CN109042340B - Rapid propagation method of safflower tissues - Google Patents

Abstract

The invention discloses a rapid propagation method of safflower tissues. The method comprises the steps of taking sterile safflower seedling stems as explants, inducing embryonic callus, culturing subculture seedlings, inducing rooting, domesticating and transplanting, and finally obtaining the regeneration plants of the safflower. The invention has high callus healing rate, high callus re-induction seedling rate and high survival rate after domestication and transplantation of tissue culture seedlings, is suitable for industrial production of safflower seedlings and lays a foundation for subsequent transgenic implementation.

Description

Rapid propagation method of safflower tissues

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a rapid propagation method of safflower tissues.

Background

Safflower (Carthamus tinctorius L.) is an annual herb of the family Compositae (Compositae), also known as safflower, crocus sativus and crocus spinosus, and is also known as a bluish flower because its leaves are indigo, also known as a bluish flower because of its yellow color, originally produced Egypt, has been cultivated in Pakistan, India, etc., the Han Dynasty was originally introduced into China, and was originally listed in "Kaibao Bencao", listed as a medium product, so far there has been a history of over 2100 years of cultivation.

Safflower mainly contains flavonoids, carthamus tinctorius polysaccharide, organic acids, carthamus tinctorius, adenophora, carotene, polyhexamethylene, 5-hydroxytryptamine, hydroxysafflor yellow A, β -oryosol, alkyl diols, lignans, carthamin yellow, carthamin red, riboflavin degradation products, riboflavin and riboflavin.

Safflower is a dual-purpose crop of oil and medicine, and the dry tubular flower is a common traditional Chinese medicine, is pungent and warm in nature, and has the effects of promoting blood circulation, removing obstruction in channels, removing blood stasis and relieving pain. It is used clinically to treat metrorrhagia, cardiac vascular diseases, thrombus and other diseases and also as pain relieving and antiphlogistic medicine. Safflower contains rich safflower yellow and a small amount of red pigment, and is widely used for coloring various foods and cosmetics such as candies, cakes, beverages, spices, wines, wheaten foods and the like after being extracted, and is a natural edible pigment. Safflower oil is widely used in pharmaceutical and chemical industries, in addition to being used as high-grade cooking oil due to the fact that safflower oil is rich in linoleic acid.

At present, although safflower is planted in a large area as a promising economic crop, diseases such as ball rot, leaf spot, root rot, rust, epidemic disease, leaf blight, powdery mildew and the like affect the production of the safflower, in order to obtain a variety which is high in yield, quality, disease and insect resistance and strong in stress resistance and can be used for oil flowers, a plant tissue culture technology can be utilized, and useful natural compounds (including medicines, pigments, spices, food additives and the like) and regenerated plants can be produced by regulating culture conditions and screening high-yield cell lines.

Plant tissue culture, also called plant in vitro culture in the broad sense, refers to a technique of separating desired tissues, organs or protoplasts, cells, etc. from a plant body, and culturing under an artificial control condition by aseptic manipulation to obtain a regenerated whole plant or produce other plant-related products with economic value. In general, plant tissue culture can be divided into direct route culture, in which a callus is formed on an explant, and indirect route culture, in which a bud is differentiated and transferred to a rooting medium, thereby growing a normal plant. The indirect approach dedifferentiates the cells to form callus that is not affected by the parent tissue, and the callus is further developed to form new plants, which not only enables rapid propagation of the plants, but also is an ideal approach for plant improvement, germplasm preservation, and production of useful compounds, as compared to the direct approach.

The present inventors have found that MS medium is easy to induce haploid callus, when 2.0 mg/L6-BA and 0.5 mg/L NAA are added, clumpy buds can grow from the haploid, the buds can grow on 1/2MS +0.1 mg/L NAA + 1% sucrose, thereby growing into normal plants, Ying, etc., using agrobacterium tumefaciens to mediate formation of transgenic american safflower variety cennial, and have studied the regenerated plant, the buds can be obtained from cotyledons and leaves, the induction rate is 26%, the research on the germination rate of transgenic american safflower variety cennial from rhizobium is not successful, and the research on the regeneration of the transgenic callus is not successful, and the research on the germination rate of the transgenic American safflower variety cennial from the callus is not successful.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a rapid propagation method of safflower tissues, which has the characteristics of convenient material acquisition and no time limitation and can improve the propagation speed and the regeneration rate of safflower.

