Culture medium for promoting germination of saussurea involucrata seeds and increasing flavone content in cell culture and application of culture medium

Abstract

The invention discloses a culture medium for promoting germination of saussurea involucrata seeds and increasing the flavone content in a cell culture and application of the culture medium. Multivitamins for promoting germination of saussurea involucrata seeds and increasing the flavone content in the cell culture comprise thiamine hydrochloride VB1, nicotinic acid and pyridoxine hydrochloride VB6; wherein the concentration of thiamine hydrochloride VB1 is 1000 mg / L, the concentration of nicotinic acid is 500 mg / L, and the concentration of pyridoxine hydrochloride VB6 is 500 mg / L. A mixed mother solution (V3 vitamin for short) of three vitamins is prepared for filtration, sterilization and addition, the concentrations of the three vitamins are thiamine hydrochloride VB11000 mg / L, nicotinic acid 500 mg / L and pyridoxine hydrochloride VB6500 mg / L respectively, the overall addition amount of V3 is 1 mL / L, the germination rate of saussurea involucrata seeds can be effectively improved, and the germination rate of the seeds is 40.58%.

Description

Technical Field

[0001] This invention belongs to the field of bioapplication technology, specifically relating to a culture medium that promotes the germination of snow lotus seeds and increases the flavonoid content in cell cultures, and its application. Background Technology

[0002] Saussurea species are rare medicinal plants whose flavonoids possess antioxidant, anti-inflammatory, and immunomodulatory activities, making them valuable in the pharmaceutical, health, and cosmetic fields. However, due to their extreme growing environments and scarcity of wild resources, traditional harvesting methods cannot meet market demand, necessitating large-scale production through bioengineering technology.

[0003] Although plant cell culture technology is a feasible approach, existing technologies suffer from bottlenecks such as low production efficiency and unstable products. This is particularly evident in the long seed germination cycle and slow seedling development, leading to low proliferation efficiency. For example, Ding Dailing et al. optimized the germination conditions of Tian Shan snow lotus seeds, finding that seed germination took 5 days, the germination rate was 20%, and the survival rate after 30 days was 57.89%. Wu Yunfang, through screening seed germination media, found that snow lotus seeds inoculated on seed germination media began to germinate after 4 days of culture, and statistical analysis of germination results after 30 days showed that the germination rate in MS+1% sucrose medium was 27.9%. Furthermore, Jia Jingming et al. found that in the aseptic seedling cultivation stage, i.e., after seed germination, it took 45 days for seedlings with leaves to emerge. These three studies all demonstrate the problems of long germination cycles and low germination rates in snow lotus seeds.

[0004] Meanwhile, flavonoid synthesis is affected by factors such as culture medium ratio and metabolic regulation, resulting in problems such as low content of target products. For example, Liu Yonggang studied the kinetics of flavonoid synthesis in snow lotus cells using both solid and liquid culture methods, finding that the flavonoid content reached its maximum at 24 days in solid culture (approximately 3%), while in liquid culture it reached its maximum at 18 days (3.63%). Liu Ying found that the total flavonoid content in the dried product of bright yellow-white, loose callus tissue was the highest in the optimal subculture medium (2.51%). Jia Jingming, through exploring the effect of callus polymorphism on total flavonoid production, found that the total flavonoid content in the dried product of callus tissue was the highest (5.95%). Ding Dailing et al. found that snow lotus callus tissue, under MS + 2.0 mg / L NAA + 1.0 mg / L After subculturing in 6-BA medium, the total flavonoid content of the dried product was 7.37%. The total flavonoid content of dried snow lotus culture reported in the above literature is between 2% and 8%. However, according to the announcement on approving five items, including Camellia chrysantha and Inula japonica (Xiao Hei Yao), as new resource foods (No. 9 of 2010), the total flavonoid content of dried snow lotus culture is ≥7%. These reports rarely meet the national standard requirements and are still some distance from the standard for new food raw materials. If large-scale production is carried out, the total flavonoid content will decrease further.

[0005] Therefore, it is urgent to find ways to quickly improve seed germination rate, shorten seed germination time, and increase the content of active ingredients in snow lotus culture to meet national standards. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a compound vitamin that promotes the germination of snow lotus seeds and increases the flavonoid content in cell cultures.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a compound vitamin that promotes the germination of snow lotus seeds and increases the flavonoid content in cell cultures, comprising thiamine hydrochloride VB1, niacin, and pyridoxine hydrochloride VB6;

[0010] The concentrations of thiamine hydrochloride (VB1) and nicotinic acid were 1000 mg / L, and the concentrations of pyridoxine hydrochloride (VB6) were 500 mg / L.

