CN112237142A - Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system - Google Patents
Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system Download PDFInfo
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- CN112237142A CN112237142A CN202011202037.4A CN202011202037A CN112237142A CN 112237142 A CN112237142 A CN 112237142A CN 202011202037 A CN202011202037 A CN 202011202037A CN 112237142 A CN112237142 A CN 112237142A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/35—Bulbs; Alliums, e.g. onions or leeks
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention relates to the field of plant culture, in particular to a tissue culture medium for lycoris, a callus culture method and a method for establishing a lycoris regeneration system. The invention provides a tissue culture medium for lycoris, which comprises an induction medium; the induction culture medium takes an MS culture medium as a basic culture medium and also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.0.0-2.5 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 0.5-2.0 mg/L of carbon nano tube; the pH value of the induction culture medium is 5.5-6.0. The tissue culture medium provided by the invention can improve the induction rate.
Description
Technical Field
The invention relates to the field of plant culture, in particular to a tissue culture medium for lycoris, a callus culture method and a method for establishing a lycoris regeneration system.
Background
Lycoris radiata (Lycoris Herb.) belongs to Amaryllidaceae (Amaryllidaceae), and is a perennial Herb. The bulbs are in an egg-shaped shape, leaves come out in spring, leaves are in a belt shape, and 4-6 flowers are arranged in an umbrella-shaped inflorescence; yellow, red, white; the quilt flap strength rolls back and collapses. The flowering period is 7-8 months. The flower stem is tall and straight, the flower shape is beautiful, and the flower color is gorgeous. All the species have high ornamental value and are known as 'Chinese tulip'. Has gradually become a new type of fresh cut flowers, garden ground cover and potted bulbous flowers.
In a natural state, the lycoris plants generally propagate by natural bulbs, but the propagation coefficient is quite low, 1-2 bulbs are averagely distributed in one flowering bulb every year, and the time for the bulbs to bloom is about 3-4 years; the sexual reproduction mode is adopted for reproduction, and the time from seed germination to flowering generally needs 5-6 years. The propagation speed is slow, the propagation period is long, the method becomes a difficult problem in the popularization and application of the whole lycoris plants, and the development of the lycoris industry in China is greatly restricted.
In recent years, papers or patents for establishing a tissue culture rapid propagation technical system of lycoris plants by taking bulbus disks as explants to induce cluster buds are successively published, but the induction rate is not high by taking the bulbus disks of lycoris as the explants, and 1/4 or 1/6 bulbus disks can generally induce 5-10 buds, have small propagation coefficient and generally have the problem of low induction rate.
Disclosure of Invention
In view of the above, the invention provides a tissue culture medium for lycoris, a callus culture method and a method for establishing a lycoris regeneration system. The induction culture medium provided by the invention can improve the induction rate.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a tissue culture medium for lycoris, which comprises an induction medium; the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.0.0-2.5 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 0.5-2.0 mg/L of carbon nano tube;
the pH value of the induction culture medium is 5.5-6.0.
Preferably, the culture medium also comprises a proliferation medium; the multiplication culture medium takes an MS culture medium as a basic culture medium and also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.5.5-3.0 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 2.0-5.0 mg/L of carbon nano-tubes;
the pH value of the proliferation culture medium is 5.5-6.0.
Preferably, the lycoris genus includes diploid lycoris, lycoris chinensis, lycoris neglecta, broccoli, lycoris longiradiata, lycoris radiata, and lycoris anhui.
The invention provides a lycoris callus culture method, which comprises the following steps:
taking the mature seed embryo of the lycoris as an explant;
inoculating the seed embryo into an induction culture medium for induction culture to obtain a callus;
inoculating the callus obtained by induction culture to a proliferation culture medium for proliferation culture to obtain a proliferation callus;
and inoculating the proliferation callus to a differentiation culture medium for differentiation culture to obtain differentiation cluster buds.
Preferably, the temperature of the induction culture is 24-26 ℃; the light source for induction culture is LED red light and blue light 1:1, and the illumination intensity is 40-100 mu mol.m-2·s-1The illumination time is 16 h;
the temperature of the proliferation culture is 24-26 ℃; the light source for proliferation culture is LED red light and blue light 1:1, and the illumination intensity is 40-100 mu mol.m-2·s-1Light ofThe illumination time is 16 h.
Preferably, the differentiation medium takes an MS culture medium as a basic culture medium, and further comprises the following components in percentage by weight: 30g/L of sucrose, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 0.5.5-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the differentiation medium is 5.5-6.0;
the temperature of the differentiation culture is 24-26 ℃; the light source for the differentiation culture is LED white light, and the illumination intensity is 100-300 mu mol.m-2·s-1The illumination time is 16 h.
The invention provides a method for establishing an lycoris regeneration system, which comprises the following steps:
inoculating the differentiated cluster buds obtained by the tissue culture medium in the technical scheme or the differentiated cluster buds obtained by the culture method in the technical scheme to a strong bud culture medium for strong bud culture to obtain strong bud seedlings;
inoculating the strong bud seedlings to a strong seedling and rooting culture medium for strong seedling and rooting culture to obtain rooted seedlings;
carrying out subculture on the rooted seedlings to obtain subcultured seedlings;
and (4) carrying out transplanting culture on the subcultured seedlings to obtain lycoris seedlings.
Preferably, the budding culture medium takes an MS culture medium as a basic culture medium, and further comprises the following components in percentage by weight: 40g/L of cane sugar, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 1.0.0-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the strong seedling culture medium is 5.5-6.0.
Preferably, the strong seedling and rooting culture medium takes an MS culture medium as a basic culture medium, and further comprises the following components in percentage by weight: 40g/L of cane sugar, 7.0-7.5 g/L of agar, 1.5-2.0 mg/L, NAA 0.5.5-1.0 mg/L, IBA 2.0.0-3.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the strong seedling and rooting culture medium is 5.5-6.0.
Preferably, the culture medium used in the transplanting culture comprises peat soil, perlite and garden soil; the volume ratio of the peat soil to the perlite to the garden soil in the culture medium is 1:1: 1.
Has the advantages that:
the invention provides a tissue culture medium for lycoris, which comprises an induction medium; the induction culture medium also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.0.0-2.5 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 0.5-2.0 mg/L of carbon nano tube; the pH value of the induction culture medium is 5.5-6.0. The invention selects glucose as the carbon source of the most plant growth of the glucose, and TDZ, 2,4-D and carbon nano tubes induce the generation, proliferation and differentiation of callus; agar is used as a coagulator of the culture medium; the invention solves the problem of low induction rate of lycoris by matching the components and using amount and relative proportion of the components.
The invention provides a method for culturing lycoris callus, which takes a seed embryo as an explant to induce and generate callus, and the callus is differentiated and cultured to germinate a large number of cluster buds. Theoretically, the buds generated by callus differentiation are almost unlimited, so that the propagation coefficient and the propagation efficiency can be greatly improved; and the seedling period can be shortened. The embryo is used as explant, the induction rate of callus is high, and the embryo can be used for various molecular biology level operations after proliferation.
