Saussurea lancea callus culture medium and culture method thereof

Abstract

The invention discloses a saussurea lancea callus culture medium as well as a preparation method and application thereof. The saussurea lancea callus culture medium comprises a proliferation culture medium, and the saussurea lancea callus proliferation culture medium comprises a basic culture medium, a plant growth regulator, potato powder, cane sugar and gellan gum; the plant growth regulator is one or more of 6-BA and NAA (Naphthalene Acetic Acid). According to the culture medium, effective proliferation of the saussurea lancea callus can be achieved, waterlogging, hardening and differentiation are not prone to occurring, and the content of flavone in active ingredients in the obtained saussurea lancea callus extract is obviously increased.

Description

Technical Field

[0001] The present invention relates to plant tissue culture technology, and specifically, to a callus culture medium for Saussurea laniceps and a culture method thereof. Background Art

[0002] Saussurea is a plant endemic to China and a national second-class protected species, with extremely high medicinal and ornamental values, and is one of the important Tibetan medicine plant resources. It is mainly distributed in the high-altitude areas in the western part of China and less in foreign countries. Research shows that Saussurea contains rich flavonoids, polyphenols, polysaccharides and secondary metabolites, and has functions such as scavenging free radicals, anti-aging and analgesia. Saussurea mainly reproduces by seeds, and predatory digging destroys the ecological balance and seriously affects its natural growth and reproduction. Saussurea laniceps, also known as Saussurea laniceps Hand.-Mazz., is a perennial herb of the genus Saussurea DC. of the family Compositae. Its flowering and fruiting periods are from August to October, and its leaves and capitula are covered with thick wool. It is distributed in Sichuan, Yunnan, Tibet and other places in China, mainly growing in the alpine scree slopes at an altitude of 3,200 - 5,280 meters, and is a very typical alpine plant in China. Therefore, using plant tissue culture technology and callus cells is an effective way to preserve seed resources and thus solve the endangered situation of Saussurea resources.

[0003] Currently, there is little research on the callus culture of Saussurea laniceps. On the one hand, because its callus proliferates slowly, it is necessary to select a suitable basic medium and continuously change the types and ratios of plant growth regulators, and it can reach a certain proliferation rate only after multiple subcultures; on the other hand, the proliferation culture process of Saussurea laniceps callus is very unstable, prone to waterlogging or hardening, or differentiation, affecting its normal proliferation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the technical problems in the prior art that the proliferation speed of Saussurea laniceps callus in the culture medium is slow during the proliferation culture process, is prone to waterlogging or hardening, and differentiates, affecting its normal proliferation, thereby providing a callus proliferation culture medium for Saussurea laniceps and a culture method thereof.

[0005] Compared with the existing culture medium, the proliferation culture medium for Saussurea laniceps callus culture of the present invention has a fast proliferation speed of Saussurea laniceps callus obtained during the proliferation culture, is not prone to waterlogging, is not prone to hardening, does not differentiate, and the flavonoid content in the active ingredients of the obtained Saussurea laniceps callus extract is significantly increased.

[0006] The present invention solves the above technical problems through the following solutions: A callus proliferation culture medium for Saussurea laniceps, which comprises the following components: Basic medium, plant growth regulator, potato powder 1 - 100 g / L, sucrose 30 g / L, gellan gum 3.5 g / L; wherein, g / L refers to the concentration in the callus proliferation medium of Saussurea laniceps.

[0007] Preferably, the concentration of the potato powder in the callus proliferation medium of Saussurea laniceps is 20 - 60 g / L. More preferably, the concentration of the potato powder is 25 - 35 g / L, for example, 25 g / L, 30 g / L, 35 g / L.

[0008] The potato powder is obtained by the following method: Select fresh and uniformly shaped potatoes, wash them clean with pure water, peel them, cut them into slices, lay them flat in a hot air dryer, set the constant temperature at 60°C for drying, crush them through a 60 - mesh sieve to obtain coarse powder; then crush them through an ultra - micro vibrating sieve and pass through a 100 - mesh sieve to obtain potato powder.

