Method for inducing and culturing saussurea longifolia callus

Abstract

The invention belongs to the field of tissue culture, and particularly relates to a saussurea longifolia callus induction and culture method which comprises the following steps: A, taking tender shoots and young leaves of wild saussurea longifolia, washing and disinfecting to obtain base tissues; b, cutting the base tissue prepared in the step A, inoculating the cut base tissue into a callus induction culture medium, and culturing to obtain an initial callus; c, carrying out subculture on the calluses prepared in the step B to obtain subcultured calluses; the tender shoots and the young leaves of the saussurea longifolia are induced to form the calluses, so that the method has the characteristics of safety, no toxicity, short period, high propagation speed, simplicity in operation and low cost, and convenience is provided for application of the saussurea longifolia in actual products.

Description

Technical Field

[0001] The invention relates to a method for inducing and culturing callus of Saussurea longifolia. Background Art

[0002] Snow lotus is a perennial herb that is cold-resistant and low-temperature-resistant. It mainly grows in areas with higher altitudes. The flowering period is generally from July to August, and the fruiting period is September. As a rare and precious alpine medicinal plant, snow lotus is the most widely used and most representative type of Tibetan medicinal material. Due to the unique growth environment of snow lotus and the difficulty of artificial cultivation and the long growth cycle, the use of plant tissue culture rapid propagation technology for artificial cultivation is currently an effective way to solve its resource shortage.

[0003] Saussurea longifolia Franch. is a plant of the genus Saussurea in the Asteraceae family. The plant is 15-30 cm tall, with an upright stem covered with long white hairs. It grows in alpine grasslands and shrubs at an altitude of 3400-4500 m. The whole plant can be used as medicine, with the effects of clearing away heat and detoxifying, reducing swelling and removing blood stasis. This variety of Saussurea is different from the Tianshan Saussurea, with unique flower colors and purple-red bracts. At present, Saussurea longifolia is still in the wild, and no one has successfully propagated (planted) Saussurea longifolia. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for inducing and culturing callus of Saussurea chinensis, by inducing tender shoots from Saussurea chinensis, selecting excellent callus for subculture, and obtaining embryonic callus culture containing rich active ingredients. The Saussurea chinensis callus culture method of the present invention has the advantages of high value-added coefficient, stable subculture, short culture cycle, green environmental protection, stable quality, etc., solves the problem that the wild plant resources of Saussurea chinensis are difficult to develop and utilize, and has good application prospects in cosmetics, health products and pharmaceutical industries.

[0005] The present invention provides a method for inducing and culturing callus of Saussurea longifolia, comprising the following steps: A: Take the young shoots and leaves of Saussurea longifolia, rinse and disinfect, and obtain the basal tissue; B: cutting the basal tissue obtained in step A and inoculating it into a callus induction medium for induction culture to obtain initial callus; C: Subculturing the initial callus obtained in step B to obtain subculture callus.

[0006] Wherein, the flushing and disinfection comprises the following steps: i) Rinse under running water, ii) disinfection with ethanol, iii) Use 1-10% (V / V, dissolved in 3x MS liquid medium) PPM solution, immerse in a shaker at 100-120 rpm for 1-3 hours, and then rinse with sterile water. Preferably, the ethanol is 75% ethanol, the PPM solution is 5% PPM solution, the rotation speed of the shaking table is preferably 110 rpm, and the treatment time is 1 h.

[0007] The components of the induction medium in step B are MS + 2,4-D 0.3-0.7 mg / L + 6-BA 1.8-2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. Preferably, the concentration of 2,4-D is 0.5 mg / L, and the concentration of 6-BA is 2.0 mg / L.

[0008] The induction and culture conditions of step B are as follows: temperature 20±1°C, humidity 50%~70%, 12 hours of light per day, and light intensity of 1500 Lx~2000 Lx. Furthermore, the culture period of step B is at least 20 days.

[0009] The induction and culture conditions in step C are as follows: temperature 20±1°C, humidity 50%-70%, 12 hours of light per day, and light intensity of 1500 Lx-2000 Lx. Furthermore, the culture period in step C is at least 28 days.

[0010] Wherein, the subculture in step C includes screening culture and / or proliferation culture; I) wherein the screening culture comprises the following steps: inoculating the callus tissue from step B into a subculture screening medium for culturing to obtain embryonic callus tissue.