In addition, in the invention, different culture mediums are adopted in different culture stages so as to meet the requirement of plants in different stages on nutrition; the saccharide substances adopted in different culture stages and the concentrations of the saccharide substances are different, glucose is used in the induction culture and the proliferation culture, and the concentration is 20-25 g/L; sucrose is used for differentiation culture, strong bud culture, strong seedling culture and rooting culture, the concentration of the sucrose in a strong bud culture medium is 40g/L, and the concentration of the sucrose in a differentiation culture medium is 30 g/L; the types and the concentrations of the saccharides adopted in different culture stages are different, and the technical problem of low induction rate can be solved to the maximum extent by adopting the culture medium and the culture method provided by the invention.
Detailed Description
The invention provides a tissue culture medium for lycoris, which comprises an induction medium; the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 20-25 g of glucose, 7.0-7.5 g/L, TDZ 2.0.0-2.5 mg/L of agar, 2, 4-D2.0-2.5 mg/L of carbon nano tube and 0.5-2.0 mg/L of carbon nano tube, preferably 22-23 g/L of glucose, 7.3g/L, TDZ (N-phenyl-N' - (1,2, 3-thiadiazole-5-yl) urea) 2.5mg/L of agar, 2,4-D2.0 mg/L of carbon nano tube and 2.0mg/L of carbon nano tube; the carbon nanotubes are preferably multi-walled carboxylated carbon nanotubes; the pH value of the induction culture medium is 5.5-6.0, and preferably 5.8. Glucose in the induction culture medium provided by the invention is used as a carbon source for plant growth; the plant growth regulator and the carbon nano tube induce the generation, proliferation and differentiation of the callus; agar is used as a coagulator of the culture medium; the invention solves the problem of low inductivity through the matching of the components and the dosage and relative proportion of each hormone.
If no special requirement exists, the raw materials adopted by the invention are all obtained by conventional purchase of the technicians in the field.
In the present invention, the tissue culture medium preferably further comprises a proliferation medium; the proliferation culture medium preferably takes an MS culture medium as a basic culture medium, and also preferably comprises the following components in percentage by weight: 20-25 g/L glucose, 7.0-7.5 g/L, TDZ 2.5.5-3.0 mg/L agar, 2, 4-D2.0-2.5 mg/L carbon nano tube, and more preferably 22-23 g/L glucose, 7.3g/L, TDZ 2.8.8 mg/L agar, 2, 4-D2.3 mg/L carbon nano tube, and 2.2mg/L carbon nano tube; the carbon nanotube is preferably a single-walled carbon nanotube, a multi-walled carboxylated carbon nanotube or a multi-walled carbon nanotube, more preferably a multi-walled carboxylated carbon nanotube or a multi-walled carbon nanotube; the pH value of the proliferation culture medium is preferably 5.5-6.0, and more preferably 5.8. In the present invention, the single-walled carbon nanotubes, multi-walled carboxylated carbon nanotubes and multi-walled carbon nanotubes are preferably obtained from Xiancheng nanomaterial science and technology, Inc.
In the present invention, the genus Lycoris preferably includes diploid Lycoris radiata, Lycoris chinensis, Lycoris neglecta, Mallotus philippinensis, Lycoris longituba, Lycoris radiata and Lycoris anhua.
The invention provides a lycoris callus culture method, which comprises the following steps:
taking the mature seed embryo of the lycoris as an explant;
inoculating the seed embryo into an induction culture medium for induction culture to obtain a callus;
inoculating the callus obtained by induction culture to a proliferation culture medium for proliferation culture to obtain a proliferation callus;
and inoculating the proliferation callus to a differentiation culture medium for differentiation culture to obtain differentiation cluster buds.
The invention takes the mature embryo of lycoris as the explant. In the present invention, the obtaining manner of the embryo preferably includes: cutting Lycoris seeds to obtain seed embryo of Lycoris. In the present invention, the cutting is preferably performed in a seed-inoculating tray; the cutting mode is preferably that a scalpel is adopted to cut the lycoris seeds; the cutting direction is preferably along the direction of the seed ridge of the lycoris seeds; by adopting the mode and the direction for cutting, the cutting of the seed embryo can be avoided, and the complete seed embryo can be obtained. In the invention, the lycoris seeds are preferably seeds harvested from 10 to 11 months; the harvesting standard of the lycoris seeds is preferably to harvest the lycoris seeds when the capsules of the lycoris capsules turn yellow from green and the seed coats turn black; the selection standard of the lycoris seeds is preferably healthy, full and consistent in size. In the invention, the harvested seeds are preferably treated as soon as possible, and more preferably treated along with picking, so that the pollution rate can be reduced; in the invention, the seeds are harvested after being mature, the harvested seeds have less bacteria and low pollution rate, and the induction rate of the callus is further improved.
Before the lycoris seeds are cut, the invention preferably further comprises the steps of sequentially cleaning and disinfecting the lycoris seeds to obtain disinfected seeds; the cleaning mode is preferably that the lycoris seeds are sequentially rinsed and washed to obtain the cleaned lycoris seeds; the rinsing is preferably carried out by using a detergent; the detergent is preferably a common household liquid detergent; the invention does not require any particular detergent, and detergents well known to those skilled in the art can be used. In the invention, the rinsing time is preferably 30-60 min, and more preferably 45 min; the flushing mode preferably adopts running water flushingWashing; the flushing time is preferably 2-2.5 h, and more preferably 2.3 h; the sterilization is preferably performed in a superclean bench with glass bottles; the sterilization preferably comprises a first sterilization and a second sterilization performed in sequence; the first disinfection mode is preferably that the lycoris seeds are soaked in ethanol; the volume concentration of the ethanol is preferably 70-100%, and more preferably 75%; the soaking time is preferably 30-45 s, and more preferably 40 s; the present invention preferably washes the first sterilized lycoris seed with sterile water; the number of times of the sterile water washing is preferably 1-3, and more preferably 2-3; the time of each time of the sterile water washing is preferably 30-45 s, and more preferably 40 s; the second disinfection method is preferably to place the lycoris seeds in the HgCl after the first disinfection2Soaking; the HgCl2The mass concentration of (b) is preferably 0.1%; the soaking time is preferably 60-70 min, and more preferably 65 min; according to the invention, the lycoris seeds after the second disinfection are preferably washed by sterile water; the number of times of the sterile water washing is preferably 4-6 times, and more preferably 5 times. After the aseptic water washing, the aseptic water absorption paper is preferably adopted to absorb the water on the surface of the lycoris seeds. The method adopts twice sterilization, so that the pollution rate can be reduced, and the induction rate of the callus can be improved.
The invention inoculates the embryo in an induction culture medium for induction culture to obtain the callus. In the present invention, the inoculation is preferably performed by placing the embryo on the induction medium; the temperature of the induction culture is preferably 24-26 ℃, and more preferably 25 ℃; the light source for induction culture is LED red light and blue light in a ratio of 1: 1; the illumination intensity of the induction culture is 40-100 mu mol.m-2·s-1More preferably 70 to 90 μm-2·s-1(ii) a The illumination time of the induction culture is preferably 16 h; the time for induction culture is preferably 30-35 d, and more preferably 33 d; the end standard of the induction culture is that the surface of the embryo is almost completely covered by the granular callus; after the induction culture is finished, the volume of the callus is preferably 2.0-2.5 times of the volume of the embryo before the induction culture. By adopting the induction culture medium and the induction mode provided by the invention, white seeds can be observed after 7-10 daysThe embryos are obviously expanded, after 15 days, granular protrusions (which are marks for callus formation) can be observed on white seed embryos, after 30-35 days, the surfaces of the seed embryos are almost completely covered by the granular calluses, and the volume of the seed embryos is 2.0-2.5 times of the original volume. The invention can improve the induction rate of the callus tissue to more than 90 percent under the specific culture condition.