[0009] The basic medium is MS medium.

[0010] The plant growth regulator is preferably selected from the mixture of 6 - BA and NAA; the concentration of 6 - BA in the callus proliferation medium of Saussurea laniceps is 0.3 - 0.7 mg / L, and the concentration of NAA in the callus proliferation medium of Saussurea laniceps is preferably 0.3 - 0.7 mg / L. More preferably, the concentration of 6 - BA is 0.3 - 0.5 mg / L, for example, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L. The concentration of NAA is 0.5 mg / L.

[0011] More preferably, the proliferation medium is MS + 6 - BA 0.3 - 0.7 mg / L + NAA 0.3 - 0.7 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6 - BA 0.1 - 0.5 mg / L + NAA 0.3 - 0.7 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L. Further, the proliferation medium is MS + 6 - BA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6 - BA 0.2 - 0.4 mg / L + NAA 0.4 - 0.6 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0012] Furthermore, the proliferation medium is MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L, MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0013] The present invention also provides a Saussurea laniceps callus induction medium, the induction medium comprising MS medium, 6-BA, NAA, sucrose and gellan gum; wherein, the concentration of 6-BA is 0.3 - 1.7 mg / L, the concentration of NAA is 0.1 - 2.2 mg / L, the concentration of sucrose is 30 g / L, the concentration of gellan gum is 3.5 g / L, mg / L and g / L refer to the concentration in the callus induction medium. Preferably, the concentration of 6-BA is 0.3 - 1.5 mg / L, and the concentration of NAA is 0.3 - 2.0 mg / L.

[0014] Further, the induction medium is MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.1 - 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.3 - 1.7 mg / L + NAA 1.8 - 2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. More preferably, the induction medium is MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.2 - 0.4 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.4 - 1.6 mg / L + NAA 1.9 - 2.1 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. For example, the induction medium is MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.5 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. When the induction medium is used for inducing callus of Saussurea laniceps, the induction rate is relatively high.

[0015] The present invention also provides a subculture medium for Saussurea laniceps callus, and the subculture medium for callus includes MS medium, 6-BA, NAA, sucrose, and gellan gum; wherein, the concentration of 6-BA is 0.3 - 0.7 mg / L, the concentration of NAA is 0.3 - 1.2 mg / L, the sucrose is 30 g / L, and the gellan gum is 3.5 g / L. mg / L and g / L refer to the concentrations in the subculture medium for callus. Preferably, the concentration of 6-BA is 0.4 - 0.6 mg / L, such as 0.5 mg / L, and the concentration of NAA is 0.3 - 0.7 mg / L or 0.8 - 1.2 mg / L, such as 0.5 mg / L or 1.0 mg / L.

[0016] Further, the subculture medium is MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.3 - 0.7 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.8 - 1.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. More preferably, the subculture medium is MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.9 - 1.1 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. For example, the subculture medium is MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. The said subculture medium can cultivate Saussurea laniceps callus that is yellow and relatively loose, and is not prone to differentiation or browning.

[0017] The present invention also provides a method for culturing Saussurea laniceps callus, which includes the following steps: Inoculate the Saussurea laniceps callus into the above-mentioned Saussurea laniceps callus proliferation medium, and then culture it.

[0018] Preferably, the Saussurea laniceps callus is obtained by the following method: Induce and culture the explants of Saussurea laniceps on the above-mentioned callus induction medium, and then inoculate them into the above-mentioned subculture medium for subculture. Preferably, the temperature for induction and subculture is 20 ± 1°C, the humidity is 50% - 70%, and it is cultured in the dark.

[0019] The preparation method of the Saussurea laniceps Hand.-Mazz. explant is as follows: After trimming the seeds of wild Saussurea laniceps Hand.-Mazz., soak them in deionized water at 4°C, rinse, and then wash with sterile water. Then, in a sterile laminar flow hood, disinfect with 75% ethanol (added with Tween 20), and rinse with sterile water; use a 2% (V / V) NaClO solution (added with Tween 20) to treat in a shaker at 110 rpm, rinse with sterile water, and then evenly place on the MS basal medium and culture in the dark at 14 ± 1°C. Transplant the germinated seeds into an MS culture bottle, culture at a temperature of 20°C with a light intensity of 1500 Lx for 12 h per day to grow into sterile seedlings.