The invention provides a rapid propagation method of safflower tissues, which comprises the following specific steps:

(1) sterile seedling culture: culturing the safflower seeds in a seedling culture medium to obtain aseptic seedlings;

(2) callus induction culture: taking a stem section of the aseptic seedling as an explant, and carrying out induction culture on a callus induction culture medium to obtain callus, wherein the callus induction culture medium comprises Phytagel, 6-BA, NAA and PVP;

(3) subculture seedling culture: culturing the callus in a redifferentiation subculture medium to obtain an adventitious seedling, wherein the redifferentiation subculture medium comprises Phytagel, 6-BA, NAA and PVP;

(4) rooting culture: inserting the adventitious seedlings into a rooting culture medium to perform rooting culture, wherein the rooting culture medium comprises Phytagel, IBA and PVP

(5) Domestication and transplantation.

Preferably, the callus induction medium consists of MS, Phytagel, 6-BA, NAA and PVP, and further preferably, the Phytagel is gellan gum, and the gellan gum is preferably G3251;

preferably, the callus induction medium comprises MS, Phytagel 1-3 g/L, PVP 1-3 g/L, 6-BA1-3 mg/L0, NAA 1-3 mg/L1, more preferably MS, Phytagel 2-3 g/L, PVP 2-3 g/L, 6-BA 2-3 mg/L, NAA 1.5-2 mg/L, even more preferably MS, Phytagel 3 g/L, PVP3 g/L, 6-BA2.0 mg/L, NAA1.8 mg/L.

Preferably, the redifferentiation subculture medium consists of MS, Phytagel, 6-BA, NAA and PVP, and further preferably, the Phytagel is gellan gum, and the gellan gum is preferably G3251;

preferably, the redifferentiation subculture medium comprises MS, Phytagel 1-3 g/L, PVP 1-3 g/L, 6-BA0.1-1 mg/L0, NAA 1-6 mg/L1, MS, Phytagel 1-3 g/L2, PVP 1-3 g/L3, 6-BA1-3 mg/L4 and NAA 1-3 mg/L5, and further preferably MS, Phytagel 2-3 g/L, PVP 2-3 g/L, 6-BA 0.1-0.2 mg/L and NAA 3-5 mg/L, and more preferably MS, Phytagel 3 g/L, PVP3 g/L, 6-BA 0.15 mg/L and A4.0 mg/L.

Preferably, the ingredients of the rooting medium are MS, indole acetic acid (IBA)0.12 mg/L, 3 g/L Phytagel and 3 g/L PVP.

Preferably, the rapid propagation method of safflower tissue comprises the following specific steps:

(1) selecting safflower seeds stored at the temperature of 20 ℃ below zero, soaking the safflower seeds in tap water at normal temperature, screening plump seeds, sucking water on the surface of the safflower seeds for later use, sterilizing the safflower seeds by using 0.1 percent of mercury bichloride in an ultra-clean workbench, rinsing the safflower seeds for 5 to 8 times by using sterile water until no disinfectant residue exists, clamping the cleaned seeds by using tweezers, placing the seeds on a sterilized inoculation dish, sucking the surface water by using sterilized filter paper, inoculating the seeds into a sterilized culture medium, namely MS culture medium +3 g/L Phytagel, and the pH value is 5.75, and performing sterile culture in a light culture box under the culture conditions that the temperature is 24 to 26 ℃, the light intensity is 3000-;

(2) the callus induction culture, namely taking aseptic seedlings with the age of 8 days, clamping and placing the aseptic seedlings on an inoculation dish by using forceps, cutting stem sections by using a scalpel, and placing the stem sections on a sterilized callus induction culture medium, wherein the induction culture medium comprises MS, Phytagel 3 g/L, PVP3 g/L, 6-BA2.0 mg/L and NAA1.8 mg/L, the culture condition is that the temperature is 24-26 ℃, the illumination intensity is 3000-;

