Suspension culture medium, suspension culture method and extraction method of ferula sinkiangensis cells

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a suspension culture medium, a suspension culture method and an extraction method of ferula sinkiang cells. The invention provides a suspension culture medium for suspension culture of ferula sinkiang cells, which is characterized in that methyl jasmonate (MeJA), spermine (Spm) and abscisic acid (ABA) are added into the suspension culture medium to promote the growth and reproduction of the ferula sinkiang cells and the accumulation of secondary metabolites. According to the suspension culture method of the ferula sinkiang cells, synthesis and accumulation of secondary metabolites of the ferula sinkiang liquid suspension cells are achieved through a suspension culture medium, the yield of medicinal secondary metabolites of the ferula sinkiang cells is increased, and a theoretical basis is provided for efficient and high-quality tissue culture and factory medicinal production of ferula sinkiang. And the liquid suspension culture is beneficial to the realization of factory scale culture, and indirectly protects the wild plant resource of ferula sinkiangensis.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and particularly relates to a suspension culture medium, a suspension culture method and an extraction method for Ferula sinkiangensis cells. Background Art

[0002] Ferula sinkiangensis K.M.Shen is a perennial once-flowering medicinal plant of the genus Ferula in the family Apiaceae. It is endemic to China and is only distributed in the Ili region of Xinjiang. This species has been listed as a critically endangered species and a national second-class protected plant. At the same time, as a unique ethnic medicine produced in Xinjiang Uygur Autonomous Region of China, its plants can secrete oleo-gum resin with a special smell, which has the effects of dissipating accumulation and resolving mass, dispelling dampness and relieving pain, and killing insects. It has a unique curative effect on rheumatoid arthritis and stomach diseases and is one of the essential Chinese herbal medicines in the international medicinal material market. Its medicinal components mainly include sesquiterpene coumarins, sesquiterpene phenylpropanoids, polysaccharides and volatile components, which have different effects such as antioxidant, anti-tumor, antiviral, regulating blood lipid and enhancing immune function. The 2022 edition of the Chinese Pharmacopoeia includes Ferula sinkiangensis as one of the medicinal original plants of Ferula. In addition to being used in traditional Chinese medicine, it is also used in Tibetan medicine and Mongolian medicine. It has important medicinal value and is a rare plant resource.

[0003] In recent years, due to the continuous growth of international trade in plant raw materials and plant extracts, the market demand for Ferula sinkiangensis has increased, resulting in a significant price increase. The huge economic benefits have intensified the incidents of wild collection and excavation, seriously damaging the natural plant resources of Ferula sinkiangensis. At the same time, Ferula sinkiangensis is an early spring ephemeral plant that reproduces by seeds. Its geographical distribution is narrow, its ecological amplitude is small, its seed germination rate is low, and its natural regeneration ability is poor. It is difficult and time-consuming to rely on seeds for artificial propagation to obtain medicinal components. At present, the tissue culture technology of Ferula sinkiangensis is only limited to the primary stage of plant regeneration system research. The formation of regenerated seedlings by tissue culture of Ferula sinkiangensis has not been broken through and is quite difficult. It is very difficult to realize the way of obtaining medicinal secondary metabolites through the later cultivation of tissue culture seedlings. There is no complete suspension culture technology system for Ferula sinkiangensis established in the existing technology. Summary of the Invention

[0004] In view of this, the present invention provides a suspension culture medium, a suspension culture method and an extraction method for Ferula sinkiangensis cells, and establishes a complete suspension culture method for Ferula sinkiangensis to improve the yield of medicinal secondary metabolites.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a suspension medium for culturing Ferula sinkiangensis cells. Based on the MS medium, it further comprises 0.1 - 23 mg / L of elicitor, 0.5 - 1 mg / L of 2,4-D, 0.5 - 1 mg / L of 6-BA, and 30 g / L of sucrose; the elicitor includes one or more of methyl jasmonate, spermine, and abscisic acid.

[0007] Preferably, when the suspension medium is used to increase the biomass of Ferula sinkiangensis cells, the elicitor includes abscisic acid or spermine, the concentration of abscisic acid in the suspension medium is 1.2 - 1.5 mg / L, and the concentration of spermine in the suspension medium is 0.3 - 1.5 mg / L;

[0008] When the medium is used to increase the content of secondary metabolites in Ferula sinkiangensis cells, the elicitor includes methyl jasmonate or spermine, the molar concentration of methyl jasmonate in the suspension medium is 11.21 - 22.43 mg / L, and the concentration of spermine in the suspension medium is 0.5 - 1.5 mg / L.

[0009] Preferably, the secondary metabolites include ferulic acid and / or total flavonoids;

[0010] When the medium is used to increase the ferulic acid content in Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is 22.43 mg / L, and the concentration of spermine in the suspension medium is 1.5 mg / L;

[0011] When the suspension medium is used to increase the total flavonoid content in Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is 11.21 - 22.43 mg / L, and the concentration of spermine in the suspension medium is 0.5 - 1.0 mg / L.

[0012] Preferably, the initial pH value of the suspension medium is 5.8 - 6.0.

[0013] The present invention provides a suspension culture method for Ferula sinkiangensis cells, comprising: inoculating Ferula sinkiangensis callus in the suspension medium described in the above technical solution for suspension culture.

[0014] Preferably, the mass - volume ratio of Ferula sinkiangensis callus to the suspension medium during inoculation is 1 g:(25 - 30) mL; the rotation speed of the suspension culture is 100 - 120 rpm.

[0015] Preferably, the temperature of the suspension culture is 21 - 23 °C.

