Preparation method and application of embryonic stem cells of dioscoreae plants

Abstract

The invention belongs to the technical field of biology, and relates to a preparation method and application of embryonic stem cells of dioscoreae plants. The culture method of the embryonic stem cells of the dioscorea family plants comprises the following steps: firstly, cleaning and disinfecting a selected dioscorea family plant body, slicing the dioscorea family plant body, and then preparing into embedded paraffin sections with the thickness of less than 0.5 mm; the method comprises the following steps: cutting a paraffin section into sections with the thickness of 1-10 microns, placing the sections under a microscope to observe the specific position of the plant embryonic stem cells, stripping the plant embryonic stem cells at the root tip part of the dioscorea family by using a surgical blade, placing the plant embryonic stem cells in a triangular flask filled with an antibiotic MS culture medium, carrying out shake cultivation, and then carrying out amplification culture by using a brown sugar MS culture medium to obtain the plant embryonic stem cells. After the culture is finished, carrying out centrifugal drying to obtain the embryonic stem cells of the dioscoreae plants; the embryonic stem cells of the dioscorea family plants can be applied to the fields of health care products, foods and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing embryogenic stem cells of Dioscoreaceae plants and their applications in the fields of health products, foods, etc. Background Art

[0002] There are many types of Dioscoreaceae plants. Among them, Chinese yam (Dioscorea opposita), also known as yam, is one of the representative crops of Dioscoreaceae. It is a herbaceous rhizomatous tuberous plant with a cultivation history of thousands of years in China and is one of the earliest cultivated crops in China. Especially the authentic medicinal materials planted in areas such as Wen County and Wuzhi in Jiaozuo, Henan, have always played an important role in traditional medicine and modern medicine. As a category of medicine and food homology, its tubers can be directly used as medicine, with the effects of strengthening the spleen, tonifying the lungs, consolidating the kidneys, and benefiting essence. It contains rich nutrients, such as various active ingredients like starch, protein, polysaccharide, amylase, choline, ergosterol, oxidase, etc., and at the same time contains various trace elements such as calcium, iron, zinc, selenium, copper, etc., with wide uses and good prospects. This is why Chinese yam has increasingly become a research hotspot and is more and more favored by consumers in recent years.

[0003] In recent years, with the development of scientific and technological levels and the improvement of living standards, the demand for Dioscorea plants represented by Chinese yam has been increasing year by year. People's pursuit of this family of plants is not only to use it as traditional Chinese medicine decoction pieces for their own treatment, but also as a leisure food for enjoyment or as a health product to protect human health. However, traditional planting methods are limited by problems such as authenticity, difficult storage, and susceptibility to heavy metal pollution, and it is difficult to meet market demand. In addition, the existing research on Dioscoreaceae plant stem cells mainly focuses on callus, and callus cells have disadvantages such as high heterogeneity and unstable culture. Therefore, it is of great significance to develop an efficient and stable method for culturing embryogenic stem cells of Dioscoreaceae plants. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing embryogenic stem cells of Dioscoreaceae plants and their applications in the fields of health products, foods, etc.

[0005] A method for culturing embryogenic stem cells of Dioscoreaceae plants includes the following steps:

[0006] Step 1, specimen pretreatment: cleaning and disinfecting the sampled Dioscoreaceae plant body.

[0007] The method for cleaning and disinfecting the Dioscoreaceae plant body taken is as follows: Wash the Dioscoreaceae plant body with running tap water, and gently brush some key parts or parts that are not easy to clean with a soft brush to brush off the attached soil or other attachments. Transfer the washed plant to a biosafety cabinet, continue cleaning and disinfecting, wash the surface of the plant to be used with an ethanol aqueous solution with a volume percentage of 75%, and the washing time is 20 - 120 seconds. After washing, pour out the ethanol aqueous solution with a volume percentage of 75%, wash the plant 3 times with sterile water, then soak it in an aqueous solution containing HgCl2 with a mass concentration of 0.1% - 0.5% for 2 - 10 minutes. After disinfection, discard the disinfectant solution, and wash the plant with sterilized water 3 times.

[0008] Step 2. Tissue section pre-experiment: Cut off the root tip of the Dioscoreaceae plant body treated in Step 1 into sections with a thickness less than 0.5 mm for standby; after melting the paraffin with an alcohol lamp, pour it into an embedding cassette, and at the same time transfer the cut sections into the embedding cassette, and continue to add melted paraffin until the embedding cassette is completely filled; after the paraffin is completely solidified, obtain the embedded paraffin sections; use a microtome to section the embedded paraffin sections, and the section thickness is 1 - 10 μm. After sectioning is completed, use a brush to pick up the paraffin sections and flatten them in water at 40°C, dry them after holding them with a glass slide, cover with a glass slide and observe under an Olympus microscope to check the position of the stem cells of the Dioscoreaceae plant.

[0009] Step 3. Specimen treatment: In the biosafety cabinet, light an alcohol lamp, pass the surgical blade over the flame of the alcohol lamp for disinfection and sterilization. After the temperature of the blade drops, according to the position of the stem cells of the Dioscoreaceae plant observed in Step 2, use the surgical blade to peel off the plant embryogenic stem cells of the root tip part of the Dioscoreaceae on the paraffin section, and place them in an antioxidant solution containing 1 mol / L sodium thiosulfate to prevent browning and oxidation. After all peeling is completed, take out the tissue block containing embryogenic stem cells from the antioxidant solution, cut it into small pieces, transfer it to the MS medium and rinse 2 - 3 times, and then transfer it to a triangular flask containing an antibiotic MS medium.

[0010] The antibiotic MS medium is to add penicillin, streptomycin and amphotericin B to the MS medium, where the addition amounts of penicillin and streptomycin are both 0.5 - 2% of the mass of the MS medium, and the addition amount of amphotericin B is 0.1 - 2 μg per milliliter of the MS medium; there are endogenous bacteria in plants, and in order to better carry out the experiment, penicillin, streptomycin and amphotericin B are added in the experiment.