Obtaining the callus, inoculating the callus obtained by induction culture to a proliferation culture medium for proliferation culture to obtain the proliferation callus. In the invention, the temperature of the proliferation culture is preferably 24-26 ℃, and more preferably 25 ℃; the light source for proliferation culture is preferably LED red light and blue light 1: 1; the illumination intensity is 40-100 mu mol.m-2·s-1More preferably 70 to 90. mu. mol/m-2·s-1(ii) a The illumination time of the proliferation culture is preferably 16 h; the time for propagation culture is preferably 60-90 d, and more preferably 80 d; the volume of the callus is preferably 50-80 times of that of a seed embryo body before induction culture. The callus can be used for molecular biology operation or differential culture to obtain a large amount of cluster buds; the molecular biological manipulation preferably comprises transgenic breeding or genetic improvement of a related trait. According to the invention, the induction culture stage and the proliferation culture stage are illuminated by LED red light and blue light in a ratio of 1:1, so that the induction rate and the proliferation speed of the callus can be improved.
After obtaining the callus, the invention inoculates the proliferation callus to a differentiation culture medium for differentiation culture to obtain differentiation cluster buds. In the invention, the differentiation culture mode is preferably to cut the callus into small pieces for culture; the volume of the small block is preferably 2.0cm3～3.0cm3More preferably 1.5cm3～2.0cm3(ii) a The differentiation medium preferably takes an MS culture medium as a basic culture medium, and also preferably comprises the following components in percentage by weight: 30g/L of sucrose, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 0.5.5-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube, more preferably 30g/L of sucrose, 7.3g/L of agar, 2.8mg/L, NAA 1.0.0 mg/L of 6-BA and 3.0mg/L of carbon nano tube; the carbon nanotubes are preferably single-walled carbonNanotubes, multi-walled carboxylated carbon nanotubes or multi-walled carbon nanotubes, more preferably multi-walled carboxylated carbon nanotubes or multi-walled carbon nanotubes; the pH value of the differentiation medium is preferably 5.5-6.0, and more preferably 5.8. In the invention, the temperature of the differentiation culture is preferably 24-26 ℃, and more preferably 25 ℃; the light source for differentiation culture is preferably LED white light; the illumination intensity of the differential culture is 100-300 mu mol.m-2·s-1More preferably 250. mu. mol. m-2·s-1(ii) a The illumination time of the differentiation culture is 16 h. In the present invention, the time for the differentiation culture is preferably 30 to 40 days, more preferably 35 days, and the height of the multiple shoots is preferably 1.0 to 1.5cm, more preferably 1.1 to 1.4 cm. By adopting the differentiation culture medium and the differentiation culture mode provided by the invention, white bud points can be observed from callus through culture for 30-40 days, and then a large number of cluster buds are differentiated. The invention adopts specific conditions in the proliferation culture stage and the differentiation culture stage to promote the rapid and mass proliferation of the callus and improve the differentiation rate.
The invention provides a method for establishing an lycoris regeneration system, which comprises the following steps:
inoculating the differentiated cluster buds obtained by the induction culture medium or the culture method in the technical scheme to a bud-strengthening culture medium for bud-strengthening culture to obtain bud-strengthening seedlings;
inoculating the strong bud seedlings to a strong seedling and rooting culture medium for strong seedling and rooting culture to obtain rooted seedlings;
carrying out subculture on the rooted seedlings to obtain subcultured seedlings;
and (4) carrying out transplanting culture on the subcultured seedlings to obtain lycoris seedlings.
The differentiated cluster buds obtained by the induction culture medium in the technical scheme or the differentiated cluster buds obtained by the culture method in the technical scheme are inoculated in a bud-strengthening culture medium for bud-strengthening culture to obtain the strong buds. In the present invention, the strong seedling culture is preferably performed in a tissue culture flask. In the present invention, the strong bud culture medium preferably uses an MS culture medium as a basal culture medium, and further preferably comprisesThe following components in percentage by weight: 40g/L of sucrose, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 1.0.0-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube, more preferably 40g of sucrose, 7.3g of agar, 2.8mg of 6-BA, 1.3mg/L of NAA and 3.0mg/L of carbon nano tube; the carbon nanotubes are preferably multi-walled carboxylated carbon nanotubes; the pH value of the strong seedling and rooting culture medium is preferably 5.5-6.0, and more preferably 5.8. In the invention, the temperature for culturing the strong seedlings is preferably 24-26 ℃, and more preferably 25 ℃; the light source for differentiation culture is preferably LED white light; the illumination intensity for strong seedling culture is 100-300 mu mol.m-2·s-1More preferably 250. mu. mol. m-2·s-1(ii) a The illumination time for strong seedling culture is 16 h; the time for culturing the strong buds is preferably 30-35 d, and more preferably 33 d; the strong bud preferably grows into 1.0-1.5 cm leaves; the circumference of the stem of the strong bud is preferably 0.5-1.0 cm. The invention adopts the specific conditions to carry out strong bud culture so that callus tissues are differentiated into a large number of cluster buds, and the larger the number is, the better the number is, the efficiency of rapid propagation is improved; if the transgenic plant is used, more transformed seedlings can be expected to be obtained.
After the strong bud seedlings are obtained, the invention inoculates the strong bud seedlings in a strong seedling and rooting culture medium for strong seedling and rooting culture, and obtains the rooting seedlings. In the present invention, the strong seedling and rooting culture is preferably performed in a tissue culture flask. In the invention, the strong seedling and rooting culture medium preferably takes an MS culture medium as a basic culture medium, and preferably further comprises the following components in percentage by weight: 40g/L of sucrose, 7.0-7.5 g/L of agar, 1.5-2.0 mg/L, NAA 0.5.5-1.0 mg/L, IBA 2.0.0-3.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano-tube, more preferably 40g of sucrose, 7.3g of agar, 6-BA1.8mg/L, NAA 0.8, 0.8mg/L, IBA 3.0.0 mg/L of 6-BAN and 3.0mg/L of carbon nano-tube; the carbon nanotubes are preferably multi-walled carboxylated carbon nanotubes; the pH value of the strong seedling and rooting culture medium is preferably 5.5-6.0; more preferably 5.8. In the invention, the temperature of strong seedling and rooting culture is preferably 24-26 ℃, and more preferably 25 ℃; the light source for differentiation culture is preferably LED white light; the illumination intensity of the strong seedling and rooting culture is 100-300 mu mol.m-2·s-1More preferably 250. mu. mol. m-2·s-1(ii) a The illumination time for the strong seedling and rooting culture is 16 h.