[0016] Preferably, the explant for culturing Ferula sinkiangensis callus includes the hypocotyl of Ferula sinkiangensis seedlings.

[0017] Preferably, the suspension culture includes dark culture; the suspension culture time is 15 days.

[0018] The invention provides a method for extracting secondary metabolites of Xinjiang Ferula cells, and extracting secondary metabolites from Xinjiang Ferula cells obtained by the suspension culture method described in the above technical scheme.

[0019] Beneficial effects of the present invention: The present invention provides a suspension culture medium for suspending and culturing Xinjiang Ferula cells, wherein methyl jasmonate (MeJA), spermine (Spm) and abscisic acid (ABA) are added to the suspension culture medium, wherein MeJA promotes the synthesis of secondary metabolites by inducing the expression of key enzyme genes in the secondary metabolic pathway, and MeJA can activate the phenylpropanoid metabolic pathway and increase the accumulation of flavonoids, phenols and terpenoid compounds. Spm affects the synthesis of secondary metabolites by regulating cell division and differentiation; Spm can also promote the accumulation of secondary metabolites by stabilizing cell membrane structure and regulating antioxidant enzyme activity. In addition, ABA can activate genes related to secondary metabolism by regulating stomatal closure and gene expression, promote the accumulation of terpenes, alkaloids and polyphenolic compounds, and quickly and massively obtain medicinal secondary metabolites by suspending and culturing callus tissue. The present invention improves the yield of medicinal secondary metabolites of Xinjiang Ferula cells under the joint action of methyl jasmonate (MeJA), spermine (Spm) and abscisic acid (ABA).

[0020] The invention provides a suspension culture method for Xinjiang Ferula cells. The suspension culture medium is used to realize the synthesis and accumulation of secondary metabolites of Xinjiang Ferula liquid suspension cells. The cell suspension culture of the invention is an important way of artificial propagation, can break through the bottleneck limitation of Ferula seed propagation, can quickly and massively obtain callus tissue or cells for extracting medicinal secondary metabolites, and provides a theoretical basis for efficient and high-quality tissue culture and factory-based medicinal production of Xinjiang Ferula. The liquid suspension culture method can rapidly proliferate a large number of cells, is conducive to realizing factory-scale culture, and indirectly protects Xinjiang Ferula wild plant resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the effect of adding different concentrations of ABA to the suspension culture medium on the fresh weight and dry weight of Xinjiang Ferula suspension cells;

[0022] Figure 2 This is a graph showing the effect of adding different concentrations of MeJA to the suspension culture medium on the fresh weight and dry weight of Xinjiang Ferula suspension cells;

[0023] Figure 3 This is a graph showing the effect of adding different concentrations of SA to the suspension culture medium on the fresh weight and dry weight of Xinjiang Ferula suspension cells;

[0024] Figure 4Effect diagram of adding different concentrations of Spm to the suspension medium on the fresh weight and dry weight of Ferula sinkiangensis suspension cells;

[0025] Figure 5 Effect diagram of adding different concentrations of ABA to the suspension medium on the ferulic acid content of Ferula sinkiangensis cells;

[0026] Figure 6 Effect diagram of adding different concentrations of MeJA to the suspension medium on the ferulic acid content of Ferula sinkiangensis cells;

[0027] Figure 7 Effect diagram of adding different concentrations of SA to the suspension medium on the ferulic acid content of Ferula sinkiangensis suspension cells;

[0028] Figure 8 Effect diagram of adding different concentrations of Spm to the suspension medium on the ferulic acid content of Ferula sinkiangensis suspension cells;

[0029] Figure 9 Effect diagram of adding different concentrations of ABA to the suspension medium on the total flavonoid content of Ferula sinkiangensis cells;

[0030] Figure 10 Effect diagram of adding different concentrations of MeJA to the suspension medium on the total flavonoid content of Ferula sinkiangensis cells;

[0031] Figure 11 Effect diagram of adding different concentrations of SA to the suspension medium on the total flavonoid content of Ferula sinkiangensis suspension cells;

[0032] Figure 12 Effect diagram of adding different concentrations of Spm to the suspension medium on the total flavonoid content of Ferula sinkiangensis suspension cells. Detailed implementation method

[0033] The present invention provides a suspension medium for culturing Ferula sinkiangensis cells, which is based on MS medium and further includes 0.1-23 mg / L elicitor, 0.5-1 mg / L 2,4-D, 0.5-1 mg / L 6-BA and 30 g / L sucrose; the elicitor includes one or more of methyl jasmonate, spermine and abscisic acid.

[0034] In the suspension medium provided by the present invention, the concentration of 2,4-D is 0.5-1 mg / L, more preferably 1 mg / L. In the specific embodiments of the present invention, the concentration of 2,4-D in the suspension medium can be 0.5 or 1 mg / L. 2,4-D in the present invention is an auxin, which is used to induce cell division and differentiation and is widely used for callus induction.

[0035] In the suspension medium provided by the present invention, the concentration of 6-BA is 0.5-1 mg / L, more preferably 1 mg / L. In the specific embodiments of the present invention, the concentration of 6-BA in the suspension medium can be 0.5 or 1 mg / L. The 6-BA of the present invention is a cytokinin, which can promote cell division and the differentiation of callus into adventitious buds.