[0020] The induction culture includes the following steps: Cut the roots, stems, and leaves of the sterile seedlings into segments with a length of 0.5 cm - 1.0 cm, inoculate them into the above induction medium, and obtain callus after dark culture. Preferably, use the stems and leaves of the sterile seedlings to induce the formation of callus.

[0021] Beneficial effects 1) Compared with the existing medium, the proliferation medium for culturing the callus of Saussurea laniceps Hand.-Mazz. in the present invention has a fast proliferation rate of the callus of Saussurea laniceps Hand.-Mazz. obtained during proliferation culture, and the proliferation coefficient can reach more than 4. During culture, the callus is not prone to waterlogging, hardening, or differentiation.

[0022] 2) In the culture method of the present invention, when culturing the callus of Saussurea laniceps Hand.-Mazz., a high proliferation rate can be achieved without multiple subcultures, and the flavonoid content in the active ingredients of the callus extract of Saussurea laniceps Hand.-Mazz. obtained is significantly increased.

[0023] 3) The callus of Saussurea laniceps Hand.-Mazz. obtained by the culture method of the present invention has the advantages of stable subculture, short culture period, environmental protection, and stable quality, and can solve the problem that it is difficult to develop and utilize the wild plant resources of Saussurea involucrata Kar. et Kir. Description of the drawings

[0024] Figure 1 : Saffron staining photo of the callus of Saussurea laniceps Hand.-Mazz. a) Photo of embryogenic callus b) Non-embryogenic callus Figure 2 : Photo of culturing Saussurea laniceps Hand.-Mazz. with sterile seedlings Figure 3 : Photo of subculturing the callus of Saussurea laniceps Hand.-Mazz. a) Waterlogged and firm-textured callus of Saussurea laniceps Hand.-Mazz. obtained from No. 8 in Table 4 b) Browning callus of Saussurea laniceps Hand.-Mazz. obtained from No. 6 in Table 4 c) Callus of Saussurea laniceps Hand.-Mazz. with a uniform granular surface and high looseness obtained from No. 2 in Table 4 Detailed implementation manners

[0025] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0026] In the following embodiments and comparative examples: Source of raw materials: Culture medium MS (M519), B5 (G398), SH (816) and plant growth regulators were purchased from Pytotechnolagy. Plant growth regulators include: Naphthaleneacetic acid ( NAA); 6-benzylaminoadenine (6-BA); 2,4- Dichlorophenoxyacetic acid (2,4-D); Kinetin (KT); Meta-topolin (mT), glacier water: glacier water is taken from Qudennima Glacier in Gamba County, Shigatse. Qudennima Glacier in Gamba County, Shigatse is located on the northern slope of the middle section of the Himalayas, 5128 meters above sea level, and the temperature of its spring mouth remains at 1.5-2℃ all year round.

[0027] The seed sterilization and sterile seedling culture is MS basic medium. The induction medium is selected from MS, B5, SH medium, and different plant growth regulators, cytokinins and other nutrients are added. The above culture media are all added with sucrose 30g / L, gellan gum 3.5g / L, adjusted to pH 5.7-5.8, and the sterilizer is set at 121℃ for 20min for standby. The daily culture conditions are temperature 20±1℃, humidity 50%-70%, and dark culture.

[0028] After pruning, the seeds of wild Saussurea edulis were used as three test batches, without pretreatment, soaked in gibberellin at room temperature for 24 h, and soaked in deionized water at 4℃ for 24 h. After rinsing under running water for 20 min, they were washed with sterile water 5 times. Then, in a sterile clean bench, they were disinfected with 75% ethanol (with Tween 20) for 15 seconds and rinsed with sterile water 5 times. They were treated with 2% (V / V) NaClO solution (with Tween 20) in a shaker at 110 rpm for 10 min, rinsed with sterile water 5 times, and then evenly placed on MS basal culture medium and cultured at 14±1℃ in the dark. When the seeds germinated and elongated to about 1 cm, the germination rate was counted and transferred to MS culture bottles for sterile seedling culture. Seed germination rate = number of germinated seeds / number of sown seeds × 100%.