After the rooted seedlings are obtained, carrying out subculture on the rooted seedlings to obtain subcultured seedlings which are used as the rooted seedlings for subsequent transplanting culture; the culture medium of the subculture is preferably a strong seedling culture medium and a rooting culture medium; consistent with the bud-strengthening culture medium, and will not be described herein; the subculture is preferably subcultured for 1 time every 30-35 d, and more preferably 33 d; the number of subcultures is preferably 1-2; more preferably 1 time. In the invention, the preferable bulb circumference of the obtained secondary culture seedling is 1.5-2.0 cm, and the preferable root system is 3-5, and more preferably 4; the weight of the subculture seedling is preferably 1.5-2.0 g, more preferably 1.8g, the invention can promote the rapid growth and rooting of the tissue culture seedling by adopting specific conditions in the strong bud culture stage, the strong seedling culture stage and the rooting culture stage, and the survival rate of later-stage transplantation can be ensured due to strong seedlings and more roots.
After obtaining the subculture seedling, the invention carries out transplanting culture on the rooted seedling to obtain the lycoris seedling. In the invention, the culture medium used in the transplanting culture preferably comprises peat soil, perlite and garden soil; the volume ratio of peat soil, perlite and garden soil in the culture medium is preferably 1:1: 1; the substrate is preferably sterilized; the disinfectant adopted during the disinfection treatment is preferably carbendazim; the dilution multiple of the disinfectant is preferably 600-1000 times, and more preferably 800 times. The matrix for transplanting is treated by the disinfectant mainly because the tissue culture seedlings are relatively weak and have weak antibacterial property, so that the infection rate of pathogenic bacteria can be reduced after treatment, and the survival rate of transplanting is improved. In the present invention, the transplanting culture is preferably performed in a plug tray; the size of the plug is preferably 10 x 10, i.e. 100 holes.
Before the transplanting culture, the invention preferably also comprises cleaning the rooting seedling; the cleaning mode is preferably to wash off the culture medium at the root part of the rooted seedling.
After the rooting seedling, the invention preferably also comprises hardening seedling treatment on the rooting seedling; the hardening treatment is preferably carried out indoors; the hardening-seedling treatment mode is preferably as follows: and (3) placing the rooted seedlings in a tissue culture buffer room for 2-3 d, and then transferring the rooted seedlings to a natural scattering light for 2-3 d to obtain the acclimatized and rooted seedlings. In the present invention, the natural astigmatism is preferably natural light. The invention transplants the sterile rooting seedling into a germ-carrying environment, exercises the resistance of the rooting seedling, can increase the rooting resistance and ensures survival after transplantation.
To further illustrate the present invention, the tissue culture medium, callus culture method and method for establishing lycoris regeneration system provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
(1) The seed collection and sterilization are carried out in 2018, 10 and 25 days, mature fruit pods of lycoris chinensis are collected, put into seed bags and taken back to a laboratory for later use.
Peeling off the fruit pod to obtain the Chinese lycoris seed. And selecting healthy and plump seeds with consistent sizes, shaking and rinsing the seeds for 40min by using a detergent, and flushing the seeds for 2h by using running water. On a clean bench, the seeds are placed in sterilized glass bottles, and a first disinfection is carried out: sterilizing with 75% ethanol for 40s, washing with sterile water for 3 times (30 ss each time); a second sterilization is then carried out: with 0.1% HgCl2Treating for 60min, washing with sterile water for 5 times, and drying surface water with sterile absorbent paper.
(2) Obtaining a seed embryo: sterile seeds were placed on the inoculation plate, the seeds were cut along the direction of the seed ridge of the lycoris seed with a scalpel, and embryos were removed and kept intact as explants for a total of 150 grains.
(3) Induction of callus: and directly inoculating the complete seed embryo to a callus induction culture medium in a flat mode to obtain the callus. The induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 22g/L glucose, 7.5g/L, TDZ 2.5.5 mg/L agar, 2,4-D2.0 mg/L multi-wall carboxylated carbon nano-tube, 1.0mg/L multi-wall carboxylated carbon nano-tube and pH value of 5.8. The light source is LED red light and blue light 1:1, and the illumination intensity is 80 mu mol.m-2·s-1And the illumination time is 16 h. After inoculation for about 8 days, obvious expansion of white seed embryos can be observed; after 15 days, a pale appearance of 91.5% white embryos was observedYellow granular protrusions, which are markers for callus formation. After 30 days, the surface of the embryos was almost completely covered with granular callus, at which time the volume of the embryos was 2.5 times the original volume.
(4) Proliferation culture of callus: after inoculating the seed embryo for 32d, the seed embryo almost completely covered with the granular callus is transferred to a proliferation medium for proliferation culture. Wherein the proliferation culture medium takes an MS culture medium as a basic culture medium and also comprises the following components by weight percent: 22g/L glucose, 7.0g/L, TDZ 2.6.6 mg/L agar, 2,4-D2.0 mg/L multiwall carboxylated carbon nanotube and 2.5mg/L multiwall carboxylated carbon nanotube, and the pH value is 5.8. The culture temperature is 25 +/-1 ℃, the light sources are LED red light and LED blue light with the ratio of 1:1, and the illumination intensity is 80 mu mol.m-2·s-1And the illumination time is 16 h. After about 90 days of culture, the volume of the callus is about 50 times of the volume of the embryo. The callus may be subjected to various molecular biological operations or to differential culture to obtain a large number of clumpy buds.
(5) Differentiation of callus: cutting callus 50 times of the embryo volume to about 1.5cm3The small blocks are transferred to the callus on a differentiation culture medium, so that the callus is differentiated into cluster buds. The differentiation culture medium takes an MS culture medium as a basic culture medium and also comprises the following components by weight percent: 30g/L of sucrose, 7.0g/L of agar, 2.5mg/L, NAA 1.5.5 mg/L of 6-BA and 2.5mg/L of multi-wall carboxylated carbon nano-tube, and the pH value is 5.8. The culture temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 38 days of culture, the pale yellow callus can be observed to differentiate white bud points, and then a large number of cluster buds are differentiated.
(6) Strong bud culture: separating cluster buds from the callus, transferring the cluster buds to a strong bud culture medium for strong bud culture to obtain strong bud test-tube plantlets. Wherein the strong bud culture medium takes an MS culture medium as a basic culture medium and also comprises the following components by weight percent: 40g/L of sucrose, 7.5g/L of agar, 3.0mg/L, NAA 1.5.5 mg/L of 6-BA and 2.5mg/L of multi-wall carboxylated carbon nano-tube, and the pH value is 5.8. The culture temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 35 days of culture, the cluster buds grow into 1.5cm leavesAnd test-tube plantlet with bulb circumference of 0.5 cm.
(7) Strong seedling and rooting culture: transferring the bud differentiated from the callus onto a strong seedling and rooting culture medium to obtain a rooting test-tube seedling. Wherein the strong seedling and rooting culture medium takes an MS culture medium as a basic culture medium, and preferably further comprises the following components in percentage by weight: 40g/L of sucrose, 7.5g/L of agar, 2.0mg/L, NAA 0.5.5 mg/L, IBA 3.0.0 mg/L of 6-BA and 2.5mg/L of multi-wall carboxylated carbon nano-tube, and the pH value is 5.8. The culture temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 32d, subculture was continued with the same formulation. And culturing for 35 days to obtain rooting test-tube plantlets with the bulb circumference of 1.5cm, 3 roots and the weight of about 1.8 g.