[0036] The suspension medium provided by the present invention includes elicitors, and the elicitors include one or more of methyl jasmonate, spermine, and abscisic acid. Elicitors are a class of special trigger factors that can cause a series of defense responses in plants, trigger the defense responses of cultured plant cells, tissues or organs, and can regulate the activity of enzymes in metabolic processes to promote the accumulation of secondary metabolites, so as to increase the yield of bioactive compounds in in vitro culture. In plant cell suspension culture, cell biomass and secondary metabolite content are two important factors that need to be considered comprehensively. As one of the key factors for establishing a plant suspension culture cell system, the type, content and ratio of hormones have crucial effects on the growth and secondary metabolism regulation of suspension culture cells. The present invention creatively discovers that adding methyl jasmonate (MeJA), abscisic acid (ABA) and spermine (Spm) to the suspension medium is beneficial to the growth of Ferula sinkiangensis cells and the accumulation of secondary metabolites. Among them, methyl jasmonate (MeJA) is an important plant signal molecule that can activate the defense responses of plants, especially against pathogen and insect attacks. In plant tissue culture, MeJA promotes the synthesis of secondary metabolites by inducing the expression of key enzyme genes in secondary metabolic pathways. For example, MeJA can activate the phenylpropanoid metabolic pathway and increase the accumulation of flavonoids, phenols and terpenoids. Salicylic acid (SA) is a key signal molecule for systemic acquired resistance (SAR) in plants and is mainly involved in the defense responses of plants against pathogens. In tissue culture, SA promotes the synthesis of secondary metabolites by activating the expression of defense-related genes. SA can also affect the secondary metabolic pathway by regulating the level of reactive oxygen species (ROS) and the activity of antioxidant enzymes. Abscisic acid (ABA) is a plant hormone that is mainly involved in plant stress responses such as drought and salt stress. In tissue culture, ABA affects the synthesis of secondary metabolites by regulating stomatal closure and gene expression. ABA can activate genes related to secondary metabolism and promote the accumulation of terpenoids, alkaloids and polyphenols. Spermine (Spm) is a polyamine compound that participates in the growth and development processes of plants. In tissue culture, Spm affects the synthesis of secondary metabolites by regulating cell division and differentiation; Spm can also promote the accumulation of secondary metabolites by stabilizing the cell membrane structure and regulating the activity of antioxidant enzymes.

[0037] As an alternative embodiment, when the suspension medium of the present invention is used to increase the biomass of Ferula sinkiangensis cells, the elicitor includes abscisic acid or spermine. The concentration of abscisic acid in the suspension medium is 1.2 - 1.5 mg / L, more preferably 1.5 mg / L. The concentration of spermine in the suspension medium is 0.3 - 1.5 mg / L, or may be 0.3 - 1.0 mg / L, more preferably 0.3 mg / L.

[0038] As an alternative embodiment, when the medium of the present invention is used to increase the content of secondary metabolites of Ferula sinkiangensis cells, the elicitor includes methyl jasmonate or spermine. The molar concentration of methyl jasmonate in the suspension medium is 50 - 100 μmol / L (i.e., 11.21 - 22.43 mg / L), and the concentration of spermine in the suspension medium is 0.5 - 1.5 mg / L. As an alternative embodiment, the secondary metabolites of the present invention include ferulic acid and / or total flavonoids; when the medium of the present invention is used to increase the content of ferulic acid in Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is preferably 100 μmol / L, and the concentration of spermine in the suspension medium is preferably 1.5 mg / L.

[0039] As an alternative embodiment, when the suspension medium of the present invention is used to increase the content of total flavonoids in Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is 50 - 100 μmol / L (i.e., 11.21 - 22.43 mg / L), or may be 50 - 70 μmol / L (i.e., 11.21 - 15.70 mg / L), more preferably 50 μmol / L ((i.e., 11.21 mg / L)); as an alternative embodiment, when the concentration of spermine in the suspension medium of the present invention is 0.5 - 1.0 mg / L, or may be 0.5 - 0.7 mg / L, more preferably 0.5 mg / L.

[0040] As an alternative embodiment, the initial pH value of the suspension medium of the present invention is 5.8 - 6.0, more preferably 5.8.

[0041] The suspension medium provided by the present invention is conducive to the growth of Ferula sinkiangensis cells and the secretion of secondary metabolites ferulic acid and total flavonoids.

[0042] The suspension medium of the present invention is applied after sterilization. The present invention has no special limitation on the sterilization method, and a conventional method can be used. The present invention has no special limitation on the source of the components in the medium combination, and conventional commercially available products can be used.

[0043] The present invention also provides a suspension culture method for Ferula sinkiangensis cells, including: inoculating Ferula sinkiangensis callus in the suspension medium described in the above technical solution for suspension culture.

[0044] As an alternative embodiment, the present invention does not particularly limit the method for culturing the callus, and conventional methods can be used. In a specific embodiment of the present invention, the explant for culturing the callus of Ferula sinkiangensis includes the hypocotyl of the Ferula sinkiangensis seedling. The method for preparing the hypocotyl of the Ferula sinkiangensis seedling in the present invention includes: disinfecting and stratifying the Ferula sinkiangensis seeds and then inoculating them into an MS medium for culturing to obtain the hypocotyl of the seedling; the composition of the MS medium includes: using MS as the basal medium, and further adding 30 g / L of sucrose and 6.5 g / L of agar.