(3) subculture of seedling, namely transferring the callus after 21 days of culture into a sterilized redifferentiation subculture medium, wherein the redifferentiation subculture medium comprises MS, Phytagel 3 g/L, PVP3 g/L, 6-BA 0.15 mg/L and NAA 4.0 mg/L: 5.75-5.85, the culture conditions comprise that the temperature is 24-26 ℃, the illumination intensity is 3000 and 5000lx, the illumination time is 16h/d, and the callus is aseptically cultured in an illumination incubator for 12 days;

(4) rooting culture, namely cutting off callus cells of the redifferentiated adventitious seedlings under aseptic conditions, and inserting the callus cells into a rooting culture medium, wherein the rooting culture medium comprises MS, indoleacetic acid (IBA)0.12 mg/L, 3 g/L Phytagel and 3 g/L PVP, and the culture conditions comprise 24-26 ℃, the illumination intensity is 3000-;

(5) domestication and transplantation: when the height of the rooting test-tube seedling after rooting culture is 5-10cm, indoor hardening of the tissue culture seedling is carried out, the hardening temperature is 24-26 ℃, the illumination intensity is 5000-; taking out the test-tube plantlet, transplanting the test-tube plantlet into a pot culture pot which uses soil and vermiculite sterilized by carbendazim as substrates according to the proportion of 1:1, watering the pot culture pot sufficiently, and ensuring the illumination intensity: 5000-10000lx, the illumination time is 16h/d, and the outdoor culture is carried out after 2 weeks, so as to ensure sufficient water.

Another object of the present invention is to provide a culture medium and a redifferentiation subculture medium which can induce the safflower stalk to produce callus and further produce adventitious buds. The callus induction culture medium consists of MS, Phytagel, 6-BA, NAA and PVP, and further preferably the Phytagel is gellan gum, and the gellan gum is preferably G3251;

preferably, the callus induction medium comprises MS, Phytagel 1-3 g/L, PVP 1-3 g/L, 6-BA1-3 mg/L0, NAA 1-3 mg/L1, more preferably MS, Phytagel 2-3 g/L, PVP 2-3 g/L, 6-BA 2-3 mg/L, NAA 1.5-2 mg/L, even more preferably MS, Phytagel 3 g/L, PVP3 g/L, 6-BA2.0 mg/L, NAA1.8 mg/L.

Preferably, the redifferentiation subculture medium consists of MS, Phytagel, 6-BA, NAA and PVP, and further preferably, the Phytagel is gellan gum, and the gellan gum is preferably G3251;

preferably, the redifferentiation subculture medium comprises MS, Phytagel 1-3 g/L, PVP 1-3 g/L, 6-BA0.1-1 mg/L0, NAA 1-6 mg/L1, MS, Phytagel 1-3 g/L2, PVP 1-3 g/L3, 6-BA1-3 mg/L4 and NAA 1-3 mg/L5, and further preferably MS, Phytagel 2-3 g/L, PVP 2-3 g/L, 6-BA 0.1-0.2 mg/L and NAA 3-5 mg/L, and more preferably MS, Phytagel 3 g/L, PVP3 g/L, 6-BA 0.15 mg/L and A4.0 mg/L.

In the above-mentioned culture medium, MS is a plant tissue culture medium commonly used in the art, and its composition includes macroelements NH4NO3, 1650 mg/L, KNO3, 1900 mg/L, CaCl 2.2H2O, 440 mg/L0, MgSO 4.7H 2O, 370 mg/L1, KH2PO 4170 mg/L2, trace elements KI, 0.83 mg/L3, H3 BO3672, 6.2 mg/3 4, MnSO 3672.4H23672, 22.3 mg/3, ZnSO 3672.7H23672, 8.6 mg/3, Na2MoO 3672.2H23672, 0.25 mg/3, CuSO 3672.5H23672, 0.025 mg/3, CoCl 72.6 H.23672, 0.025 mg/3, FeO 3, 0.0.0.025 mg/3, Na 2H 72, sodium chloride, sodium.