[0011] Step 4. Shaking culture: Place the Erlenmeyer flask containing Dioscoreaceae embryogenic stem cells in a vertical constant temperature oscillator, with a rotation speed of 80 - 120 rpm and a temperature of 20 - 25 °C, and conduct shaking culture for 24 - 36 h. After the culture is completed, first filter with a No. 2 sieve to remove the fragments of the larger root tip part, and then filter and separate the obtained filtrate with a 40 μm cell filter membrane to obtain the embryogenic stem cells of Dioscoreaceae plants;

[0012] Since the aggregation degree of embryogenic stem cells is lower and smaller compared with callus, mainly presenting single cells or small cell clumps, therefore, the filtrate can be filtered and separated with a 40 μm cell filter membrane to obtain the embryogenic stem cells of Dioscoreaceae plants;

[0013] Step 5. Subculture and cell preservation: Expand the culture of the Dioscoreaceae plant embryogenic stem cells filtered in Step 4 in a brown sugar MS medium, with a culture rotation speed of 80 - 120 rpm and a temperature of 20 - 25 °C, and conduct shaking culture; Monitor the growth of Dioscoreaceae plant embryogenic stem cells during the culture process. Before the Dioscoreaceae plant embryogenic stem cells enter the plateau phase of growth, when the cell growth rate is fast and the activity is high, take a part of the medium containing Dioscoreaceae plant embryogenic stem cells, add dimethyl sulfoxide with a volume of 5 - 10% of its volume, and cryopreserve the Dioscoreaceae plant embryogenic stem cells to obtain cryopreserved Dioscoreaceae plant embryogenic stem cells; Continue the culture, take 10 μL of Dioscoreaceae stem cells on a disposable cell counting plate to measure the cell number, and when the order of magnitude of the cell number is 10 7 or more, end the subculture of Dioscoreaceae stem cells to obtain a medium containing Dioscoreaceae plant embryogenic stem cells;

[0014] Step 6. Collection, drying and pulverization: Pour the medium containing Dioscoreaceae plant embryogenic stem cells into a centrifuge bottle, balance it, place it in a centrifuge, with a rotation speed of 1500 - 2500 rpm and a centrifugation time of 5 - 10 minutes. After the centrifugation is completed, discard the supernatant to obtain Dioscoreaceae plant embryogenic stem cells; Freeze-dry or microwave-dry the obtained Dioscoreaceae plant embryogenic stem cells. After complete drying, use a small pulverizer to pulverize and sieve to obtain the powder of Dioscoreaceae plant embryogenic stem cells.

[0015] For the brown sugar MS medium, the mass ratio of brown sugar to the mass of MS medium powder is 3:1 - 1:3; Penicillin and streptomycin are added to the brown sugar MS medium, and the addition amounts of penicillin and streptomycin are both 0.5 - 2% of the mass of the MS medium;

[0016] The prepared cryopreserved Dioscoreaceae plant embryogenic stem cells can be applied to cell resuscitation and subculture, subsequent cell experiments, etc.;

[0017] The specific formulation of the MS medium is shown in the following table:

[0018]

[0019] The prepared powder of Dioscoreaceae plant embryonic stem cells can be applied in the fields of agriculture, food, health products, medicine, etc., such as the following two applications;

[0020] A health product composition containing Dioscoreaceae plant embryonic stem cells, comprising the following components in mass ratio: 20-30% of Dioscoreaceae plant embryonic stem cells, 20-25% of lentinus edodes powder, 15-20% of pumpkin seed powder, 10-15% of barley powder, 5-10% of pea powder, 5-10% of lotus root powder, 2-5% of ginseng powder, 2-5% of soybean phospholipid, 2-5% of papain and 2-5% of red bean powder. This composition has the effect of reducing blood lipid.

[0021] A compound nutritional noodle containing Dioscorea opposita thumb embryonic stem cell powder. In every 100g of flour, the addition amount of Dioscorea opposita thumb embryonic stem cell powder is 10-40% of the flour quality, the addition amount of carrot is 5-10% of the flour quality, the addition amount of beef is 10-15% of the flour quality, the addition amount of skimmed milk is 1-8% of the flour quality, and the addition amount of salt is 0.5-2% of the flour quality. After mixing evenly, it is pressed.

[0022] The beneficial technical effects of the present invention are as follows:

[0023] 1. The present invention innovatively uses the root tip of Dioscoreaceae plants for pre-experiment. By making tissue sections and observing under a microscope, the position of embryonic stem cells is accurately located. Since the root tip of Dioscoreaceae plants is small and embryonic stem cells are only distributed in thin-layer tissues, it is extremely difficult to obtain by traditional methods. Through the pre-experiment, the difficulty of obtaining embryonic stem cells can be effectively reduced, and the problem of low cell acquisition efficiency in the prior art can be solved.

[0024] 2. The present invention uses brown sugar to partially replace the MS medium for large-scale culture, significantly reducing the cost. In a large fermenter, the addition amount of brown sugar can reach 3 / 4 of the total amount of the medium, and the cost of brown sugar is much lower than that of the MS medium (about 10 yuan for 500g of brown sugar, about 240 yuan for 500g of MS medium). This innovative medium formula not only saves costs but also improves the commercial application value of the present invention.

[0025] 3. In the present invention, corresponding embryogenic stem cells of Dioscoreaceae plants are obtained. Although there is a cell wall on the cell surface of plant stem cells compared with animal stem cells, which is beneficial to their survival to a certain extent, however, compared with the cruel external adverse environment, the protective effect that this cell wall can play is still minimal. Therefore, in order to preserve the obtained plant embryogenic stem cells, it is necessary to cryopreserve the plant stem cells. The main reasons for the death of plant stem cells usually include two types. One is cell dehydration, and the other is serious damage to biological membranes caused by extracellular ice formation. Therefore, a certain protective agent can be added during the process of cryopreserving cells to avoid this situation.

