the tissue culture medium provided by this invention to rapidly propagate Lycoris radiata callus regeneration tissue using single scales of Lycoris radiata as explants, the propagation coefficient of Lycoris radiata can be improved.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] This invention provides a tissue culture medium for the regeneration of Lycoris radiata, the tissue culture medium comprising a callus induction medium, a callus proliferation medium, and a callus differentiation medium;

[0008] The callus induction medium uses B5 medium as the basic medium and also includes: 20-30 mg / L 6-BA, 15-20 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 1.0-3.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0009] The callus proliferation culture medium uses B5 medium as the basic medium and also includes: 10-15 mg / L 6-BA, 3.0-5.0 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 0.5-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0010] The callus differentiation medium uses modified MS medium as the basic medium and also includes: 2.0–4.0 mg / L 6-BA, 1.0–3.0 mg / L NAA, 0.5–1.0 g / L acid-hydrolyzed casein, 1.0–2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5–7.0 g / L agar;

[0011] The modified MS medium comprises: ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium chloride dihydrate 880 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, sodium molybdate dihydrate 0.25 mg / L, cobalt chloride hexahydrate 0.025 mg / L, copper sulfate pentahydrate 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, nicotinic acid 5.0 mg / L, vitamin B1 10.0 mg / L, vitamin B6 1.0 mg / L, glycine 2 mg / L, and inositol 100 mg / L.

[0012] This invention provides a method for culturing induced shoots of Lycoris radiata, wherein the culturing method uses the tissue culture medium described in the above technical solution and includes the following steps:

[0013] Single scales of Lycoris radiata were inoculated onto callus induction medium for induction culture to obtain single scales that had produced callus.

[0014] The single scales that have produced callus are transferred to a callus proliferation culture medium for proliferation culture to obtain callus tissue.

[0015] The callus mass was transferred to a callus differentiation medium for differentiation culture to obtain buds; the Lycoris radiata was Lycoris radiata and / or Lycoris chinensis.

[0016] Preferably, the single scale is a slice cut from a single leaf of the Lycoris bulb, and the single scale retains the tissue on the bulb disc; before preparing the single scale, the outermost 2 to 3 layers of leaves of the Lycoris bulb are peeled off.

[0017] Preferably, the length and width of the single scale are (0.5~1.5)cm×(0.5~1.5)cm.

[0018] Preferably, the induction culture is a dark culture; the induction culture time is 65-75 days; the proliferation culture is a dark culture; the proliferation culture time is 30-40 days; the differentiation culture is a light culture, the light culture time is 12-16 h / d, the light culture intensity is 800-1200 lx; ​​the differentiation culture time is 50-60 days.

[0019] The temperature for the induction culture, proliferation culture and differentiation culture was 25±1℃.

[0020] Preferably, the diameter of the callus mass is 1.5 to 2.5 cm.

[0021] This invention provides a method for regenerating Lycoris callus tissue through tissue culture, comprising the following steps:

[0022] The buds obtained by the cultivation method described above are transferred to a bud-strengthening culture medium for bud-strengthening culture to obtain rootless seedlings.

[0023] The rootless seedlings were transferred to a strong seedling rooting medium for strong seedling rooting culture to obtain tissue culture seedlings.

[0024] Preferably, the bud-strengthening culture is a light culture, with a light culture time of 12-16 h / d and a light culture intensity of 800-1200 lx; ​​the bud-strengthening culture time is 30-40 days.

[0025] The seedling rooting culture is a light culture, with a light culture time of 12-16 h / d and a light intensity of 800-1200 lx; ​​the seedling rooting culture time is 30-40 days.

[0026] Preferably, the bud-strengthening culture medium uses MS medium as the basic culture medium and further includes: 1.5-3.0 mg / L NAA, 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L carbon nanotubes, 40-60 g / L sucrose and 6.5-7.0 g / L agar;

[0027] The seedling rooting medium uses MS medium as the basic medium and also includes: 1.5-2.0 mg / L 6-BA, 0.5-1.0 mg / L NAA, 2.0-2.5 mg / L IBA, 1.0-2.0 mg / L carbon nanotubes, 40-60 g / L sucrose and 6.5-7.0 g / L agar.

[0028] Preferably, the temperature for both the bud cultivation and seedling rooting cultivation is 25±1℃.

[0029] The beneficial effects of the present invention are as follows: The present invention provides a tissue culture medium for the regeneration of Lycoris radiata and Lycoris radiata in China, wherein the tissue culture medium includes a callus induction medium, a callus proliferation medium and a callus differentiation medium;

[0030] In the tissue culture medium, the added NAA (naphthaleneacetic acid) and 6-BA (6-benzylaminopurine) can stimulate cell division, promote root and shoot formation, and induce callus formation in Lycoris radiata. Acid-hydrolyzed casein promotes the formation, proliferation, and differentiation of callus in Lycoris radiata bulbs. Carbon nanotubes can promote the differentiation, shoot growth, and rooting of callus in Lycoris radiata. The combined effects of 6-BA, NAA, carbon nanotubes, and acid-hydrolyzed casein facilitate the induction of callus formation, proliferation, differentiation, and rooting in Lycoris radiata. Combined with B5 medium and modified MS medium, the absorption capacity of Lycoris radiata to nutrients is enhanced, ultimately increasing the propagation coefficient of Lycoris radiata and obtaining a large number of Lycoris radiata tissue culture seedlings. The B5 medium, with its increased nicotinic acid and calcium content, facilitates nutrient absorption by Lycoris radiata. The modified MS medium, with its increased amounts of calcium, vitamin B1, vitamin B6, and organic matter such as nicotinic acid, promotes the absorption of nitrate nitrogen from the medium, thus aiding callus formation. The increased organic matter content in the MS medium enhances cell membrane permeability and strengthens nutrient absorption by Lycoris radiata. The results of these examples demonstrate that the tissue culture medium of this invention can yield a large number of Lycoris radiata tissue culture seedlings, increasing the propagation coefficient of Lycoris radiata. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0032] Figure 1 This is the situation on day 28 of induction of callus tissue from the bulb of Lycoris radiata in Example 1;

[0033] Figure 2 This is the situation on day 42 of induction of callus tissue from the bulb of Lycoris radiata in Example 1;

[0034] Figure 3 This is the proliferation of Lycoris radiata callus on day 35 of Example 1;

[0035] Figure 4 This is the differentiation of Lycoris radiata callus on day 35 of Example 1;

[0036] Figure 5 This is a longitudinal section of the bulb of Lycoris radiata. Detailed Implementation

[0037] This invention provides a tissue culture medium for the regeneration of Lycoris radiata, wherein the tissue culture medium includes a callus induction medium, a callus proliferation medium, and a callus differentiation medium;

