Basic culture medium, tissue culture medium and regeneration tissue culture method for regeneration of long-tube Lycoris radiata

Abstract

The present invention belongs to the technical field of plant propagation and cultivation, and in particular relates to a basal culture medium, a tissue culture medium, and a regeneration tissue culture method for regenerating long-barreled Lycoris radiata. The present invention provides a tissue culture medium for regenerating long-barreled Lycoris radiata, the tissue culture medium comprising an induction medium, a proliferation medium, a differentiation medium, a bud-strengthening medium, and a seedling-strengthening and rooting medium; in the tissue culture medium provided by the present invention, a modified MS medium and a modified B5 medium are used as the basal culture medium, and the combined action of 2,4-D, 6-BA, IBA, NAA, carbon nanotubes, and casein hydrolyzate facilitates the induction of long-barreled Lycoris radiata callus formation, proliferation, differentiation, and rooting, and sucrose, an energy substance, is combined to jointly improve the propagation coefficient of the long-barreled Lycoris radiata, thereby obtaining a large number of long-barreled Lycoris radiata tissue culture seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant propagation and cultivation, and particularly relates to a basic culture medium, a tissue culture medium and a regeneration tissue culture method for regenerating long-tube Lycoris radiata. Background Art

[0002] Lycoris longituba (Y. Hsu & G. J. Fan) is a perennial herbaceous plant in the Amaryllis family. Its basal, tapering, rounded leaves are green with a distinct central pale band. Its umbels contain 5 to 7 flowers. Its buds are light purple, its petals white, and its perianth lobes slightly streaked with reddish tints on the ventral side. They are oblong, slightly recurved at the apex, and not wrinkled at the margins. The corolla tube is 4 to 6 cm long, and the stamens are slightly shorter than the perianth. It blooms from July to August and produces capsules with black seeds. It prefers sunny, humid environments, such as shady hillsides, rocky slopes, and under cliffs, but it can also tolerate semi-shade and drought conditions. It is somewhat cold-tolerant, resilient, and has no particular soil requirements. It has a narrow distribution, occurring only on shady, humid hillsides within Jiangsu Province, and its population is relatively small.

[0003] The bulbs of Lycoris radiata contain alkaloids such as lycorine, galantamine, and lycorine, making them useful as pharmaceutical ingredients. The high concentrations of galantamine and lycorine are known to detoxify, dissipate pharyngeal congestion, and treat sore throats, boils, carbuncles, and scrofula. The bulbs, typically two to three years old, are harvested in spring and autumn, washed and sun-dried, or sliced ​​and sun-dried. For external use, mash the dried bulbs and apply to the affected area.

[0004] In gardens, Lycoris radiata can be used as ground cover under forests, planted in clusters in flower borders, or grown naturally among rocks. Its robust scapes and tall stems make it an ideal cut flower. Lycoris radiata is typically propagated by seed and bulb division. However, due to its unique biological characteristics, seed yields are low, germination takes a long time, and the vegetative growth period is short (for example, leaves appear from early to mid-January to mid-to-late April). The time from sowing to flowering is five to six years, resulting in a long bulb production cycle and high costs. With bulb division, a flowering bulb naturally produces only one to two young bulbs per year, resulting in a very low reproduction coefficient.

[0005] In recent years, tissue culture and rapid propagation technology systems for Lycoris radiata using bulb disks as explants have been established, but they generally suffer from the problem of low propagation coefficient. Therefore, there is an urgent need to improve the regeneration tissue culture medium for Lycoris radiata. Summary of the Invention

[0006] The purpose of the present invention is to provide a basic culture medium, tissue culture medium and regeneration tissue culture method for regenerating long-tube Lycoris radiata. The basic culture medium and tissue culture medium provided by the present invention are used to carry out long-tube Lycoris radiata callus regeneration tissue culture rapid propagation with long-tube Lycoris radiata seed embryos as explants to improve the reproduction coefficient of long-tube Lycoris radiata.

[0007] In order to solve the above problems, the present invention provides the following technical solutions:

[0008] The invention provides a basal culture medium for regeneration tissue culture of Lycoris radiata. The basal culture medium is a modified B5 culture medium and / or a modified MS culture medium. The modified B5 culture medium comprises: 134 mg / L of ammonium sulfate, 3 mg / L of boric acid, 880 mg / L of calcium chloride dihydrate, 0.025 mg / L of cobalt chloride hexahydrate, 0.025 mg / L of copper sulfate pentahydrate, 37.3 mg / L of disodium ethylenediaminetetraacetic acid, 27.8 mg / L of ferrous sulfate heptahydrate, 500 mg / L of magnesium sulfate heptahydrate, 10 mg / L of manganese sulfate monohydrate, 0.25 mg / L of sodium molybdate dihydrate, 0.75 mg / L of potassium iodide, 2500 mg / L of potassium nitrate, 150 mg / L of sodium dihydrogen phosphate, 2 mg / L of zinc sulfate heptahydrate; 100 mg / L of inositol, 1 mg / L of vitamin B6, 10 mg / L of vitamin B1, and 5.0 mg / L of nicotinic acid.

[0009] The modified MS medium includes: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 880 mg / L calcium chloride dihydrate, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 5.0 mg / L niacin, 10.0 mg / L vitamin B1, 1.0 mg / L vitamin B6, 2 mg / L glycine, and 100 mg / L inositol.

[0010] The present invention provides a tissue culture medium for regeneration of long-tube Lycoris radiata, wherein the tissue culture medium comprises an induction medium, a proliferation medium, a differentiation medium, a bud-strengthening medium, and a seedling-strengthening and rooting medium;

[0011] The induction medium uses the improved B5 medium described in the above technical solution as the basic medium, and also includes: 1.0-2.0 mg / L 6-BA, 6.0-10.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0012] The proliferation culture medium uses the improved B5 culture medium described in the above technical solution as the basic culture medium, and further includes: 1.0-2.0 mg / L 6-BA, 3.0-5.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0013] The differentiation medium uses the improved MS medium described in the above technical solution as the basic medium, and also includes: 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L NAA, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0014] The bud-strengthening culture medium uses the improved MS culture medium described in the above technical solution as the basic culture 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;

[0015] The seedling-strengthening and rooting culture medium uses MS culture medium as a basic culture medium and further comprises: 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.

[0016] The present invention provides a regeneration tissue culture method for Lycoris radiata callus, wherein the regeneration tissue culture method adopts the tissue culture medium described in the above technical solution and comprises the following steps:

[0017] Inoculating the embryos of Lycoris radiata on an induction medium for callus induction culture to obtain embryos that have produced callus tissue;

[0018] transferring the embryos that have produced callus tissue to a proliferation medium for callus proliferation culture to obtain callus tissue masses;

[0019] transferring the callus mass to a differentiation medium for callus differentiation culture to obtain clustered buds;

[0020] Transferring the clustered buds to a bud-strengthening medium for bud-strengthening culture to obtain rootless seedlings;

[0021] The rootless seedlings are transferred to a seedling-strengthening and rooting culture medium for seedling-strengthening and rooting culture to obtain tissue culture seedlings.

[0022] Preferably, before callus induction culture, the method further comprises: cleaning and disinfecting the embryo; the cleaning and disinfection comprises: first washing with sterile water 2 to 3 times, then disinfecting with a sodium hypochlorite solution having an effective chlorine content of 0.1% to 0.3% for 1 to 2 minutes, and then washing with sterile water 4 to 5 times.