[0026] 4. In the present invention, the protective agent used is dimethyl sulfoxide (DMSO), which is a permeating protective agent and is currently widely used in the process of protecting animal cells, but there are few reports on its application in the preservation of plant cells. Cryopreserving plant embryogenic stem cells has the following advantages: (1) reducing gene drift; (2) slowing down the senescence of cell lines; (3) stabilizing phenotypes; (4) reducing the opportunities of microbial contamination and cross-contamination, etc. The principle of cell cryopreservation lies in minimizing the formation of crystals inside cells, reducing the damage to cells by free radicals, changing the permeability of biological membranes to electrolytes, drugs, poisons and metabolites, and reducing the cryogenic damage to cells caused by the high-concentration solutes formed by the solidification of intracellular water, thereby increasing the survival rate of cells during resuscitation. The protective mechanism of dimethyl sulfoxide is that before the cell cryopreservation suspension completely solidifies, it penetrates into the cells, generates a certain molar concentration inside and outside the cells, reduces the concentration of electrolytes in the unfrozen solution inside and outside the cells, thereby lowering the freezing point and protecting the cells from the damage of high-concentration electrolytes. At the same time, the intracellular water will not exude excessively, avoiding excessive dehydration and shrinkage of the cells, and it has no obvious toxicity to the cells.

[0027] 5. In the present invention, the addition amount of the protective agent dimethyl sulfoxide is 5 - 10%, and the remaining is the liquid culture medium as a nutrient substance, with an addition amount of 90 - 95%. The best period for cryopreservation is before the growth of plant embryogenic stem cells enters the plateau phase. At this time, the cell growth rate is fast, the cell activity is high, the cryopreservation effect is good, and the survival rate of cells after resuscitation is high. In the present invention, the commonly used and inexpensive protective agent dimethyl sulfoxide is selected, and no additional macromolecular substances such as polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, and hydroxyethyl starch are added. This cryopreservation solution is the MS medium except for DMSO. Because the cryopreserved are plant stem cells and there is a cell wall outside the plant cells that can provide basic protection to the cells, and the various components in the culture medium are already sufficient to supply energy. This cryopreservation method is simple, the materials are inexpensive, and it can eliminate some biases against DMSO cryopreservation solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The order of magnitude of the number of Dioscorea opposita stem cells in Example 1 of the present invention is 10 7 Result diagram;

[0029] Figure 2 It is the culture result of Dioscoreaceae plant stem cells using brown sugar MS media with various ratios in Experiment 1 of the present invention. Specific implementation manners

[0030] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] Example 1

[0032] A method for culturing plant embryogenic stem cells of Dioscorea opposita, comprising the following steps:

[0033] Step 1. Specimen pretreatment: Clean and disinfect the taken Dioscorea opposita plant body;

[0034] The method for cleaning and disinfecting the taken Dioscorea opposita plant body is as follows: Wash the Dioscorea opposita plant body with running tap water, and gently brush some key parts or parts that are not easy to clean with a soft brush to brush off the attached soil or other attachments. Transfer the washed plant to a biosafety cabinet, continue to clean and disinfect, wash the surface of the plant to be used with an ethanol aqueous solution with a volume percentage of 75% for 20 seconds. After the washing is completed, pour out the ethanol aqueous solution with a volume percentage of 75%, wash the plant 3 times with sterile water, then soak it in an aqueous solution containing HgCl2 with a mass concentration of 0.1% for 2 minutes. After the disinfection is completed, discard the disinfectant solution, and wash the plant with sterilized water for 3 times.

[0035] Step 2. Tissue section pre-experiment: Cut off the root tip of the Dioscorea opposita plant body processed in Step 1 into sections with a thickness less than 0.5 mm for standby; Melt the paraffin with an alcohol lamp and pour it into an embedding cassette. At the same time, transfer the cut sections into the embedding cassette, and continue to add melted paraffin until the embedding cassette is completely filled; After the paraffin is completely solidified, obtain the embedded paraffin sections; Use a microtome to section the embedded paraffin sections with a thickness of 1 μm. After the sectioning is completed, use a brush to pick up the paraffin sections and flatten them in water at 40°C, dry them after holding them with a glass slide, cover the glass slide and observe under an Olympus microscope to check the position of the Dioscorea opposita plant stem cells;

[0036] Step 3. Specimen processing: In a biosafety cabinet, light an alcohol lamp, pass the surgical blade through the flame of the alcohol lamp for disinfection and sterilization. After the temperature of the blade drops, according to the position of the stem cells of the yam plant observed in Step 2, use the surgical blade to peel the embryonic stem cells at the root tip of the yam on the paraffin section, and place them in an antioxidant solution of 1 mol / L sodium thiosulfate to prevent browning and oxidation. After all the peeling is completed, take out the tissue block containing embryonic stem cells from the antioxidant solution, cut it into small pieces, transfer it to the MS medium and rinse it twice, and then transfer it to an Erlenmeyer flask containing antibiotic MS medium;

[0037] The antibiotic MS medium is prepared by adding penicillin, streptomycin and amphotericin B to the MS medium. The addition amounts of penicillin and streptomycin are both 0.5% of the mass of the MS medium, and the addition amount of amphotericin B is 0.1 μg per milliliter of the MS medium. There are endogenous bacteria in plants, and in order to better conduct the experiment, penicillin, streptomycin and amphotericin B are added in the experiment.

[0038] Step 4. Shaking culture: Place the Erlenmeyer flask containing the embryonic stem cells of the yam in a vertical constant temperature oscillator, with a rotation speed of 80 rpm and a temperature of 20 °C, and conduct shaking culture for 24 h. After the culture is completed, first filter it with a No. 2 sieve to remove the larger fragments at the root tip part, and then filter and separate the obtained filtrate with a 40-μm cell filter membrane to obtain the embryonic stem cells of the yam plant;

[0039] Since the aggregation degree of embryonic stem cells is lower and smaller compared with callus, mainly presenting single cells or small cell clusters, therefore, the filtrate can be filtered and separated with a 40-μm cell filter membrane to obtain the embryonic stem cells of the yam plant;

[0040] Step 5. Subculture and cell preservation: Expand the embryonic stem cells of the yam plant obtained by filtration in Step 4 in the brown sugar MS medium, with a culture rotation speed of 80 rpm and a temperature of 20 °C, and conduct shaking culture; Monitor the growth of the embryonic stem cells of the yam plant during the culture process. Before the growth of the embryonic stem cells of the yam plant enters the plateau phase, when the cell growth rate is fast and the activity is high, take a part of the medium containing the embryonic stem cells of the yam plant, add 5% of its volume of dimethyl sulfoxide, and cryopreserve the embryonic stem cells of the yam plant to obtain cryopreserved embryonic stem cells of the yam plant; Continue the culture, take 10 μL of the yam stem cells on a disposable cell counting plate to measure the cell number, and when the order of magnitude of the cell number is 10 7 or more (as shown in Figure 1 ), end the subculture of the yam stem cells to obtain the medium containing the embryonic stem cells of the yam plant;