(8) Hardening and transplanting seedlings: hardening seedlings are carried out indoors. And (4) loosening the cover of the tissue culture bottle, placing the tissue culture bottle in a buffer room of a tissue culture room for 3 days, then moving the tissue culture bottle into an indoor room, placing the tissue culture bottle in natural scattered light, and placing the tissue culture bottle for 2 days to obtain the seedling exercising and rooting test-tube plantlet. After the hardening-off, the rooted test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the rooted test-tube plantlet is transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 800 times of carbendazim. And then performing normal water and fertilizer management.
The whole period of obtaining the Chinese lycoris seedlings in the embodiment is about 6-7 months.
Example 2
(1) The seeds are collected and sterilized in 2018, 11 and 15 days, mature fruit pods of smilax glabra are collected in a nursery garden of the laboratory, and the mature fruit pods are filled into seed bags and taken back to the laboratory for later use.
And (4) opening the fruit pods to obtain the smile seeds. Selecting healthy and plump seeds with consistent size, shaking and rinsing with detergent for 45min, and washing with running water for 2h 15 min. On a clean bench, the seeds are placed in sterilized glass bottles, and a first disinfection is carried out: sterilizing with 75% ethanol for 40s, washing with sterile water for 3 times (about 40s each time); a second sterilization is then carried out: with 0.1% HgCl2Treating for 65min, washing with sterile water for 6 times, and drying surface water with sterile absorbent paper.
(2) Obtaining a seed embryo: the sterile seeds are placed on a seed inoculating tray, the lycoris seeds are longitudinally cut along the ridge direction of the lycoris seeds by using a scalpel, and the whole embryos are taken out to be explants for standby, and 138 seeds are used.
(3) And (3) induction culture of the callus, directly inoculating the complete seed embryo to a callus induction culture medium in a flat mode to obtain the callus. The induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 24g/L glucose, 7.2g/L, TDZ 2.2.2 mg/L agar, 2, 4-D2.5 mg/L multiwalled carbon nanotube and pH 5.8. The culture temperature is 25 +/-1 ℃, the light sources are LED red light and LED blue light with the ratio of 1:1, and the illumination intensity is 80 mu mol.m-2·s-1And the illumination time is 16 h. After inoculation for about 8 days, obvious expansion of white seed embryos can be observed; after 15 days, a yellowish granular protrusion was observed on 93.6% of the white embryos, which is indicative of callus formation. After 35d, the surface of the embryos was almost completely covered with granular callus, at which time the volume of the embryos was about 1.2 times the original volume.
(4) After inoculation of callus proliferation culture embryos for 33d, the embryo covered with granular callus is transferred to a callus proliferation culture medium to perform callus proliferation culture. Wherein the proliferation culture medium takes an MS culture medium as a basic culture medium and also comprises the following components by weight percent: 23g/L glucose, 7.3g/L, TDZ 2.8.8 mg/L agar, 2,4-D2.0 mg/L multiwalled carbon nanotube and 2.5mg/L multiwalled carbon nanotube, and the pH value is 5.7. The culture temperature is 25 +/-1 ℃, the light sources are LED red light and LED blue light with the ratio of 1:1, and the illumination intensity is 80 mu mol.m-2·s-1And the illumination time is 16 h. After about 80 days of culture, the callus volume was about 70 times the embryo volume. The callus may be subjected to various molecular biological operations or to differential culture to obtain a large number of clumpy buds.
(5) Differentiation of callus 70 times the volume of the embryo was cut into about 2.0cm3The small blocks are transferred to a callus differentiation culture medium to lead the callus to differentiate into cluster buds. The differentiation culture medium takes an MS culture medium as a basic culture medium and also comprises the following components by weight percent: 30g of glucose, 7.3g/L of agar, 2.8mg/L, NAA 1.3.3 mg/L of 6-BA and 3.0mg/L of multi-wall carbon nano-tube, and the pH value is 5.8. CulturingThe temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 40 days of culture, the yellowish callus can be observed to differentiate into green bud points, and then a large number of cluster buds.
(6) And (4) separating cluster buds from the callus, transferring the cluster buds to a strong bud culture medium for strong bud culture to obtain a strong bud test-tube plantlet. Wherein the strong seedling culture is preferably carried out in a tissue culture bottle. In the invention, the budding culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 40g/L of sucrose, 7.3g/L of agar, 2.5mg/L of 6-BA, 1.2mg/L of NAA1, 3.0mg/L of multi-wall carbon nano-tube and the pH value of 5.8. The culture temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 40 days of culture, the cluster buds grow into strong bud test-tube seedlings with leaf length of 1.3cm and bulb circumference of about 0.6 cm.
(7) And (3) strong seedling and rooting culture, transferring the bud differentiated from the callus onto a strong bud and rooting culture medium to obtain a rooting test-tube seedling. Wherein the strong seedling and rooting culture medium takes an MS culture medium as a basic culture medium and also comprises the following components in percentage by weight: 40g/L of sucrose, 7.5g/L of agar, 1.8mg/L, NAA 0.5.5 mg/L, IBA 2.5.5 mg/L of 6-BA and 3.0mg/L of multi-wall carbon nano-tube, and the pH value is 5.8. The culture temperature is 25 +/-1 ℃, the light source is LED white light, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h. After 32d, subculture was continued with the same formulation. After 35 days of culture, rooting test-tube plantlets with the bulb circumference of about 1.6cm, 4 roots and the weight of 1.6g can be obtained.
(8) Hardening seedlings and transplanting the hardened seedlings are carried out indoors. And (4) loosening the cover of the tissue culture bottle, placing the tissue culture bottle in a buffer room of a tissue culture room for 3 days, then moving the tissue culture bottle into an indoor room, placing the tissue culture bottle in natural scattered light, and placing the tissue culture bottle for 2 days to obtain the seedling exercising and rooting test-tube plantlet. After the hardening-off, the rooted test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the rooted test-tube plantlet is transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 800 times of carbendazim. And then performing normal water and fertilizer management.
The whole period of obtaining the seedling of the lycoris aurea is about 6-6.5 months.
Comparative example 1
Taking healthy underground lycoris radiata bulbs with the diameter of 2.0cm, peeling off outer-layer bulbs, cutting off all roots and the upper half part 1/2 of the bulbs, reserving a bulb disc, cleaning soil on the surface of the roots by a brush, cutting into 4 blocks according to the size of the bulb disc, wherein each block is 1.0cm2, washing with 84 detergents in a shaking way for 30min, and washing with running water for 30min for later use.
The above materials were rinsed on a clean bench with 75% alcohol for 30s, 0.1% HgCl2Sterilizing for 40min, washing with sterile water for 4 times, and drying surface water with sterile absorbent paper.
Directly inoculating the sterilized bulb blocks onto a bud induction culture medium in a manner that a basal disc is downward, wherein the culture medium comprises the following components: 30g/L of sucrose, 7.5g/L of agar, 3.0mg/L of MS, 6-BA and 1.5 mg/LNAA.
After 28 days of culture, shoots grew from the scale of the explants. Subculture was carried out after 40 days. The culture temperature is 25 ℃, and the illumination intensity is 200 mu mol.m-2·s-1And the illumination time is 12 h/d.