[0023] Preferably, the embryo is obtained by removing the pericarp of the Lycoris radiata capsule and cutting the seed along the longitudinal diameter to obtain the embryo.

[0024] Preferably, the callus induction culture and callus proliferation culture are both dark cultures; the callus differentiation culture, bud culture and seedling rooting culture are all light-dark alternating cultures, the illumination time is 12 to 16 h / d, and the illumination intensity is 800 to 1200 Lx.

[0025] Preferably, the diameter of the callus mass is 1.5 to 2.5 cm; the length of the clustered buds is 1.0 to 1.5 cm.

[0026] Preferably, the callus induction culture time is 28 to 45 days; the callus proliferation culture time is 30 to 40 days; the callus differentiation culture time is 30 to 40 days; the bud culture time is 60 to 80 days; and the seedling rooting culture time is 30 to 40 days.

[0027] Preferably, the strong bud culture process further includes transferring the culture product of the strong bud culture for 30 to 40 days to a new strong bud culture medium for subculture to obtain rootless seedlings.

[0028] Preferably, it is characterized in that the temperature of the callus induction culture, callus proliferation culture, callus differentiation culture, bud strengthening culture and seedling rooting culture is 25±1°C.

[0029] The invention has the beneficial effects of providing a basal culture medium for regeneration tissue culture of Lycoris radiata, wherein the basal culture medium is a modified B5 culture medium and / or a modified MS culture medium; the modified B5 culture medium comprises: 134 mg / L of ammonium sulfate, 3 mg / L of boric acid, 880 mg / L of calcium chloride dihydrate, 0.025 mg / L of cobalt chloride hexahydrate, 0.025 mg / L of copper sulfate pentahydrate, 37.3 mg / L of disodium ethylenediaminetetraacetic acid, 27.8 mg / L of ferrous sulfate heptahydrate, 500 mg / L of magnesium sulfate heptahydrate, 10 mg / L of manganese sulfate monohydrate, 0.25 mg / L of sodium molybdate dihydrate, 0.75 mg / L of potassium iodide, 2500 mg / L of potassium nitrate, 150 mg / L of sodium dihydrogen phosphate, 2 mg / L of zinc sulfate heptahydrate; 100 mg / L of inositol, 1 mg / L of vitamin B6, 10 mg / L of vitamin B1, and 5.0 mg / L of niacin;

[0030] The modified MS medium includes: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 880 mg / L calcium chloride dihydrate, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium edetate, 27.8 mg / L ferrous sulfate heptahydrate, 5.0 mg / L nicotinic acid, 10.0 mg / L vitamin B1, 1.0 mg / L vitamin B6, 2 mg / L glycine, and 100 mg / L inositol.

[0031] In the basic culture medium for regeneration tissue culture provided by the present invention, the amount of CaCl2·2H2O in the improved MS culture medium is increased, and the amount of calcium element is increased, which can promote the absorption of nitrate nitrogen in the culture medium, thereby facilitating the induction of callus tissue and the morphological establishment of the plant; the increased amount of organic matter such as vitamin B1, vitamin B6 and niacin can improve the permeability of the cell membrane and promote the absorption of nutrients; the addition of calcium element and the organic matter niacin in the improved B5 culture medium are intended to promote nutrient absorption; the use of the basic culture medium of the present invention can significantly improve the callus induction rate and differentiation rate of Lycoris radiata, and ultimately improve the reproduction coefficient.

[0032] The present invention also provides a tissue culture medium for regeneration of Lycoris radiata, wherein the tissue culture medium comprises an induction medium, a proliferation medium, a differentiation medium, a bud-strengthening medium, and a seedling-strengthening and rooting medium;

[0033] The induction medium uses the improved B5 medium as the basic medium and further comprises: 1.0-2.0 mg / L 6-BA, 6.0-10.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0034] The proliferation culture medium uses a modified B5 culture medium as a basic culture medium and further comprises: 1.0-2.0 mg / L 6-BA, 3.0-5.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar;

[0035] The differentiation medium uses a modified MS medium as a basic medium and further comprises: 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L NAA, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar;

[0036] The bud-strengthening culture medium uses a modified MS culture medium as a basic culture medium and further comprises: 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;

[0037] The seedling-strengthening and rooting culture medium uses MS culture medium as a basic culture medium and further comprises: 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.

[0038] In the tissue culture medium, the added 2,4-D (2,4-dichlorophenoxyacetic acid) promotes the formation of Lycoris radiata callus; 6-BA (6-benzylaminopurine) stimulates cell division to promote the growth and development of Lycoris radiata and the formation of callus; NAA (α-naphthaleneacetic acid) promotes the germination, growth and rooting of Lycoris radiata; IBA plays a role in rooting, and casein hydrolyzate promotes the differentiation of Lycoris radiata embryos and clustered buds; carbon nanotubes can promote the growth of Lycoris radiata callus, bud growth and rooting; under the joint action of 2,4-D, 6-BA, IBA, NAA, carbon nanotubes and casein hydrolyzate, it is beneficial to induce the formation, proliferation, differentiation and rooting of Lycoris radiata callus, and combined with sucrose, an energy substance, to jointly improve the callus induction rate and differentiation rate of Lycoris radiata, and ultimately improve the reproduction coefficient and obtain a large number of Lycoris radiata tissue culture seedlings. The results of the examples show that the tissue culture medium of the present invention can produce a large number of long-tube Lycoris tissue culture seedlings and improve the propagation coefficient of long-tube Lycoris. This shows that the present invention improves the propagation coefficient of long-tube Lycoris and shortens the tissue culture cycle by synergizing the components of the induction medium, proliferation medium, differentiation medium, bud growth medium, and seedling growth and rooting medium under appropriate component concentration control. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0040] Figure 1 This is the situation on the 12th day of induction of Lycoris radiata embryo callus in Example 1;

[0041] Figure 2 This is the 30th day of induction of Lycoris radiata embryo callus in Example 1;

[0042] Figure 3 This is the 28th day of differentiation of Lycoris radiata callus in Example 1;

[0043] Figure 4 This is the 25th day of the strong seedling and rooting culture of the rootless seedlings of the long-tube Lycoris radiata in Example 1. DETAILED DESCRIPTION

[0044] The invention provides a basic culture medium for regeneration tissue culture of Lycoris radiata. The basic culture medium is a modified B5 culture medium and / or a modified MS culture medium.

[0045] The improved B5 culture medium of the present invention comprises: 134 mg / L ammonium sulfate, 3 mg / L boric acid, 880 mg / L calcium chloride dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 500 mg / L magnesium sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 0.75 mg / L potassium iodide, 2500 mg / L potassium nitrate, 150 mg / L sodium dihydrogen phosphate, 2 mg / L zinc sulfate heptahydrate; 100 mg / L inositol, 1 mg / L vitamin B6, 10 mg / L vitamin B1 and 5.0 mg / L niacin. More preferably, the modified B5 medium contains only: 134 mg / L ammonium sulfate, 3 mg / L boric acid, 880 mg / L calcium chloride dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium edetate, 27.8 mg / L ferrous sulfate heptahydrate, 500 mg / L magnesium sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 0.75 mg / L potassium iodide, 2500 mg / L potassium nitrate, 150 mg / L sodium dihydrogen phosphate, 2 mg / L zinc sulfate heptahydrate; 100 mg / L inositol, 1 mg / L vitamin B6, 10 mg / L vitamin B1, and 5.0 mg / L niacin. The modified B5 medium is supplemented with 880 mg / L CaCl2·2H2O and 5 mg / L organic niacin.