[0041] Step 6. Collection, drying and pulverization: Pour the culture medium containing Dioscorea opposita thumb plant embryogenic stem cells into a centrifuge bottle. After balancing, place it in a centrifuge with a rotation speed of 1500 rpm and a centrifugation time of 5 minutes. After centrifugation, discard the supernatant to obtain Dioscorea opposita thumb plant embryogenic stem cells; subject the obtained Dioscorea opposita thumb plant embryogenic stem cells to freeze-drying or microwave drying. After complete drying, use a small pulverizer to pulverize and sieve to obtain the powder of Dioscorea opposita thumb plant embryogenic stem cells.

[0042] For the brown sugar MS culture medium, the mass ratio of brown sugar to MS culture medium powder is 3:1; penicillin and streptomycin are added to the brown sugar MS culture medium, and the addition amounts of penicillin and streptomycin are both 0.5% of the mass of the MS culture medium.

[0043] The cryopreserved Dioscorea opposita thumb plant embryogenic stem cells prepared can be applied to cell resuscitation and expansion culture, subsequent cell experiments, etc.

[0044] The powder of Dioscorea opposita thumb plant embryogenic stem cells prepared can be applied to fields such as agriculture, food, health products, pharmaceuticals, etc., for example, the following two applications;

[0045] A composition of a health product containing Dioscorea opposita thumb plant embryogenic stem cells, including the following components in mass ratio: Dioscorea opposita thumb plant embryogenic stem cells 20%, mushroom powder 20%, pumpkin seed powder 15%, barley powder 10%, pea powder 10%, lotus root powder 5%, ginseng powder 5%, soy lecithin 5%, papain 5% and red bean powder 5%. This composition has the effect of reducing blood lipid.

[0046] A composite nutritious noodle containing the powder of Dioscorea opposita thumb embryogenic stem cells. In every 100 g of flour, the addition amount of the powder of Dioscorea opposita thumb embryogenic stem cells is 10% of the mass of the flour, the addition amount of carrots is 5% of the mass of the flour, the addition amount of beef is 10% of the mass of the flour, the addition amount of skim milk is 1% of the mass of the flour, and the addition amount of salt is 0.5% of the mass of the flour. After mixing evenly, press.

[0047] Example 2

[0048] A culture method for Dioscorea nipponica Makino plant embryogenic stem cells, including the following steps:

[0049] Step 1. Specimen pretreatment: Clean and disinfect the Dioscorea nipponica Makino plant body from which the materials are taken.

[0050] The method for cleaning and disinfecting the Dioscorea nipponica plant body from which materials are taken is as follows: Wash the Dioscorea nipponica plant body with running tap water, and gently brush some key parts or parts that are not easy to clean with a soft brush to brush off the attached soil or other attachments. Transfer the washed plant to a biosafety cabinet for continued cleaning and disinfection. Wash the surface of the plant to be used with an ethanol aqueous solution with a volume percentage of 75% for 120 seconds. After the washing is completed, pour out the ethanol aqueous solution with a volume percentage of 75%, wash the plant 3 times with sterile water, then soak it in an aqueous solution containing 0.5% (mass concentration) of HgCl2 for 10 minutes. After the disinfection is completed, discard the disinfectant solution and wash the plant with sterilized water 3 times.

[0051] Step 2: Preliminary experiment on tissue sectioning: Cut off the root tip of the Dioscorea nipponica plant body processed in Step 1 into sections with a thickness less than 0.5 mm for standby; melt the paraffin with an alcohol lamp and pour it into an embedding cassette. At the same time, transfer the cut sections into the embedding cassette and continue to add melted paraffin until the embedding cassette is completely filled; after the paraffin is completely solidified, obtain the embedded paraffin sections; use a microtome to section the embedded paraffin sections with a thickness of 10 μm. After the sectioning is completed, use a writing brush to pick up the paraffin sections and flatten them in water at 40 °C, dry them after holding them with a glass slide, cover the glass slide and observe under an Olympus microscope to check the position of the stem cells of the Dioscorea nipponica plant.

[0052] Step 3: Specimen treatment: In the biosafety cabinet, light an alcohol lamp, pass the surgical blade through the flame of the alcohol lamp for disinfection and sterilization. After the temperature of the blade drops, according to the position of the stem cells of the Dioscorea nipponica plant observed in Step 2, use the surgical blade to peel off the plant embryogenic stem cells of the root tip part of the Dioscorea nipponica on the paraffin section and place them in an antioxidant solution containing 1 mol / L of sodium thiosulfate to prevent browning and oxidation. After all the peeling is completed, take out the tissue block containing embryogenic stem cells from the antioxidant solution, cut it into small pieces, transfer it to the MS medium and wash it 3 times, and then transfer it to a triangular flask containing an antibiotic MS medium.

[0053] The antibiotic MS medium is the MS medium added with penicillin, streptomycin and amphotericin B. The addition amounts of penicillin and streptomycin are both 2% of the mass of the MS medium, and the addition amount of amphotericin B is 2 μg per milliliter of the MS medium; there are endogenous bacteria in plants, and in order to make the experiment better operate, penicillin, streptomycin and amphotericin B are added in the experiment.