[0011] II) The proliferation culture comprises the following steps: inoculating the initial callus or embryonic callus into a proliferation culture medium for culturing to obtain subculture callus.

[0012] The subculture screening medium comprises the following components: 1 / 2MS + 2,4-D 2.8-3.2 mg / L + KT 0.3-0.7 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum. Furthermore, the concentration of 2,4-D is 3.0 mg / L, and the concentration of KT is 0.5 mg / L.

[0013] The components of the proliferation culture medium are basal culture medium, plant growth regulator, sucrose and gellan gum.

[0014] The basal culture medium is selected from any one of B5, MS and SH culture media.

[0015] The plant growth regulator is selected from any one of “6-BA and 2,4-D” or “mT and 2,4-D”; When the basal culture medium is B5 or MS culture medium.

[0016] The plant growth regulator is selected from any one of “6-BA and 2,4-D” or “mT and 2,4-D”; When the plant growth regulator is "6-BA and 2,4-D", the concentration range of the 6-BA is 0.2-1.2 mg / L, and the concentration of the 2,4-D is 0.2-0.8 mg / L.

[0017] When the plant growth regulator is "mT and 2,4-D", the concentration range of the mT is 0.8-1.2 mg / L, and the concentration of the 2,4-D is 0.3-0.8 mg / L.

[0018] When the basic culture medium is SH culture medium, the plant growth regulators are "6-BA and 2,4-D", the concentration range of the 6-BA is 0.8-1.2 mg / L, and the concentration of the 2,4-D is 0.3-0.8 mg / L.

[0019] Further, the proliferation culture medium is preferably any one of the following components: B5 / MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, 1 / 2 MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, B5 / MS+6-BA 0.5 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, B5 / MS+ mT 1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, SH+6-BA 1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0020] The present invention also provides a long-leaf snow lotus callus induction medium, wherein the induction medium comprises the following components: MS + 2,4-D 0.3-0.7 mg / L + 6-BA 1.8-2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, preferably, the 2,4-D concentration is 0.5 mg / L, and the 6-BA concentration is 2.0 mg / L.

[0021] The present invention also provides a subculture screening medium for long-leaf snow lotus callus, wherein the subculture screening medium comprises the following components: 1 / 2MS + 2,4-D 2.8-3.2 mg / L + KT 0.3-0.7 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, preferably, the 2,4-D concentration is 3.0 mg / L, and the KT concentration is 0.5 mg / L.

[0022] The present invention also provides a subculture and proliferation culture medium for callus tissue of long-leaf snow lotus, wherein the proliferation culture medium comprises the following components: a basic culture medium, a plant growth regulator, sucrose, and gellan gum; further, the basic culture medium is selected from any one of B5, MS, and SH culture media, and the plant growth regulator is selected from any one of 6-BA and 2,4-D or mT and 2,4-D. When the basic culture medium is B5 or MS culture medium, the plant growth regulator is selected from "6-BA and 2,4-D" or "mT and 2,4-D"; when the plant growth regulator is "6-BA and 2,4-D", the concentration range of 6-BA is 0.2-1.2 mg / L, and the concentration of 2,4-D is 0.2-0.8 mg / L; when the plant growth regulator is "mT and 2,4-D", the concentration range of mT is 0.8-1.2 mg / L, and the concentration of 2,4-D is 0.3-0.8 mg / L; when the basic culture medium is SH culture medium, the plant growth regulator is selected from 6-BA and 2,4-D, the concentration range of 6-BA is 0.8-1.2 mg / L, and the concentration of 2,4-D is 0.3-0.8 mg / L.

[0023] Preferably, the proliferation medium is preferably the following components: any one of B5 / MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, B5 / MS+6-BA 0.5 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, 1 / 2 MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, B5 / MS+ mT 1.0 mg / l +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, and SH+6-BA 1.0 mg / L+2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0024] The positive and progressive effects of the present invention are: (1) The induction and cultivation method of the present invention can efficiently cultivate long-leaf snow lotus callus tissue, reduce the browning rate of induced callus tissue cells, and has strong proliferation ability, providing a basis for the induction of adventitious buds and the production of secondary metabolites. It is safe and non-toxic, has a short cycle, fast reproduction, simple operation and low cost.