[0011] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a culture medium that promotes the germination of snow lotus seeds, comprising,

[0012] Mix MS medium, sucrose, agar and distilled water, bring the volume to 1 L and stir well. Adjust the pH to 5.8-6.0, autoclave at 121℃ for 20 minutes, and cool to obtain the basic culture medium solution.

[0013] The compound vitamins described in claim 1 were added to the basal culture medium solution to prepare the culture medium for promoting the germination of snow lotus seeds.

[0014] In a preferred embodiment of the preparation method described in this invention, the basic culture medium solution contains 4.43 g / L MS, 30 g / L sucrose, and 6 g / L agar.

[0015] In a preferred embodiment of the preparation method described in this invention, the amount of the compound vitamin added relative to the basal culture medium solution is 1 mL / L.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a culture medium in promoting the germination of snow lotus seeds during the cultivation of snow lotus seeds.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a culture medium that increases the flavonoid content in snow lotus callus, comprising,

[0018] Mix MS medium, sucrose, agar and distilled water, bring the volume to 1 L and stir well. Adjust the pH to 5.8-6.0, autoclave at 121℃ for 20 minutes, and cool to obtain the basic culture medium solution.

[0019] Add the aforementioned compound vitamins, trans-zeatin nucleoside, and indoleacetic acid to the basal culture medium solution, mix well, pour into plates, and cool to obtain a culture medium that increases the flavonoid content in snow lotus callus.

[0020] In a preferred embodiment of the preparation method described in this invention, the basic culture medium solution contains 4.43 g / L MS, 30 g / L sucrose, and 7 g / L agar.

[0021] As a preferred embodiment of the preparation method described in this invention, the amount of compound vitamins added to the basic culture medium solution is 1 mL / L, the amount of trans-zeatin nucleoside added is 5 mL / L, and the amount of indoleacetic acid added is 100 μL / L.

[0022] The concentration of trans-zeatin nucleotide was 350 mg / L, and the concentration of indoleacetic acid was 1000 mg / L.

[0023] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a culture medium in increasing the flavonoid content in snow lotus callus.

[0024] Beneficial effects of this invention:

[0025] (1) This invention provides a compound vitamin that promotes the germination of snow lotus seeds and increases the flavonoid content in cell cultures. Thiamine hydrochloride VB1 (1000 mg / L), nicotinic acid (500 mg / L), and pyridoxine hydrochloride VB6 (500 mg / L) were selected as the three vitamins to be added to the culture medium. By comparing and analyzing the effects of adding single vitamins, two vitamin combinations, and three mixed vitamins on the germination of snow lotus seeds, it was found that the germination rate of adding single vitamins was about 18.36%, and the germination rate of adding two vitamins was about 20.77%, which was not much different from the germination rate of adding single vitamins. However, by preparing a mixed mother liquor of the three vitamins (referred to as V3 vitamins) and filtering and sterilizing it before adding it, with the amount of V3 added being 1 mL / L, the germination rate of snow lotus seeds reached the highest level, with a germination rate of about 40.58%.

[0026] (2) The snow lotus seed germination medium established in this invention is MSV3 medium, which can effectively shorten the germination time of snow lotus seeds. Germination occurs in three days, and the germination rate is about 40.58%. MSV3 medium can effectively improve the germination time and germination rate of snow lotus seeds. The culture cycle is short, and cotyledons grow in six days, which is fast.

[0027] (3) By improving the MS medium, the present invention adds trans-zeatin nucleoside and indoleacetic acid to the MSV3 medium, which can synergistically and significantly increase the biomass of snow lotus callus, and the increase is 5 times that of the existing reports. In addition, the total flavonoid content of the dried snow lotus culture cultured in the MS improved medium reaches up to 10%, which is about 2% higher than that of the MS basic medium (8.40%), and is 1-4 times higher than the existing reports, which can significantly increase the total flavonoid content of snow lotus culture.