[0054] Step 4. Shaking culture: Place the Erlenmeyer flask containing Dioscorea nipponica embryonic stem cells in a vertical constant temperature shaker, with a rotation speed of 120 rpm and a temperature of 25 °C, and conduct shaking culture for 36 h. After the culture is completed, first filter with a No. 2 sieve to remove the fragments of the larger root tip part, and then perform secondary filtration separation on the obtained filtrate with a 40-μm cell filter membrane to obtain the embryonic stem cells of Dioscorea nipponica plants;

[0055] Since the aggregation degree of embryonic stem cells is lower and smaller compared with callus, mainly presenting single cells or small cell clumps, therefore, the filtrate can be subjected to secondary filtration separation with a 40-μm cell filter membrane to obtain the embryonic stem cells of Dioscorea nipponica plants;

[0056] Step 5. Subculture and cell preservation: Expand the culture of the Dioscorea nipponica plant embryonic stem cells filtered in Step 4 in a brown sugar MS medium, with a culture rotation speed of 120 rpm and a temperature of 25 °C, and conduct shaking culture; Monitor the growth of Dioscorea nipponica plant embryonic stem cells during the culture process. Before the Dioscorea nipponica plant embryonic stem cells enter the plateau phase of growth, when the cell growth rate is fast and the activity is high, take a part of the medium containing Dioscorea nipponica plant embryonic stem cells, add dimethyl sulfoxide accounting for 10% of its volume, and perform cryopreservation on the Dioscorea nipponica plant embryonic stem cells to obtain cryopreserved Dioscorea nipponica plant embryonic stem cells; Continue the culture, take 10 μL of Dioscorea nipponica stem cells on a disposable cell counting plate, measure the cell number, and when the order of magnitude of the cell number is 10 7 or more, end the subculture of Dioscorea nipponica stem cells to obtain the medium containing Dioscorea nipponica plant embryonic stem cells;

[0057] Step 6. Collection, drying and pulverization: Pour the medium containing Dioscorea nipponica plant embryonic stem cells into a centrifuge bottle, balance it, place it in a centrifuge, with a rotation speed of 2500 rpm and a centrifugation time of 10 minutes. After the centrifugation is completed, discard the supernatant to obtain Dioscorea nipponica plant embryonic stem cells; Perform freeze-drying or microwave drying on the obtained Dioscorea nipponica plant embryonic stem cells. After complete drying, use a small pulverizer for pulverization and sieving to obtain the powder of Dioscorea nipponica plant embryonic stem cells.

[0058] For the brown sugar MS medium, the mass ratio of brown sugar to the mass of MS medium powder is 1:3; Penicillin and streptomycin are added to the brown sugar MS medium, and the addition amounts of penicillin and streptomycin are both 2% of the mass of the MS medium;

[0059] The prepared cryopreserved Dioscorea nipponica plant embryonic stem cells can be applied to cell resuscitation and subculture, subsequent cell experiments, etc.;

[0060] The prepared powder of Dioscorea nipponica plant embryogenic stem cells can be applied in the fields of agriculture, food, health products, pharmaceuticals, etc., such as the following two applications;

[0061] A health product composition containing Dioscorea nipponica plant embryogenic stem cells, including the following components in mass ratio: Dioscorea nipponica plant embryogenic stem cells 30%, lentinus edodes powder 20%, pumpkin seed powder 20%, barley powder 12%, pea powder 5%, lotus root powder 5%, ginseng powder 2%, soy lecithin 2%, papain 2% and red bean powder 2%. This composition has the effect of reducing blood lipid.

[0062] A compound nutritional noodle containing Dioscorea nipponica embryogenic stem cell powder. In every 100g of flour, the addition amount of Dioscorea nipponica embryogenic stem cell powder is 40% of the flour mass, the addition amount of carrot is 10% of the flour mass, the addition amount of beef is 15% of the flour mass, the addition amount of skim milk is 8% of the flour mass, and the addition amount of salt is 2% of the flour mass. After mixing evenly, it is pressed.

[0063] Example 3

[0064] A cultivation method of Dioscorea bulbifera plant embryogenic stem cells, including the following steps:

[0065] Step 1. Specimen pretreatment: Clean and disinfect the collected Dioscorea bulbifera plant body;

[0066] The method for cleaning and disinfecting the collected Dioscorea bulbifera plant body is as follows: Wash the Dioscorea bulbifera plant body with running tap water, and gently brush some key parts or parts that are not easy to clean with a soft brush to brush off the attached soil or other attachments. Transfer the washed plant to a biosafety cabinet and continue cleaning and disinfecting. Wash the surface of the prepared plant with an ethanol aqueous solution with a volume percentage of 75% for 80 seconds. After washing, pour out the ethanol aqueous solution with a volume percentage of 75%, wash the plant 3 times with sterile water, then soak it in an aqueous solution containing HgCl2 with a mass concentration of 0.3% for 6 minutes. After the disinfection is completed, discard the disinfectant solution and wash the plant with sterilized water for 3 times.

[0067] Step 2. Pre-experiment of tissue section: Cut off the root tips of the Dioscorea bulbifera L. plant bodies processed in Step 1, and cut them into sections with a thickness less than 0.5 mm for standby; After melting the paraffin with an alcohol lamp, pour it into an embedding cassette. At the same time, transfer the cut sections into the embedding cassette, and continue to add the melted paraffin until the embedding cassette is completely filled; After the paraffin is completely solidified, obtain the embedded paraffin sections; Use a microtome to section the embedded paraffin sections with a thickness of 5 μm. After the sectioning is completed, use a brush to pick up the paraffin sections and flatten them in water at 40 °C. After drying them with a glass slide, cover the glass slide and observe under an Olympus microscope to check the position of the stem cells of the Dioscorea bulbifera L. plant;

[0068] Step 3. Specimen treatment: In a biosafety cabinet, light an alcohol lamp, pass the surgical blade over the flame of the alcohol lamp for disinfection and sterilization. After the temperature of the blade drops, according to the position of the stem cells of the Dioscorea bulbifera L. plant observed in Step 2, use the surgical blade to peel off the plant embryonic stem cells of the root tip part of the Dioscorea bulbifera L. on the paraffin section, and place them in an antioxidant solution of 1 mol / L sodium thiosulfate to prevent browning and oxidation. After all the peeling is completed, take out the tissue block containing embryonic stem cells from the antioxidant solution, cut it into small pieces, transfer it to the MS medium and rinse it 3 times, and then transfer it to a triangular flask containing the antibiotic MS medium;

[0069] The antibiotic MS medium is to add penicillin, streptomycin and amphotericin B to the MS medium. The addition amounts of penicillin and streptomycin are both 1% of the mass of the MS medium, and the addition amount of amphotericin B is 1 μg per milliliter of the MS medium; There are endogenous bacteria in plants, and in order to better carry out the experiment, penicillin, streptomycin and amphotericin B are added in the experiment.