[0025] (2) The induction and cultivation method of the present invention can obtain a large amount of long-leaf Saussurea callus containing active substances in a short time, overcoming the disadvantage that the plant chemical components are greatly different due to different growth conditions such as geographical environment, climate, soil, etc., which directly affect the quality of the medicinal material. On the other hand, it also protects rare wild plant resources from destruction and promotes the rational utilization and sustainable development of resources.

[0026] (3) The induction culture medium of the present invention can efficiently induce Saussurea ovata, the screening culture medium can screen out embryonic callus suitable for the proliferation of Saussurea ovata, and the proliferation culture medium can achieve effective proliferation of the embryonic callus of Saussurea ovata, with strong proliferation ability and a proliferation coefficient of more than 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Table 3 Example 2 Longye Saussurea callus in different states and its safranin stained sections a Table 3 Photographs of callus tissue of No. 1 and its safranin-stained sections b Table 3 Photographs of callus tissue of No. 3 and its safranin-stained sections c: Photograph of callus tissue No. 4 in Table 3 and its safranin-stained section Figure 2 This is a photo of the callus morphology in culture. a is the water-soaked callus grown on medium No. 3 in Example Table 7 b is slightly water-soaked callus grown on medium No. 7 in Table 5 of Example c is the callus tissue that grows well on medium No. 1 in Example Table 7 Implementation

[0028] Example 1 Induction of callus tissue of Saussurea longifolia The basal culture media MS, B5, White, Heller, SH and plant growth regulator, PPM, in this example were purchased from Pytotechnology. NaClO was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The MS culture medium model was M519, the B5 culture medium model was G398, the White culture medium model was W898, the Heller culture medium model was H393, and the SH culture medium model was 816.

[0029] Description of plant growth regulators: NAA: naphthylacetic acid, 6-BA: 6-benzylaminoadenine, 2,4-D: 2,4-dichlorophenoxyacetic acid, KT: kinetin, mT: Meta-topolin, cytokinin, PPM is Plant Preservative Mixture (PPM) plant tissue culture antibacterial agent, the induction and culture conditions of the following examples are all temperature 20±1°C, humidity 50%~70%, 12 hours of light per day, and light intensity of 1500 Lx~2000 Lx.

[0030] Take one of the MS, B5, White, Heller or SH culture media, mix with different plant growth regulators and cytokinins, make the culture medium in a certain ratio, add 30g / L sucrose and 3.5g / L gellan gum to each, adjust the pH to 5.7-5.8, sterilize at 121℃ in a sterilizer for 20min and set aside.

[0031] Take the young shoots and leaves of wild long-leaf snow lotus, rinse them under running water for 2 hours, then disinfect them with 75% ethanol for 30 seconds in a sterile clean bench, and rinse them with sterile water three times. Use 10% (V / V) NaClO aqueous solution to treat it in a shaker at 110 rpm for 10 minutes, or use 5% (V / V, dissolved in 3 times MS liquid culture medium) PPM (Plant Preservation Mixture) solution, immerse it in a shaker at 110 rpm for 1 hour, and finally rinse it with sterile water three times for use.

[0032] In a sterile clean bench, the sterilized young shoots of Saussurea longifolia were cut into 0.5 cm-1.0 cm segments with a scalpel and inoculated into the culture medium designed in Table 1. 30 g / L sucrose and 3.5 g / L gellan gum were added to each culture medium. After 20 days of culture, the callus induction rate was calculated. Callus induction rate = number of induced explants / number of inoculated explants × 100%.

[0033] The results are shown in Table 1: After disinfection with 10% (V / V) NaClO solution, the explants turned dark brown, and the induction rate when inoculated on the culture medium was 0. When disinfected with 5% PPM, the explants hardly changed color, and callus tissue could be induced in the three designed culture media, with the highest induction rate of 98%. According to the induction rate in Table 1, it can be seen that the preferred induction medium formula is: MS + 2,4-D0.5 mg / L + 6-BA2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. Under this medium formula, the induction rate can reach 98%, and callus tissue can be induced efficiently.

[0034] If NAA is used, callus of Saussurea longifolia cannot be induced efficiently. If the 2,4-D content is too high or the 6-BA content is too low, the callus induction rate will be low and the long-leaf snow lotus callus cannot be induced efficiently.