[0038] The callus induction medium uses B5 medium as the basic medium and also includes: 20-30 mg / L 6-BA, 15-20 mg / L NAA, acid-hydrolyzed casein 0.5-1.0 g / L, carbon nanotubes 1.0-3.0 mg / L, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0039] The callus induction culture medium of the present invention uses B5 medium as the basic culture medium and further includes: 20-30 mg / L 6-BA, 15-20 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 1.0-3.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar; preferably, B5 medium is used as the basic culture medium, and the following are also added: 20-30 mg / L 6-BA, 15-20 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 1.0-3.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar. The concentration of NAA in the callus induction medium of this invention is 15-20 mg / L, preferably 17-19 mg / L, and more preferably 18 mg / L; the concentration of 6-BA in the callus induction medium is 20-30 mg / L, preferably 13-18 mg / L, and more preferably 16 mg / L. The concentration of agar in the callus induction medium is 6.5-7.0 g / L, and more preferably 6.7 g / L. The concentration of acid-hydrolyzed casein in the callus induction medium is 0.5-1.0 g / L, preferably 0.7-0.9 g / L, and more preferably 0.8 g / L. The NAA, 6-BA, acid-hydrolyzed casein, and carbon nanotubes added in this invention can promote callus formation and improve the callus induction rate.

[0040] In this invention, the pH value of the callus induction culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0041] The callus proliferation culture medium of this invention uses B5 medium as the basic culture medium and further includes: 10-15 mg / L 6-BA, 3.0-5.0 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 0.5-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar; preferably, it uses B5 medium as the basic culture medium and further adds: 10-15 mg / L 6-BA, 3.0-5.0 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 0.5-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar. The concentration of NAA in the callus proliferation culture medium of this invention is 3.0-5.0 mg / L, preferably 3.3-4.6 mg / L, and more preferably 4.0 mg / L. The concentration of 6-BA in the callus proliferation medium is 10-15 mg / L, preferably 11-14 mg / L, and more preferably 13 mg / L. The concentration of carbon nanotubes in the callus proliferation medium is 0.5-2.0 mg / L, preferably 1.3-1.8 mg / L, and more preferably 1.5 mg / L. The concentration of acid-hydrolyzed casein in the callus proliferation medium is 0.5-1.0 g / L, preferably 0.6-0.8 g / L, and more preferably 0.8 g / L. The concentration of agar in the callus proliferation medium is 6.5-7.0 g / L, and more preferably 6.8 g / L. In this invention, 6-BA, NAA, acid-hydrolyzed casein, and carbon nanotubes work together to promote the proliferation of Lycoris radiata callus and shorten the proliferation culture time.

[0042] In this invention, the pH value of the callus proliferation culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0043] The callus differentiation culture medium of the present invention uses modified MS medium as the basic culture medium and further includes: 2.0-4.0 mg / L 6-BA, 1.0-3.0 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar; preferably, it uses modified MS medium as the basic culture medium and further adds: 2.0-4.0 mg / L 6-BA, 1.0-3.0 mg / L NAA, 0.5-1.0 g / L acid-hydrolyzed casein, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0044] The concentration of NAA in the callus differentiation medium of the present invention is 1.0–2.0 mg / L, preferably 1.3–1.7 mg / L, and more preferably 1.5 mg / L. The concentration of 6-BA in the callus differentiation medium is 2.0–4.0 mg / L, preferably 2.5–3.5 mg / L, and more preferably 3.0 mg / L. The concentration of acid-hydrolyzed casein in the callus differentiation medium is 0.5–1.0 g / L, preferably 0.6–0.8 g / L, and more preferably 0.7 g / L. The concentration of carbon nanotubes in the callus differentiation medium is 1.0–2.0 mg / L, preferably 1.1–1.6 mg / L, and more preferably 1.4 mg / L. The concentration of agar in the callus differentiation medium is 6.5–7.0 g / L, and more preferably 6.7 g / L. This invention promotes callus differentiation and yields a large number of clustered shoots by using appropriate concentrations of 6-BA, NAA, acid-hydrolyzed casein, and carbon nanotubes.

[0045] In this invention, the pH value of the callus differentiation culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0046] The modified MS culture medium of this invention comprises: ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium chloride dihydrate 880 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, sodium molybdate dihydrate 0.25 mg / L, cobalt chloride hexahydrate 0.025 mg / L, copper sulfate pentahydrate 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, nicotinic acid 5.0 mg / L, vitamin B1 10.0 mg / L, vitamin B6 1.0 mg / L, glycine 2 mg / L, and inositol 100 mg / L.

[0047] This invention increases the amount of CaCl2·2H2O in the modified MS medium, which is beneficial to improving the absorption rate of nitrate nitrogen in the medium by Lycoris radiata, thereby promoting the induction of callus and the growth of tissue culture seedlings; at the same time, the increased amount of vitamin B1 and niacin facilitates the increase of cell membrane permeability, which helps the absorption of nutrients in the medium and promotes the differentiation of callus and the growth of buds.

[0048] The callus induction medium, callus proliferation medium, and callus differentiation medium provided by this invention all contain sucrose, acid-hydrolyzed casein, and carbon nanotubes. Sucrose, acid-hydrolyzed casein, and carbon nanotubes can provide the carbon and nitrogen sources required for plant growth, promoting callus formation and the germination and growth of clustered shoots. This invention, with appropriate concentrations of 6-BA and NAA, promotes the growth and differentiation of Lycoris radiata shoots and the rooting of rootless seedlings. The tissue culture medium provided by this invention has good application effects, improves the propagation coefficient, and shortens the tissue culture time.

[0049] This invention does not have any particular limitations on the source of the B5 medium, MS medium, 6-BA, NAA, carbon nanotubes, acid-hydrolyzed casein, sucrose, and agar; conventional commercially available products can be used. In the embodiments of this invention, the acid-hydrolyzed casein used was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0050] The callus induction culture medium, callus proliferation culture medium and callus differentiation culture medium provided by the present invention all contain carbon nanotubes. The carbon nanotubes mentioned in the present invention are preferably any one of single-walled carbon nanotubes, multi-walled carbon nanotubes and carboxylated multi-walled carbon nanotubes; from a cost perspective, multi-walled carbon nanotubes are preferred.

[0051] This invention provides a method for culturing induced shoots of Lycoris radiata, wherein the culturing method uses the tissue culture medium described in the above technical solution and includes the following steps:

[0052] Single scales of Lycoris radiata were inoculated onto callus induction medium for induction culture to obtain single scales that had produced callus.

[0053] The single scales that have produced callus are transferred to a callus proliferation culture medium for proliferation culture to obtain callus tissue.

[0054] The callus mass was transferred to a callus differentiation medium for differentiation culture to obtain buds; the Lycoris radiata was Lycoris radiata and / or Lycoris chinensis.

[0055] This invention involves inoculating single scales of Lycoris radiata onto a callus induction medium for induction culture to obtain single scales that have produced callus. The Lycoris radiata described in this invention includes Lycoris radiata and / or Lycoris chinensis. Preferably, single scales of Lycoris radiata and Lycoris chinensis are used as explants for seedling cultivation.