[0070] Step 4. Shaking culture: Place the triangular flask containing the embryonic stem cells of Dioscorea bulbifera L. in a vertical constant temperature oscillator with a rotation speed of 100 rpm and a temperature of 22 °C for shaking culture for 30 h. After the culture is completed, first filter it with a No. 2 sieve to remove the larger fragments of the root tip part, and then filter and separate the obtained filtrate with a 40-μm cell filter membrane to obtain the embryonic stem cells of the Dioscorea bulbifera L. plant;

[0071] Since the aggregation degree of embryonic stem cells is lower and smaller compared with callus, mainly presenting single cells or small cell aggregates, therefore, the filtrate can be filtered and separated with a 40-μm cell filter membrane to obtain the embryonic stem cells of the Dioscorea bulbifera L. plant;

[0072] Step 5. Subculture and cell preservation: Expand the Dioscorea bulbifera L. plant embryogenic stem cells obtained by filtration in Step 4 in a brown sugar MS medium. The culture rotation speed is 100 rpm, the temperature is 22 °C, and shake flask culture is carried out. Monitor the growth of Dioscorea bulbifera L. plant embryogenic stem cells during the culture process. Before the growth of Dioscorea bulbifera L. plant embryogenic stem cells enters the plateau phase, when the cell growth rate is fast and the activity is high, take a part of the medium containing Dioscorea bulbifera L. plant embryogenic stem cells, add dimethyl sulfoxide accounting for 8% of its volume, and cryopreserve the Dioscorea bulbifera L. plant embryogenic stem cells to obtain cryopreserved Dioscorea bulbifera L. plant embryogenic stem cells. Continue the culture, take 10 μL of Dioscorea bulbifera L. stem cells on a disposable cell counting plate, measure the cell number, and when the order of magnitude of the cell number is 7 above, end the subculture of Dioscorea bulbifera L. stem cells to obtain a medium containing Dioscorea bulbifera L. plant embryogenic stem cells;

[0073] Step 6. Collection, drying and pulverization: Pour the medium containing Dioscorea bulbifera L. plant embryogenic stem cells into a centrifuge bottle, balance it, place it in a centrifuge, with a rotation speed of 2000 rpm and a centrifugation time of 8 minutes. After centrifugation, discard the supernatant to obtain Dioscorea bulbifera L. plant embryogenic stem cells. Freeze-dry or microwave-dry the obtained Dioscorea bulbifera L. plant embryogenic stem cells. After complete drying, use a small pulverizer to pulverize and sieve to obtain the powder of Dioscorea bulbifera L. plant embryogenic stem cells.

[0074] For the brown sugar MS medium, the mass ratio of brown sugar to MS medium powder is 2:1; penicillin and streptomycin are added to the brown sugar MS medium, and the addition amounts of penicillin and streptomycin are both 1% of the mass of the MS medium;

[0075] The prepared cryopreserved Dioscorea bulbifera L. plant embryogenic stem cells can be applied to cell resuscitation and subculture, subsequent cell experiments, etc.;

[0076] The prepared powder of Dioscorea bulbifera L. plant embryogenic stem cells can be applied to fields such as agriculture, food, health products, pharmaceuticals, etc., for example, the following two applications;

[0077] A composition of a health product containing Dioscorea bulbifera L. plant embryogenic stem cells, including the following components in mass ratio: Dioscorea bulbifera L. plant embryogenic stem cells 22%, mushroom powder 25%, pumpkin seed powder 15%, barley powder 15%, pea powder 5%, lotus root powder 10%, ginseng powder 2%, soybean phospholipid 2%, papain 2% and red bean powder 2%. This composition has the effect of reducing blood lipid.

[0078] A composite nutritional noodle containing the powder of Dioscorea bulbifera L. embryogenic stem cells. In every 100 g of flour, the addition amount of Dioscorea bulbifera L. embryogenic stem cell powder is 20% of the mass of the flour, the addition amount of carrot is 8% of the mass of the flour, the addition amount of beef is 12% of the mass of the flour, the addition amount of skim milk is 5% of the mass of the flour, and the addition amount of salt is 1% of the mass of the flour. After mixing evenly, press.

[0079] Experiment 1. Effects of different brown sugar MS media and different culture times on the yield of embryonic stem cells:

[0080] During the experiment, it was found that under certain conditions, if sufficient nutrients were given to the stem cells of Dioscoreaceae, their growth rate was relatively fast, and they could even grow exponentially. However, the price of the medium might be a prominent bottleneck restricting the development of the plant stem cell industry. Therefore, we considered using some inexpensive and nutrient-rich substances to replace part of the medium.

[0081] Brown sugar is a traditional sugar product with a consumption habit of over a thousand years in China. It is inexpensive and mainly prepared by simple processing of sugarcane or beets. The main component of brown sugar is sucrose, and it also contains various mineral elements such as K, Ca, P, Fe, Zn, Mg, Cu, etc., as well as a large number of amino acids such as lysine, threonine, valine, etc., and also contains various vitamins such as carotene and higher carbon plant alcohols. Therefore, we believe that brown sugar can be used as a partial substitute for MS medium, and relevant experiments were carried out.