[0028] (4) The subculture medium for snow lotus callus established in this invention is made by adding (0.05-0.15) mmol / L tryptophan to MS medium or using MS modified medium. The inoculum size is 2.00 gFW / L. After 30 days of culture, the biomass is (8-12) gFW / L and (25-35) gFW / L, which are 4-6 times and 12-17 times the initial inoculum size, respectively. This greatly increases the biomass of snow lotus culture, which is 2-5 times the growth rate reported in traditional studies. At the same time, the total flavonoid content of dried product is 12%-13% and 9%-10%, respectively, which can meet the national standard requirements (total flavonoid content of dried product ≥7%). The subculture medium for snow lotus callus established in this invention can effectively increase the total flavonoid content of snow lotus cell culture. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0030] Figure 1 This shows the germination of snow lotus seeds in Example 1 of the present invention.

[0031] Figure 2 This shows the growth status of callus induced by snow lotus leaves in Example 2 of the present invention after 25 days.

[0032] Figure 3 The growth status of the snow lotus callus in Example 3 of the present invention after being subcultured three times (i.e., cultured for 30 days) on subculture medium ① (a), subculture medium ② (b), and MS modified medium ③ (c).

[0033] Figure 4 The growth status of the snow lotus callus in Example 4 of the present invention after three subcultures (i.e., cultured for 30 days) in subculture medium ① (a), subculture medium ④ (d), and MS modified medium ⑤ (e).

[0034] Figure 5 The growth status of the snow lotus callus in Example 5 of the present invention after three subcultures (i.e., cultured for 30 days) in subculture medium ① (a), subculture medium ⑥ (f), and MS modified medium ⑦ (g). Detailed Implementation

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0036] In this embodiment of the invention, the effects of culture medium on the biomass and total flavonoid content of snow lotus cell cultures were determined.

[0037] (1) Determine the fresh weight of snow lotus cell culture: Remove the agar from the callus tissue after the culture period ends, absorb the excess water on dry filter paper, and weigh it to obtain the fresh weight.

[0038] (2) Determination of total flavonoid content:

[0039] (a) Construction of standard curve

[0040] Rutin reference standard stock solution: Accurately weigh 20 mg of rutin reference standard, dissolve it in methanol, transfer it to a 50 mL volumetric flask, dilute to the mark, and shake well. The concentration of this solution is 0.4 mg / mL.

[0041] Accurately measure 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL of rutin reference stock solution and place them in 25 mL volumetric flasks. Add water to 6.0 mL, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide solution, add water to the mark, shake well, and let stand for 15 min to prepare reference solutions with rutin concentrations of 0.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, 48.0 μg / mL, 64.0 μg / mL, 80.0 μg / mL, and 96.0 μg / mL. Using the 0.0 μg / mL rutin reference solution as a blank, measure the absorbance of each concentration of reference solution at a wavelength of 510 nm.

[0042] A standard curve was plotted with absorbance as the ordinate and concentration of the reference solution as the abscissa. The standard curve for total flavonoids was obtained as follows: y = 0.0066x - 0.0076, R² = 0.999.

[0043] (ii) Determination of the content of the sample

[0044] Accurately weigh 0.10 g of the sample to be tested (particle size less than 80 mesh), place it in a Soxhlet extractor, add an appropriate amount of methanol, heat under reflux until the extract is colorless, cool to room temperature, quantitatively transfer the extract into a 50 mL volumetric flask, wash the container with a small amount of methanol, add the washings to the same volumetric flask, add methanol to the mark, make up to volume, and shake well; after filtration, take the supernatant as the test solution.

[0045] Accurately measure 5 mL of the test solution and place it in a 25 mL volumetric flask. Add water to 6.0 mL, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide solution, add water to the mark, shake well, and let stand for 15 min to prepare the test solution. Use a rutin concentration of 0.0 μg / mL as a blank. Measure the absorbance at a wavelength of 510 nm. Calculate the total flavonoid content in the test solution using a linear regression equation.

[0046] Note: The sample quantity and volume of the sample solution can be adjusted appropriately according to the total flavonoid content in the sample to ensure that the measured absorbance value is within the linear range.

[0047] (III) Calculation of Results

[0048] The total flavonoid content in the sample is calculated using the following formula:

[0049]

[0050] In the formula:

[0051] X—The total flavonoid content in the sample, expressed as rutin;

[0052] A—Absorbance of the test solution;

[0053] V—the dilution factor during sample extraction;

[0054] k, b — slope and intercept of the standard curve;

[0055] M—Sample size of the sample to be tested, in micrograms (μg).