Indoleacetic acid is an organic substance. The pure product is colorless leaf-shaped crystal or crystalline powder, which is usually used as plant growth hormone to regulate the growth of plants and promote the rooting and sprouting of plants.

Polyvinylpyrrolidone (PVP) is a nonionic polymer compound and one of N-vinyl amide polymers, and PVP is a synthetic water-soluble polymer compound, has the general properties of the water-soluble polymer compound, and has the colloid protection effect, film forming property, cohesiveness, hygroscopicity, solubilization or agglomeration effect, and can be widely applied to the fields of medicine and health and food processing.

In the culture medium of the present invention, the Phytagel is Gelzan gel, specifically G3251 (a plant gel produced by Phytechlab, CAS: 71010-52-1). G3251 is one of Gelzan gel, which is prepared by fermenting microorganism, and compared with the similar Gelzan gel product, G3251 has the advantages of small dosage (the dosage of 0.25% can reach the gel strength of agar 1.5%), adjustable freezing point, melting point, elasticity and hardness, small product difference, good compatibility with nutritional additives, good thermal stability, acid resistance and the like.

The third purpose of the invention is to provide the application of the culture medium which can induce the safflower stalk to generate callus, and the culture medium can induce the safflower stalk to generate callus; further provides an application of the redifferentiation subculture medium, which can induce the safflower stem to generate callus to grow adventitious buds.

Compared with the prior art, the invention has the following advantages:

1. the invention adopts the safflower stem as the explant, combines the 6-BA and the NAA with specific proportion, can achieve good healing rate and multiplication multiple, has the characteristics of convenient material acquisition and no time limitation, and can improve the reproduction speed and the regeneration rate of the safflower.

2. The culture medium is added with only 6-BA, NAA, G3251 and PVP on the basis of the MS culture medium, has simple components, is easy to prepare, has high homogenization degree and is easy to realize industrialized operation.

3. Gellan gum G3251(Gelzan gum) was used instead of traditional agar. The G3251 gellan gum has higher transparency than traditional agar, can be better compatible with various nutrient substances, promotes root growth, has better elasticity, has small growth obstruction of roots, and ensures that the regeneration plant of safflower has better rooting effect.

4. The invention obtains the complete plant by utilizing the regeneration of the callus, finishes the tissue culture rapid propagation and the transgenic downstream technical system at one time, and lays a foundation for the subsequent transgenic work.

Drawings

FIG. 1 shows a 10-day-old aseptic seedling in the aseptic seedling culture step;

FIG. 2 shows embryogenic callus induced by Medium 2;

FIG. 3 shows the clumped seedlings induced by the medium 14;

FIG. 4 shows adventitious roots induced by rooting culture;

FIG. 5 shows the growth of indoor seedlings after transplanting them out of the room for 2 weeks;

Detailed Description

In order to better understand the technical scheme of the invention, the technical scheme provided by the invention is described in detail by combining the embodiment.

Example 1 Effect of different hormone ratios of the culture Medium for callus induction of safflower Stem on the recovery ratio

In order to compare the influence of hormones with different proportions in a safflower stem callus induction culture medium on the callus growth rate of the safflower stems, the components of the culture medium are adjusted according to the conventional culture medium preparation technology in the field, so that the culture medium consisting of the following different components is obtained:

the culture medium 1 comprises MS, G32513G/L, PVP 3G/L, NAA 1.5 mg/L, 6-BA2.0 mg/L;

culture medium 2MS, G32513G/L, PVP 3G/L, NAA1.8 mg/L, 6-BA2.0 mg/L;

culture medium 3 MS, G32513G/L, PVP 3G/L, NAA 2.0 mg/L, 6-BA2.0 mg/L;

the culture medium 4 comprises MS, G32513G/L, PVP 3G/L, NAA 1.5 mg/L, 6-BA 2.5 mg/L;