[0082] Prepare No. 1 MS medium according to the formula of MS medium. Weigh 17.23 g of MS medium powder and 17.22 g of brown sugar to prepare No. 2 semi-MS semi-brown sugar medium. Weigh 8.61 g of MS medium powder and 25.84 g of brown sugar to prepare No. 3 1 / 4MS + 3 / 4 brown sugar medium. Weigh 4.31 g of MS medium powder and 30.14 g of brown sugar to prepare No. 4 1 / 8MS + 7 / 8 brown sugar medium. Take 20 mL of Chinese yam stem cell culture solution from the same shaken Erlenmeyer flask and add it to 3 280-mL No. 1 MS culture solutions, 3 280-mL No. 2 semi-MS semi-brown sugar culture solutions, 3 280-mL No. 3 1 / 4MS + 3 / 4 brown sugar culture solutions, and 3 280-mL No. 4 1 / 8MS + 7 / 8 brown sugar culture solutions respectively. Expand the culture according to the above method. After culturing for 24 h, 48 h, and 72 h respectively, centrifuge to collect Chinese yam stem cells and weigh to obtain the wet weight of Chinese yam stem cells. (See Figure 2 shown)

[0083] Table 1 Weights (wet weight) of stem cells obtained with different media and different culture times (g)

[0084]

[0085] From the relevant experimental data in Table 1, it can be concluded that after culturing with the culture media No. 1-4 for 72 hours, the wet weight of the cells will increase significantly, indicating that 72 hours of culture time is better. At the same time, the increase in the No. 3 culture medium (1 / 4MS + 3 / 4 brown sugar culture medium) is more, indicating that brown sugar can be used to partially replace the MS culture medium, and the wet weight of the obtained stem cells may even be more than that of the full MS culture medium. The rich nutritional components in brown sugar improve the cell growth amount of the cells and can greatly save costs.

[0086] Experiment 2: Animal function experiment. The Dioscorea opposita Thunb. embryogenic stem cells prepared in Example 1 of this application were used to obtain the powder of Dioscorea opposita Thunb. plant embryogenic stem cells, and then the animal function test was carried out according to the provisions of the "Technical Specification for Health Food Inspection and Evaluation (2003 Edition)" of the Ministry of Health.

[0087] Adaptation period: Wistar rats were purchased and a hyperlipidemia animal model was established for relevant research. They were normally fed in the animal breeding room for 1 week to adapt to the environment, and were allowed to freely eat and drink water.

[0088] Model establishment period: They were randomly divided into 5 groups according to body weight. Among them, 10 rats were used as the blank control group and were normally fed, and were always given the rat maintenance feed purchased from the market. The remaining 40 rats were given the model feed as the model group. After the model group was given the model feed for 2 weeks, the rats in the blank control group and the model group were bled from the tail without fasting. After blood collection, the serum was separated as soon as possible, and the levels of serum TC, TG, LDL-C, and HDL-C were measured.

[0089] The model feed is the maintenance feed purchased and added with 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, appropriate amounts of casein, calcium hydrogen phosphate, and stone powder.

[0090] Administration of the test sample: According to the TC level, the 40 rats in the model group were randomly divided into 4 groups, namely the model control group, the positive control group, the Dioscorea opposita Thunb. embryogenic stem cell group, and the health care sample group. There were no significant differences in TC, TG, LDL-C, and HDL-C among the 4 groups. After grouping, the Dioscorea opposita Thunb. embryogenic stem cell group and the health care sample group were given the test sample orally every day, the positive control group was given simvastatin tablets, the blank control group and the model control group were given the same volume of water, the blank control group continued to be given the maintenance feed, and the model control group, the positive control group, the Dioscorea opposita Thunb. embryogenic stem cell group, and the health care sample group continued to be given the model feed, and the body weight was measured regularly.

[0091] Index observation: At the end of the experiment, blood was collected without fasting. After blood collection, the serum was separated as soon as possible, and the levels of serum TC, TG, LDL-C, and HDL-C were measured.

[0092] The gavage dose of the positive control group (simvastatin tablets) was 10 mg / kg.bw, and the gavage doses of the Dioscorea opposita embryogenic stem cell group and the health care sample group (Dioscorea opposita embryogenic stem cell composition) were 20 mg / kg.bw. The specific results are shown in Table 2 as follows:

[0093] Table 2 Effects of Dioscorea opposita embryogenic stem cell composition health care products on hyperlipidemia animal models

[0094]

[0095]

[0096] ▲p < 0.05, ▲▲p < 0.01;

[0097] According to the results in Table 2, after establishing a hyperlipidemia animal model, rats were fed simvastatin, Dioscorea opposita embryogenic stem cells or a health care product composition of Dioscorea opposita embryogenic stem cells for four weeks. Compared with the model control group, both the Dioscorea opposita embryogenic stem cell group and the positive control group (simvastatin) could extremely significantly inhibit the increase in the contents of TC, TG, and LDL-C (p < 0.01), and extremely significantly inhibit the decrease in the content of HDL-C in the serum (p < 0.01), making their contents close to the normal level. For the health care sample group (the health care product composition of Dioscorea opposita embryogenic stem cells), it could significantly inhibit the increase in the content of TC (p < 0.05), extremely significantly inhibit the increase in the contents of TG and LDL-C (p < 0.01), and extremely significantly inhibit the decrease in the content of HDL-C in the serum (p < 0.01), indicating that both Dioscorea opposita embryogenic stem cells and the health care product composition containing Dioscorea opposita embryogenic stem cells have good effects on reducing blood lipids.

[0098] Unless otherwise specified, the test methods used in the present invention are all conventional methods; the materials, reagents, etc. used can all be obtained through commercial channels.