[0046] In the basic culture medium for regeneration tissue culture provided by the present invention, CaCl2·2H2O and organic nicotinic acid are added to the improved B5 culture medium in order to promote the absorption of nutrients, especially nitrate nitrogen, by the explants.

[0047] The improved MS culture medium of the present invention comprises: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 880 mg / L calcium chloride dihydrate, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 5.0 mg / L niacin, 10.0 mg / L vitamin B1, 1.0 mg / L vitamin B6, 2 mg / L glycine, and 100 mg / L inositol. Preferably, the modified MS medium contains only the following: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 880 mg / L calcium chloride dihydrate, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium edetate, 27.8 mg / L ferrous sulfate heptahydrate, 5.0 mg / L niacin, 10.0 mg / L vitamin B1, 1.0 mg / L vitamin B6, 2 mg / L glycine, and 100 mg / L inositol.

[0048] In the basic culture medium for regeneration tissue culture provided by the present invention, adding CaCl2·2H2O to the improved MS culture medium and increasing its dosage can promote the absorption of nitrate nitrogen in the culture medium, thereby facilitating the induction of callus tissue and the morphological establishment of plants. Increasing the dosage of vitamin B1 and niacin can improve the permeability of cell membranes and thus promote nutrient absorption.

[0049] The invention provides a tissue culture medium for regeneration of long-tube Lycoris radiata. The tissue culture medium comprises an induction medium, a proliferation medium, a differentiation medium, a bud-strengthening medium, and a seedling-strengthening and rooting medium.

[0050] The induction culture medium of the present invention uses the improved B5 culture medium described in the above technical solution as the basic culture medium, and further includes: 1.0-2.0 mg / L 6-BA, 6.0-10.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 30 g / L sucrose and 6.5-7.0 g / L agar; preferably, the improved B5 culture medium is used as the basic culture medium, and further only contains: 1.0-2.0 mg / L 6-BA, 6.0-10.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0051] The concentration of 6-BA in the induction medium of the present invention is 1.0-2.0 mg / L, preferably 1.2-1.8 mg / L, more preferably 1.5 mg / L. The concentration of 2,4-D in the induction medium is 6.0-10.0 mg / L, preferably 7.0-9.0 mg / L, more preferably 8 mg / L. The concentration of casein hydrolyzate in the induction medium is 0.5-1.0 g / L, preferably 0.7-0.9 g / L, more preferably 0.8 g / L. The concentration of agar in the induction medium is 6.5-7.0 g / L, more preferably 6.7 g / L. The 2,4-D, 6-BA and casein hydrolyzate added in the present invention can promote the formation of callus tissue. There is no special limitation on the source of the casein hydrolyzate, and conventional commercially available products can be used. In the embodiment of the present invention, the casein hydrolyzate used was purchased from Gaojiaoyan (Beijing) Technology Co., Ltd.

[0052] In the present invention, the pH value of the induction medium is preferably 5.75-5.85, more preferably 5.80.

[0053] The proliferation culture medium of the present invention uses the modified B5 medium described in the above technical solution as a basic culture medium, and further includes: 1.0-2.0 mg / L 6-BA, 3.0-5.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar. Preferably, the modified B5 medium is used as a basic culture medium, and further contains only: 1.0-2.0 mg / L 6-BA, 3.0-5.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar. The concentration of 6-BA in the proliferation culture 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 2,4-D in the proliferation medium is 3.0 to 5.0 mg / L, preferably 3.5 to 4.5 mg / L, and more preferably 4.0 mg / L. The concentration of casein hydrolyzate in the proliferation medium is 0.5 to 1.0 g / L, preferably 0.7 to 0.9 g / L, and more preferably 0.8 g / L. The concentration of carbon nanotubes in the proliferation medium is 1.0 to 2.0 mg / L, preferably 1.2 to 1.7 mg / L, and more preferably 1.5 mg / L. The carbon nanotubes described in the present invention are preferably multi-walled carbon nanotubes. The concentration of agar in the proliferation medium is 6.5 to 7.0 g / L, and more preferably 6.7 g / L. In the present invention, 6-BA, 2,4-D, casein hydrolyzate, and carbon nanotubes can jointly promote the proliferation of Lycoris radiata callus at appropriate concentration ratios.

[0054] In the present invention, the pH value of the proliferation medium is preferably 5.75-5.85, more preferably 5.80.

[0055] The differentiation medium of the present invention uses the improved MS medium described in the above technical solution as the basic medium, and also includes: 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L NAA, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar; preferably, the improved MS medium is used as the basic medium, and only contains: 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L NAA, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0056] The concentration of 6-BA in the differentiation medium of the present invention is 2.0-4.0 mg / L, preferably 2.3-3.3 mg / L, and more preferably 3.0 mg / L. The concentration of NAA in the differentiation medium is 1.0-2.0 mg / L, preferably 1.2-1.8 mg / L, and more preferably 1.4 mg / L. The concentration of casein hydrolysate in the 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 differentiation medium is 1.0-2.0 mg / L, preferably 1.1-1.6 mg / L, and more preferably 1.4 mg / L. The carbon nanotubes of the present invention are preferably multi-walled carbon nanotubes. Currently, there are three types of commercially available carbon nanotubes: single-walled carbon nanotubes, multi-walled carbon nanotubes, and carboxylated multi-walled carbon nanotubes. Preliminary experiments have shown that the three types of carbon nanotubes have similar effects, and multi-walled carbon nanotubes are the cheapest, so multi-walled carbon nanotubes are selected. The concentration of agar in the differentiation medium is 6.5-7.0 g / L, more preferably 6.7 g / L. The present invention promotes callus differentiation into a large number of clustered shoots under the specific action of appropriate components of 2,4-D, 6-BA, NAA, casein hydrolyzate and carbon nanotubes.

[0057] In the present invention, the pH value of the differentiation medium is preferably 5.75 to 5.85, more preferably 5.80.

[0058] The bud-strengthening culture medium of the present invention uses the improved MS culture medium described in the above technical solution as a 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; preferably, the improved MS culture medium is used as a basic culture medium, and further only contains: 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 2.0-4.0 mg / L, 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 1.5 to 3.0 mg / L, preferably 1.8 to 2.5 mg / L, and more preferably 2.0 mg / L. The concentration of carbon nanotubes in the bud-strengthening medium is 1.0 to 2.0 mg / L, preferably 1.3 to 1.8 mg / L, and more preferably 1.5 mg / L. The carbon nanotubes described in the present invention are preferably multi-walled carbon nanotubes. Currently, there are three types of commercially available carbon nanotubes: single-walled carbon nanotubes, multi-walled carbon nanotubes, and carboxylated multi-walled carbon nanotubes. After preliminary experiments, the three types of carbon nanotubes have similar effects, and multi-walled carbon nanotubes are the cheapest, so multi-walled carbon nanotubes are selected. The concentration of agar in the bud-strengthening medium is 6.5 to 7.0 g / L, and more preferably 6.7 g / L. The present invention promotes the growth of clustered buds under the action of modified MS medium, NAA, 6-BA, and carbon nanotubes, thereby obtaining rootless seedlings.