culture medium 5 MS, G32513G/L, PVP 3G/L, NAA1.8 mg/L, 6-BA 2.5 mg/L;

the culture medium 6 comprises MS, G32513G/L, PVP 3G/L, NAA 2.0 mg/L, 6-BA 2.5 mg/L;

culture medium 7 including MS, G32513G/L, PVP 3G/L, NAA 1.5 mg/L, 6-BA 3.0 mg/L;

culture medium 8 including MS, G32513G/L, PVP 3G/L, NAA1.8 mg/L, 6-BA 3.0 mg/L;

culture medium 9 MS, G32513G/L, PVP 3G/L, NAA 2.0 mg/L, 6-BA 3.0 mg/L;

the specific experimental procedure is as follows:

(1) selecting safflower seeds stored at the temperature of 20 ℃ below zero, soaking the safflower seeds in tap water at normal temperature, screening full seeds, absorbing surface water on absorbent paper for standby, sterilizing the safflower seeds by using 0.1 percent of mercury bichloride in a super clean workbench, rinsing the safflower seeds for 8 times by using sterile water until no disinfectant remains, clamping the cleaned seeds by using tweezers, placing the seeds on a sterilized inoculation dish, absorbing the surface water by using sterilized filter paper, inoculating the seeds into a sterilized culture medium, namely MS culture medium +3 g/L Phytagel, wherein the pH is 5.75, and culturing the safflower seeds in a light culture box under the conditions of the temperature of 26 ℃, the light intensity of 5000lx and the light time of 16 h/d.

(2) Callus induction culture: taking aseptic seedlings with the age of 8 days, clamping the aseptic seedlings by using forceps, placing the aseptic seedlings on an inoculation dish, cutting stem sections by using a scalpel for 1-2cm, and placing the stem sections in the sterilized callus induction culture media 1-9, wherein 3 parallel experiments are designed for each culture medium. The culture conditions are as follows: temperature 25 ℃, light intensity: 4500lx, illumination time 16h/d, sterile culturing in illumination incubator for 21 days. The induction rate was counted and the results are shown in table 1 and fig. 2.

TABLE 1 Effect of different Induction media on the Induction of callus

From the results in Table 1, it can be seen that the concentration ratio of the hormone has a great influence on the culture of the safflower callus with the stem as the explant, and a higher concentration of 6-BA, such as 3.0 mg/L, or a lower concentration of NAA, such as 1.5 mg/L, results in a lower callus, which is a non-embryogenic callus, and does not benefit the later culture of the regenerated plant.

Example 2: influence of different hormone ratios of redifferentiation subculture medium on differentiated seedling

In order to compare the influence of hormones with different proportions in a redifferentiation subculture medium on differentiated seedlings, the components of the culture medium are adjusted according to the conventional culture medium preparation technology in the field, so that the culture medium with the following different components is obtained:

culture medium 10 including MS, G32513G/L, PVP 3G/L, NAA 3.0 mg/L, 6-BA0.1 mg/L;

culture medium 11 MS, G32513G/L, PVP 3G/L, NAA 4.0 mg/L, 6-BA0.1 mg/L;

culture medium 12 MS, G32513G/L, PVP 3G/L, NAA 5.0 mg/L, 6-BA0.1 mg/L;

culture medium 13 MS, G32513G/L, PVP 3G/L, NAA 3.0 mg/L, 6-BA 0.15 mg/L;

culture medium 14 including MS, G32513G/L, PVP 3G/L, NAA 4.0 mg/L, 6-BA 0.15 mg/L;

the culture medium 15 comprises MS, G32513G/L, PVP 3G/L, NAA 5.0 mg/L, 6-BA 0.15 mg/L;

the culture medium 16 comprises MS, G32513G/L, PVP 3G/L, NAA 3.0 mg/L, 6-BA 0.2 mg/L;

culture medium 17 including MS, G32513G/L, PVP 3G/L, NAA 4.0 mg/L, 6-BA 0.2 mg/L;

culture medium 18 including MS, G32513G/L, PVP 3G/L, NAA 5.0 mg/L, 6-BA 0.2 mg/L;

the specific experimental procedure is as follows:

callus cultured in the culture medium 2 for 21 days is transferred into a sterilized redifferentiation subculture medium 10-18, and 3 parallel experiments are designed for each culture medium. The culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 4500lx, the illumination time is 16h/d, the sterile cultivation is carried out in an illumination incubator for 12 days, and the proliferation rate is calculated. The results are shown in Table 2 and FIG. 3.