[0029] The results are shown in Table 1: Seeds germinated in all three batches, and one batch was easier to operate without pretreatment, with the highest germination rate and less susceptible to bacteria. The germinated seeds were transplanted into MS culture bottles at 20°C and 12 h / day light. Cultured at 1500 Lx, All can be grown into sterile seedlings.

[0030] Table 1 Germination rate of Saussurea edulis seeds

[0031] In a sterile laminar flow hood, use a scalpel to cut the roots, stems, and leaves of the sterile seedlings into segments 0.5 cm - 1.0 cm in length, and inoculate them into the following 3 media. Each medium is added with 30 g / L sucrose and 3.5 g / L gellan gum. After about two weeks of dark culture, it can be observed that the cut segments swell significantly and dense tissue appears at the incision. After 30 days of culture Visible yellowish-green, relatively dense granular callus can be seen, and at the same time Statistical callus induction rate. Callus induction rate = number of explants induced to form callus / number of inoculated explants × 100%。

[0032] The results are shown in Table 2 Table 2 Induction of Callus from Different Explants of Saussurea laniceps

[0033] Except that the induction rate of roots on Medium 2 is 0, light yellowish callus was induced on the other types of media. Among them, the callus induction rate on Medium 1 is the highest, and the induction effect is the best with the young leaves of sterile seedlings as the explants. Overall, the induction effect of Medium 1 is the best, and the induction rate using leaves is significantly higher than that of stems and significantly higher than that of roots.

[0034] To further study the effect of different types of media on the induction of callus from Saussurea laniceps, select the stems and leaves of sterile seedlings with a higher induction rate, cut them into segments and inoculate them into the following 9 media. Each medium is added with 30 g / L sucrose and 3.5 g / L gellan gum, and 4 dishes are inoculated for each type of medium. 20 ± 1°C, humidity 50% - 70%, After about two weeks of dark culture, it can be observed that the cut segments swell significantly and dense tissue appears at the incision. After 30 days of culture Visible yellowish-green, relatively dense granular callus can be seen. Statistical callus induc tion rate. Callus induction rate = number of explants induced to form callus / number of inoculated explants × 100%.

[0035] To evaluate the effect of different media on the induction of callus from Saussurea laniceps, the results are shown in Table 3: Table 3 Induction of Callus from Saussurea laniceps

[0036] As can be seen from Table 3, when using stems and leaves as explants to induce callus on 9 different types of media, the highest induction rate is on Medium 5. The callus shows light yellow and yellowish-green, is in a looser state, and no browning tissue appears. The lowest induction rate is on Medium 9, and the tissue is severely browned and is more moist than other brittle callus tissues. Therefore, when culturing and inducing callus from Saussurea laniceps, the selection of the components of the medium is very important. If the content of plant growth regulators is too high, too low, or the types are not suitable, it will lead to a lower induction rate of callus from Saussurea laniceps and may cause problems such as browning and waterlogging of the callus during induction.

[0037] MS No.5 + 6 - BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L is the preferred induction medium, and MS No.2 + 6 - BA 1.5 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L is the second preferred induction medium.

[0038] (1) To screen out the subculture medium that can maintain the callus state of Saussurea laniceps, the callus induced in the above steps was taken and inoculated into media containing MS and B5, supplemented with different ratios of 6 - BA, 2,4 - D, NAA, and KT for subculture. Each medium was added with 30 g / L sucrose and 3.5 g / L gellan gum, and 4 dishes were inoculated for each treatment. 20 ± 1°C, humi dity 50% - 70%, Dark culture for 30 Observe the cell state after [days].

[0039] Callus in different states was obtained, and its browning degree and texture were judged by visual inspection combined with the method of picking with sterile forceps. The results are shown in Table 4.