[0035] Table 1 Induction of callus tissue of Saussurea longifolia

[0036]

[0037] Example 2 Screening of embryonic callus culture medium In order to screen out the medium for subculture that can maintain the state of callus tissue of Saussurea longifolia, the callus tissue induced by No. 4# in Table 1 was taken and inoculated into the medium containing different proportions of 6-BA and NAA in Table 2 for subculture. Each medium was added with 30g / L sucrose and 3.5g / L gellan gum. Five bottles were inoculated for each treatment, and the cell state was observed after 28 days of culture. The results are shown in Table 2, and callus tissues of different morphologies were obtained: among them, in medium No. 2, the callus tissue with yellow-green edges and relatively loose was the best state medium, which could be subcultured and screened.

[0038] Table 2 Effects of different culture medium formulas on subculture of callus tissue of Saussurea longifolia

[0039] Note: The more “+” there are, the more severe the browning is. (2) In order to further screen out a culture medium suitable for subculture of Saussurea longifolia callus, the yellow-green crisp callus numbered 2# in Table 2 was selected and inoculated into culture media with different formulations in Table 3 for subculture. Each culture medium was supplemented with 30 g / L sucrose and 3.5 g / L gellan gum. Three bottles were inoculated for each treatment and the cell status was observed after 28 days of culture.

[0040] The results are shown in Table 3. The calli cultured in No. 1, No. 2 and No. 4 mediums all had browning phenomenon. Moreover, the calli cultured in No. 1 and No. 2 mediums were hard and showed signs of differentiation, which did not meet the characteristics of embryonic callus. The calli cultured in No. 3 medium were yellow-green, loose in texture, without differentiation, browning and vitrification. Therefore, No. 3 medium, 1 / 2MS+3.0 mg / L 2,4-D+0.5 mg / L KT+30 g / L sucrose+3.5 g / L gellan gum, was the best medium formula for subculture selection of embryonic callus characteristics.

[0041] Table 3 Effects of different culture medium formulas on callus growth of Saussurea longifolia

[0042] Note: The more “+” there are, the more severe the browning is. Callus tissue is divided into embryonic callus and non-embryonic callus. There are obvious differences in morphology and cytology between these two types of callus tissue. Embryonic callus refers to callus tissue with the ability to produce embryos. It has the characteristics of plant stem cells, with spherical particles on the surface, small cells, neatly and tightly arranged, large nuclei, and rich contents such as starch and soluble sugars. It is a group of cell clusters with super strong growth ability and division potential, and can be continuously propagated. Non-embryonic callus refers to cells in callus tissue that do not have the ability to produce embryos. They are mostly viscous, easily water-stained or browned, with loose and irregular cell tissue structure, relatively large cells, no organelles, and do not have cell clusters that can be continuously propagated. Safranin, molecular formula is C 20 H 19 N4Cl is a natural dye extracted from the saffron stigma. It is a commonly used dye in cytology and plant and animal histology. It can dye the lignified, suberized and keratinized parts of plants and cell nuclei red.

[0043] To further identify the callus tissues of different colors and textures in the above steps, the three different forms of callus tissues in Table 3 were sliced ​​and stained using safranin reagent. The results are shown in Table 4. The callus tissues with yellow-green color, loose texture and granular surface are embryonic callus cell clusters.

[0044] Table 4 Identification of different types of Saussurea longifolia callus

[0045] Example 3: Screening of callus proliferation medium The embryonic callus obtained by selecting No. 3 in Table 3 was cultured in the culture medium as shown in Table 5 with 30 g / L sucrose and 3.5 g / L gellan gum added, and subcultured 3 times, each time for 28 days, and the state and growth rate of the callus were observed.

[0046] 1) Waterlogging: Waterlogging refers to the high degree of vacuolation of callus cells, irregular surface shape, not granular, and the callus surface is observed to be covered with water, and there is even so much water that it can be observed around the callus culture medium. Judgment standard of water staining: visual inspection shows that 50%-100% of the callus tissue has water stains on its surface Judgment standard of slight water staining: visual inspection shows that less than 50% of the callus tissue surface has water stains.

[0047] Waterlogging of callus tissue can cause cells to become loose and unstable, affecting the normal growth and differentiation of tissues, and reducing the regeneration capacity of callus tissue.