TABLE 2 Effect of different subculture media on embryogenic callus differentiation and seedling

As can be seen from the results in Table 2, the concentration ratio of the hormone has a great influence on the proliferation rate of differentiated seedlings, compared with the prior art, the 6-BA and the NAA with great concentration difference are adopted, the generation of adventitious buds is facilitated, and the investigation finds that the combination of the hormone concentration of NAA 4.0 mg/L and the hormone concentration of 6-BA 0.15 mg/L is selected, all calluses can form the adventitious buds, and further the adventitious seedlings are grown, the proliferation rate of the adventitious buds reaches 4-5 times, and a good basis is provided for the subsequent culture of regenerated plants.

Example 3: rooting and domestication transplanting of differentiated seedlings

Under the aseptic condition, callus cells of the adventitious seedlings obtained from the culture medium 14 are cut off and inserted into a rooting culture medium, 20 adventitious seedlings are adopted in the experiment, the rooting culture medium is MS + indoleacetic acid (IBA)0.12 mg/L + 3G/L G3251+ 3G/L PVP, the pH is 5.75, the culture condition is that the temperature is 25 ℃, the illumination intensity is 4000lx, the illumination time is 16h/d, the adventitious roots are cultured in an illumination culture box in an aseptic mode, the rooting rate is counted, all the 20 adventitious seedlings grow adventitious roots, and the rooting rate reaches 100%, as shown in figure 4.

Culturing the 20 adventitious seedlings in a rooting culture medium to 5-10cm, carrying out indoor hardening-seedling on the tissue culture seedlings (i.e. seedlings with adventitious roots), wherein the hardening-seedling temperature is 26 ℃, the illumination intensity is 8500lx, the illumination time is 16h/d, and the indoor bottleneck opening is opened for hardening-seedling for 2 days. Taking out the tissue culture seedlings, transplanting the tissue culture seedlings into a pot culture bowl which is prepared by sterilizing soil and vermiculite by using carbendazim and taking a substrate according to the proportion of 1:1, watering enough water, and illuminating intensity: 10000lx, illumination time 16h/d, outdoor culture after 2 weeks, and guarantee sufficient water. The effect of transplantation was observed after 2 weeks, as shown in fig. 5. All 20 tissue culture seedlings grow into healthy plants, and the transplanting survival rate reaches 100%.

As can be seen from the above rooting and transplanting results, gellan gum G3251 was used instead of conventional agar. The G3251 gellan gum has higher transparency than traditional agar, can be better compatible with various nutrient substances, promotes root growth, has better elasticity, has small growth obstruction of roots, and ensures that the regeneration plant of safflower has better rooting effect. Furthermore, the excellent rooting effect ensures that the excellent transplanting survival rate is obtained.

The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (4)

1. A rapid propagation method of safflower tissue comprises the following specific steps:

(1) sterile seedling culture: culturing the safflower seeds in a seedling culture medium to obtain aseptic seedlings;

(2) callus induction culture: taking a stem section of the aseptic seedling as an explant, and carrying out induction culture on a callus induction culture medium to obtain callus, wherein the callus induction culture medium comprises Phytagel, 6-BA, NAA and PVP;

(3) subculture seedling culture: culturing the callus in a redifferentiation subculture medium to obtain an adventitious seedling, wherein the redifferentiation subculture medium comprises Phytagel, 6-BA, NAA and PVP;

(4) rooting culture: inserting the adventitious seedlings into a rooting culture medium to perform rooting culture, wherein the rooting culture medium comprises Phytagel, IBA and PVP

(5) Domesticating and transplanting;

the callus induction culture medium comprises MS, Phytagel 3G/L, PVP 3G/L, 6-BA2.0 mg/L0 and NAA 1.8mg/L, the redifferentiation subculture medium comprises MS, Phytagel 3G/L, PVP 3G/L, 6-BA 0.15 mg/L and NAA 4.0 mg/L, the Phytagel is G3251, and the rooting culture medium comprises MS, IBA 0.12 mg/L, 3G/L Phytagel and 3G/L PVP.