[0045] Since the wild population of Ferula sinkiangensis is scarce and the seed maturity period is short (25 - 30 days), the collection work is extremely difficult. The applicant cooperated with the local protected area management department and adopted methods such as fixed-point monitoring and timely harvesting to collect a sufficient amount of experimental seeds, laying an important foundation for subsequent research. The method for preparing the callus in the present invention includes: inoculating the hypocotyl of the Ferula sinkiangensis into an induction medium for induction culture to obtain callus. The composition of the induction medium includes: using MS as the basal medium, and further including 2.5 mg / L of 2,4-D, 30 g / L of sucrose, and 6.5 g / L of agar. The induction culture time in the present invention is 15 - 30 d. The reason for choosing 30 d at the longest is that the callus will turn brown and the cells will die due to the long culture time. After obtaining the callus, the present invention inoculates the callus into a subculture proliferation medium for subculture to obtain loose callus. As an alternative embodiment, when subculturing in the present invention, the callus is cut into small pieces and then inoculated into a new subculture proliferation medium for subculture. The subculture proliferation medium for the callus of the present invention is based on the MS medium and further includes 1.0 mg / L of 2,4-D, 1.0 mg / L of 6-BA, 30 g / L of sucrose, and 6.5 g / L of agar, and the pH value is 5.8 - 6.0. As an alternative embodiment, the present invention does not particularly limit the small pieces of the callus. In a specific embodiment of the present invention, the size of the small pieces of the callus is 1 * 1 cm. The present invention also does not particularly limit the inoculation quantity and method, and conventional methods can be used. In a specific embodiment of the present invention, 10 small pieces of callus are inoculated into each culture dish with a diameter of 90 mm.

[0046] The subculture time in the present invention is 30 d, and the number of subculture times is 5 - 6 times, more preferably 6 times. After each subculture in the present invention, the subculture proliferation medium is replaced. The callus obtained from the previous subculture is cut into small pieces and then transferred to a new subculture proliferation medium, and the composition of the subculture proliferation medium remains unchanged. The subculture in the present invention can provide more sufficient nutrients for the callus of Ferula sinkiangensis.

[0047] After the subculture is completed, the present invention gently crushes the obtained loose callus with tweezers and inoculates it into the suspension culture medium for suspension culture. As an optional embodiment, the mass volume ratio of Xinjiang Ferula callus and suspension culture medium during inoculation is 1g: (25-30) mL, more preferably 1g: 30 mL. As an optional embodiment, the rotation speed of the suspension culture of the present invention is 100-120rpm, more preferably 120rpm. As an optional embodiment, the temperature of the suspension culture of the present invention is 21-23°C, more preferably 23°C. The setting of the inoculation amount and the volume ratio of the culture medium, the rotation speed and the temperature of the present invention is conducive to the proliferation of Xinjiang Ferula cells, and improves the yield and the accumulation of secondary metabolites. As an optional embodiment, the suspension culture of the present invention includes dark culture. As an optional embodiment, the suspension culture time of the present invention is 15d. The dark culture of the present invention can avoid the decomposition of light-sensitive substances and promote the accumulation of metabolites. The limitation of the culture time can reduce the risks of nutrient exhaustion, accumulation of metabolic waste, pH changes, insufficient oxygen, excessive cell density and contamination.

[0048] The present invention provides a method for extracting secondary metabolites from Xinjiang Ferula cells, wherein the Xinjiang Ferula cells obtained by the suspension culture method described in the above technical solution are subjected to secondary metabolites extraction. The present invention has no special limitation on the extraction method, and conventional methods can be used.

[0049] In the research of Xinjiang Ferula tissue culture technology, traditional tissue culture rapid propagation technology faces many bottlenecks: it is difficult to establish a sterile seedling regeneration system, the culture cycle is long, and no breakthrough has been achieved so far. In response to this technical problem, the present invention innovatively uses successfully induced callus tissue as material, and through the regulation of culture conditions, such as inoculation amount, culture medium volume, shaker speed and temperature, etc., the conditions suitable for Xinjiang Ferula cell growth are found, and exogenous signal substances are added to stimulate plants and affect the synthesis and accumulation of secondary metabolites in plants. Through the suspension culture technology system of the present invention, efficient acquisition of medicinal active ingredients is achieved. This technological breakthrough has significant advantages: first, a large number of suspension cells can be obtained in a relatively short period of time (2 to 3 months), greatly improving production efficiency; second, by optimizing culture conditions, the content of target medicinal ingredients can be directed to increase; third, the production cost is greatly reduced compared with traditional extraction methods, with good economic benefits; most importantly, this technology can quickly culture and obtain a large number of Xinjiang Ferula cells, which provides a new way for the sustainable utilization of Xinjiang Ferula medicinal resources, effectively reduces dependence on wild resources, and has important practical significance for the protection of rare plants.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] 1. Test materials

[0052] Plant materials: Callus induced from hypocotyls of seedlings after germination of Ferula sinkiangensis seeds was selected.

[0053] The composition of the induction medium was: Based on MS medium, 2.5 mg / L 2,4-D, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8 - 6.0.

[0054] The subculture and proliferation medium for callus was based on MS medium, and 1.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8 - 6.0.

[0055] Main drugs and reagents: MS medium of Coolabr brand, MS powder containing 3% sucrose by mass concentration, 2,4-D, 6-BA, sodium hydroxide (NaOH), hydrochloric acid (HCl), alcohol, distilled water, salicylic acid (SA), polyethylene glycol (PEG 6000), abscisic acid (ABA), spermine (Spm), methyl jasmonate (MeJA), 60% methanol, 60% ethanol, ferulic acid standard of SIGMA brand for chromatography.

[0056] Main instruments: Sujing Antai clean bench, YD-ZD-Z-5 type distilled water boiler produced by Shanghai Lichen Bangxi, constant temperature shaker, electrothermal blast drying oven, light incubator, electronic balance, HT165R type high-speed refrigerated centrifuge of Xiangyi brand, chromatographic grade organic mobile phase of THEMO brand, M2 type multifunctional microplate reader of American MD brand, DH200 type dry thermostat of Ruicheng brand, pH meter, alcohol lamp, forceps, beaker, petri dish, conical flask (100 mL), stainless steel cell sieve.