[0056] In this invention, the single scale is a slice cut from a single leaf of the Lycoris radiata bulb, and the single scale retains the tissue on the bulb disc; before preparing the single scale, the outermost 2-3 layers of leaves of the Lycoris radiata bulb are peeled off. The preferred length and width of the single scale in this invention are (0.5-1.5) cm × (0.5-1.5) cm, more preferably (1.2-1.5) cm × (0.9-1.0) cm.

[0057] In this invention, the single scales of the Lycoris radiata are preferably obtained by the following method: The outermost 2-3 layers of scale leaves are removed from the bulb; all roots and the lower 1 / 3 and upper 1 / 2-2 / 3 of the scale leaves are removed; the remaining bulb disc and scale leaves are then divided to obtain a bulb disc block; the innermost 2 layers of scale leaves of the bulb disc block are removed to obtain a bulb block; the bulb block is then cut into single scales and inoculated. The single scales of this invention preferably retain a small amount of tissue from the bulb disc, which is beneficial for inducing callus formation. The diameter of the bulb of this invention is preferably 3-5 cm, more preferably 4 cm; healthy Lycoris radiata bulbs that have grown for more than 3 years are preferably collected; the collection time is preferably from late April to late June during the leafless period. The bulb includes roots, a bulb disc, and scale leaves; in this invention, the outermost 2-3 layers of scale leaves are preferably removed, more preferably the outermost 2 layers of scale leaves are removed. After obtaining the bulb with the outermost 2-3 layers of scales removed, preferably, all roots are removed, the lower part of the bulb disc (representing 1 / 3 of its volume) is removed, and the upper part of the scale leaves (representing 1 / 2-2 / 3 of their volume) is removed, resulting in a bulb disc with scales. This bulb disc with scales comprises a bulb disc representing 2 / 3 of the original bulb disc volume and scale leaves representing 1 / 3-1 / 2 of the original scale leaves volume. Preferably, the upper and lower parts of the Lycoris bulb are positioned with the bulb disc at the bottom and the scale leaves at the top. Preferably, the bulb disc with scales is divided into bulb disc blocks; the division is preferably equal. Preferably, the innermost 2 layers of scales are removed from the bulb disc block to obtain bulb blocks; these bulb blocks preferably have 2-3 layers of scales. The bulb blocks are cut into single scales and then inoculated. Preferably, a small amount of tissue from the bulb disc is retained in the single scales, which is beneficial for inducing callus formation. The timing of bulb collection and the setting of bulb diameter in this invention are intended to improve the callus induction rate. A longitudinal section of the Lycoris radiata bulb in the embodiment is shown below. Figure 5 .

[0058] In this invention, prior to induction culture, it is preferable to further include: cleaning and disinfecting the bulb tubers before cutting them into single scales. The cleaning process preferably includes: first washing with liquid detergent and agitation for 20-40 minutes, followed by rinsing with running water for 60-90 minutes. Then, rinsing with 75% alcohol for 30-45 seconds, and then rinsing with sterile water 3-4 times. Preferably, the first washing with liquid detergent and agitation for 20-40 minutes, more preferably 30-40 minutes; rinsing with running water for 60-90 minutes, more preferably 75 minutes; rinsing with 75% alcohol for 30-45 seconds, more preferably 40 seconds; and then rinsing with sterile water 3-4 times, more preferably 4 times.

[0059] The disinfection method of this invention preferably includes: disinfecting with a sodium hypochlorite solution with an effective chlorine content of 2.0% to 3.0% for 10 to 15 minutes, followed by rinsing with sterile water 4 to 5 times. Preferably, the invention uses a sodium hypochlorite solution with an effective chlorine content of 2.0% to 3.0%, more preferably a sodium hypochlorite solution with an effective chlorine content of 2.5%, and the disinfection time with the sodium hypochlorite solution is preferably 10 to 15 minutes, more preferably 12 to 13 minutes; finally, preferably rinsing with sterile water 4 to 5 times, more preferably 4 times.

[0060] Existing techniques for rapid propagation of Lycoris radiata via tissue culture mostly use the double scales of the bulb disc as explants. A single Lycoris radiata bulb can only be divided into 6-8 bulb discs for tissue culture seedlings. The bulb discs can only induce bud formation, which then leads to seedling development, resulting in low propagation efficiency. This invention uses single scales as explants for rapid propagation through tissue culture. One Lycoris radiata bulb can yield more than 30 single scales. Single scales can induce callus tissue, resulting in more differentiated buds than bulb discs, thus significantly increasing the propagation coefficient and shortening the tissue culture cycle.

[0061] After cleaning and disinfecting the bulb tubers, the present invention preferably cuts the bulb tubers into single scales and inoculates them onto a callus induction medium for induction culture to obtain single scales that have produced callus tissue. The preferred size of the single scale in the present invention is 1.0–1.5 μm. 2 More preferably 1.3m 2 The preferred method for inoculating the single scales in this invention is to place them horizontally, concave side down, on the callus induction culture medium. In this invention, the induction culture temperature is preferably 24–26°C, more preferably 25°C. The induction culture is preferably dark culture; the induction culture time is preferably 65–75 days, more preferably 68–73 days, and more preferably 70 days. The induction culture time is calculated from the time the single scales are inoculated into the callus induction culture medium until the single scales that produce pale yellow callus tissue are obtained.

[0062] After obtaining single scales of callus tissue that appear pale yellow, the present invention transfers the single scales that have produced callus tissue to a callus tissue proliferation culture medium for proliferation culture to obtain callus tissue clumps.

[0063] In this invention, the temperature for the proliferation culture is preferably 24–26°C, more preferably 25°C. In this invention, the proliferation culture is preferably conducted in the dark. The proliferation culture time is preferably 30–40 days, more preferably 35–40 days. The proliferation culture time in this invention is calculated from the time the single scale that has produced callus is transferred to the callus proliferation medium until the diameter of the callus mass reaches 1.5–2.5 cm.

[0064] After obtaining callus tissue masses with a diameter of 1.5 to 2.5 cm through proliferation culture, the present invention transfers the callus tissue masses to callus differentiation culture medium for differentiation culture to obtain buds.

[0065] In this invention, callus tissue masses with a diameter of 1.5–2.5 cm are preferably transferred to callus differentiation medium for differentiation culture, more preferably with a diameter of 1.8–2.0 cm. The preferred temperature for differentiation culture is 24–26°C, more preferably 25°C. In this invention, the differentiation culture is preferably light-induced; the light-induced culture time is preferably 12–16 h / d, more preferably 14–16 h / d, more preferably 16 h / d; the light intensity is preferably 800–1200 lx, more preferably 1000–1100 lx. The preferred culture time for differentiation culture is 50–60 days, more preferably 55–58 days.