[0056] D—Volume of the extract, in mL.

[0057] The results are expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions, and are retained to three significant figures.

[0058] Example 1

[0059] Germination of snow lotus seeds:

[0060] (1) Preparation method of MSV3 medium:

[0061] Preparation method of 1L MSV3 medium: Accurately weigh 4.43 g MS, 30 g sucrose and 6 g agar into a 1 L Erlenmeyer flask, add an appropriate amount of distilled water and stir thoroughly, make up to 1 L and stir evenly, adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclave at 121℃ for 20 minutes, after sterilization, add 1 mL of pre-filtered and sterilized vitamin V3 (formulation see Table 1) and mix thoroughly, finally pour into plates, cool and set aside.

[0062] Table 1

[0063]

[0064] (2) Cultivation of sterile seedlings of Xinjiang snow lotus:

[0065] Select plump, disease-free natural Xinjiang snow lotus seeds, soak them in purified water for 48 hours, treat them with 75% ethanol for 30 seconds, then disinfect them with sodium hypochlorite solution for 10 minutes, rinse them repeatedly with sterile water 3-4 times, and finally place them on sterile filter paper to absorb the surface moisture. Inoculate them on MSV3 medium with sterile forceps at a temperature of 20℃ and a light exposure of 12 h / d. Germination occurs in three days, with a germination rate of 40.58%.

[0066] Figure 1 This describes the germination status of snow lotus seeds.

[0067] The results are shown in Table 2. The cotyledons grew out in six days, which means that the seedlings were sterile.

[0068] Table 2. Germination results of Saussurea involucrata seeds in MSV3 medium.

[0069]

[0070] Example 2: Induction and culture of snow lotus callus

[0071] (1) Preparation of callus induction culture medium:

[0072] 1L of callus induction medium consists of: accurately weighing 4.43 g MS, 30 g sucrose, 2 mg NAA (naphthaleneacetic acid), 0.5 mg 6-BA (6-benzylpurine), and 7 g agar into a 1 L Erlenmeyer flask, adding an appropriate amount of distilled water and stirring thoroughly, bringing the volume to 1 L and stirring evenly, adjusting the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclaving at 121℃ for 20 minutes, finally pouring into plates, cooling and storing for later use.

[0073] (2) Induction and culture of callus tissue

[0074] Select cotyledons of sterile seedlings cultured for 10 days as explants. Under sterile conditions, use a sterile scalpel and sterile forceps to cut the cotyledons of sterile seedlings into small segments of 2-5 mm in length to avoid mechanical damage to the cotyledons. Remove the tips and bases of the cotyledons.

[0075] When inoculated into callus induction medium and cultured in the dark at 24°C, yellowish-white callus tissue will form at the cut or leaf edge after about 20-30 days. Figure 2 The growth of callus induced from snow lotus leaves for 25 days is shown. It can be clearly seen that the two ends of the snow lotus leaves swell, and the cut surface produces dense, yellowish-white clumps of callus tissue.

[0076] Example 3: Screening of subculture culture medium for snow lotus callus

[0077] (1) Preparation of subculture medium:

[0078] 1L subculture proliferation medium① includes: accurately weigh 4.43 g MS, 30 g sucrose, 2 mg NAA (naphthaleneacetic acid), 0.5 mg 6-BA (6-benzylpurine), and 7 g agar into a 1 L Erlenmeyer flask, add an appropriate amount of distilled water and stir thoroughly, then bring the volume to 1 L and stir evenly.

[0079] 1L subculture proliferation medium ② includes: accurately weigh 4.43 g MS, 30 g sucrose, 2 mg NAA (naphthaleneacetic acid), 0.5 mg 6-BA (6-benzylpurine), 0.02 g tryptophan and 7 g agar into a 1 L Erlenmeyer flask, add an appropriate amount of distilled water and stir thoroughly, then bring the volume to 1 L and stir evenly.

[0080] 1L MS modified medium ③ consists of: accurately weighing 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask, adding an appropriate amount of distilled water and stirring thoroughly, bringing the volume to 1 L and stirring evenly, adjusting the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclaving at 121℃ for 20 minutes, and after sterilization, adding 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1), 5 mL of 350 mg / L trans-zeatin nucleoside, and 100 μL of 1000 mg / L IAA (indoleacetic acid) and mixing thoroughly, finally pouring into plates, cooling and storing for later use.