[0048] 2) Hardness: Callus tissue generally has a certain hardness. Soft or loose callus tissue means that when touched with tweezers, it can be easily separated without using a knife. Soft callus tissue is often accompanied by water stains.

[0049] Soft callus tissue will affect the normal growth and division of cells. Slow cell growth will lead to poor proliferation culture results. If the callus tissue is too soft, the callus tissue may die during the culture process.

[0050] 3) The growth rate was evaluated visually by the multiple of callus enlargement. A + means that the callus enlarged by approximately 1 time.

[0051] The results showed that the callus of Saussurea longifolia was in the best condition in the plant growth regulators with 6-BA and 2,4-D, with a granular surface and fast growth. However, if the 2,4-D content was too high, such as 1.5 mg / L, the surface of Saussurea longifolia callus would become waterlogged, soft, and the growth rate would be greatly reduced.

[0052] Table 5 Effects of different culture media on subculture of callus tissue of Saussurea longifolia

[0053] Note: The more +'s there are, the faster the growth will be. Callus tissue was cultured using MS medium supplemented with different concentrations of plant growth regulators 6-BA, 2,4-D, 30 g / L sucrose and 3.5 g / L gellan gum.

[0054] Select callus tissues in the same state as in Table 5, inoculate 3 bottles of each culture medium, observe cell morphology after 28 days of culture and calculate the proliferation coefficient. Proliferation coefficient = weight of callus tissue at harvest / weight of callus tissue at inoculation. The results are shown in Table 6.

[0055] Table 6. Effects of different culture medium formulas on callus growth of Saussurea longifolia

[0056] Among them, the proliferation rate of medium formula No. 2 MS+6-BA 1.0mg / L+2,4-D 0.5mg / L+sucrose 30g / L+gellan gum 3.5g / L was higher, followed by medium formula No. 1 MS+6-BA 0.5mg / L+2,4-D 0.5mg / L+sucrose 30g / L+gellan gum 3-3.5 g / L. After culture, the callus tissue was crisp and could be subcultured. The callus tissues of No. 4 and No. 5 were crisp, and the proliferation rate was above 3.5, slightly worse than No. 1.

[0057] To further screen culture medium with high callus proliferation rate, plant growth regulators at concentrations of 6-BA 0.5-1.0 mg / l and 2,4-D 0.5 mg / l or mT+6-BA 1.0 mg / l and 2,4-D 0.5 mg / l were combined and added to different basal culture media including MS, B5, White, Heller or SH. Sucrose 30 g / L and gellan gum 3.5 g / L were added. The culture medium formula is shown in Table 7. Three bottles were inoculated with each culture medium. After 30 days of culture, the callus status was observed and the proliferation coefficient was calculated.

[0058] The results are shown in Table 7: Among them, medium No. 1 in Table 7, i.e., B5+6-BA 1.0 mg / l +2,4-D 0.5 mg / l + sucrose 30 g / L + gellan gum 3-3.5 g / L, has a higher proliferation rate, a proliferation coefficient of 6.3, and a better callus tissue state.

[0059] Table 7. Effects of different culture medium formulations on the growth of Saussurea longifolia callus.

[0060]

[0061] In summary, the proliferation culture medium formula of Saussurea ovata can be selected as follows: B5 / MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L +sucrose 30 g / L +gellan gum 3.5 g / L; B5 / MS+6-BA 0.5 mg / L +2,4-D 0.5 mg / L +sucrose 30 g / L +gellan gum 3.5 g / L; B5 / MS+ mT 1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L; 1 / 2 MS+6-BA1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L; SH+6-BA 1.0 mg / L +2,4-D 0.5 mg / L +sucrose 30 g / L+gellan gum 3.5 g / L.

[0062] The proliferation coefficients were high and the callus was in good condition and could be further cultured.

[0063] Example 4: Detection of components in callus extract (1) Extraction of callus tissue 50 g wet weight of the callus tissue of Saussurea involucrata obtained in Table 7 was weighed, crushed, added with 75% ethanol (solid-liquid ratio of 1:10), extracted by ultrasound (600 W) for 20 min, filtered and the filtrate was collected, and the concentrations of polyphenols, flavonoids and polysaccharides in the extract were tested.