[0066] This invention also provides a method for regenerating Lycoris callus tissue through tissue culture, wherein the regeneration tissue culture method uses the tissue culture medium described in the above technical solution and includes the following steps:

[0067] The buds obtained by the cultivation method described above are transferred to a bud-strengthening culture medium for bud-strengthening culture to obtain rootless seedlings.

[0068] The rootless seedlings were transferred to a strong seedling rooting medium for strong seedling rooting culture to obtain tissue culture seedlings; the Lycoris radiata was Lycoris radiata and / or Lycoris chinensis.

[0069] In this invention, the buds preferably include clustered buds, and after differentiation culture to obtain clustered buds with a height of 1.5 to 2.5 cm; preferably, the clustered buds are separated from the callus tissue and transferred to a bud strengthening culture medium for bud strengthening culture to obtain rootless seedlings.

[0070] The preferred temperature for bud-strengthening culture in this invention is 24–26°C, more preferably 25°C. In this invention, the bud-strengthening culture is preferably light-based; the light-based culture time is preferably 12–16 h / d, more preferably 14–16 h / d, and more preferably 16 h / d; the light-based culture intensity is preferably 800–1200 lx, more preferably 1000 lx. The preferred culture time for bud-strengthening culture in this invention is 30–40 days, more preferably 33–35 days.

[0071] The bud-strengthening culture medium of the present invention preferably uses MS medium as the basal medium and further includes: 1.5–3.0 mg / L NAA, 2.0–4.0 mg / L 6-BA, 1.0–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose, and 6.5–7.0 g / L agar; more preferably, it uses a modified MS medium as the basal medium and further adds: 1.5–3.0 mg / L NAA, 2.0–4.0 mg / L 6-BA, 1.0–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose, and 6.5–7.0 g / L agar. The concentration of 6-BA in the bud-strengthening culture medium of the present invention is preferably 2.0–4.0 mg / L, more preferably 2.5–3.6 mg / L, and more preferably 2.0–3.0 mg / L. The concentration of NAA in the bud-strengthening medium is preferably 1.5–3.0 mg / L, more preferably 1.8–2.5 mg / L, and even more preferably 2.0 mg / L. The concentration of carbon nanotubes in the bud-strengthening medium is preferably 1.0–2.0 mg / L, more preferably 1.3–1.8 mg / L, and even more preferably 1.5 mg / L. The carbon nanotubes used in this invention are preferably multi-walled carbon nanotubes. The concentration of agar in the bud-strengthening medium is preferably 6.5–7.0 g / L, and even more preferably 6.7 g / L. This invention promotes the growth of clustered buds under the action of modified MS medium, NAA, 6-BA, and carbon nanotubes, thereby obtaining rootless seedlings.

[0072] In this invention, the pH value of the bud-strengthening culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0073] The time for cultivating strong shoots in this invention is calculated from the time of transfer to the strong shoot culture medium until rootless seedlings with 3-4 leaves or a bulb circumference of 0.8-1.0 cm are obtained.

[0074] After obtaining rootless seedlings with 3-4 leaves or a bulb circumference of 0.8-1.5 cm through strong bud culture, the present invention transfers the rootless seedlings to a strong seedling rooting culture medium for strong seedling rooting culture to obtain tissue culture seedlings.

[0075] The seedling rooting medium of the present invention preferably uses MS medium as the basic medium and further includes: 1.5-2.0 mg / L 6-BA, 0.5-1.0 mg / L NAA, 2.0-2.5 mg / L IBA, 1.0-2.0 mg / L carbon nanotubes, 40-60 g / L sucrose and 6.5-7.0 g / L agar; preferably, the seedling rooting medium uses MS medium as the basic medium and further adds: 1.5-2.0 mg / L 6-BA, 0.5-1.0 mg / L NAA, 2.0-2.5 mg / L IBA, 1.0-2.0 mg / L carbon nanotubes, 40-60 g / L sucrose and 6.5-7.0 g / L agar.

[0076] The concentration of 6-BA in the seedling rooting medium of the present invention is preferably 1.5–2.0 mg / L, more preferably 1.6–1.9 mg / L, and even more preferably 1.8 mg / L. The concentration of NAA in the seedling rooting medium is preferably 0.5–1.0 mg / L, more preferably 0.7–0.9 mg / L, and even more preferably 0.8 mg / L. The concentration of IBA in the seedling rooting medium is preferably 2.0–2.5 mg / L, more preferably 2.1–2.4 mg / L, and even more preferably 2.3 mg / L. The concentration of carbon nanotubes in the seedling rooting medium is preferably 1.0–2.0 mg / L, more preferably 1.1–1.7 mg / L, and even more preferably 1.6 mg / L. The carbon nanotubes used in the present invention are preferably multi-walled carbon nanotubes. The concentration of agar in the seedling rooting medium is preferably 6.5–7.0 g / L, and even more preferably 6.7 g / L. The NAA, 6-BA, IBA and carbon nanotubes of the present invention can promote the rooting and growth of rootless Lycoris radiata seedlings, resulting in tissue culture seedlings.

[0077] In this invention, the pH value of the seedling rooting culture is preferably 5.75 to 5.85, more preferably 5.80.

[0078] In this invention, the preferred temperature for the seedling rooting culture is 24–26°C, more preferably 25°C. The seedling rooting culture is preferably conducted under light conditions, with the light culture duration preferably 12–16 h / d, more preferably 13–16 h / d, and more preferably 16 h / d; the light intensity is preferably 800–1200 lx, more preferably 1000 lx. The preferred culture time for the seedling rooting culture is 30–40 days, more preferably 30–35 days.

[0079] In this invention, the induction culture, proliferation culture, differentiation culture, shoot strengthening culture, and seedling rooting culture are preferably carried out in tissue culture bottles. There are no special limitations on the specifications of the tissue culture bottles; conventional products are acceptable. The callus induction culture medium, callus differentiation culture medium, shoot strengthening culture medium, and seedling rooting culture medium described in this invention are sterilized and then poured into the tissue culture bottles. This invention does not have special limitations on the sterilization method; conventional methods are acceptable.

[0080] The present invention obtains Lycoris radiata test-tube seedlings with a bulb circumference of 1.5-2.0 cm, 3-5 roots, and a weight of 0.5-1.0 g after strong seedling rooting culture.

[0081] The bulb circumference of the test-tube seedlings described in this invention is preferably 1.5–2.0 cm, more preferably 1.6–1.8 cm. The root system of the test-tube seedlings described in this invention is preferably 3–5, more preferably 4. The weight of the test-tube seedlings described in this invention is preferably 0.5–1.0 g, more preferably 0.7–0.8 g.

[0082] The seedlings described in this invention are cultured to obtain test-tube seedlings. After the test-tube seedlings meet the three conditions of bulb diameter 0.3-0.5cm, root system 3-5, and weight 0.5-1.0g, they are hardened off, transplanted, and cultured to improve the survival rate of the test-tube seedlings.