[0040] Table 4 Effects of different medium formulations on the subculture of Saussurea laniceps callus

[0041] Note: The more '+' for the browning degree, the higher it represents.

[0042] As can be seen from Table 4, during the subculture of Saussurea laniceps callus, browning, waterlogging, and differentiation are likely to occur. Therefore, the selection of the components of the subculture medium is very important. If the content of plant growth regulators is too high, too low, or the types are inappropriate, it is not conducive to the subculture of Saussurea laniceps callus.

[0043] In the No.1 and No.2 media, the yellow and relatively loose callus is in a better state of the medium and can be used for subculture screening. Therefore, comprehensively evaluating, the preferred medium is MS No.2 + 6 - BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L, and the second preferred medium is MS No.1 + 6 - BA 0.5 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

[0044] (2)Callus is divided into embryogenic callus and non-embryogenic callus, and there are obvious differences between these two types of callus in morphology and cytology. Embryogenic callus refers to callus with the ability of embryogenesis, and its morphological characteristics are spherical particles on the surface, mostly loose, with small cells and large nuclei, arranged neatly and tightly, containing rich contents such as starch and soluble sugars, and it is a cell mass with strong proliferation and division ability, which can be continuously and stably subcultured. Non-embryogenic callus refers to callus cells that do not have the ability of embryogenesis. Its morphology is mostly viscous, easy to be waterlogged or browned, the cells are irregular, relatively large and the tissue structure is loose, without obvious cell organelles, and it cannot be stably and continuously subcultured. Safranin, with the molecular formula C20H19N4Cl, is a natural dye extracted from the stigma of saffron. It is a commonly used dye in cytology and animal and plant histology, and can stain the lignified, suberized and cutinized parts of plants and cell nuclei red.

[0045] To further identify the callus with different colors and textures in the above steps, safranin reagent was used to section and stain the two main types of callus. The results are shown in Table 5. The callus with light yellow color, loose texture and granular surface is the embryogenic callus cell mass, which can be used for subsequent subculture screening.

[0046] Table 5 Identification of Saussurea laniceps callus with different morphologies

[0047] (3)To continue to screen the medium suitable for the proliferation culture of Saussurea laniceps callus, the above-mentioned yellow, loose and granular callus was selected and inoculated into the medium with different formulations for culture. Each medium was added with 30 g / L sucrose and 3.5 g / L gellan gum, and 4 dishes were inoculated for each treatment. 20 ± 1°C, humidity 50% - 70%, Dark Culture 30 After [days] obs erve the cell state. Weigh the initial fresh weight at 0 day, and weigh the fresh weight after one stage of culture (30 d) and calculate the proliferation coefficient for each category.

[0048] Proliferation coefficient = final wet cell weight / initial wet cell weight.

[0049] The preparation methods of natural organic additives such as potato powder, banana powder and coconut juice in some media are as follows. Potato powder: Select fresh and uniform potatoes, wash them clean with pure water, peel them, cut them into slices with a thickness of 5 mm, lay them flat in a hot air dryer, set the constant temperature at 60 °C and dry for 48 h, and obtain the coarse powder by passing through a 60-mesh sieve; then through ultra-fine vibration pulverization and passing through a 100-mesh sieve to obtain potato powder.

[0050] Banana powder: Bananas with 70% ripeness are selected, peeled and cut into 1 cm slices, which are laid flat in a hot air dryer. The temperature is set at a constant 60 °C and dried for 48 h. The crude banana powder is obtained by crushing and sieving, and the banana powder is obtained by ultra-fine crushing and then passing through a 100-mesh sieve.

[0051] Coconut juice: Commercially available coconuts produced in Hainan are selected, opened at the coconut stalk to extract coconut juice, and sealed and stored at 4 °C for later use. Callus in different states is obtained, and it is visually inspected and combined with the method of clamping with sterile forceps to evaluate the browning degree and texture looseness during the proliferation culture of callus. The results are shown in Table 6: Table 6 Effects of different medium formulations on the growth of Saussurea laniceps callus

[0052] Note: The more “+” for looseness represents the crisper texture, and the more “-” represents the tighter texture. Among them, the more obvious, uniform and loose the granular shape is, the better the state of the callus, and it is more suitable for subsequent targeted breeding and stable subculture.