[0081] (2) Take the same amount of yellowish-white callus (2.0 gFW / L) and inoculate it into the above culture medium. The temperature is 24℃, the light is 16 h / d, and the culture is subcultured once every 15 days. After three subcultures, vigorous growth and compact structure of yellowish-white or red callus can be obtained. Figure 3 The growth of *Saussurea involucrata* callus in Example 3 of this invention was observed after three subcultures on subculture medium ① (a), subculture medium ② (b), and MS modified medium ③ (c). Biomass and total flavonoid content were measured after 30 days of culture, and the results are shown in Tables 5 and 6. Figure 3 The growth status of snow lotus callus after three subcultures on subculture medium ① (a), subculture medium ② (b), and MS modified medium ③ (c).

[0082] Table 5 Comparison of callus biomass from three culture media for Saussurea involucrata

[0083]

[0084] Table 6. Comparison of total flavonoid content in dried snow lotus callus from three different culture media.

[0085]

[0086] Example 4: Screening of subculture culture medium for snow lotus callus

[0087] (1) Preparation of subculture medium:

[0088] 1L subculture proliferation medium ④ includes: accurately weigh 4.43 g MS, 30 g sucrose, 2 mg NAA (naphthaleneacetic acid), 0.5 mg 6-BA (6-benzylpurine), 0.01 g tryptophan and 7 g agar into a 1 L Erlenmeyer flask, add an appropriate amount of distilled water and stir thoroughly, then bring the volume to 1 L and stir evenly.

[0089] 1L MS modified medium ⑤ consists of: accurately weighing 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask, adding an appropriate amount of distilled water and stirring thoroughly, bringing the volume to 1 L and stirring evenly, adjusting the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclaving at 121℃ for 20 minutes, and after sterilization, adding 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1), 5 mL of 200 mg / L trans-zeatin nucleoside, and 50 μL of 1000 mg / L IAA (indoleacetic acid) and mixing thoroughly, finally pouring into plates, cooling and storing for later use.

[0090] (2) Take the same amount of yellowish-white callus (2.0 gFW / L) and inoculate it into the above culture medium. The temperature is 24℃, the light is 16 h / d, and the culture is subcultured once every 15 days. After three subcultures, vigorous growth and compact structure of yellowish-white or red callus can be obtained. Figure 4 The growth of the snow lotus callus in Example 4 of this invention was observed after three subcultures in subculture medium ④ (d) and MS modified medium ⑤ (e). Biomass and total flavonoid content were measured after 30 days of culture, and the results are shown in Tables 7 and 8. Figure 4 The growth status of snow lotus callus after three subcultures in subculture medium ① (a), subculture medium ④ (d), and MS modified medium ⑤ (e).

[0091] Table 7 Comparison of callus biomass from three culture media for Saussurea involucrata

[0092]

[0093] Table 8. Comparison of total flavonoid content in dried snow lotus callus from two different culture media.

[0094]

[0095] Example 5: Screening of subculture culture medium for snow lotus callus

[0096] (1) Preparation of subculture medium:

[0097] 1L subculture proliferation medium⑥ includes: accurately weigh 4.43 g MS, 30 g sucrose, 2 mg NAA (naphthaleneacetic acid), 0.5 mg 6-BA (6-benzylpurine), 0.03 g tryptophan and 7 g agar into a 1 L Erlenmeyer flask, add an appropriate amount of distilled water and stir thoroughly, then bring the volume to 1 L and stir evenly.

[0098] 1L MS modified medium⑦ consists of: accurately weighing 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask, adding an appropriate amount of distilled water and stirring thoroughly, bringing the volume to 1 L and stirring evenly, adjusting the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclaving at 121℃ for 20 minutes, and after sterilization, adding 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1), 5 mL of 500 mg / L trans-zeatin nucleoside, and 200 μL of 1000 mg / L IAA (indoleacetic acid) and mixing thoroughly, finally pouring into plates, cooling and storing for later use.

[0099] (2) Take the same amount of yellowish-white callus (2.0 gFW / L) and inoculate it into the above culture medium. The temperature is 24℃, the light is 16 h / d, and the culture is subcultured once every 15 days. After three subcultures, vigorous growth and compact structure of yellowish-white or red callus can be obtained. Figure 5 The growth of *Saussurea involucrata* callus in Example 5 of this invention was observed after three subcultures in subculture medium ⑥ (f) and MS modified medium ⑦ (g). Biomass and total flavonoid content were measured after 30 days of culture, and the results are shown in Tables 9 and 10. Figure 5 The growth status of snow lotus callus after three subcultures in subculture medium ① (a), subculture medium ⑥ (f), and MS modified medium ⑦ (g).