[0083] After obtaining the test-tube seedlings, the present invention preferably performs hardening-off and transplanting on the seedlings. In the present invention, the hardening-off is preferably completed indoors, the hardening-off temperature is preferably the temperature at which Lycoris radiata grows, and the hardening-off time is preferably 4-6 days, more preferably 5-6 days. Preferably, during hardening-off, the caps of the rooting culture bottles of strong seedlings are loosened, and the bottles are first placed in the buffer room of the tissue culture room for 2-3 days, and then placed under natural diffused light for 2-3 days.

[0084] In this invention, during transplanting, the roots of the hardened test-tube seedlings are preferably washed and then transplanted into a substrate for cultivation. The substrate for transplanting and cultivation preferably includes peat moss, perlite, and garden soil. The preferred volume ratio of peat moss, perlite, and garden soil in this invention is (1-2):(0.5-1.0):(1-2). The preferred transplanting temperature is 18-30℃, more preferably 20-25℃, and even more preferably 25℃. Before transplanting, this invention preferably disinfects the substrate, preferably using an 800-1000 times dilution of a broad-spectrum fungicide, more preferably 800 times. The preferred broad-spectrum fungicide in this invention is chlorothalonil.

[0085] This invention does not have any special limitations on the source and specifications of the seedling trays used in the transplanting and cultivation process; conventional products are acceptable. The preferred specifications of the seedling trays used in this invention are 10 rows × 10 columns. After transplanting, sufficient water should be applied to help the roots establish, followed by normal water and fertilizer management.

[0086] The innovation of this invention lies in using single scales of Lycoris radiata as explants and employing a specific combination of growth regulators to induce callus tissue, thereby increasing the propagation coefficient of Lycoris radiata.

[0087] This invention uses single scales of *Lycoris radiata* and *Lycoris radiata* as explants to induce callus formation. After proliferation culture, callus is induced to differentiate into clusters of shoots. Following seedling and rooting cultures, uniform and robust plants are obtained, ensuring both ornamental value and medicinal quality. This provides technical support for their large-scale cultivation and lays the technical foundation for establishing an Agrobacterium-mediated genetic transformation system for *Lycoris radiata* and *Lycoris radiata*. Furthermore, using single scales as explants significantly improves the propagation efficiency and propagation coefficient of tissue culture propagation compared to using bulb discs. Moreover, this technique preserves only a small amount of tissue from the bulb disc, making sterilization easier and eliminating the need for heavy metal mercuric chloride sterilization; common chlorine-based disinfectants can achieve sterilization.

[0088] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0089] The modified MS medium was used in the following examples and comparative examples, and its specific composition is as follows:

[0090] The modified MS medium consisted of: ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium chloride dihydrate 880 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, sodium molybdate dihydrate 0.25 mg / L, cobalt chloride hexahydrate 0.025 mg / L, copper sulfate pentahydrate 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, nicotinic acid 5.0 mg / L, vitamin B1 10.0 mg / L, vitamin B6 1.0 mg / L, glycine 2 mg / L, and inositol 100 mg / L.

[0091] The method for improving MS culture medium is to increase the amount of calcium chloride dihydrate to twice the amount in the original formula, that is, to add 880 mg / L of calcium chloride dihydrate; and to increase the amount of organic components vitamin B1 and niacin to 10 times the original amount, namely vitamin B1 10.0 mg / L and niacin 5.0 mg / L; and to increase the concentration of vitamin B6 to twice the original amount, to 1.0 mg / L.

[0092] Example 1: Method for establishing a regeneration system for Lycoris radiata using single scales

[0093] (1) Obtaining explants: During the leafless stage of Lycoris radiata, i.e., late April, healthy 4-year-old bulbs with a diameter of about 4.5 cm were dug up. The outermost two layers of scale leaves were peeled off, all roots were removed, 1 / 3 of the lower part of the bulb plate was removed, and 1 / 2 to 2 / 3 of the upper part of the scale leaves were removed. The surface soil was washed off with a brush, and the bulb plate was divided into 8 equal parts according to its size, resulting in 8 bulbs with scale leaves. Then, the innermost two layers of scale leaves of each bulb were removed, and the surface area of ​​each bulb was 1.5 to 2.0 cm². 2 Each bulb contains two layers of scales; the bulbs are washed with liquid detergent and shaken for 15 minutes, then rinsed with running water for 30 minutes; the resulting material is then transferred to a clean bench and rinsed with 75% alcohol for 40 seconds, then rinsed three times with sterile water for later use.

[0094] (2) Disinfection of explants: The bulbs obtained in step (1) were sterilized for 15 minutes using sodium hypochlorite solution on a clean bench, rinsed 4 times with sterile water, and the surface moisture was absorbed with sterile absorbent paper before use. The effective chlorine content of the sodium hypochlorite solution was approximately 2.5%.

[0095] (3) Callus induction: The bulb masses disinfected in step (2), i.e., double scales with a small amount of bulb disc, were transferred to an inoculation tray. The double scales were cut into single scales about 1.5 cm long and 1.0 cm wide using a scalpel, while retaining a small amount of tissue from the bulb disc. The single scales were placed horizontally (concave side down) on the callus induction medium for dark culture. The callus induction medium consisted of B5 medium as the basic medium, with the addition of 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar. The pH of the callus induction medium was 5.8. The culture temperature was 25 ± 1℃. After 28 days of culture, the scales began to sprout, and a protrusion appeared at the lower end. After another 42 days of culture, pale yellow callus tissue appeared at the protrusion. The induction culture time was 70 days.

[0096] (4) Callus proliferation: After 70 days of induction culture of callus in step (3), the single scales along with the callus were transferred to proliferation medium for proliferation culture. The proliferation medium consisted of B5 medium as the basic medium, supplemented with 15 mg / L 6-BA, 5.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 1.0 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar; the pH of the proliferation medium was 5.75–5.85. The proliferation culture was conducted in the dark at a temperature of 25 ± 1 °C. After 35 days of culture, the callus proliferated into clumps with a diameter of 1.5–2.5 cm.

[0097] (5) Callus differentiation: The callus obtained in step (4), along with the single scales, was transferred to a callus differentiation medium for differentiation culture. The differentiation medium consisted of modified MS medium as the basic medium, with the addition of 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes. The pH of the callus differentiation medium was 5.75–5.85, the light intensity was 800–1200 lx, and the photoperiod was 16 h / d. The temperature for differentiation culture was 25 ± 1℃. After 25 days of differentiation culture, white buds were observed to differentiate from the pale yellow callus. After another 10 days of differentiation culture, clustered buds differentiated from the callus. After another 20 days of culture, the clustered buds grew into bud masses with a height of 1.5–2.5 cm. From inoculation into the differentiation medium until the clustered shoots grow to a height of 1.5–2.5 cm, the culture time is 55 days. The culture temperature is 25 ± 1℃.