[0057] 2. Index determination methods

[0058] (1) Fresh weight: The suspension cells of Ferula sinkiangensis under different treatments were filtered through a 100 μm pore size sieve, the water was blotted with filter paper, and then weighed to obtain the fresh weight.

[0059] (2) Dry weight: The suspension cells of Ferula sinkiangensis obtained after filtration through a 100 μm pore size sieve were dried in a 60 °C drying oven for 12 h until constant weight, which was the dry weight.

[0060] (3) Determination of ferulic acid content: Grind the dried callus of Ferula sinkiangensis into powder, weigh 0.20 g of the sample, add 1.00 mL of methanol-water with a volume concentration of 60%, ultrasonically extract at 60 °C for 30 min, heat at 80 °C for 1.5 h, then make up the volume to 1 mL with methanol-water with a volume concentration of 60%, finally centrifuge at 4 °C and 12,000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm filter membrane and then determine. Chromatographic conditions: Shimadzu LC-20A, chromatographic column: C18 column (250 mm * 4.6 mm, 5 μm); detector: PDA diode array detector; detection wavelength: 320 nm, flow rate: 1.0 mL / min; column temperature: 25 °C; automatic injection, injection volume: 10 μL; mobile phase: methanol (A), 0.1% phosphoric acid aqueous solution (B), isocratic: A:B = 20:80.

[0061] (4) Determination of total flavonoid content: It is carried out using a kit. The kit uses the NaNO2-Al(NO3)3-NaOH colorimetric method to determine the total flavonoid content. That is, in an alkaline nitrite solution, flavonoid compounds form a red complex with aluminum ions that has a characteristic absorption peak at 510 nm. Measure the absorbance value of the reaction product at 510 nm, and then the total flavonoid content in the sample can be calculated. The specific operation steps are carried out according to the kit instructions.

[0062] 3. Data processing

[0063] Use Microsoft Office Excel 2019 for data sorting, use SPSS 25.0 for data variance analysis, and use Origin 2022 for graph drawing.

[0064] Example 1

[0065] 1. Preparation of callus: Disinfect the seeds of Ferula sinkiangensis with a 0.1% mercuric chloride solution by mass concentration. After stratification for 90 days, inoculate the seeds of Ferula sinkiangensis or the germinated sterile seedlings on the MS medium in a sterile environment (the composition of the MS medium is: MS + 30 g / L sucrose + 6.5 g / L agar, pH value is 5.8 - 6.0). After the seeds germinate, select the hypocotyls of the seedlings and inoculate them into the induction medium to induce the formation of callus. The composition of the induction medium is: based on the MS medium, it also adds 2.5 mg / L 2,4-D, 30 g / L sucrose and 6.5 g / L agar, pH value is 5.8 - 6.0.

[0066] If the callus aggregates too densely, browning will occur and the cells will die. Therefore, the induction culture time is 30 days. After cutting the callus obtained from the induction culture, small pieces of callus are obtained, and the area of each piece of callus is maintained at about 1 cm × 1 cm. The subculture proliferation medium of the callus is poured into a petri dish to prepare a culture medium plate. The diameter of the petri dish is 90 mm. The obtained small pieces are inoculated on the culture medium plate for subculture, and the culture medium plate is placed in an incubator. About 10 small pieces of callus are inoculated in each culture medium plate, and the subculture time for each time is 30 days. Loose callus is obtained after 6 subcultures.

[0067] The subculture proliferation medium of the callus is based on MS medium and also contains 1.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 30 g / L sucrose and 6.5 g / L agar, and the pH value is 5.8 - 6.0.

[0068] 2. Select the loose callus obtained after 6 subcultures in step 1. Use forceps to gently crush the callus cells and inoculate them into a 100 mL Erlenmeyer flask. The composition of the suspension medium is: based on MS medium and also contains 1 mg / L 2,4-D, 1 mg / L 6-BA and 30 g / L sucrose. A 5-factor 5-level L 25 (5 5 ) orthogonal experimental design is used to study the effects of 5 factors, namely inoculation amount, medium volume, shaker speed, culture temperature, and initial pH value of the medium, on the fresh weight and dry weight of Ferula sinkiangensis suspension cells, without considering the interaction between factors. Each treatment has 5 replicate experiments and is placed in a constant temperature shaker for dark culture. The cell biomass is measured on the 15th day. The experimental factors and level gradients are shown in Table 1, and the specific treatments and biomass measurement results of the orthogonal experiment are shown in Table 2.

[0069] Table 1 Factors and levels of the orthogonal experimental design

[0070]

[0071] Table 2 Comparison of the biomass of Ferula sinkiangensis suspension cells under different treatments

[0072]

[0073]

[0074] Note: Different lowercase English letters after the data indicate significant differences (P < 0.05). The same applies hereinafter.

[0075] As can be seen from Table 2, the fresh weight of Ferula sinkiangensis K. M. Shen was the highest in Treatment 17 (inoculum amount 2.00 g + culture medium volume 60.00 mL + shaker speed 120.00 rpm + temperature 23.00 °C + initial pH value 5.80), which was 6.94 g, significantly higher than that of the other treatments. The dry weight was the highest in Treatment 17, which was 0.32 g, significantly higher than that of the other treatments.