[0100] Table 9 Comparison of callus biomass from three culture media for Saussurea involucrata

[0101]

[0102] Table 10 Comparison of total flavonoid content in dried snow lotus callus from three different culture media

[0103]

[0104] Comparative Example 1

[0105] Preparation method of 1L MSV3 medium: Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, add 1 mL of pre-filtered and sterilized Vitamin V3 (formulation shown in Table 1) and mix thoroughly. Finally, pour the mixture into plates and cool for later use (this is the best example). Based on this, set up the following experiments:

[0106] Experiment 1 (Single Thiamine Hydrochloride VB1): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 2 g / L thiamine hydrochloride VB1 that has been filtered and sterilized beforehand. Mix thoroughly and then pour into plates. Cool and set aside for later use.

[0107] Experiment 2 (Nicotinic Acid Single): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 2 g / L nicotinic acid that has been filtered and sterilized beforehand. Mix thoroughly and then pour into plates. Cool and set aside for later use.

[0108] Experiment 3 (Pyridoxine VB6 hydrochloride alone): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 2 g / L pyridoxine VB6 hydrochloride that has been filtered and sterilized beforehand. Mix thoroughly and then pour into plates. Cool and set aside for later use.

[0109] Experiment 4 (Thymine VB1 Hydrochloride + Nicotinic Acid): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 1 g / L thiamine VB1 hydrochloride and 1 g / L nicotinic acid that have been filtered and sterilized beforehand. Mix thoroughly and then pour into plates. Cool and set aside for later use.

[0110] Experiment 5 (Thymine VB1 hydrochloride + Pyridoxine VB6 hydrochloride): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 1 g / L thiamine VB1 hydrochloride and 1 g / L pyridoxine VB6 hydrochloride that have been filtered and sterilized beforehand. Mix thoroughly and pour into plates. Cool and set aside for later use.

[0111] Experiment 6 (Pyridoxine Hydrochloride VB6 + Nicotinic Acid): Accurately weigh 4.43 g MS, 30 g sucrose, and 6 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop, add 1 g / L pyridoxine hydrochloride VB6 and 1 g / L nicotinic acid that have been filtered and sterilized beforehand, and mix thoroughly. Finally, pour the mixture into plates, cool, and set aside for later use.

[0112] The cultivation conditions for the germination of snow lotus seeds are the same as in Example 1.

[0113] The experimental results are as follows:

[0114]

[0115] Comparative experiments showed that adding the same concentration (2 g / L) of thiamine hydrochloride VB1, nicotinic acid, or pyridoxine hydrochloride VB6 alone resulted in a germination rate of only 27.54% (thiamine hydrochloride VB1), with a germination time of approximately 8 days. This indicates that while single vitamins have a certain promoting effect, they cannot achieve rapid and efficient germination. Furthermore, among the dual-vitamin combinations, nicotinic acid + pyridoxine hydrochloride VB6 had the highest germination rate (34.78%), but this was still lower than the optimal embodiment (40.58%), and the germination time was 6 days. Particularly noteworthy is that the germination rate of the thiamine hydrochloride VB1 + nicotinic acid combination (18.84%) was even lower than that of thiamine hydrochloride VB1 alone (27.54%), suggesting that there may be mutual inhibition or an imbalance in the ratio of vitamins. Similarly, combining thiamine hydrochloride VB1 with pyridoxine hydrochloride VB6 (Experiment 5) resulted in a sharp decrease in germination rate (8.7%), far lower than the effect of using thiamine hydrochloride VB1 alone (27.54%). This clearly demonstrates that the interactions between vitamins in the snow lotus seed germination system are complex, and not all simple mixtures of "active ingredients" produce positive effects; some combinations may even produce antagonistic effects. Among the dual-vitamin combinations, Experiment 6 (nicotinic acid + pyridoxine hydrochloride VB6) achieved relatively good results (germination rate 34.78%), suggesting a possible positive interaction between nicotinic acid and pyridoxine hydrochloride VB6. Only a compound vitamin V3 solution containing thiamine hydrochloride (VB1), niacin, and pyridoxine hydrochloride (VB6) at specific concentrations (1 g / L thiamine hydrochloride (VB1), 0.5 g / L niacin, and 0.5 g / L pyridoxine hydrochloride (VB6)) achieved a significant synergistic effect, shortening the germination time to 3 days and increasing the germination rate to over 40%. This indicates that the three vitamins have a non-additive positive interaction in promoting snow lotus seed germination; none can be omitted, and the ratio is crucial.