[0098] (6) Bud Strengthening Culture: The clustered buds from step (5) were separated from the callus tissue and transferred to a bud strengthening culture medium for bud strengthening culture. The bud strengthening culture medium consisted of MS as the basic medium, with only 50 g / L sucrose, 6.5–7.0 g / L agar, 3.0 mg / L 6-BA, 1.5 mg / L NAA, and 1.5 mg / L multi-walled carbon nanotubes added. After 30 days of bud strengthening culture, the clustered buds grew into test-tube seedlings with 3–4 leaves or a bulb circumference of 1.0 cm. The culture temperature was 25 ± 1 °C.

[0099] (7) Seedling Strengthening and Rooting Culture: Seedling strengthening and rooting culture can be completed in one step. Specifically, the test-tube seedlings obtained after the bud strengthening culture are transferred to a bud strengthening and rooting culture medium for co-culture. The components of the bud strengthening and rooting culture medium are: MS as the basic medium, supplemented with only 50 g / L sucrose, 6.8 g / L agar, 2.0 mg / L 6-BA, 1.0 mg / L NAA, 2.0 mg / L IBA, and 1.5 mg / L multi-walled carbon nanotubes. The culture temperature is 25 ± 1℃, the light intensity is 800–1200 lx, and the photoperiod is 16 h / d. After 35 days of culture, test-tube seedlings with a bulb circumference of approximately 1.3 cm, 3–5 roots, and a weight of 0.8 g can be obtained.

[0100] (8) Hardening off and transplanting: Hardening off is carried out indoors. After loosening the caps of the tissue culture bottles obtained in step (7), place them in the buffer room of the tissue culture room for 2 days; then move them indoors and place them under natural diffused light for another 3 days. After hardening off, use tweezers to remove the test tube seedlings, wash off the culture medium from the roots, transplant them into 10×10 seedling trays, and water them thoroughly. The cultivation substrate is peat moss: perlite and garden soil, with a volume ratio of peat moss, perlite and garden soil of 1.2:0.5:1. The cultivation substrate must be disinfected with a 900-fold broad-spectrum fungicide. Then, normal water and fertilizer management is carried out.

[0101] Example 2: Method for establishing a Lycoris radiata regeneration system using the single-scale method

[0102] (1) Obtaining explants: During the leafless period of Lycoris radiata in late June, healthy bulbs of Lycoris radiata that are 3 years old or older and have a diameter of about 3.5 cm were dug up. The outermost two layers of scale leaves were peeled off, all roots were removed, 1 / 3 of the lower part of the bulb plate was removed, and 1 / 2 of the upper part of the scale leaves were removed to obtain the bulb plate with scale leaves. The surface soil was washed off with a brush, and the bulb plate was divided into 6 equal parts according to its size. Then, the innermost two layers of scale leaves of each bulb were removed. After removal, the surface size of each bulb was about 2.0 cm. 2 Small pieces containing three layers of scales were collected; the bulb pieces were washed with liquid detergent and shaken for 20 minutes, then rinsed with running water for 25 minutes; the resulting material was then transferred to a clean bench and rinsed with 75% alcohol for 35 seconds, then rinsed three times with sterile water for later use.

[0103] (2) Disinfection of explants: The phosphorus stems obtained in step (1) were sterilized for 12 minutes on a clean bench with sodium hypochlorite solution containing 2.5% available chlorine, rinsed 4 times with sterile water, and dried with sterile absorbent paper before use.

[0104] (3) Callus induction: Transfer the sterilized bulb masses (i.e., double scales with a small amount of bulb disc) from step 2 to an inoculation tray. Use a scalpel to cut the double scales into single scales approximately 1.2 cm long and 0.9 cm wide, retaining a small amount of tissue from the bulb disc. Place the single scales horizontally (concave side down) on the callus induction medium for induction culture. The culture temperature is 25±1℃.

[0105] The callus induction medium consisted of B5 medium as the basal medium, supplemented with 20 mg / L 6-BA, 15 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 3.0 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 7.0 g / L agar, with a pH of 5.8. Induction culture was conducted in the dark. Day 1 of induction culture was defined as the first day after horizontal inoculation of bulblets into the callus induction medium. After 28 days of induction culture, scales began to sprout, and protrusions appeared at the lower ends. After another 14 days of culture, pale yellow callus tissue appeared at the protrusions. Culture was continued for another 28 days to complete the callus induction culture. The total induction culture period was 70 days.

[0106] (4) Callus proliferation: After the induction culture is completed, the single scales and callus tissue are transferred together to the callus proliferation culture medium for proliferation culture.

[0107] The callus proliferation medium consisted of B5 medium as the basal medium, supplemented with 6.9 g / L agar, 30 g / L sucrose, 10 mg / L 6-BA, 3.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 2.0 mg / L multi-walled carbon nanotubes. The pH was 5.8, and the medium was cultured in the dark. After 35 days of proliferation culture, the callus tissue proliferated into clumps approximately 2.0 cm in diameter. The proliferation culture was conducted in the dark at a temperature of 25 ± 1 °C.

[0108] (5) Callus differentiation: 2.0 cm diameter clumps were transferred together to callus differentiation medium for differentiation culture. The callus differentiation medium consisted of modified MS as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.5 mg / L NAA, 0.75 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes. The pH of the callus differentiation medium was 5.8.

[0109] The light intensity for differentiation culture was 800–1200 lx, and the light duration was 16 h. After 23 days of culture, pale yellow callus tissue differentiated into white buds, and after 35 days, clustered buds differentiated from the callus tissue. After another 20 days of culture, the clustered buds grew into bud blocks with a height of 1.5–2.5 cm. The culture temperature was 25 ± 1℃. The culture time from inoculation into the differentiation medium to obtaining bud blocks with a height of 1.5–2.5 cm was 55 days.

[0110] (6) Shoot Enhancement Culture: The clustered shoots, with a length of 1.5–2.5 cm, obtained from the culture were isolated from the callus tissue and transferred to a shoot enhancement culture medium for further cultivation. The shoot enhancement culture medium consisted of MS as the basic medium, supplemented only with 60 g / L sucrose, 6.5 g / L agar, 3.0 mg / L 6-BA, 1.5 mg / L NAA, and 2.0 mg / L multi-walled carbon nanotubes; the pH was 5.80. After 35 days of cultivation, the clustered shoots grew into test-tube seedlings with 3–4 leaves or a bulb circumference of 0.8–1.5 cm; the culture temperature was 25 ± 1 °C.