[0064] (2) Determination of total polyphenols Standard curve: Gallic acid 0.33 mg / mL solution, diluted to 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, 0.3300 mg / mL concentration gradient. Take 100 μL of long-leaf Saussurea callus extract, add 500 μL of 10% Folin-Ciocalteu reagent, mix thoroughly, let stand for 5 minutes, then add 400 μL of 7.5% Na2CO3, mix thoroughly, room temperature for 60 minutes, and finally take 100 μL to measure the 765nm OD value on the microplate reader. The concentration of total polyphenols in long-leaf Saussurea callus was calculated according to the standard curve. The results are shown in Table 8: The total polyphenol concentration of long-leaf Saussurea callus is 0.30±0.067 mg / g fresh weight.

[0065] (3) Determination of total flavonoids Standard solution: Use quercetin to prepare an ethanol solution of 0.2315 mg / mL, and dilute it into a concentration gradient of 2-10 times. Take 300μL of the extract of long-leaf Saussurea callus, first add 50μL 5% NaNO2, mix, and place at room temperature for 6 min; then add 50μL 10% Al(NO3)3, mix, and place at room temperature for 6 min; finally add 500 μL 4% NaOH and 140 μL water, mix, and place at room temperature for 15 min. Take 100μL of the mixed solution to measure the OD value at 500nm. The concentration of total polyphenols in long-leaf Saussurea was calculated according to the standard curve. The results are shown in Table 8: The total flavonoids of long-leaf Saussurea callus were 0.73±0.086mg / g fresh weight.

[0066] (4) Polysaccharide determination The concentration of polysaccharides was detected by the phenol-sulfuric acid method: 2 mL of the sample was added with 1 mL of 5% phenol reagent, and then 5 mL of concentrated sulfuric acid was added, and the sample was allowed to stand for 5 min, and then heated in a 40°C water bath for 30-40 min, taken out, cooled to room temperature, and finally the absorbance was tested at 490 nm. The polysaccharide concentration was calculated based on the glucose standard solution curve. The results are shown in Table 8: The polysaccharide concentration of the long-leaf snow lotus callus was 13.07±0.12 mg / g fresh weight In conclusion, the callus of Saussurea involucrata contains rich polyphenols, flavonoids and polysaccharides.

[0067] Table 8. Concentration of active ingredients in Saussurea longifolia callus project Total polyphenol concentration (mg / g) Total flavonoids concentration (mg / g) Polysaccharide (mg / g) Saussurea longifolia callus 0.30±0.067 0.73±0.086 13.07±0.12 The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that for ordinary technicians in this field, the present invention can be further modified in several ways without departing from the principle of the present invention. Improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for inducing and culturing callus of Saussurea involucrata, characterized in that: The following steps are involved: A: Take the young shoots and leaves of Saussurea longifolia, rinse and disinfect, and obtain the basal tissue; B: cutting the basal tissue obtained in step A and inoculating it into a callus induction medium for induction culture to obtain initial callus; C: Subculturing the initial callus obtained in step B to obtain subculture callus.

2. The method for inducing and culturing according to claim 1, characterized in that: The flushing and disinfection comprises the following steps: i) Rinse under running water, ii) disinfection with ethanol, iii) Use 1-10% (V / V, dissolved in 3x MS liquid medium) PPM solution, immerse in a shaker at 100-120 rpm for 1-3 hours, and then rinse with sterile water. Preferably, the ethanol is 75% ethanol, the PPM solution is 5% PPM solution, the rotation speed of the shaking table is preferably 110 rpm, and the treatment time is 1 h.

3. The method for inducing and culturing according to claim 1, characterized in that: The induction medium in step B comprises the following components: MS + 2,4-D 0.3-0.7 mg / L + 6-BA 1.8-2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. Preferably, the concentration of 2,4-D is 0.5 mg / L, and the concentration of 6-BA is 2.0 mg / L.

4. The method for inducing and culturing according to claim 1, characterized in that: The culture conditions in step B are as follows: temperature 20±1°C, humidity 50%-70%, illumination for 12 hours per day, and illumination intensity of 1500 Lx-2000 Lx. Preferably, the culture period in step B is at least 20 days. The culture conditions in step C are a temperature of 20±1° C., a humidity of 50% to 70%, 12 hours of light per day, and a light intensity of 1500 Lx to 2000 Lx. Preferably, the culture period in step C is at least 28 days.