[0053] From the perspective of the proliferation coefficient, medium No. 13 is the highest and medium No. 11 is the second. Considering the state of the callus, there is differentiation to varying degrees in medium Nos. 5, 7, 8 and 13, and the texture is tightly packed to varying degrees in medium Nos. 3, 4, 9 and 12. The callus states of medium Nos. 1, 10, 11 and 13 have a higher looseness. Among them, inositol, coconut juice, and banana powder have no obvious promoting effect on the proliferation of Saussurea laniceps, and are prone to cause differentiation of the callus of Saussurea laniceps, and are not suitable for the proliferation culture of Saussurea laniceps.

[0054] Adding organic nutrient potato powder to the basic medium formulation can increase the proliferation rate of callus, and the state shows yellow and loose granular shape. The volume of the callus expands significantly, and there is no differentiation or browning phenomenon. By comparison, the optimal addition concentration of potato powder is 30 g / L. Through comprehensive evaluation, the best medium formulation for callus proliferation is No. 11 MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L; the sub-optimal medium formulation is No. 13 MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L. Using the optimized proliferation medium, without complex stepwise temperature control, after a short-term subculture, the proliferation coefficient of Saussurea laniceps callus can reach more than 4 times, significantly promoting the proliferation of Saussurea laniceps callus.

[0055] (1) Extraction of callus Weigh separately 10 g (wet weight) of the callus of Saussurea laniceps that was subcultured with the number 3 in Table 6 (MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L), 10 g of the callus of Saussurea laniceps that was subcultured with the number 11 (MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + 30 g / L potato powder), and 10 g of the callus of Saussurea laniceps that was subcultured with the number 13 (MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + 30 g / L potato powder). Add deionized water (with a solid-liquid ratio set at the standard of 1:10), pulverize in a homogenizer 5 times, 2 minutes each time, then extract by ultrasound (600W) for 30 minutes, with the extraction temperature below 50 °C, centrifuge at 8000 rmp for 60 minutes, and filter the supernatant to obtain the extract of the callus of Saussurea laniceps.

[0056] Solid content detection method and results: Weigh separately 6 ± 0.1 g of the extract of the callus of Saussurea laniceps obtained by extraction. Set the oven temperature at 105 °C and keep drying at a constant temperature for 3.5 h. Finally, weigh and calculate to obtain that its solid content is approximately 0.6%.

[0057] (2) Determination of total flavonoids The aluminum nitrate colorimetric method was used to detect the total flavonoid content. Standard solution: Prepare an ethanol solution of 0.2315 mg / mL with quercetin and dilute it successively into concentration gradients of 2 - 10 times. Take 300 μL of the extract of the callus of Saussurea laniceps respectively, first add 50 μL of 5% NaNO2 solution, mix well and let it stand at room temperature for 6 min; then add 50 μL of 10% Al(NO3)3 solution, mix and let it stand at room temperature for 6 min; finally add 500 μL of 4% NaOH and 140 μL of deionized water, mix evenly and let it stand at room temperature for reaction for 15 min. Take 100 μL of the mixed solution and measure the absorbance value at 500 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the total polyphenol content of Saussurea laniceps according to the standard curve. The results are shown in Table 7. For the callus of Saussurea laniceps cultured in the basic medium, the total flavonoids in the extract are 0.10 mg / mL. For the callus of Saussurea laniceps cultured with better proliferation effect by adding 30 g / L potato powder, the detected total flavonoid contents in the extracts are 0.13 mg / mL and 0.14 mg / mL, both higher than the total flavonoid content in the extract of the callus cultured in the medium without added potato powder.

[0058] Table 7 Contents of active ingredients in the callus of Saussurea laniceps

[0059] Note: * represents P < 0.05, that is, there are significant differences between No. 2 and No. 3 and No. 1 respectively.