[0076] The range analysis of the biomass of Ferula sinkiangensis K. M. Shen suspension cells under different treatments is shown in Table 3. From the range value R in Table 3, it can be seen that the influence degree of the five culture factors on the biomass of Ferula sinkiangensis K. M. Shen suspension cells is inoculum amount > shaker speed > temperature > culture medium volume > pH value. The best combination to promote cell biomass is A4B5C4D2E4, that is, inoculum amount 2.00 g + culture medium volume 60.00 mL + shaker speed 120.00 rpm + temperature 23.00 °C + pH value 6.2. However, the pH value of the culture medium is generally more suitable for the growth of Ferula sinkiangensis K. M. Shen cells at 5.8 - 6.0. Therefore, the culture conditions for subsequent treatments were set as callus inoculum amount 2.00 g, culture medium volume 60.00 mL, shaker speed 120.00 rpm, temperature 23.00 °C, and initial pH value of the culture medium 5.8.

[0077] Table 3 Range analysis of the biomass of Ferula sinkiangensis K. M. Shen suspension cells under different treatments

[0078]

[0079] Note: K1 - K5 are the total biomass of suspension cells of the five factors at the same level respectively, k1 - k5 are the average biomass of suspension cells of the five factors at the same level respectively, and R is the range of biomass at different levels of the five factors.

[0080] Example 2 Four elicitor addition tests

[0081] The culture medium composition was: based on the MS culture medium, 1 mg / L 2,4 - D, 1 mg / L 6 - BA, and 30 g / L sucrose were also added, and the pH value was 5.8 - 6.0, denoted as Culture Medium 1.

[0082] Treatment 1: Methyl Jasmonate (MeJA) was added to Culture Medium 1. The molar concentrations of MeJA in Culture Medium 1 were 0 (CK group), 50 μmol / L (i.e., 11.21 mg / L), 100 μmol / L (i.e., 22.43 mg / L), 150 μmol / L, and 200 μmol / L respectively; three replicate experiments were set for each concentration gradient;

[0083] Treatment 2: Add the inducer salicylic acid (SA) to Medium 1. The SA molar concentrations in Medium 1 are 0 (CK group), 25, 50, 75, and 100 μmol / L respectively; three replicate experiments are set for each concentration gradient.

[0084] Treatment 3: Add the inducer abscisic acid (ABA) to Medium 1. The ABA mass concentrations in Medium 1 are 0 (CK group), 0.3, 0.5, 1.0, and 1.5 mg / L respectively; three replicate experiments are set for each concentration gradient.

[0085] Treatment 4: Add the inducer spermine (Spm) to Medium 1. The Spm mass concentrations in Medium 1 are 0 (CK group), 0.3, 0.5, 1.0, and 1.5 mg / L respectively; three replicate experiments are set for each concentration gradient.

[0086] The callus is the loose callus obtained after the subculture of Example 1 for 6 times. The callus is inoculated into each of the media of Treatments 1 - 4 and placed in a constant temperature shaker for dark culture. When culturing, the inoculation amount of the callus is 2.00 g, the volume of the medium is 60.00 mL, the shaker speed is 120.00 rpm, the culture temperature is 23.00 °C, and the initial pH value of the medium is 5.80. To study the effects of the four inducers on the biomass, ferulic acid content, and total flavonoid content of Ferula sinkiangensis suspension-cultured cells, the inducers are added on the 0th day of culture for all treatments, and the cell biomass and metabolite content are measured on the 15th day of dark culture.

[0087] 1. Among the treatments with different concentrations of ABA, as the ABA concentration increases, the cell biomass shows a trend of first decreasing and then increasing. The fresh weight and dry weight of Ferula sinkiangensis cells under the treatment of 1.50 mg / L are the highest, reaching 5.78 g and 0.25 g respectively, as Figure 1 shown in Table 4.

[0088] Among the treatments with different concentrations of MeJA, as the MeJA concentration increases, the cell biomass of Ferula sinkiangensis gradually decreases. The fresh weight and dry weight of Ferula sinkiangensis cells in the CK group are the highest, reaching 5.26 g and 0.26 g respectively, which are significantly higher than those of the other treatments. The treatment of 50.00 μmol / L ranks second. The fresh weight of Ferula sinkiangensis cells under the treatment of 100.00 μmol / L is the lowest, being 2.00 g, and the dry weight of Ferula sinkiangensis cells under the treatment of 200.00 μmol / L is the lowest, being 0.06 g, as Figure 2 shown in Table 5.

[0089] Among the treatments with different concentrations of SA, as the SA concentration increased, the cell biomass of Ferula sinkiangensis gradually decreased. The fresh weight and dry weight of the Ferula sinkiangensis cells in the CK group were the highest, reaching 5.70 g and 0.22 g respectively, which were significantly higher than those of the other treatments. The treatment with 25.00 μmol / L was the second, and the fresh weight and dry weight of the Ferula sinkiangensis cells under the 100.00 μmol / L treatment were the lowest, being 1.40 g and 0.03 g respectively, lower than those of the other treatments, as Figure 3 shown in Table 6

[0090] Among the treatments with different concentrations of Spm, as the Spm concentration increased, the cell biomass of Ferula sinkiangensis showed an upward trend. The fresh weight of the Ferula sinkiangensis cells under the 0.30 mg / L treatment was the highest, reaching 4.29 g, higher than those of the other treatments. The dry weight of the Ferula sinkiangensis cells under the 1.50 mg / L treatment was the highest, reaching 0.17 g, higher than those of the other treatments. The fresh weight and dry weight of the Ferula sinkiangensis cells in the CK group were the lowest, being 2.54 g and 0.08 g respectively, lower than those of the other treatments, as Figure 4 shown in Table 7 Figures 1 - 4 Different lowercase English letters indicate significant differences (P < 0.05), the same below

[0091] 2. The results of the content of ferulic acid, a cell metabolite, are shown in Figures 5 - 8 Table 4