[0116] In summary, the compound vitamin culture medium provided by this invention significantly exceeds the reasonable expectations of those skilled in the art. Data shows that the relationship between the three vitamins in the germination of snow lotus seeds is complex, not a simple dose-response or additive relationship. Some two-component combinations (thiamine hydrochloride VB1 + nicotinic acid, thiamine hydrochloride VB1 + pyridoxine hydrochloride VB6) even exhibit inhibitory effects, highlighting the unpredictable and strong positive synergistic effect produced by the coexistence of thiamine hydrochloride VB1 + nicotinic acid + pyridoxine hydrochloride VB6 in a specific ratio. Therefore, this invention provides not only a specific culture medium formulation, but also reveals a novel technical means to efficiently break the dormancy of snow lotus seeds and simultaneously improve germination rate and germination percentage.

[0117] Comparative Example 2

[0118] The 1L MS modified medium consists of: accurately weighing 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask, adding an appropriate amount of distilled water and stirring thoroughly, bringing the volume to 1 L and stirring evenly. Adjusting the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid, autoclaving at 121℃ for 20 minutes, and after sterilization, adding 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1), 5 mL of 350 mg / L trans-zeatin nucleoside, and 100 μL of 1000 mg / L IAA (indoleacetic acid) and mixing thoroughly. Finally, pouring the mixture into plates and cooling it for later use (this is the best embodiment). Based on this, the following experiments were conducted:

[0119] Experiment 7 (Vitamin V3 + trans-zeatin nucleoside): Accurately weigh 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1) and 5.3 mL of 350 mg / L trans-zeatin nucleoside. Mix thoroughly and pour into plates. Cool and set aside for later use.

[0120] Experiment 8 (Vitamin V3 + IAA): Accurately weigh 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1) and 1.85 mL of 1000 mg / L IAA (indoleacetic acid). Mix thoroughly and pour into plates. Cool and set aside for later use.

[0121] Experiment 9 (trans-zeatin nucleoside + IAA): Accurately weigh 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 6 mL of pre-filtered and sterilized 350 mg / L trans-zeatin nucleoside and 1.75 mL of 1000 mg / L IAA (indoleacetic acid). Mix thoroughly and pour into plates. Cool and set aside for later use.

[0122] Experiment 10 (Vitamin V3): Accurately weigh 4.43 g MS, 30 g sucrose, and 7 g agar into a 1 L Erlenmeyer flask. Add an appropriate amount of distilled water and stir thoroughly. Make up to 1 L and stir evenly. Adjust the pH to 5.8-6.0 with 1 mol / L KOH and dilute hydrochloric acid. Autoclave at 121℃ for 20 minutes. After sterilization, wait for the temperature to drop and add 1 mL of pre-filtered and sterilized Vitamin V3 (formula shown in Table 1) and mix thoroughly. Finally, pour the mixture into plates and let it cool before use.

[0123] The subculture conditions for callus were the same as in Example 3.

[0124] The experimental results are as follows:

[0125]

[0126] Comparative experiments showed that, compared to the single hormone group, with an initial inoculum of 2.0 gFW / L, after 30 days of culture, the final biomass of the optimal embodiment was 2.7 times that of Experiment 7 (13.17 gFW / L) with only trans-zeatin nucleotides added, and 3 times that of Experiment 8 (11.67 gFW / L) with only IAA added. This demonstrates that the effect of using any single hormone alone is limited. Compared to the vitamin-free group, the final biomass of the optimal embodiment was 1.7 times that of Experiment 9 (20.67 gFW / L) with only two hormones but no vitamin V3 added. This clearly shows that the lack of vitamin V3 leads to a significant reduction in biomass. Compared to the hormone-free group, the final biomass of the optimal embodiment was 3.4 times that of Experiment 10 (10.5 gFW / L) with only vitamin V3 but no hormones, confirming the driving role of plant hormones in cell proliferation.