[0060] (3) Determination of total polyphenols The total polyphenol content was detected by the Folin-Ciocalteu method. Standard curve: Prepare a gallic acid solution with a concentration of 0.3300 mg / mL, and sequentially dilute it into concentration gradients of 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, and 0.3300 mg / mL. Take 100 μL of the callus extract of Saussurea laniceps, add 500 μL of 10% Folin-Ciocalteu reagent, mix well, let stand for 5 min (not exceeding 8 min), then add 400 μL of 7.5% Na2CO3 solution, mix well and let stand at room temperature for 60 min. Finally, take 100 μL of the mixed solution and measure the absorbance at 765 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the total polyphenol content of the callus of Saussurea laniceps according to the standard curve. The total polyphenol content of the extract of the callus of Saussurea laniceps was detected to be 0.10 mg / mL.

[0061] (4)Determination of polysaccharide The polysaccharide content was detected by the phenol-sulfuric acid method. Take 2 mL of the sample, add 1 mL of 5% phenol reagent, then add 5 mL of concentrated sulfuric acid, let stand for 5 min, then heat in a water bath at 40 °C for 30 - 40 min, take out, cool to room temperature, and finally detect the absorbance at 490 nm. Calculate the polysaccharide content according to the glucose standard solution curve. The average polysaccharide content of the extract of the callus of Saussurea laniceps was detected to be 3.94 mg / mL.

[0062] (5)Determination of protein content The protein concentration of the sample was detected using the BCA Protein Assay Kit from Beyotime. Prepare the standard product and working solution according to the instructions, add 100 μL of the sample and mix well, let stand at 60 °C for 30 min, and finally detect the absorbance at 562 nm on an ELISA reader. Calculate the protein concentration of the sample according to the standard curve and the volume of the sample used. The average protein content of the extract of the callus of Saussurea laniceps was detected to be 0.66 mg / mL.

[0063] In summary, the callus of Saussurea laniceps contains rich polyphenols, flavonoids, polysaccharides, and protein components. Adding potato powder to the basal medium may affect the accumulation of its components. Among them, culturing the callus of Saussurea laniceps in a medium supplemented with 30 g / L potato powder can effectively increase its total flavonoid content.

[0064] The above examples only represent several embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present invention. These all belong to the protection scope of the present invention and should be subject to the appended claims.

Claims

1. A callus proliferation medium for Saussurea laniceps, characterized in that, It includes the following components: MS medium, plant growth regulator, 1 - 100 g / L of potato powder, 30 g / L of sucrose, 3.5 g / L of gellan gum; wherein, g / L refers to the concentration in the callus proliferation medium of Saussurea laniceps Hand.-Mazz.

2. The proliferative culture medium according to claim 1, wherein The concentration of the potato powder is 20 - 60 g / L. Preferably, the concentration of the potato powder is 25 - 35 g / L. For example, 25 g / L, 30 g / L, 35 g / L; The plant growth regulator is selected from the mixture of 6 - BA and NAA. The concentration of 6 - BA is 0.3 - 0.7 mg / L, and the concentration of NAA is 0.3 - 0.7 mg / L. Preferably, the concentration of 6 - BA is 0.3 - 0.5 mg / L. For example, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, and the concentration of NAA is 0.5 mg / L.

3. The proliferation medium according to claim 1, wherein, The potato powder is obtained by the following method: 1) After the potatoes are cleaned and peeled, they are cut into slices. 2) They are laid flat in a hot air dryer and dried at a constant temperature, then crushed and sieved through a 60 - mesh sieve to obtain coarse powder. 3) They are crushed by an ultramicro vibrating sieve and sieved through a 100 - mesh sieve to obtain potato powder.