[0092] Among the treatments with different concentrations of ABA, the content of ferulic acid in the Ferula sinkiangensis cells in the CK group was the highest, reaching 95.44 μg / g, significantly higher than those of the other treatments. The treatment with 1.50 mg / L was the second, and the content of ferulic acid in the Ferula sinkiangensis cells under the 0.30 mg / L treatment was the lowest, being 59.16 μg / g, significantly lower than those of the other treatments, see Figure 5 Table 4

[0093] Among the treatments with different concentrations of MeJA, the content of ferulic acid in the Ferula sinkiangensis cells under the 100.00 μmol / L treatment was the highest, reaching 79.32 μg / g, significantly higher than those of the other groups except the CK group. The content of ferulic acid in the Ferula sinkiangensis cells under the 200.00 μmol / L treatment was the lowest, being 4.65 μg / g, significantly lower than those of the other treatments, see Figure 6 Table 5

[0094] Among the treatments with different concentrations of SA, as the SA concentration increased, the content of ferulic acid gradually decreased. The content of ferulic acid in the Ferula sinkiangensis cells in the CK group was the highest, reaching 92.87 μg / g, significantly higher than those of the other treatments. The content of ferulic acid in the Ferula sinkiangensis cells under the 100 μmol / L treatment was the lowest, being 3.58 μg / g, significantly lower than those of the other treatments, see Figure 7 Table 6

[0095] Among the treatments with different concentrations of Spm, the content of ferulic acid in Ferula sinkiangensis cells was the highest under the treatment of 1.50 mg / L, reaching 115.66 μg / g, which was higher than that of the other treatments. The content of ferulic acid in Ferula sinkiangensis cells was the lowest under the treatment of 0.50 mg / L, being 78.46 μg / g, which was significantly lower than that of the other treatments. See Figure 8 and Table 7.

[0096] 3. The results of the total flavonoid content of cell metabolites are as Figures 9 - 12 shown.

[0097] Among the treatments with different concentrations of ABA, with the increase of ABA concentration, the total flavonoid content first increased and then decreased. The total flavonoid content in Ferula sinkiangensis cells was the highest under the treatment of 0.30 mg / L, reaching 43.33 mg / g, which was significantly higher than that of the other treatments. The treatment of 1.00 mg / L ranked second. The total flavonoid content in Ferula sinkiangensis cells was the lowest under the treatment of 1.50 mg / L, being 24.58 mg / g, which was significantly lower than that of the other treatments. See Figure 9 and Table 4.

[0098] Among the treatments with different concentrations of MeJA, with the increase of MeJA concentration, the total flavonoid content first rose and then decreased. The total flavonoid content in Ferula sinkiangensis cells was the highest under the treatment of 50.00 μmol / L, reaching 81.70 mg / g, which was significantly higher than that of the other treatments. See Figure 10 and Table 5.

[0099] Among the treatments with different concentrations of SA, with the increase of SA concentration, the total flavonoid content gradually decreased. The total flavonoid content in Ferula sinkiangensis cells in the CK group was the highest, reaching 35.67 mg / g, which was significantly higher than that of the other treatments. See Figure 11 and Table 6.

[0100] Among the treatments with different concentrations of Spm, with the increase of Spm concentration, the total flavonoid content first rose and then decreased. The total flavonoid content in Ferula sinkiangensis cells was the highest under the treatment of 0.50 mg / L, reaching 39.40 mg / g. See Figure 12 and Table 7.

[0101] Table 4 Determination results of fresh weight, dry weight, ferulic acid content and total flavonoid content of Ferula sinkiangensis suspension cells under different concentrations of ABA

[0102]

[0103]

[0104] Note: Different lowercase English letters indicate significant differences (P < 0.05), the same below.

[0105] Table 5 Determination results of fresh weight, dry weight, ferulic acid content and total flavonoid content of Ferula sinkiangensis suspension cells under different concentrations of MeJA

[0106]

[0107] Table 6 Determination results of fresh weight, dry weight, ferulic acid content and total flavonoid content of Ferula sinkiangensis suspension cells under different concentrations of SA

[0108]

[0109] Table 7 Determination results of fresh weight, dry weight, ferulic acid content and total flavonoid content of Ferula sinkiangensis suspension cells under different concentrations of Spm

[0110]

[0111] Comparative Example 1

[0112] The culture medium composition is as follows: based on MS medium, 1 mg / L of 2,4-D, 1 mg / L of 6-BA, 30 g / L of sucrose and 6.5 g / L of agar are also added; it is denoted as Medium 2.

[0113] SA was added to Medium 2 to make the final molar concentrations of SA be 0, 5, 10, 20, 50, 100, 200 μmol / L respectively; 3 replicate experiments were set for each concentration gradient;

[0114] The callus was the loose callus obtained after subculture of Example 1 for 6 times. The callus was inoculated into Medium 2 with different molar concentrations of SA. 3 bottles were inoculated for each treatment, 3 replicates were set, 2.0 g of callus was inoculated into each bottle, and the initial pH value of the medium was 5.8. It was placed in an incubator for culture, the culture temperature was 25°C during the day and 15°C at night; the photoperiod was 12 h / d for light and 12 h / d for darkness. The effects of SA on the biomass, ferulic acid content and total flavonoid content of Ferula sinkiangensis callus were studied. An elicitor was added to all treatments on the 0th day of culture, and the callus was taken out of the culture flask on the 14th day, 28th day and 42nd day of culture respectively, and the cell biomass and metabolite content were measured. See Table 8 and Table 9.