[0127] Under identical hormonal conditions, the addition of only a trace amount of vitamin V3 (1 mL / L) resulted in a significant increase in biomass, jumping from 20.67 gFW / L to 35.33 gFW / L, an increase of over 70%. This is not a simple "additional effect" because if the effect were simply additive, the theoretical value of "Experiment 9 (20.67 gFW / L) + Experiment 10 (10.5 gFW / L)" would be approximately 31.17 gFW / L, still lower than the actual 35.33 gFW / L. Therefore, vitamin V3 significantly amplified the physiological effects of the two plant hormones (trans-zeatin nucleotide and IAA), fully stimulating the cell's growth potential, thus producing a synergistic effect of "1+1+1 >> 3." These three elements constitute a highly efficient synergistic system of "signal (hormones) - metabolic support (vitamins) - growth response (high biomass)."

[0128] This invention addresses the pain points of long seed germination cycles and low levels of target products in snow lotus cell cultures by developing an innovative culture medium system and process. First, a novel seed germination culture medium is designed. By precisely controlling nutrient and environmental parameters, the germination cycle of snow lotus seeds is effectively shortened, significantly improving germination rate and seedling vigor compared to traditional methods. Second, to address the low flavonoid content in snow lotus cell cultures, two synergistic strategies are proposed: one is to add tryptophan as a key precursor to activate the flavonoid biosynthesis pathway; the other is to construct a composite optimized culture medium, dynamically controlling the ratio of multiple factors and metabolic pathways to achieve a synergistic increase in cell biomass and flavonoid content.

[0129] In summary, this invention overcomes the problems of low efficiency and poor yield in snow lotus cell culture through the synergistic innovation of seed germination acceleration technology and flavonoid synthesis enhancement strategy. It provides an efficient and controllable solution for the industrial production of high-value-added flavonoids and has significant industrial application potential and socio-economic benefits.

[0130] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A compound vitamin that promotes the germination of snow lotus seeds and increases the flavonoid content in cell cultures, characterized in that: Including thiamine hydrochloride (VB1), nicotinic acid, and pyridoxine hydrochloride (VB6); The concentrations of thiamine hydrochloride (VB1) and nicotinic acid were 1000 mg / L, and the concentrations of pyridoxine hydrochloride (VB6) were 500 mg / L.

2. A method for preparing a culture medium to promote the germination of snow lotus seeds, characterized in that: include, Mix MS medium, sucrose, agar and distilled water, bring the volume to 1 L and stir well. Adjust the pH to 5.8-6.0, autoclave at 121℃ for 20 minutes, and cool to obtain the basic culture medium solution. The compound vitamins described in claim 1 were added to the basal culture medium solution to prepare the culture medium for promoting the germination of snow lotus seeds.

3. The preparation method according to claim 2, characterized in that: The basal culture medium solution contained 4.43 g / L MS, 30 g / L sucrose, and 6 g / L agar.

4. The preparation method according to claim 2 or 3, characterized in that: The amount of the compound vitamin added to the basal culture medium solution is 1 mL / L.

5. The application of the culture medium prepared by any of the preparation methods described in claims 2 to 4 in promoting the germination of snow lotus seeds during the cultivation of snow lotus seeds.

6. A method for preparing a culture medium to increase the flavonoid content in snow lotus callus, characterized in that: include, Mix MS medium, sucrose, agar and distilled water, bring the volume to 1 L and stir well. Adjust the pH to 5.8-6.0, autoclave at 121℃ for 20 minutes, and cool to obtain the basic culture medium solution. Add the compound vitamins, trans-zeatin nucleoside and indoleacetic acid described in claim 1 to the basal culture medium solution, mix well, pour into plates, and cool to obtain a culture medium that increases the flavonoid content in snow lotus callus.

7. The preparation method according to claim 6, characterized in that: The basal culture medium solution contained 4.43 g / L MS, 30 g / L sucrose, and 7 g / L agar.

8. The preparation method according to claim 6 or 7, characterized in that: The basic culture medium solution contains 1 mL / L of compound vitamins, 5 mL / L of trans-zeatin nucleoside, and 50-200 μL / L of indoleacetic acid. The concentration of trans-zeatin nucleoside was 200-500 mg / L, and the concentration of indoleacetic acid was 1000 mg / L.

9. The application of the culture medium prepared by any one of the preparation methods described in claims 6 to 8 in increasing the flavonoid content in snow lotus callus.

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