[0111] (7) Seedling strengthening and rooting culture: Seedling strengthening and rooting culture can be completed in one step. The specific method is to transfer the test-tube seedlings obtained after the seedling strengthening culture to the seedling strengthening and rooting culture medium for co-culture of seedling strengthening and rooting. The composition of the culture medium is: the basic medium is MS, with only 50 g / L sucrose, 6.7 g / L agar, 1.5 mg / L 6-BA, 0.8 mg / L NAA, 2.5 mg / L IBA and 1.0 mg / L multi-walled carbon nanotubes added; the pH value is 5.8, the culture temperature for seedling strengthening and rooting co-culture is 25±1℃, the light intensity is 800~1200lx, and the photoperiod is 16h / d. After 30 days of culture, test-tube seedlings with a bulb circumference of 1.5 cm, 3-5 roots, and a weight of 0.7 g can be obtained.

[0112] (8) Hardening off and transplanting: Hardening off is carried out indoors. The process is as follows: loosen the caps of the tissue culture bottles, place them in the buffer room of the tissue culture room for 3 days, then move them indoors and place them under natural diffused light for another 3 days. After hardening off, use tweezers to remove the test-tube seedlings, wash off the culture medium from the roots, and transplant them into 10×10 seedling trays, watering thoroughly. The cultivation substrate is peat moss, perlite, and garden soil, with a peat moss:perlite:garden soil ratio of 1:0.8:1 (by volume). The cultivation substrate must be disinfected with an 800-fold broad-spectrum fungicide. Normal water and fertilizer management is then carried out.

[0113] Example 3

[0114] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 25 mg / L 6-BA, 15 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0115] Example 4

[0116] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 30 mg / L 6-BA, 15 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0117] Example 5

[0118] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 20 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0119] Example 6

[0120] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 25 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0121] Comparative Example 1

[0122] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 15 mg / L 6-BA, 10 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0123] Comparative Example 2

[0124] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 20 mg / L 6-BA, 10 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0125] Comparative Example 3

[0126] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 25 mg / L 6-BA, 10 mg / L NAA, 1.0 g / L acid hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0127] Comparative Example 4

[0128] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 30 mg / L 6-BA, 10 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0129] Comparative Example 5

[0130] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 35 mg / L 6-BA, 10 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0131] Comparative Example 6

[0132] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 15 mg / L 6-BA, 15 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0133] Comparative Example 7

[0134] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 35 mg / L 6-BA, 15 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0135] Comparative Example 8

[0136] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 15 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0137] Comparative Example 9

[0138] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 35 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0139] Comparative Example 10

[0140] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 15 mg / L 6-BA, 25 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0141] Comparative Example 11

[0142] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 20 mg / L 6-BA, 25 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0143] Comparative Example 12

[0144] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 25 mg / L 6-BA, 25 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0145] Comparative Example 13

[0146] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 30 mg / L 6-BA, 25 mg / L NAA, 1.0 g / L acid hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0147] Comparative Example 14

[0148] Only three steps were performed: (1) obtaining explants, (2) disinfecting explants, and (3) inducing callus. All three steps were the same as in Example 1. The only difference was that the induction medium used was B5 medium as the basic medium, with the addition of 35 mg / L 6-BA, 25 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose, and 6.8 g / L agar.

[0149] The callus induction rates of Examples 1-6 and Comparative Examples 1-14 were calculated according to the formula for calculating callus induction rate, and the results are shown in Table 1.

[0150] Callus induction rate (%) = Number of single scales induced to form callus * 100 / Number of single scales inoculated

[0151] Table 1. Effects of different hormone concentrations on callus induction rate of single scales of Lycoris radiata.

[0152]

[0153]

[0154] Comparative Example 15

[0155] Only three steps were performed: (1) explant acquisition, (2) explant disinfection, and (3) callus induction. All three steps were the same as in Example 1, with the only difference being the composition of the induction culture medium:

[0156] MS medium was used as the basic medium, with the addition of only 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose and 6.8 g / L agar;

[0157] Comparative Example 16

[0158] Only three steps were performed: (1) explant acquisition, (2) explant disinfection, and (3) callus induction. All three steps were the same as in Example 1, with the only difference being the composition of the induction culture medium:

[0159] Using 1 / 2 MS medium as the basic medium, only 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose and 6.8 g / L agar were added;

[0160] Comparative Example 17

[0161] Only three steps were performed: (1) explant acquisition, (2) explant disinfection, and (3) callus induction. All three steps were the same as in Example 1, with the only difference being the composition of the induction culture medium:

[0162] Using WPM medium as the basic medium, only 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose and 6.8 g / L agar were added;

[0163] Comparative Example 18

[0164] Only three steps were performed: (1) explant acquisition, (2) explant disinfection, and (3) callus induction. All three steps were the same as in Example 1, with the only difference being the composition of the induction culture medium:

[0165] Using N6 medium as the basic medium, only 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose and 6.8 g / L agar were added;

[0166] Comparative Example 19

[0167] Only three steps were performed: (1) explant acquisition, (2) explant disinfection, and (3) callus induction. All three steps were the same as in Example 1, with the only difference being the composition of the induction culture medium:

[0168] Using DKW medium as the basic medium, only 30 mg / L 6-BA, 20 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose and 6.8 g / L agar were added.

[0169] The callus induction rate was calculated for Example 1 and Comparative Examples 15-19 according to the formula for calculating the callus induction rate. The results are shown in Table 2.

[0170] Callus induction rate (%) = Number of single scales induced to form callus * 100 / Number of single scales inoculated

[0171] Table 2. Effects of basic culture medium on callus induction rate of Lycoris radiata single scales.

[0172]

[0173] Comparative Example 20

[0174] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0175] The differentiation medium consisted of modified MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA and 1.5 mg / L multi-walled carbon nanotubes.

[0176] Comparative Example 21

[0177] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0178] The differentiation medium consisted of modified MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 0.25 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0179] Comparative Example 22

[0180] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0181] The differentiation medium consisted of modified MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.25 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0182] Example 7

[0183] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0184] The differentiation medium consisted of modified MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 0.50 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0185] Example 8

[0186] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0187] The differentiation medium consisted of modified MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 0.75 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0188] The callus differentiation rate of Examples 1, 7, and 8, and Comparative Examples 20-22 was calculated using the following formula, and the results are shown in Table 3.

[0189] Callus differentiation rate (%) = Number of differentiated callus tissues * 100 / Number of inoculated callus tissues

[0190] Table 3. Effects of acid-hydrolyzed casein on callus differentiation of single scales of Lycoris radiata.

[0191]

[0192]

[0193] As shown in Table 3, the differentiation rate of callus tissue is high when the amount of acid-hydrolyzed casein added is 0.5–1.25 g / L. Considering the cost, the amount of acid-hydrolyzed casein added is 0.5–1 g / L.

[0194] Comparative Example 23

[0195] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0196] The differentiation medium consisted of B5 medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0197] Comparative Example 24

[0198] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0199] The differentiation medium consisted of MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0200] Comparative Example 25

[0201] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0202] The differentiation medium consisted of 1 / 2 MS medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein and 1.5 mg / L multi-walled carbon nanotubes.