After the adventitious bud is subcultured for 40d, the adventitious bud is transferred to a bud multiplication medium for multiplication culture. The formula of the culture medium is sucrose 30g/L, agar 7.5g/L, MS, 3.0mg/L6-BA and 1.0mg/L NAA. The culture temperature is 25 ℃, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h/d. The culture temperature is 25 ℃, and the illumination intensity is 200 mu mol.m-2·s-1And the illumination time is 16 h/d.
After the multiple shoots are cultured for 40 days, strong shoot culture is carried out, the multiple shoots are inoculated on a culture medium of sucrose 40g/L + agar 7.5g/L + MS +2.0 mg/L6-BA +0.2mg/LNAA, subculture is carried out once every 30 days, and the multiple shoots are cultured into new bulbs with the diameter of 3mm for 90 days. The culture temperature is 25 ℃, and the illumination intensity is 270 mu mol.m-2·s-1And the illumination time is 16 h/d.
Transferring the new bulb grown after the strong bud culture into the following culture medium: after 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of MS, 6-BA and 1.5mg/LNAA2 weeks, white protrusions appear around the bulblets and are the buds of the callus, light yellow tissues appear around the white protrusions after 20 days, light yellow callus masses appear after 30 days, and at the moment, subculture is carried out again until the callus masses with the diameter of more than 1cm are induced. The culture temperature was 25 ℃ and the culture was performed in the dark.
Cutting the callus, dividing into small pieces with diameter of 1.5cm, transferring to the bud induction culture medium, and inducing cluster buds. The culture method is the same as the bud induction step. Each callus differentiated into 9 shoots.
And (4) transferring the cluster buds to a strong bud rooting culture medium for strong bud rooting culture. The culture temperature is 26 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. The strong bud rooting culture medium is sucrose 30g/L, agar 7.5g/L, MS, 2.0 mg/L6-BA, 0.2mg/L NAA and 2.0mg/L IBA, and after culturing for 30 days, the tissue culture seedling with the diameter of 4mm and 4 roots can be grown.
And (4) hardening and transplanting the tissue culture seedlings indoors. And opening the tissue culture bottle, and placing the tissue culture bottle under natural scattered light for 3 d. After hardening, the lycoris radiata seedlings are taken out by using tweezers, the culture medium at the root is washed off, and the lycoris radiata seedlings are transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 800 times of carbendazim. Then performing normal water and fertilizer management to obtain the mature Chinese lycoris.
The whole period of the garlic sprouts obtained by the comparative example is about 11-12 months.
Comparative example 2
Taking healthy Chinese lycoris bulbus underground with diameter of 3.0cm, peeling off outer layer of bulbus, cutting off all roots and upper half part 2/3 of bulbus, keeping bulbus disk, cleaning soil on root surface with brush, cutting into 4 pieces according to the size of bulbus disk, each piece is 1.0cm in size284 washing agent is washed for 28min by shaking and is washed for 33min by running water for standby.
The above materials were rinsed on a clean bench with 75% alcohol for 33s, 0.1% HgCl2Sterilizing for 42min, washing with sterile water for 5 times, and drying surface water with sterile absorbent paper.
Directly inoculating the sterilized bulb blocks onto a bud induction culture medium in a manner that a basal disc is downward, wherein the culture medium comprises the following components: sucrose 30g/L + agar7.5g/L + MS +3.0mg/L6-BA +1.5 mg/LNAA. After 28 days of culture, shoots grew from the scale of the explants. Subculture was carried out after 40 days. The culture temperature is 26 ℃, and the illumination intensity is 220 mu mol.m-2·s-1And the illumination time is 12 h/d.
After the adventitious bud is subcultured for 40d, the adventitious bud is transferred to a bud multiplication medium for multiplication culture. The formula of the culture medium is sucrose 30g/L, agar 7.5g/L, MS, 3.0mg/L6-BA and 1.0mg/L NAA. The culture temperature is 24 ℃, and the illumination intensity is 250 mu mol.m-2·s-1And the illumination time is 16 h/d. The culture temperature is 25 ℃, and the illumination intensity is 200 mu mol.m-2·s-1And the illumination time is 16 h/d.
And (3) performing strong bud culture after the propagation culture of the cluster buds for 40 days, inoculating the cluster buds to a sucrose 40g/L + agar 7.5g/L + MS +2.0 mg/L6-BA +0.2mg/LNAA culture medium, subculturing every 30 days, and culturing for 60 days in total to grow the cluster buds into new bulbs with the diameter of 3 mm. The culture temperature is 25 ℃, and the illumination intensity is 270 mu mol.m-2·s-1And the illumination time is 16 h/d.
Transferring the new bulb grown after the strong bud culture into a culture medium: after 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of MS, 6-BA and 1.5mg/LNAA2 weeks, white protrusions appear around the bulblets and are the buds of the callus, light yellow tissues appear around the white protrusions after 20 days, light yellow callus masses appear after 30 days, and at the moment, subculture is carried out again until the callus masses with the diameter of 5cm are induced. The culture temperature was 25 ℃ and the culture was performed in the dark.
Cutting the callus, dividing into small pieces with diameter of 1.5cm, transferring to the bud induction culture medium, and inducing cluster buds. The culture method is the same as the bud induction step. Each callus differentiated to give 11 shoots.
And (4) transferring the cluster buds to a strong bud rooting culture medium for strong bud rooting culture. The culture temperature is 25 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. The strong bud rooting culture medium is sucrose 30g/L, agar 7.5g/L, MS, 2.0mg/L IBA, 0.2mg/L NAA and 2.0 mg/L6-BA, and after culturing for 30 days, the tissue culture seedling with the diameter of 3mm and 4 roots can be grown.
Hardening and transplanting the tissue culture seedlings, and hardening the seedlings indoors. And opening the tissue culture bottle, and placing the tissue culture bottle under natural scattered light for 2 d. After hardening, the lycoris radiata seedlings are taken out by using tweezers, the culture medium at the root is washed off, and the lycoris radiata seedlings are transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 900 times of carbendazim. Then performing normal water and fertilizer management to obtain the mature Chinese lycoris.
The whole period of the garlic sprouts obtained by the comparative example is about 11-12 months.
Comparative example 3
Taking underground Bulbus Lycoridis Radiatae of healthy pair with diameter of 4.0cm, removing outer layer of bulb, cutting off all roots and upper half 3/5 of bulb, keeping bulb disc, cleaning soil on root surface with brush, cutting into pieces with size of 1.2cm according to the size of bulb disc284 washing agent is washed for 33min by shaking and is washed for 30min by running water for standby.
Rinsing the above materials with 75% ethanol on a clean bench for 28s, sterilizing with 0.1% HgCl2 for 35min, washing with sterile water for 3 times, and drying surface water with sterile absorbent paper.
Directly inoculating the sterilized bulb blocks onto a bud induction culture medium in a manner that a basal disc is downward, wherein the culture medium comprises the following components: 30g/L of sucrose, 7.5g/L of agar, 3.0mg/L of MS, 6-BA and 1.5 mg/LNAA. After 28 days of culture, shoots grew from the scale of the explants. Subculture was carried out after 40 days. The culture temperature is 26 ℃, and the illumination intensity is 220 mu mol.m-2·s-1And the illumination time is 12 h/d.