2. The method for rapid propagation of safflower tissue according to claim 1, comprising the steps of:

(1) sterile seedling culture, namely selecting safflower seeds stored at the temperature of 20 ℃ below zero, soaking the safflower seeds in tap water at normal temperature, screening plump seeds, sucking water on the surface of the safflower seeds for later use by absorbent paper, sterilizing the safflower seeds by using 0.1 percent of mercury bichloride in an ultra-clean workbench, rinsing the safflower seeds for 5 to 8 times by using sterile water until no disinfectant residue exists, clamping and cleaning the cleaned seeds by using tweezers, placing the seeds on a sterilized inoculation dish, sucking the surface water by using sterilized filter paper, and finally inoculating the seeds into a sterilized culture medium, wherein the components of the culture medium are MS, 3 g/L Phytagel and pH is 5.75, the culture condition is that the temperature is 24 to 26 ℃, the illumination intensity is 3000-;

(2) the callus induction culture, namely taking aseptic seedlings with the age of 8 days, clamping and placing the aseptic seedlings on an inoculation dish by using forceps, cutting stem sections by using a scalpel, and placing the stem sections on a sterilized callus induction culture medium, wherein the induction culture medium comprises MS, Phytagel 3 g/L, PVP3 g/L, 6-BA2.0 mg/L and NAA1.8 mg/L, the culture condition is that the temperature is 24-26 ℃, the illumination intensity is 3000-;

(3) subculture of seedling, namely transferring the callus after 21 days of culture into a sterilized redifferentiation subculture medium, wherein the redifferentiation subculture medium comprises MS, Phytagel 3 g/L, PVP3 g/L, 6-BA 0.15 mg/L and NAA 4.0 mg/L: 5.75-5.85, the culture conditions comprise that the temperature is 24-26 ℃, the illumination intensity is 3000 and 5000lx, the illumination time is 16h/d, and the callus is aseptically cultured in an illumination incubator for 12 days;

(4) rooting culture, namely cutting off callus cells of the redifferentiated adventitious seedlings under aseptic conditions, and inserting the callus cells into a rooting culture medium, wherein the rooting culture medium comprises MS, indoleacetic acid (IBA)0.12 mg/L, 3 g/L Phytagel and 3 g/L PVP, and the culture conditions comprise 24-26 ℃, the illumination intensity is 3000-;

(5) domestication and transplantation: when the height of the rooting test-tube seedling after rooting culture is 5-10cm, indoor hardening of the tissue culture seedling is carried out, the hardening temperature is 24-26 ℃, the illumination intensity is 5000-; taking out the test-tube plantlet, transplanting the test-tube plantlet into a pot culture pot which uses soil and vermiculite sterilized by carbendazim as substrates according to the proportion of 1:1, watering the pot culture pot sufficiently, and ensuring the illumination intensity: 5000-10000lx, the illumination time is 16h/d, and the outdoor culture is carried out after 2 weeks, so as to ensure sufficient water.

3. The use of a culture medium capable of inducing the stem of the safflower to generate callus is characterized in that the components of the culture medium are MS, Phytagel 3 g/L, PVP3 g/L, 6-BA2.0 mg/L and NAA1.8 mg/L, and the culture medium can induce the stem of the safflower to generate callus.

4. The use of a culture medium capable of inducing the differentiation and seedling growth of safflower callus is characterized in that the components of the culture medium are MS, Phytagel 3 g/L, PVP3 g/L, 6-BA 0.15 mg/L and NAA 4.0 mg/L, the safflower callus is induced by using a stem section of safflower as an explant, and the culture medium can induce the differentiation and seedling growth of the safflower callus.

Images