4. The proliferative culture medium according to claim 1, wherein, The proliferation medium is MS + 0.3 - 0.7 mg / L of 6 - BA + 0.3 - 0.7 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum or MS + 0.1 - 0.5 mg / L of 6 - BA + 0.3 - 0.7 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum; preferably, the proliferation medium is MS + 0.4 - 0.6 mg / L of 6 - BA + 0.4 - 0.6 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum or MS + 0.2 - 0.4 mg / L of 6 - BA + 0.4 - 0.6 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum; further, the proliferation medium is MS + 0.5 mg / L of 6 - BA + 0.5 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum or MS + 0.3 mg / L of 6 - BA + 0.5 mg / L of NAA + 30 g / L of potato powder + 30 g / L of sucrose + 3.5 g / L of gellan gum.

5. A callus induction medium for Saussurea laniceps, characterized in that, The induction medium comprises MS medium, 6-BA, NAA, sucrose and gellan gum; wherein, the concentration of 6-BA is 0.3 - 1.7 mg / L, the concentration of NAA is 0.1 - 2.2 mg / L, the concentration of sucrose is 30 g / L, and the concentration of gellan gum is 3.5 g / L. mg / L and g / L refer to the concentrations in the callus induction medium. Preferably, the concentration of 6-BA is 0.3 - 1.5 mg / L, and the concentration of NAA is 0.3 - 2.0 mg / L. Preferably, the induction medium is MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.1 - 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.3 - 1.7 mg / L + NAA 1.8 - 2.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. More preferably, the induction medium is MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.2 - 0.4 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.4 - 1.6 mg / L + NAA 1.9 - 2.1 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. For example, the induction medium is MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 1.5 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

6. A subculture medium for Saussurea laniceps callus, characterized in that, The callus subculture medium comprises MS medium, 6-BA, NAA, sucrose, and gellan gum; wherein, the concentration of 6-BA is 0.3 - 0.7 mg / L, the concentration of NAA is 0.3 - 1.2 mg / L, the sucrose is 30 g / L, and the gellan gum is 3.5 g / L. mg / L and g / L refer to the concentrations in the callus subculture medium. Preferably, the concentration of 6-BA is 0.4 - 0.6 mg / L, such as 0.5 mg / L, and the concentration of NAA is 0.3 - 0.7 mg / L or 0.8 - 1.2 mg / L, such as 0.5 mg / L or 1.0 mg / L; Preferably, the subculture medium is MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.3 - 0.7 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.3 - 0.7 mg / L + NAA 0.8 - 1.2 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L; More preferably, the subculture medium is MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.4 - 0.6 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.4 - 0.6 mg / L + NAA 0.9 - 1.1 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L. For example, the subculture medium is MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L or MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L.

7. A method for culturing Saussurea laniceps callus, comprising the following steps: Inoculate the Saussurea laniceps callus into the Saussurea laniceps callus proliferation medium according to any one of claims 1 - 4, and after culturing, the culturing temperature is 20 ± 1°C, the humidity is 50% - 70%, and perform dark culturing.

8. The cultivation method according to claim 7, characterized in that, The Saussurea laniceps callus is obtained by the following method: Induce and culture the Saussurea laniceps explant on the callus induction medium according to claim 5, and then inoculate it into the subculture medium according to claim 6 for subculture. The temperatures of the induction and subculture are 20 ± 1°C, the humidity is 50% - 70%, and perform dark culturing.

9. The cultivation method according to claim 8, characterized in that, The preparation method of the Saussurea laniceps explant is as follows: 1) Take the seeds of wild Saussurea laniceps, trim them, soak them in deionized water at 4°C, and after rinsing, wash them with sterile water; 2) In a sterile laminar flow hood, disinfect with 75% ethanol (added with Tween 20), and rinse with sterile water; 3) Treat with 2% (V / V) NaClO solution (added with Tween 20) at 110 rpm in a shaker, rinse with sterile water, and evenly place them on the MS basal medium for culturing in the dark at 13°C. 4) Transplant the germinated seeds into an MS culture bottle, culture at a temperature of 20 °C with a light intensity of 1500 Lx for 12 h per day to grow into sterile seedlings.

10. The culturing method according to claim 8, wherein, The induction culture includes the following steps: Cut the roots, stems, and leaves of the sterile seedlings into segments with a length of 0.5 cm - 1.0 cm, and inoculate them into the induction medium to obtain callus.

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