[0115] Table 8 Determination results of fresh weight and dry weight of cells obtained by solid culture of Ferula sinkiangensis under different concentrations of SA

[0116]

[0117] Table 9 Determination results of ferulic acid content and total flavonoid content of cells obtained by solid culture of Ferula sinkiangensis under different concentrations of SA

[0118]

[0119] Comparative Example 2

[0120] Same as Comparative Example 1, the only difference is that polyethylene glycol (PEG 6000, abbreviated as PEG) is added to Medium 2 so that the final mass concentration of PEG is 0, 5%, 10%, 15%, 20%, and 30% respectively; three replicate experiments are set for each concentration gradient. After the solid medium (i.e., Medium 2) solidifies, equal volumes of PEG solutions with different concentrations are poured into tissue culture flasks (50 mL per flask) for soaking, allowing the water potential of the medium and the solution to fully equilibrate. After soaking for 12 h, the solution in the tissue culture flask is poured out, and 2.0 g of callus is inoculated into each flask. The initial pH value of the medium is 5.8. The medium is placed in an incubator for cultivation, with the cultivation temperature being 25 °C during the day and 15 °C at night; the photoperiod is 12 h of light / d and 12 h of darkness / d.

[0121] Inducers are added on the 0th day of cultivation for all treatments. On the 30th day of cultivation, the callus is taken out from the tissue culture flask, and the cell biomass and metabolite content are measured. The results are shown in Table 10.

[0122] Table 10 Determination results of fresh weight, dry weight, ferulic acid content, and total flavonoid content of cells obtained from solid culture of Ferula sinkiangensis under different concentrations of SA

[0123]

[0124] In summary, in the examples of the present invention, the ferulic acid and total flavonoid contents of Ferula sinkiangensis cells obtained under the conditions of suspension culture are significantly higher than those in the solid medium. Under the suspension culture conditions, different combinations of inoculation amount, medium volume, shaker speed, temperature, and pH value can affect the cell biomass of Ferula sinkiangensis to varying degrees. Among them, the inoculation amount is the dominant factor, the influence of the shaker speed is the second, and the influence of the temperature ranks third. The optimal combination of culture conditions suitable for suspension culture of Ferula sinkiangensis is A4B5C4D2E4, combined with the optimal pH of 5.8 - 6.0, that is, the mass-to-volume ratio of callus to medium is 1 g:30 mL, the shaker speed is 120.00 r / min, the temperature is 23.00 °C, and the pH value of the suspension medium is 5.8 - 6.0. It is recommended to promote and apply it in practical production.

[0125] The cell biomass and secondary metabolite content vary with the addition of different concentrations of inducers. Adding 1.50 mg / L ABA to the suspension medium results in the highest fresh weight and dry weight of Ferula sinkiangensis cells under this treatment. Adding 1.50 mg / L Spm to the suspension medium results in the highest ferulic acid content of Ferula sinkiangensis cells under this treatment. Adding 50.00 μmol / L MeJA to the suspension medium results in the highest total flavonoid content of Ferula sinkiangensis cells under this treatment, which is significantly higher than the other treatments. The use of inducers can also be optimized to extract target products.

[0126] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A suspension medium for culturing Ferula sinkiangensis cells, characterized in that, Based on MS medium, it also includes 0.1 - 23 mg / L elicitor, 0.5 - 1 mg / L 2,4-D, 0.5 - 1 mg / L 6-BA and 30 g / L sucrose; the elicitor includes one or more of methyl jasmonate, spermine and abscisic acid.

2. The suspension medium according to claim 1, wherein When the suspension medium is used to increase the biomass of Ferula sinkiangensis cells, the elicitor includes abscisic acid or spermine, the concentration of abscisic acid in the suspension medium is 1.2 - 1.5 mg / L, and the concentration of spermine in the suspension medium is 0.3 - 1.5 mg / L; When the medium is used to increase the content of secondary metabolites in Ferula sinkiangensis cells, the elicitor includes methyl jasmonate or spermine, the molar concentration of methyl jasmonate in the suspension medium is 11.21 - 22.43 mg / L, and the concentration of spermine in the suspension medium is 0.5 - 1.5 mg / L.

3. The suspension medium according to claim 2, characterized in that, The secondary metabolites include ferulic acid and / or total flavonoids; When the medium is used to increase the ferulic acid content of Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is 22.43 mg / L, and the concentration of spermine in the suspension medium is 1.5 mg / L; When the suspension medium is used to increase the total flavonoid content of Ferula sinkiangensis cells, the molar concentration of methyl jasmonate in the suspension medium is 11.21 - 22.43 mg / L, and the concentration of spermine in the suspension medium is 0.5 - 1.0 mg / L.

4. The suspension medium according to claim 1, wherein The initial pH value of the suspension medium is 5.8 - 6.

0.

5. A method for suspension culture of Ferula sinkiangensis cells, characterized in that, It includes: Inoculate the callus of Ferula sinkiangensis in the suspension medium according to any one of claims 1 - 4 for suspension culture.

6. The suspension culture method according to claim 5, wherein, When inoculating, the mass - volume ratio of the callus of Ferula sinkiangensis to the suspension medium is 1 g:(25 - 30) mL; the rotation speed of the suspension culture is 100 - 120 rpm.

7. The suspension culture method according to claim 5, wherein The temperature of the suspension culture is 21 - 23 °C.

8. The suspension culture method according to claim 5, wherein The explant for culturing the callus of Ferula sinkiangensis includes the hypocotyl of Ferula sinkiangensis seedlings.

9. The suspension culture method according to claim 5, characterized in that, The suspension culture includes dark culture; the time of the suspension culture is 15 d.

10. A method for extracting secondary metabolites of Ferula sinkiangensis cells, characterized in that, Extract the secondary metabolites from the Ferula sinkiangensis cells obtained by the suspension culture method according to any one of claims 5 - 9.

Images