[0059] In the present invention, the pH value of the bud-strengthening culture medium is preferably 5.75-5.85, more preferably 5.80.

[0060] The seedling-strengthening and rooting culture medium of the present invention uses MS culture medium as a basic culture medium and further comprises: 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-strengthening and rooting culture medium uses MS culture medium as a basic culture medium and further only contains: 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.

[0061] The concentration of 6-BA in the seedling-growing and rooting medium of the present invention is 1.5-2.0 mg / L, preferably 1.6-1.9 mg / L, and more preferably 1.8 mg / L. The concentration of NAA in the seedling-growing and rooting medium is 0.5-1.0 mg / L, preferably 0.7-0.9 mg / L, and more preferably 0.8 mg / L. The concentration of IBA in the seedling-growing and rooting medium is 2.0-2.5 mg / L, preferably 2.1-2.4 mg / L, and more preferably 2.3 mg / L. The concentration of carbon nanotubes in the seedling-growing and rooting medium is 1.0-2.0 mg / L, preferably 1.1-1.7 mg / L, and more preferably 1.6 mg / L. The carbon nanotubes of the present invention are preferably multi-walled carbon nanotubes. Currently, there are three types of commercially available carbon nanotubes: single-walled carbon nanotubes, multi-walled carbon nanotubes, and carboxylated multi-walled carbon nanotubes. Preliminary experiments have shown that the three types of carbon nanotubes are similar in effectiveness, with multi-walled carbon nanotubes being the cheapest, hence the choice. The agar concentration in the seedling and rooting medium is 6.5-7.0 g / L, 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.

[0062] In the present invention, the pH value of the seedling strengthening and rooting culture is preferably 5.75-5.85, more preferably 5.80.

[0063] The induction culture medium, proliferation culture medium, differentiation culture medium, bud-strengthening culture medium, and seedling-strengthening and rooting culture medium provided by the present invention all contain sucrose. Sucrose can be a carbon source required for plant growth, promoting the occurrence of callus tissue and the germination and growth of clustered buds. The present invention promotes the growth and differentiation of buds of long-tube Lycoris radiata and the rooting of rootless seedlings under the joint action of 2,4-D, 6-BA, NAA, IBA, carbon nanotubes, casein hydrolyzate, and sucrose. The tissue culture culture medium provided by the present invention has a good application effect, can simultaneously obtain a large number of tissue culture seedlings, shortens the time for long-tube Lycoris radiata seedling cultivation and reduces the cost of seedling cultivation, improves the callus induction rate and differentiation rate, ultimately improves the reproduction coefficient, and shortens the tissue culture cycle.

[0064] The present invention has no particular limitation on the sources of the B5 medium, MS medium, 2,4-D, 6-BA, NAA, IBA, carbon nanotubes, casein hydrolysate, sucrose and agar, and conventional commercially available products can be used.

[0065] The present invention also provides a regeneration tissue culture method for Lycoris radiata callus, wherein the regeneration tissue culture method adopts the tissue culture medium described in the above technical solution, and comprises the following steps:

[0066] Inoculating the embryos of Lycoris radiata on an induction medium for callus induction culture to obtain embryos that have produced callus tissue;

[0067] transferring the embryos that have produced callus tissue to a proliferation medium for callus proliferation culture to obtain callus tissue masses;

[0068] transferring the callus mass to a differentiation medium for callus differentiation culture to obtain clustered buds;

[0069] Transferring the clustered buds to a bud-strengthening medium for bud-strengthening culture to obtain rootless seedlings;

[0070] The rootless seedlings are transferred to a seedling-strengthening and rooting culture medium for seedling-strengthening and rooting culture to obtain tissue culture seedlings.

[0071] The long-tube Lycoris radiata embryos of the present invention are inoculated onto an induction medium for callus induction culture to obtain embryos that have produced callus tissue. The present invention preferably uses the long-tube Lycoris radiata embryos as explants for seedling cultivation. In the present invention, the long-tube Lycoris radiata embryos are preferably obtained by removing the pericarp of the long-tube Lycoris radiata capsule and cutting the seed along the longitudinal diameter to obtain the embryos. The embryos of the present invention are preferably complete, mature embryos.

[0072] The capsules of the long-tube Lycoris radiata of the present invention are preferably collected in September to October when the pericarp turns yellow and the seed coat turns black. The selection of the time for collecting the capsules of the present invention is to increase the induction rate of embryo callus tissue.

[0073] In the present invention, before callus induction culture, it is preferred that the embryos are cleaned and disinfected. The cleaning of the present invention is preferably completed using sterile water. The present invention does not specifically limit the cleaning method, as long as it is clean. The present invention preferably first washes with sterile water 2 to 3 times, then disinfects with a sodium hypochlorite solution with an effective chlorine content of 0.1% to 0.3% for 1 to 2 minutes, and then washes with sterile water 4 to 5 times. The present invention preferably first washes with sterile water 2 to 3 times, more preferably 3 times; the disinfection using a sodium hypochlorite solution with an effective chlorine content of 0.1% to 0.3% by mass is more preferred, and the disinfection time of the present invention is preferably 1 to 2 minutes, more preferably 1 minute; finally, it is preferably washed with sterile water 4 to 5 times, more preferably 4 times.

[0074] In the prior art, the rapid propagation of Lycoris radiata through tissue culture mostly uses bulb discs as explants to induce the production of buds, which in turn strengthen the buds and induce rooting, thereby establishing a rapid propagation technology system. The biggest disadvantage of this technology is its low propagation coefficient and low callus induction rate. The present invention uses embryos as explants for tissue culture rapid propagation and seedling cultivation, solving the technical problems of low propagation coefficient and low callus induction rate when using bulb discs as explants for tissue culture seedling cultivation in the prior art. It can obtain a large number of consistent and robust Lycoris radiata plants while shortening the tissue culture cycle.

[0075] After the embryos are cleaned and disinfected, the present invention preferably inoculates the embryos of Lycoris radiata on an induction medium for callus induction culture to obtain embryos that have produced callus tissue. In the present invention, one embryo is preferably inoculated in each induction medium. The inoculation method of the embryos of the present invention is preferably to place them horizontally on the induction medium. In the present invention, the temperature of the callus induction culture is preferably 24-26°C, more preferably 25°C. The callus induction culture of the present invention is preferably dark culture; the time of the callus induction culture is preferably 28-45d, more preferably 30-43d, more preferably 32-40d. The time of the induction culture of the present invention is calculated from the inoculation of the embryos on the induction medium until the embryos with light yellow callus tissue are obtained.

[0076] After the embryos of light yellow callus tissue appear, the present invention transfers the embryos that have produced callus tissue into a proliferation medium for callus tissue proliferation culture to obtain callus tissue masses;

[0077] In the present invention, the temperature of the proliferation culture is preferably 24-26°C, more preferably 25°C. In the present invention, the proliferation culture is preferably dark culture. The proliferation culture duration is preferably 30-40 days, more preferably 35-40 days. The proliferation culture duration is calculated from the transfer of embryos that have produced callus tissue to the proliferation culture medium until a callus mass with a diameter of 1.5-2.5 cm is obtained.