[0203] Comparative Example 26

[0204] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0205] The differentiation medium consisted of WPM medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0206] Comparative Example 27

[0207] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0208] The differentiation medium consisted of N6 medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0209] Comparative Example 28

[0210] Only five steps were performed: (1) explant acquisition, (2) explant disinfection, (3) callus induction, (4) callus proliferation, and (5) callus differentiation. All five steps were the same as in Example 1, with the only difference being the composition of the differentiation medium.

[0211] The differentiation medium consisted of DKW medium as the basic medium, supplemented with 7.0 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, 2.0 mg / L NAA, 1.0 g / L acid-hydrolyzed casein, and 1.5 mg / L multi-walled carbon nanotubes.

[0212] The callus differentiation rate of Example 1 and Comparative Examples 23-28 was calculated using the same formula as above, and the results are shown in Table 4. Table 4 shows that the callus differentiation rate of *Lycoris radiata* cultured on the modified MS medium was the highest.

[0213] Table 4. Effects of basic culture medium on the differentiation rate of callus from single scales of Lycoris radiata.

[0214]

[0215] In summary, the technical solution of this invention enables efficient and rapid propagation of *Lycoris radiata* and *Lycoris chinensis*, and also facilitates the exploration of establishing their transgenic technology system. This invention uses single scales as explants to establish a callus-based regeneration technology system for the medicinal and ornamental plants *Lycoris radiata* and *Lycoris chinensis*, providing technical support for bulb propagation and genetic transformation using tissue culture methods, and laying the foundation for their industrial application in ornamental and medicinal fields.

[0216] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture medium for the regeneration of Lycoris radiata, characterized in that, The tissue culture medium includes callus induction medium, callus proliferation medium, and callus differentiation medium; The callus induction medium uses B5 medium as the basic medium and also adds: 20~30 mg / L 6-BA, 15~20 mg / L NAA, 1.0 g / L acid hydrolyzed casein, 2.5 mg / L carbon nanotubes, 30 g / L sucrose and 6.5~7.0 g / L agar; The callus proliferation culture medium uses B5 medium as the basic medium and also adds: 10~15mg / L 6-BA, 3.0~5.0mg / L NAA, 0.5~1.0g / L acid-hydrolyzed casein, 0.5~2.0mg / L carbon nanotubes, 30g / L sucrose and 6.5~7.0g / L agar; The callus differentiation medium used modified MS medium as the basic medium, and also added with: 4.0 mg / L 6-BA, 1.0~3.0 mg / L NAA, 0.5~1.0 g / L acid hydrolyzed casein, 1.5 mg / L carbon nanotubes, 30 g / L sucrose and 6.5~7.0 g / L agar; The modified MS medium comprises: ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium chloride dihydrate 880 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, sodium molybdate dihydrate 0.25 mg / L, cobalt chloride hexahydrate 0.025 mg / L, copper sulfate pentahydrate 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, nicotinic acid 5.0 mg / L, vitamin B1 10.0 mg / L, vitamin B6 1.0 mg / L, glycine 2 mg / L, and inositol 100 mg / L. The explants cultured in the tissue culture medium are single scales; the preparation method of the single scales is as follows: the outermost 2-3 layers of scale leaves are removed from the bulb, all roots are removed, and the lower 1 / 3 and upper 1 / 2-2 / 3 of the scale leaves are removed. The remaining bulb disc and scale leaves are then divided to obtain a bulb disc block. The innermost 2 layers of scale leaves of the bulb disc block are removed to obtain a bulb block. The bulb block is then cut into single scales. A small amount of tissue from the bulb disc must be retained in each single scale. The length and width of each single scale are (0.5-1.5) cm × (0.5-1.5) cm. The Lycoris radiata mentioned are Lycoris radiata and Lycoris chinensis.

2. A method for cultivating induced buds of Lycoris radiata, characterized in that, The culture method uses the tissue culture medium described in claim 1 and includes the following steps: Single scales of Lycoris radiata were inoculated onto callus induction medium for induction culture to obtain single scales that had produced callus. The single scales that have produced callus tissue are transferred to a callus proliferation culture medium for proliferation culture to obtain callus tissue. The callus mass was transferred to a callus differentiation medium for differentiation culture to obtain shoots; the Lycoris radiata was Lycoris longituba and Lycoris chinensis. The method for preparing the single scale is as follows: peel off the outermost 2-3 layers of scale leaves from the bulb, remove all roots and the lower 1 / 3 of the bulb disc and the upper 1 / 2-2 / 3 of the scale leaves, then cut the remaining bulb disc and scale leaves to obtain a bulb disc block; remove the innermost 2 layers of scale leaves from the bulb disc block to obtain a bulb block; cut the bulb block into single scales; each single scale must retain a small amount of tissue from the bulb disc; the length and width of each single scale are (0.5-1.5) cm × (0.5-1.5) cm.

3. The cultivation method according to claim 2, characterized in that, The induction culture is a dark culture; the induction culture time is 65-75 days; the proliferation culture is a dark culture; the proliferation culture time is 30-40 days; the differentiation culture is a light culture, the light culture time is 12-16 h / d, the light culture intensity is 800-1200 lx; ​​the differentiation culture time is 50-60 days. The temperature for the induction culture, proliferation culture and differentiation culture was 25±1℃.

4. The cultivation method according to claim 2, characterized in that, The diameter of the callus mass is 1.5~2.5cm.

5. A method for regenerating Lycoris callus tissue, characterized in that, Includes the following steps: The buds obtained by the cultivation method according to any one of claims 2 to 4 are transferred to a bud-strengthening culture medium for bud-strengthening culture to obtain rootless seedlings; The rootless seedlings were transferred to a seedling rooting culture medium for seedling rooting culture to obtain tissue culture seedlings.

6. The regeneration tissue culture method according to claim 5, characterized in that, The bud strengthening culture is a light culture, with a light culture time of 12-16 h / d and a light intensity of 800-1200 lx; ​​the bud strengthening culture time is 30-40 days. The seedling rooting culture is a light culture, with a light culture time of 12-16 h / d and a light intensity of 800-1200 lx; ​​the seedling rooting culture time is 30-40 days.

7. The regeneration tissue culture method according to claim 5, characterized in that, The bud-strengthening medium uses MS medium as the basic medium and also includes: 1.5~3.0 mg / L NAA, 2.0~4.0 mg / L 6-BA, 1.0~2.0 mg / L carbon nanotubes, 40~60 g / L sucrose and 6.5~7.0 g / L agar; The seedling rooting medium uses MS medium as the basic medium and also includes: 1.5~2.0 mg / L 6-BA, 0.5~1.0 mg / L NAA, 2.0~2.5 mg / L IBA, 1.0~2.0 mg / L carbon nanotubes, 40~60 g / L sucrose and 6.5~7.0 g / L agar.

8. The regeneration tissue culture method according to claim 5, characterized in that, The temperature for both the strong bud culture and the strong seedling rooting culture was 25±1℃.

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