After the adventitious bud is subcultured for 40d, the adventitious bud is transferred to a bud multiplication medium for multiplication culture. The formula of the culture medium is sucrose 30g/L, agar 7.5g/L, MS, 3.0mg/L6-BA and 1.0mg/L NAA. The culture temperature is 24 ℃, and the illumination intensity is 150 mu mol.m-2·s-1And the illumination time is 16 h/d. The culture temperature is 25 ℃, and the illumination intensity is 260 mu mol.m-2·s-1And the illumination time is 16 h/d.
Culturing the multiple shoots for 40 days, inoculating the multiple shoots to sucrose 40g/L + agar 7.5g/L + MS +2.0 mg/L6-BA +0.2mg/LNAA medium, subculturing for 90 days, and growing the multiple shoots into diameter2.5mm of fresh bulb. The culture temperature is 25 ℃, and the illumination intensity is 300 mu mol.m-2·s-1And the illumination time is 16 h/d.
Transferring the new bulb grown after the strong bud culture into a culture medium: after 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of MS, 6-BA and 1.5mg/LNAA2 weeks, white protrusions appear around the bulblets and are the buds of the callus, light yellow tissues appear around the white protrusions after 20 days, light yellow callus masses appear after 30 days, and at the moment, subculture is carried out again until the callus masses with the diameter of 5cm are induced. The culture temperature was 25 ℃ and the culture was performed in the dark.
Cutting the callus, dividing into small pieces with diameter of 1.2cm, transferring to the bud induction culture medium, and inducing multiple shoots, wherein each callus has 10 buds. The culture method is the same as the bud induction step.
Transferring the bud to a strong bud rooting culture medium for strong bud rooting culture. The culture temperature is 25 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. The strong bud rooting culture medium is sucrose 30g/L, agar 7.5g/L, MS, 2.0mg/L IBA, 0.2mg/L NAA and 2.0 mg/L6-BA, and after culturing for 30 days, the tissue culture seedling with the diameter of 5mm and 3 roots can be grown.
Hardening and transplanting the tissue culture seedlings, and hardening the seedlings indoors. And opening the tissue culture bottle, and placing the tissue culture bottle under natural scattered light for 2 d. After the hardening-off, the seedlings are taken out by tweezers, the culture medium at the root is washed off, and the seedlings are transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 1000 times of carbendazim. Then performing normal water and fertilizer management to obtain the mature Chinese lycoris.
The whole period of the garlic sprouts obtained by the comparative example is about 11-12 months.
Comparative example 4
Harvesting the seeds of the Lycoris chinensis, sterilizing and obtaining embryo in the same manner as in example 1, and sequentially performing callus induction, proliferation culture and differentiation, bud strengthening culture, seedling strengthening, rooting and seedling hardening. Except that the callus induction and proliferation were carried out according to the method of comparative example 1, in which the induction medium was sucrose 30g/L + agar 7.5g/L + MS +3.0mg/L6-BA +1.5mg/LNAA, and the proliferation medium was sucrose 30g/L + agar 7.5g/L + MS +3.0mg/L6-BA +1.0 mg/LNAA. Meanwhile, the callus induction rate is only 15%. The whole period of the garlic sprouts obtained by the comparative example is about 6-7 months.
Comparative example 5
The bulbodium caucasiae of the Diels button of the China-grass was obtained as an explant in the manner of comparative example 3, and induction culture and the like were performed according to the steps (3) to (8) of example 1, during which the callus induction rate was only 5%. The whole period of the garlic sprouts obtained by the comparative example is about 8-9 months.
The callus induction rates, differentiation coefficients, and seedling stages of examples 1 and 2 and comparative examples 1 to 5 were examined, and the results are shown in Table 1.
TABLE 1 comparison of callus or clumpy bud induction rates for different explants of different species
As can be seen from Table 1, the culture medium and the culture method provided by the invention can significantly improve the callus induction rate by 16.5-88.6%; the differentiation coefficient can be remarkably improved and is about 5.33-15 times of that of comparative examples 1-3 and 5; the seedling period is obviously shortened compared with comparative examples 1-3 and 5.
As can be seen from the above examples and comparative examples, the culture medium and the culture method provided by the invention can solve the technical problem of low induction rate to the maximum extent; the culture medium provided by the invention solves the problem of low induction rate of lycoris by matching of all components and the using amount and relative proportion of all components; according to the lycoris callus culture method provided by the invention, the seed embryo is used as the explant to induce and generate callus, and the callus is subjected to differentiation culture to germinate a large number of cluster buds, so that the propagation coefficient and the propagation efficiency can be greatly improved. The embryo is used as explant, the induction rate of callus is high, and the embryo can be used for various molecular biology level operations after proliferation.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A tissue culture medium for Lycoris comprises an induction medium; the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.0.0-2.5 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 0.5-2.0 mg/L of carbon nano tube;
the pH value of the induction culture medium is 5.5-6.0.
2. The tissue culture medium of claim 1, further comprising a propagation medium; the multiplication culture medium takes an MS culture medium as a basic culture medium and also comprises the following components in percentage by weight: 20-25 g/L of glucose, 7.0-7.5 g/L, TDZ 2.5.5-3.0 mg/L of agar, 2.0-2.5 mg/L of 2,4-D and 2.0-5.0 mg/L of carbon nano-tubes;
the pH value of the proliferation culture medium is 5.5-6.0.
3. The tissue culture medium of claim 1 or 2, wherein the genus Lycoris comprises diploid Lycoris radiata, Lycoris chinensis, Lycoris neglecta, Lycoris longiradiata, Lycoris rubra, and Lycoris anhua.
4. A lycoris callus culture method is characterized by comprising the following steps:
taking the mature seed embryo of the lycoris as an explant;
inoculating the seed embryo into an induction culture medium for induction culture to obtain a callus;
inoculating the callus obtained by induction culture to a proliferation culture medium for proliferation culture to obtain a proliferation callus;
and inoculating the proliferation callus to a differentiation culture medium for differentiation culture to obtain differentiation cluster buds.
5. The culture method according to claim 4, wherein the temperature of the induction culture is 24-26 ℃; the light source for induction culture is LED red light and blue light 1:1, and the illumination intensity is 40-100 mu mol.m-2·s-1The illumination time is 16 h;
the temperature of the proliferation culture is 24-26 ℃; the light source for proliferation culture is LED red light and blue light 1:1, and the illumination intensity is 40-100 mu mol.m-2·s-1The illumination time is 16 h.
6. The culture method according to claim 4, wherein the differentiation medium is a MS medium as a basal medium, and further comprises the following components in percentage by weight: 30g/L of sucrose, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 0.5.5-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the differentiation medium is 5.5-6.0;
the temperature of the differentiation culture is 24-26 ℃; the light source for the differentiation culture is LED white light, and the illumination intensity is 100-300 mu mol.m-2·s-1The illumination time is 16 h.
7. A method for establishing a lycoris regeneration system is characterized by comprising the following steps:
inoculating the differentiated cluster buds obtained by the tissue culture medium of any one of claims 1 to 3 or the differentiated cluster buds obtained by the culture method of any one of claims 4 to 6 to a strong bud culture medium for strong bud culture to obtain strong bud seedlings;
inoculating the strong bud seedlings to a strong seedling and rooting culture medium for strong seedling and rooting culture to obtain rooted seedlings;
carrying out subculture on the rooted seedlings to obtain subcultured seedlings;
and (4) carrying out transplanting culture on the subcultured seedlings to obtain lycoris seedlings.