5. The method for inducing and culturing according to claim 1, characterized in that: The subculture in step C includes screening culture and / or proliferation culture ； The screening culture comprises the following steps: inoculating the initial callus tissue in step B into a subculture screening medium for culture to obtain embryonic callus tissue; The value-added culture comprises the following steps: inoculating the initial callus or embryonic callus into a value-added culture medium for culture to obtain a subculture callus; Wherein, the screening medium comprises the following components: 1 / 2MS + 2,4-D 2.8-3.2 mg / L + KT 0.3-0.7 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum, preferably, the concentration of 2,4-D is 3.0 mg / L, and the concentration of KT is 0.5 mg / L; The components of the proliferation culture medium are basal culture medium, plant growth regulator, sucrose and gellan gum; Wherein, the basal culture medium is selected from any one of B5, MS, and SH culture medium, and the plant growth regulator is selected from any one of "6-BA and 2,4-D" or "mT and 2,4-D"; When the basal medium is B5 or MS medium, the plant growth regulator is selected from any one of "6-BA and 2,4-D" or "mT and 2,4-D"; When the plant growth regulator is "6-BA and 2,4-D", the concentration of the 6-BA is 0.2-1.2 mg / L, and the concentration of the 2,4-D is 0.2-0.8 mg / L; When the plant growth regulator is "mT and 2,4-D", the concentration of mT is 0.8-1.2 mg / L, and the concentration of 2,4-D is 0.3-0.8 mg / L; When the basic culture medium is SH culture medium, the plant growth regulator is "6-BA and 2,4-D", wherein the concentration of 6-BA is 0.8-1.2 mg / L, and the concentration of 2,4-D is 0.3-0.8 mg / L; Preferably, the proliferation medium is preferably the following components: any one of B5 / MS+6-BA1.0 mg / L +2,4-D 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, 1 / 2 MS+6-BA1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, B5 / MS+6-BA 0.5 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, B5 / MS+ mT1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, SH+6-BA 1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L.

6. A callus induction medium for Saussurea involucrata, characterized in that: The induction medium comprises the following components: MS + 2,4-D 0.3-0.7 mg / L + 6-BA 1.8-2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. Preferably, the concentration of 2,4-D is 0.5 mg / L, and the concentration of 6-BA is 2.0 mg / L.

7. A medium for subculture screening of callus tissue of Saussurea involucrata, characterized in that: The subculture screening medium comprises the following components: 1 / 2MS + 2,4-D 2.8-3.2 mg / L + KT 0.3-0.7 mg / L + 30 g / L sucrose + 3.5 g / L gellan gum. Preferably, the concentration of 2,4-D is 3.0 mg / L, and the concentration of KT is 0.5 mg / L.

8. A culture medium for increasing callus tissue of Saussurea longifolia, characterized in that: The components of the proliferation culture medium include the following components: basic culture medium, plant growth regulator, sucrose and gellan gum.

9. The growth medium according to claim 8, characterized in that The basal culture medium is selected from any one of B5, MS, and SH culture medium, and the plant growth regulator is selected from any one of "6-BA and 2,4-D" or "mT and 2,4-D"; Wherein, when the basic culture medium is B5 or MS culture medium, the plant growth regulator is selected from any one of "6-BA and 2,4-D" or "mT and 2,4-D"; When the plant growth regulator is "6-BA and 2,4-D", the concentration range of the 6-BA is 0.2 to 1.2 mg / L, and the concentration of the 2,4-D is 0.2 to 0.8 mg / L; When the plant growth regulator is "mT and 2,4-D", the concentration range of mT is 0.8-1.2 mg / L, and the concentration of 2,4-D is 0.3-0.8 mg / L; When the basic culture medium is SH culture medium, the plant growth regulators are "6-BA and 2,4-D", wherein the "6-BA concentration range is 0.8 to 1.2 mg / L, and the 2,4-D concentration is 0.3 to 0.8 mg / L".

10. The proliferation medium according to claim 9, characterized in that Selected from the following components: any one of B5 / MS+6-BA1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, 1 / 2 MS+6-BA1.0 mg / L+2,4-D 0.5 mg / L +sucrose 30 g / L+gellan gum 3.5 g / L, B5 / MS+6-BA 0.5 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, B5 / MS+ mT 1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L, SH+6-BA1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+gellan gum 3.5 g / L.

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