[0078] After the callus mass with a diameter of 1.5 to 2.5 cm is obtained through proliferation culture, the present invention transfers the callus mass to a differentiation medium for callus differentiation culture to obtain clustered buds.

[0079] The present invention preferably transfers callus clumps with a diameter of 1.5 to 2.5 cm to a differentiation medium for callus differentiation culture, and more preferably transfers callus clumps with a diameter of 1.8 to 2.0 cm to a differentiation medium for callus differentiation culture. The temperature of the differentiation culture of the present invention is preferably 24 to 26°C, more preferably 25°C. In the present invention, the differentiation culture is preferably alternating light and dark culture; the illumination time of the light culture is preferably 12 to 16 h / d, more preferably 14 to 16 h / d, more preferably 16 h / d; the intensity of the illumination is preferably 800 to 1200 Lx, more preferably 1000 Lx. The culture time of the differentiation culture of the present invention is preferably 30 to 40 d, more preferably 35 to 40 d.

[0080] After obtaining clustered buds with a height of 1.0 to 1.5 cm through differentiation culture, the present invention preferably separates the clustered buds from the callus tissue and transfers them to a bud-strengthening culture medium for bud-strengthening culture to obtain rootless seedlings.

[0081] The temperature for the strong bud culture of the present invention is preferably 24-26°C, more preferably 25°C. In the present invention, the strong bud culture is preferably a light-dark alternating culture; the illumination time of the light culture is preferably 12-16 hours / day, more preferably 14-16 hours / day, and more preferably 16 hours / day; the intensity of the illumination is preferably 800-1200 lux, more preferably 1000 lux. The incubation time of the strong bud culture of the present invention is preferably 60-80 days, more preferably 70-80 days.

[0082] The bud-strengthening culture process of the present invention preferably further comprises transferring the culture product of the bud-strengthening culture for 30 to 40 days to a new bud-strengthening culture medium for subculture to obtain rootless seedlings; more preferably, transferring the culture product of the bud-strengthening culture for 35 to 40 days to a new bud-strengthening culture medium for subculture to obtain rootless seedlings. The subculture process of the present invention preferably involves replacing the culture medium, wherein the culture medium for subculture is the same as the bud-strengthening culture medium. The bud-strengthening culture process of the present invention is calculated from the time of transfer to the bud-strengthening culture medium until a rootless seedling having 3 to 4 leaves and a bulb circumference of 0.8 to 1.5 cm is obtained.

[0083] After the strong buds are cultured to obtain rootless seedlings with 3 to 4 leaves or bulb circumferences of 0.8 to 1.5 cm, the present invention transfers the rootless seedlings to a strong seedling and rooting culture medium for strong seedling rooting culture to obtain tissue culture seedlings.

[0084] In the present invention, the temperature of the seedling rooting culture is preferably 24-26°C, more preferably 25°C. In the present invention, the seedling rooting culture is preferably carried out under light-dark alternating culture conditions. The illumination time of the present invention is preferably 12-16 h / d, more preferably 14-16 h / d, more preferably 16 h / d; the intensity of the illumination is preferably 800-1200 Lx, more preferably 1000 Lx. The cultivation time of the seedling rooting culture of the present invention is preferably 30-40 d, more preferably 35-40 d, more preferably 40 d.

[0085] In the present invention, the callus induction culture, callus proliferation culture, callus differentiation culture, bud growth culture, and seedling rooting culture are preferably carried out in tissue culture bottles. The specifications of the tissue culture bottles are not particularly limited, and conventional products can be used. The callus induction medium, induction medium, differentiation medium, bud growth medium, seedling growth medium, and rooting medium described in the present invention are sterilized and poured into the tissue culture bottles. The sterilization method is not particularly limited, and conventional methods can be used.

[0086] After rooting and culturing the seedlings of the present invention, preferably, a long-tube Lycoris radiata test-tube plantlet having a bulb circumference of 1.5 to 2.0 cm, 3 to 5 roots, and a weight of 1.5 to 2.0 g is obtained. The bulb circumference of the test-tube plantlet of the present invention is preferably 1.5 to 2.0 cm, more preferably 1.6 to 1.8 cm, and the root system of the test-tube plantlet of the present invention is preferably 3 to 5, more preferably 4. The weight of the test-tube plantlet of the present invention is preferably 1.5 to 2.0 g, more preferably 1.6 to 1.8 g.

[0087] The invention provides a test tube seedling after rooting and culturing the seedlings. The test tube seedlings meet the following three conditions: bulb diameter of 0.3-0.5 cm, 3-5 root systems and weight of 1.5-2.0 g. Then, the seedlings are hardened and transplanted, thereby improving the survival rate of the test tube seedlings.

[0088] After obtaining the test tube seedlings, the present invention preferably hardens and transplants the test tube seedlings. In the present invention, the hardening is preferably completed indoors, the hardening temperature is preferably the temperature at which Lycoris radiata grows, and the hardening time is preferably 4 to 6 days, more preferably 6 days. During hardening, the present invention preferably loosens the cap of the tissue culture bottle for rooting and then places it in the buffer room of the tissue culture room for 2 to 3 days, and then places it under natural scattered light for 2 to 3 days.

[0089] In the present invention, when transplanting, it is preferred that the roots of the test tube seedlings after hardening are washed and then transplanted into a substrate for cultivation. The transplanting and cultivation substrate preferably includes the following components by volume: 0.5-1.5 parts of peat soil, 0.5-1.5 parts of perlite, and 0.5-1.5 parts of garden soil. The volume ratio of peat soil, perlite, and garden soil in the present invention is preferably (0.5-1.5): (0.5-1.5): (0.5-1.5), more preferably (0.8-1.3): (0.8-1.3): (0.8-1.3), and more preferably 1:1:1; the temperature for transplanting is preferably 15-28°C, more preferably 20-28°C, and more preferably 25°C. Before transplanting, the present invention preferably disinfects the substrate. The present invention does not specifically limit the method of disinfection. The present invention preferably disinfects the substrate with 800-1000 times of a broad-spectrum fungicide, more preferably 800 times. The broad-spectrum fungicide of the present invention is preferably thiophanate-methyl.

[0090] The present invention does not specifically limit the source and specifications of the plug trays used for transplanting and culturing; conventional products can be used. The plug trays used in the present invention preferably have 10 rows x 10 columns. In the present invention, 1 to 2 test tube seedlings are preferably planted in each plug tray. After transplanting, sufficient water should be applied to establish rooting, followed by normal water and fertilizer management.

[0091] The existing long-tube Lycoris radiata uses bulb discs as explants, which results in low callus induction rates and low propagation coefficients. The innovation of the present invention lies in using embryos as explants and employing a specific combination of growth regulators to induce callus, thereby increasing the callus induction rate and differentiation rate, and ultimately the propagation coefficient.