8. The method as claimed in claim 7, wherein the strong bud culture medium takes MS culture medium as basic culture medium, and further comprises the following components in percentage by weight: 40g/L of cane sugar, 7.0-7.5 g/L of agar, 2.5-3.0 mg/L, NAA 1.0.0-1.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the strong seedling culture medium is 5.5-6.0.
9. The method of claim 7, wherein the strong seedling and rooting culture medium takes an MS culture medium as a basic culture medium, and further comprises the following components in percentage by weight: 40g/L of cane sugar, 7.0-7.5 g/L of agar, 1.5-2.0 mg/L, NAA 0.5.5-1.0 mg/L, IBA 2.0.0-3.5 mg/L of 6-BA and 2.0-5.0 mg/L of carbon nano tube;
the pH value of the strong seedling and rooting culture medium is 5.5-6.0.
10. The method according to claim 7, wherein the cultivation substrate used in the transplanting cultivation comprises peat soil, perlite and garden soil; the volume ratio of the peat soil to the perlite to the garden soil in the culture medium is 1:1: 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115843691A (en) * | 2022-12-22 | 2023-03-28 | 天津师范大学 | Induction method for regulating and controlling garlic test tube bulb by using multi-walled carbon nanotube |
| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116369203A (en) * | 2023-03-20 | 2023-07-04 | 江苏省中国科学院植物研究所 | Lycoris plant inflorescence regeneration medium and inflorescence regeneration method |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596600A (en) * | 2004-08-18 | 2005-03-23 | 南京林业大学 | Long tube lycoris fast breeding method |
| CN101366357A (en) * | 2008-09-17 | 2009-02-18 | 杭州植物园 | Method for tissue culture and quick propagate technique of reddish blue spider lily |
| CN102217540A (en) * | 2011-05-19 | 2011-10-19 | 南京林业大学 | Quick propagation method for lycoris chinensis |
| CN102960243A (en) * | 2012-06-28 | 2013-03-13 | 浙江农林大学 | Method for tissue culture and rapid propagation by basal disc-free scales of Lycoris chinensis |
| CN105475143A (en) * | 2016-01-26 | 2016-04-13 | 广西植物研究所 | Method for obtaining regenerated plant through longtube stonegarlic tissue culture |
| CN105830920A (en) * | 2016-04-01 | 2016-08-10 | 浙江大学 | In-vitro probulb division growth method for realizing rapid proliferation of Lycoris herbs |
| CN106605596A (en) * | 2016-12-16 | 2017-05-03 | 江苏省中国科学院植物研究所 | Method for mass propagation of lycoris aurea through somatic embryogenesis |
| CN106665357A (en) * | 2017-01-04 | 2017-05-17 | 江苏省中国科学院植物研究所 | Method for establishing lycoris regeneration system |
| CN108575755A (en) * | 2018-05-03 | 2018-09-28 | 上饶师范学院 | A kind of method of morning pears androgenesis |
| CN108901844A (en) * | 2018-07-09 | 2018-11-30 | 江苏省中国科学院植物研究所 | A method of building Lycoris genetic conversion system |
| CN110583488A (en) * | 2019-10-24 | 2019-12-20 | 杭州植物园（杭州市园林科学研究院） | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink |
| CN109006472B (en) * | 2018-06-12 | 2020-06-16 | 杭州电子科技大学 | Method for tissue culture and rapid propagation of ornamental plants by using carbon nano tubes with specific concentrations |
| KR102140050B1 (en) * | 2019-12-05 | 2020-07-31 | 충남대학교 산학협력단 | Production method of Lycoris radiata adventitious roots for producing galantamine |
| CN111727882A (en) * | 2020-06-22 | 2020-10-02 | 中国科学院植物研究所 | Method for promoting peppermint callus induction by using multi-walled carbon nanotubes |
-
2020
- 2020-11-02 CN CN202011202037.4A patent/CN112237142B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596600A (en) * | 2004-08-18 | 2005-03-23 | 南京林业大学 | Long tube lycoris fast breeding method |
| CN101366357A (en) * | 2008-09-17 | 2009-02-18 | 杭州植物园 | Method for tissue culture and quick propagate technique of reddish blue spider lily |
| CN102217540A (en) * | 2011-05-19 | 2011-10-19 | 南京林业大学 | Quick propagation method for lycoris chinensis |
| CN102960243A (en) * | 2012-06-28 | 2013-03-13 | 浙江农林大学 | Method for tissue culture and rapid propagation by basal disc-free scales of Lycoris chinensis |
| CN105475143A (en) * | 2016-01-26 | 2016-04-13 | 广西植物研究所 | Method for obtaining regenerated plant through longtube stonegarlic tissue culture |
| CN105830920A (en) * | 2016-04-01 | 2016-08-10 | 浙江大学 | In-vitro probulb division growth method for realizing rapid proliferation of Lycoris herbs |
| CN106605596A (en) * | 2016-12-16 | 2017-05-03 | 江苏省中国科学院植物研究所 | Method for mass propagation of lycoris aurea through somatic embryogenesis |
| CN106665357A (en) * | 2017-01-04 | 2017-05-17 | 江苏省中国科学院植物研究所 | Method for establishing lycoris regeneration system |
| CN108575755A (en) * | 2018-05-03 | 2018-09-28 | 上饶师范学院 | A kind of method of morning pears androgenesis |
| CN109006472B (en) * | 2018-06-12 | 2020-06-16 | 杭州电子科技大学 | Method for tissue culture and rapid propagation of ornamental plants by using carbon nano tubes with specific concentrations |
| CN108901844A (en) * | 2018-07-09 | 2018-11-30 | 江苏省中国科学院植物研究所 | A method of building Lycoris genetic conversion system |
| CN110583488A (en) * | 2019-10-24 | 2019-12-20 | 杭州植物园（杭州市园林科学研究院） | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink |
| KR102140050B1 (en) * | 2019-12-05 | 2020-07-31 | 충남대학교 산학협력단 | Production method of Lycoris radiata adventitious roots for producing galantamine |
| CN111727882A (en) * | 2020-06-22 | 2020-10-02 | 中国科学院植物研究所 | Method for promoting peppermint callus induction by using multi-walled carbon nanotubes |
Non-Patent Citations (1)
| Title |
|---|
| 吕学思: "离体条件下的换锦花小鳞茎发生研究", 《中国优秀硕士学位论文全文数据库 农业科技辑 D048-234》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115843691A (en) * | 2022-12-22 | 2023-03-28 | 天津师范大学 | Induction method for regulating and controlling garlic test tube bulb by using multi-walled carbon nanotube |
| CN115843691B (en) * | 2022-12-22 | 2023-08-08 | 天津师范大学 | Induction method for regulating garlic test tube bulb by using multiwall carbon nanotubes |
| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116267612B (en) * | 2023-03-16 | 2024-01-09 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116369203A (en) * | 2023-03-20 | 2023-07-04 | 江苏省中国科学院植物研究所 | Lycoris plant inflorescence regeneration medium and inflorescence regeneration method |
| CN116369203B (en) * | 2023-03-20 | 2024-03-15 | 江苏省中国科学院植物研究所 | Lycoris plant floret regeneration medium and floret regeneration method |
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