[0092] This technology uses the long-barreled Lycoris embryo as an explant to induce the production of callus tissue. After proliferation culture, it is induced to differentiate into clustered buds. After the steps of strong bud culture, strong seedling rooting culture, etc., a long-barreled Lycoris plant with good consistency and robustness is obtained. The present invention establishes a regeneration technology system for the long-barreled Lycoris radiata, a dual-purpose plant for medicinal and ornamental purposes, based on callus tissue, to provide technical support for the establishment of a bulb propagation and genetic transformation system for the long-barreled Lycoris radiata, and also lays the foundation for its industrial application in ornamental and medicinal aspects. The long-barreled Lycoris radiata plant germplasm obtained by tissue culture of the present invention does not degenerate, which can ensure the ornamental effect and the quality of the medicinal material, and will provide technical support for its large-scale cultivation, and also lay a technical foundation for the establishment of an Agrobacterium-mediated genetic transformation system for the long-barreled Lycoris radiata.

[0093] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0094] The modified B5 medium and the modified MS medium were used in the following examples and comparative examples, and their specific compositions are as follows:

[0095] The method for improving the B5 culture medium comprises the following steps: adding 880 mg / L calcium chloride dihydrate to the B5 culture medium formula, and increasing the amount of organic matter nicotinic acid to 5 mg / L; the composition of the improved B5 culture medium is as follows: 134 mg / L ammonium sulfate, 3 mg / L boric acid, 880 mg / L calcium chloride dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 500 mg / L magnesium sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 0.75 mg / L potassium iodide, 2500 mg / L potassium nitrate, 150 mg / L sodium dihydrogen phosphate, 2 mg / L zinc sulfate heptahydrate; 100 mg / L inositol, 5 mg / L nicotinic acid, 1 mg / L vitamin B6, and 10 mg / L vitamin B1.

[0096] The method for improving the MS medium is to add calcium chloride dihydrate to the MS medium, increase its dosage to 880 mg / L in the original composition, and increase the dosage of organic components vitamin B1 and niacin in the MS medium to 10 times the original dosage, and increase vitamin B6 to 2 times the original dosage, to 10.0 mg / L B1 and 1.0 mg / L respectively. VB6 and nicotinic acid 5.0 mg / L; the composition of the modified MS medium is ammonium nitrate (NH4NO3) 1650 mg / L, potassium nitrate (KNO3) 1900 mg / L, potassium dihydrogen phosphate (KH2PO4) 170 mg / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 370 mg / L, calcium chloride dihydrate (CaCl2·2H2O) 880 mg / L, manganese sulfate tetrahydrate (MnSO4·4H2O) 22.3 mg / L, zinc sulfate heptahydrate (ZnSO4·7H2O) 8.6 mg / L, potassium iodide (KI) 0.83 mg / L, boric acid (H3BO3) 6.2 mg / L, sodium molybdate dihydrate (NaMoO4·2H2O) 0.25 mg / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.025 mg / L, copper sulfate pentahydrate (CuSO4·5H2O) 0.025 mg / L, disodium ethylenediaminetetraacetic acid (Na2-EDTA) 37.3 mg / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 27.8 mg / L, niacin (VB5) 5.0 mg / L, thiamine hydrochloride (VB1) 10.0 mg / L, pyridoxine hydrochloride (vitamin B6) 1.0 mg / L, glycine 2 mg / L, inositol 100 mg / L.

[0097] Example 1

[0098] 1. Obtaining explants: At the end of September 2022, when the pericarp of Lycoris radiata turns yellow and the seed coat turns black, the capsules are picked and brought back to the laboratory. The pericarp is removed and the seeds are cut along the longitudinal diameter to obtain the complete seed embryos of Lycoris radiata.

[0099] 2. Disinfection of explants: Wash the embryos obtained in step 1 three times with sterile water on a clean bench, then disinfect with a sodium hypochlorite solution with an effective chlorine content of 0.1% for 1.0 min, and wash four times with sterile water for later use.

[0100] 3. Callus induction: The embryos disinfected in step 2 were horizontally inoculated onto callus induction medium. The induction medium consisted of a modified B5 medium supplemented with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar, with a pH of 5.8. The induction culture was performed in the dark. The first day after the embryos were horizontally inoculated onto the callus induction medium was the first day of induction culture. After 12 days of induction culture, the embryos began to germinate. After 28 days of induction culture, light yellow callus appeared on the surface of the embryos.

[0101] 4. Callus Proliferation: After 32 days of induction culture in step 3, embryos that have produced callus are transferred to callus proliferation medium for proliferation culture. The callus proliferation medium consists of a modified B5 medium supplemented with 1.0 mg / L 6-BA, 5.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 1.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar; the pH is 5.8, and the proliferation culture is carried out in the dark. After 35 days of proliferation culture, the callus has proliferated into clumps with a diameter of 2.0 cm.

[0102] 5. Callus Differentiation: Transfer the 2.0 cm diameter clumps from step 3 to callus differentiation medium for differentiation culture. The differentiation medium consists of a modified MS basal medium supplemented with 2.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 g / L casein hydrolysate, 1.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar. The pH is 5.8, and the proliferation culture is alternating light and dark. The light culture duration is 16 hours per day, the dark culture duration is 8 hours per day, and the light intensity for light culture is 800-1200 lx. After 35 days of culture, the pale yellow callus can be observed to differentiate into white buds, which then differentiate into a large number of clustered buds.

[0103] 6. Bud Culture: Separate the 1.0-1.5 cm tall clustered buds from step 5 from the callus tissue and transfer them to a bud culture medium for bud culture. The bud culture medium consists of a modified MS basal medium supplemented with 2.0 mg / L 6-BA, 1.5 mg / L NAA, 1.0 mg / L carbon nanotubes, 50 g / L sucrose, and 6.5 g / L agar; the pH is 5.8.

[0104] After 35 days of culture, the same bud-strengthening medium is used for subculture. After another 35 days of culture, the clustered buds grow into rootless test tube seedlings with 3 to 4 leaves and a bulb circumference of 0.8 to 1.5 cm.

[0105] 7 Seedling growth and rooting culture: Seedling growth and rooting culture can be completed in one step. The specific method is to transfer the rootless test tube seedlings obtained after the bud growth culture in step 6 to the bud growth and rooting culture medium for bud growth and rooting co-culture. The components of the bud growth and rooting culture medium are: MS as the basic culture medium, and only 1.5mg / L 6-BA, 0.5mg / L NAA, 2.0mg / L IBA, 1.0mg / L carbon nanotubes, 40g / L sucrose and 6.5g / L agar are added. The temperature for bud growth and rooting co-culture is 25±1℃, the light intensity is 800~1200lx, and the light duration is 16h / d. After 40d of culture, test tube seedlings with a bulb circumference of 1.5~2.0cm, 3~5 roots, and a fresh weight of 0.5~1.0g can be obtained.

[0106] 8. Hardening and transplanting: Hardening is done indoors. Specifically, loosen the cap of the tissue culture bottle and place it in the buffer room of the tissue culture room for 3 days. Then move it indoors and place it under natural scattered light for another 3 days. After hardening is completed, use tweezers to remove the test tube seedlings, wash off the culture medium on the roots, transplant them into a 10×10 hole tray, and water them enough to establish roots (the criterion for establishing roots is that water leaks from the bottom of the hole tray). The cultivation medium is peat soil, perlite and garden soil, and the volume ratio of peat soil, perlite and garden soil is 1:1:1. The cultivation medium must be disinfected with 800 times chlorothalonil before transplanting the test tube seedlings. Then carry out normal water and fertilizer management.

[0107] Example 2

[0108] 1. Obtaining explants: In early October 2022, peel the fruits of Lycoris radiata that had been harvested and stored in the refrigerator for about half a month, obtain their mature seeds, cut them along the longitudinal diameter with a scalpel, and obtain the complete seed embryos of Lycoris radiata.

[0109] 2. Disinfection of explants: Wash the embryos obtained in step 1 three times with sterile water on a clean bench, then disinfect with a sodium hypochlorite solution with an effective chlorine content of 0.3% for 1.0 min, and wash four times with sterile water for later use.

[0110] 3. Callus induction: The embryos sterilized in step 2 were horizontally inoculated onto callus induction medium. The induction medium consisted of a modified B5 medium supplemented with 2.0 mg / L 6-BA, 8.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar, with a pH of 5.8. The induction culture was performed in the dark. The first day after the embryos were horizontally inoculated onto the callus induction medium was the first day of induction culture. After 15 days of induction culture, the embryos began to germinate. After 30 days of induction culture, pale yellow callus appeared on the embryo surface.

[0111] 4. Callus proliferation: After 40 days of induction culture, the embryos that have produced callus are transferred to callus proliferation medium for proliferation culture.

[0112] The callus proliferation medium consisted of a modified B5 medium supplemented with 2.0 mg / L 6-BA, 4.0 mg / L 2,4-D, 0.5 g / L casein hydrolysate, 2.0 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar. The pH was 5.8, and the proliferation culture was carried out in the dark. After 40 days of proliferation culture, the callus proliferated into clumps approximately 1.8 cm in diameter.

[0113] 5. Callus differentiation: The clumps with a diameter of approximately 1.8 cm from step 3 were transferred to callus differentiation medium for differentiation culture. The components of the differentiation medium are: modified MS as the basic medium, with only 3.0 mg / L 6-BA, 2.0 mg / L NAA, 0.5 g / L casein hydrolysate, 1.5 mg / L multi-walled carbon nanotubes, 30 g / L sucrose, and 7.0 g / L agar added; the pH value is 5.8, and the proliferation culture is a light-dark alternating culture. The light culture time of the light-dark alternating culture is 16 h / d, the dark culture time is 8 h / d, and the light intensity of the light culture is 800-1200 lx; ​​after 40 days of culture, it can be observed that the pale yellow callus differentiates into white buds, and then differentiates into a large number of clustered buds.

[0114] 6. Bud Culture: Separate the 1.0-1.5 cm tall clustered buds from step 5 from the callus tissue and transfer them to a bud culture medium for bud culture. The bud culture medium consists of a modified MS basal medium supplemented with 4.0 mg / L 6-BA, 2.0 mg / L NAA, 2.0 mg / L multi-walled carbon nanotubes, 60 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0115] After 40 days of culture, the same bud-strengthening medium is used for subculture. After another 40 days of culture, the clustered buds grow into rootless test tube seedlings with 3 to 4 leaves and a bulb circumference of 0.8 to 1.5 cm.

[0116] 7 Seedling and rooting culture: Seedling and rooting culture can be completed in one step. The specific method is to transfer the rootless test tube seedlings obtained after the bud culture in step 6 to the bud and rooting culture medium for seedling and rooting culture. The components of the seedling and rooting culture medium are: MS as the basic culture medium, and only 2.0mg / L 6-BA, 1.0mg / L NAA, 2.5mg / L IBA, 2.0mg / L carbon nanotubes, 40g / L sucrose and 7.0g / L agar are added, and the pH value is 5.8. The culture temperature for seedling and rooting culture is 25±1℃, the light intensity is 800~1200lx, and the light time is 16h / d. After 40d of culture, test tube seedlings with a bulb circumference of 1.5~2.0cm, 3~5 roots, and a weight of 1.5~2.0g can be obtained.

[0117] 8. Hardening and Transplanting: Hardening is performed indoors. Loosely cap the tissue culture flask and place it in the buffer room of the tissue culture room for 3 days. Then, move it indoors and place it under natural diffuse light for another 3 days. After hardening, remove the test tube seedlings with tweezers, wash off the culture medium from the roots, and transplant them into a 10×10 hole tray. Water thoroughly to establish roots. The cultivation medium is peat moss, perlite, and garden soil in a 1:1:1 volume ratio. The cultivation medium must be disinfected with an 800x dilution of broad-spectrum fungicide before transplanting. After transplanting, follow normal watering and fertilization procedures.

[0118] Comparative Example 1

[0119] The same as steps 1 to 3 in Example 1, the only difference is that the explant is a long-tube Lycoris radiata bulb disk.

[0120] Callus induction rate (%) = number of embryos induced to callus * 100 / number of embryos inoculated

[0121] Callus bud differentiation rate (%) = number of differentiated callus blocks * 100 / number of inoculated callus blocks

[0122] Table 1 Effects of different explants on callus induction and differentiation

[0123]

[0124] Example 3

[0125] The same steps as in Example 1, Steps 1 to 5, differ only in that the composition of the differentiation medium is as follows: modified MS as the basal medium, supplemented with 2.0 mg / L 6-BA, 1.0 mg / L NAA, 0.75 g / L casein hydrolysate, 1.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar.

[0126] Comparative Example 2

[0127] The same steps as in Example 1, step 1 to step 5, differ only in that the composition of the differentiation medium is as follows: modified MS is used as the basic medium, and only 2.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5 g / L agar are added.

[0128] Comparative Example 3

[0129] The same steps as in Example 1, Steps 1 to 5, differ only in that the composition of the differentiation medium is as follows: modified MS as the basal medium, supplemented with 2.0 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L carbon nanotubes, 1.25 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0130] Differentiation coefficient = the number of buds that can be differentiated from each callus

[0131] Table 2 Effect of casein hydrolyzate concentration (nitrogen source) on callus bud differentiation

[0132]

[0133] Comparative Example 4

[0134] The same steps as in Example 1, Steps 1 to 3, were performed except that the basal medium of the induction medium was MS medium; the induction medium composition was as follows: MS as the basal medium, supplemented with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0135] Comparative Example 5

[0136] The same steps as in Example 1, Steps 1 to 3, were performed except that the basal medium of the induction medium was 1 / 2 MS medium. The induction medium was composed of 1 / 2 MS as the basal medium, supplemented with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0137] Comparative Example 6

[0138] The same steps as in Example 1, Steps 1 to 3, were performed except that the basal medium of the induction medium was B5 medium; the induction medium composition was as follows: B5 as the basal medium, supplemented with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0139] Comparative Example 7

[0140] The same steps as in Example 1, Steps 1 to 3, differ only in that the basal medium of the induction medium is DKW medium; the induction medium composition is: DKW as the basal medium, supplemented only with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0141] Comparative Example 8

[0142] The same steps as in Example 1, Steps 1 to 3, were performed except that the basal medium of the induction medium was N6 medium; the induction medium composition was as follows: N6 as the basal medium, supplemented with 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

[0143] The calculation formula for callus induction rate (%) is the same as above. The results are shown in Table 5. It can be seen that the callus induction rate of Example 1 using the improved B5 as the basic medium is the highest, which is 66.67%.

[0144] Table 3 Effects of different basic culture media on the callus induction rate of Lycoris radiata embryos

[0145] serial number Basic culture medium Number of inoculated embryos Callus induction rate (%) Comparative Example 4 MS 30 40.00 Comparative Example 5 1 / 2MS 30 36.33 Example 1 Improved B5 30 66.67 Comparative Example 6 B5 30 36.67 Comparative Example 7 DKW 30 33.33 Comparative Example 8 N6 30 30.00

[0146] Examples 4 to 7 and Comparative Examples 9 to 17 only carried out callus induction culture, that is, only steps 1 to 3 in Example 1 were performed.

[0147] Example 4

[0148] The same steps as in Example 1 were performed in Steps 1 to 3, except that the 2,4-D concentration in the induction medium in Step 3 was changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0149] Example 5

[0150] The same steps as in Example 1 were performed in Steps 1 to 3, except that the 2,4-D concentration in the induction medium in Step 3 was changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0151] Example 6

[0152] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0153] Example 7

[0154] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0155] Comparative Example 9

[0156] The same steps as in Example 1 were performed in Steps 1 to 3, except that the 2,4-D concentration in the induction medium in Step 3 was changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0157] Comparative Example 10

[0158] The same steps as in Example 1 were performed in Steps 1 to 3, except that the 2,4-D concentration in the induction medium in Step 3 was changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0159] Comparative Example 11

[0160] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0161] Comparative Example 12

[0162] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0163] Comparative Example 13

[0164] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0165] Comparative Example 14

[0166] The same steps as in Example 1, except that the 6-BA concentration of the induction medium in step 3 was changed. The culture medium was modified B5 medium as the basal medium, supplemented with 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0167] Comparative Example 15

[0168] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0169] Comparative Example 16

[0170] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0171] Comparative Example 17

[0172] The same steps as in Example 1, except that the concentrations of 6-BA and 2,4-D in the induction medium in step 3 were changed. The culture medium was modified B5 medium as the basal medium, supplemented with only 6-BA, 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar. The amounts of 6-BA and 2,4-D added are shown in Table 4.

[0173] The calculation formulas for the callus induction rates (%) of Examples 1 to 2, Examples 4 to 7, and Comparative Examples 9 to 17 are the same as above. The results are shown in Table 4.

[0174] Table 4 Effects of 6-BA and 2,4-D concentrations on callus induction rate of Lycoris radiata embryos

[0175]

[0176]

[0177] In summary, the technical solution of the present invention can improve the callus induction rate and differentiation rate of Lycoris radiata, and ultimately improve the reproduction efficiency and reproduction coefficient.

[0178] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A tissue culture medium for regeneration of Lycoris radiata, characterized in that: The tissue culture medium includes an induction medium, a proliferation medium, a differentiation medium, a bud growth medium, and a seedling growth and rooting medium; The induction culture medium uses the improved B5 culture medium as the basic culture medium and further comprises: 1.0-2.0 mg / L 6-BA, 6.0-10.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 30 g / L sucrose and 6.5-7.0 g / L agar; The proliferation culture medium uses a modified B5 culture medium as a basic culture medium and further comprises: 1.0-2.0 mg / L 6-BA, 3.0-5.0 mg / L 2,4-D, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar; The modified B5 medium comprises: 134 mg / L ammonium sulfate, 3 mg / L boric acid, 880 mg / L calcium chloride dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 500 mg / L magnesium sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 0.75 mg / L potassium iodide, 2500 mg / L potassium nitrate, 150 mg / L sodium dihydrogen phosphate, 2 mg / L zinc sulfate heptahydrate; 100 mg / L inositol, 1 mg / L vitamin B6, 10 mg / L vitamin B1, and 5.0 mg / L niacin. The differentiation medium uses a modified MS medium as a basic medium and further comprises: 2.0-4.0 mg / L 6-BA, 1.0-2.0 mg / L NAA, 0.5-1.0 g / L casein hydrolysate, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose, and 6.5-7.0 g / L agar; The modified MS medium comprises: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 880 mg / L calcium chloride dihydrate, 22.3 mg / L manganese sulfate tetrahydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L disodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate heptahydrate, 5.0 mg / L nicotinic acid, 10.0 mg / L vitamin B1, 1.0 mg / L vitamin B6, 2 mg / L glycine, and 100 mg / L inositol. The bud-strengthening culture medium uses a modified MS culture medium as a basic culture medium and further comprises: 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-strengthening and rooting culture medium uses MS culture medium as a basic culture medium and further comprises: 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.

2. The tissue culture medium according to claim 1, wherein The induction medium uses a modified B5 medium as a basic medium and further includes: 1.0 mg / L 6-BA, 6.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar; or, the induction medium uses a modified B5 medium as a basic medium and further includes: 1.0 mg / L 6-BA, 8.0 mg / L 2,4-D, 1.0 g / L casein hydrolysate, 30 g / L sucrose, and 6.5 g / L agar.

3. A method for regenerating callus tissue of Lycoris radiata, characterized in that: The regeneration tissue culture method adopts the tissue culture medium according to claim 1 or 2, comprising the following steps: Inoculating the embryos of Lycoris radiata on an induction medium for callus induction culture to obtain embryos that have produced callus tissue; transferring the embryos that have produced callus tissue to a proliferation medium for callus proliferation culture to obtain callus tissue masses; transferring the callus mass to a differentiation medium for callus differentiation culture to obtain clustered buds; Transferring the clustered buds to a bud-strengthening medium for bud-strengthening culture to obtain rootless seedlings; The rootless seedlings are transferred to a seedling-strengthening and rooting culture medium for seedling-strengthening and rooting culture to obtain tissue culture seedlings.

4. The regeneration tissue culture method according to claim 3, wherein Before callus induction culture, the method further includes: cleaning and disinfecting the embryo; the cleaning and disinfection include: first cleaning with sterile water 2-3 times, then disinfecting with sodium hypochlorite solution with an effective chlorine content of 0.1%-0.3% for 1-2 minutes, and cleaning with sterile water 4-5 times.

5. The regeneration tissue culture method according to claim 3 or 4, characterized in that: The embryo is obtained by removing the pericarp of the long-tube Lycoris radiata capsule and cutting the seed along the longitudinal diameter to obtain the embryo.

6. The regeneration tissue culture method according to claim 3, characterized in that: The callus induction culture and callus proliferation culture are both dark cultures; the callus differentiation culture, bud culture and seedling rooting culture are all light-dark alternating cultures, with the illumination time being 12-16h / d and the illumination intensity being 800-1200Lx.

7. The regeneration tissue culture method according to claim 3, characterized in that: The diameter of the callus mass is 1.5 to 2.5 cm; the length of the clustered buds is 1.0 to 1.5 cm.

8. The regeneration tissue culture method according to claim 3, characterized in that: The time for the callus induction culture is 28 to 45 days; the time for the callus proliferation culture is 30 to 40 days; the time for the callus differentiation culture is 30 to 40 days; the time for the strong bud culture is 60 to 80 days; and the time for the strong seedling rooting culture is 30 to 40 days.

9. The regeneration tissue culture method according to claim 3, characterized in that: The strong bud culture process also includes transferring the culture product of the strong bud culture for 30 to 40 days to a new strong bud culture medium for subculture to obtain rootless seedlings.

10. The regeneration tissue culture method according to claim 3, characterized in that: The temperatures for the callus induction culture, callus proliferation culture, callus differentiation culture, bud strengthening culture and seedling rooting culture are all 25±1°C.

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