Claims

1. A method for culturing plant embryonic stem cells of Dioscoreaceae, characterized in that: It includes the following steps: Step 1, specimen pretreatment: Clean and disinfect the obtained Dioscoreaceae plant body. Step 2, tissue section pre-experiment: Cut off the root tip of the Dioscoreaceae plant body processed in Step 1, cut it into sections with a thickness less than 0.5 mm, and make embedded paraffin sections; Cut the embedded paraffin sections into sections with a thickness of 1-10 μm. After the sectioning is completed, observe under a microscope to check the position of the stem cells of the Dioscoreaceae plant. Step 3, specimen treatment: In a biosafety cabinet, light an alcohol lamp, pass the surgical blade over the flame of the alcohol lamp for disinfection and sterilization. After the temperature of the blade drops, according to the position of the stem cells of the Dioscoreaceae plant observed in Step 2, use the surgical blade to peel off the embryonic stem cells of the root tip part of the Dioscoreaceae on the paraffin section, and place them in an antioxidant solution containing sodium thiosulfate with a concentration of 1 mol / L. After all the peeling is completed, take out the tissue block containing embryonic stem cells from the antioxidant solution, cut it into small pieces, transfer it to an MS medium for rinsing 2-3 times, and then transfer it to an Erlenmeyer flask containing an antibiotic MS medium. The said antibiotic MS medium is to add penicillin, streptomycin and amphotericin B to the MS medium, where the addition amounts of penicillin and streptomycin are both 0.5-2% of the mass of the MS medium, and the addition amount of amphotericin B is 0.1-2 μg per milliliter of the MS medium. Step 4, shaker culture: Place the Erlenmeyer flask containing the embryonic stem cells of the Dioscoreaceae in a vertical constant temperature oscillator, with a rotation speed of 80-120 rpm and a temperature of 20-25 °C, for shaker culture. The culture time is 24-36 h. After the culture is completed, first filter with a No. 2 sieve to remove the larger fragments of the root tip part, and then filter and separate the obtained filtrate with a 40-μm cell filter membrane to obtain the embryonic stem cells of the Dioscoreaceae plant. Step 5. Subculture and cell preservation: Expand the Dioscoreaceae plant embryogenic stem cells obtained by filtration in Step 4 in a brown sugar MS medium at a culture rotation speed of 80 - 120 rpm and a temperature of 20 - 25°C; Monitor the growth of the Dioscoreaceae plant embryogenic stem cells during the culture process. When the cell growth rate is fast and the cell activity is high, cryopreserve the Dioscoreaceae plant embryogenic stem cells to obtain cryopreserved Dioscoreaceae plant embryogenic stem cells; Continue the culture. Take 10 μL of Dioscoreaceae stem cells on a disposable cell counting plate to measure the cell number. When the order of magnitude of the cell number is 10 7 or more, end the subculture of the Dioscoreaceae stem cells to obtain a medium containing Dioscoreaceae plant embryogenic stem cells; Step 6, collection, drying and pulverization: Pour the medium containing the embryonic stem cells of the Dioscoreaceae plant into a centrifuge bottle, balance it, place it in a centrifuge, with a rotation speed of 1500-2500 rpm and a centrifugation time of 5-10 minutes. After the centrifugation is completed, discard the supernatant to obtain the embryonic stem cells of the Dioscoreaceae plant; Freeze-dry or microwave-dry the obtained embryonic stem cells of the Dioscoreaceae plant. After complete drying, pulverize and sieve to obtain the powder of the embryonic stem cells of the Dioscoreaceae plant. In the said brown sugar MS medium, the mass ratio of brown sugar to MS medium powder is 3:1 to 1:3, and the addition amounts of penicillin and streptomycin are both 0.5%-2% of the mass of the MS medium.

2. The culture method of the plant embryonic stem cells of Dioscoreaceae according to claim 1, characterized in that: The method for cleaning and disinfecting the collected plant body in Step 1 is as follows: Wash the collected Dioscoreaceae plant body with running tap water, and gently brush the key parts with a soft brush to remove soil and attachments;; Transfer the washed Dioscoreaceae plant to a biosafety cabinet for continuous cleaning and disinfection. Wash the surface of the Dioscoreaceae plant with an ethanol aqueous solution with a volume percentage of 75% for 20 - 120 seconds. After the washing is completed, pour out the ethanol aqueous solution with a volume percentage of 75%, wash the plant 3 times with sterile water, then soak it in an HgCl2 aqueous solution with a mass concentration of 0.1% - 0.5% for 2 - 10 minutes. After the disinfection is completed, discard the disinfectant solution and wash the plant with sterile water 3 times.

3. The method for culturing embryonic stem cells of Dioscoreaceae plants as described in claim 1, characterized in that: The operation method of the tissue section pre-experiment described in Step 2 is as follows: Cut off the root tip of the Dioscoreaceae plant body treated in Step 1 and cut it into sections with a thickness less than 0.5 mm for standby; After melting the paraffin with an alcohol lamp, pour it into an embedding cassette. At the same time, transfer the cut sections into the embedding cassette and continue to add melted paraffin until the embedding cassette is completely filled; After the paraffin is completely solidified, obtain the embedded paraffin sections; Use a microtome to section the embedded paraffin sections with a thickness of 1 - 10 μm. After the sectioning is completed, use a brush to pick up the paraffin sections and flatten them in water at 40°C. After drying them with a glass slide, cover them with a glass slide and observe them under an Olympus microscope to determine the position of the stem cells of Dioscoreaceae plants.

4. The culture method of plant embryonic stem cells of Dioscoreaceae according to claim 1, characterized in that: For the described MS medium, the components and addition amounts added per liter of the medium are: potassium nitrate 1900 mg, ammonium nitrate 1650 mg, potassium dihydrogen phosphate 170 mg, magnesium sulfate 370 mg, calcium chloride 440 mg, potassium iodide 0.83 mg, boric acid 6.2 mg, manganese sulfate 22.3 mg, zinc sulfate 8.6 mg, copper sulfate 0.025 mg, sodium molybdate 0.25 mg, cobalt chloride 0.025 mg, inositol 100 mg, glycine 2 mg, thiamine hydrochloride (VB1) 0.1 mg, pyridoxine hydrochloride (VB6) 0.5 mg, nicotinic acid (VB5) 0.5 mg, sucrose 30000 mg, 6-benzylaminopurine (6-BA) 1 mg, naphthaleneacetic acid (NAA) 1 mg, ferrous sulfate 27.8 mg, disodium ethylenediaminetetraacetate 37.3 mg, and the balance is water.

5. The culture method of plant embryonic stem cells of Dioscoreaceae according to claim 1, characterized in that: The method for cryopreserving embryonic stem cells of Dioscoreaceae plants: When the growth rate of embryonic stem cells of Dioscoreaceae plants is fast and their activity is high, take the medium containing embryonic stem cells of Dioscoreaceae plants, add dimethyl sulfoxide with a volume of 5 - 10% of its volume, and cryopreserve the embryonic stem cells of Dioscoreaceae plants to obtain cryopreserved embryonic stem cells of Dioscoreaceae plants.

6. The method for culturing plant embryonic stem cells of Dioscoreaceae according to any one of claims 1-5, characterized in that: The described Dioscoreaceae plants are Chinese yam, Dioscorea nipponica or Dioscorea bulbifera.

7. The application of the embryonic stem cells of Dioscoreaceae plants prepared as described in any one of claims 1 - 5 in health products.

8. Use of the Dioscoreaceae plant embryonic stem cells prepared according to claim 7 in health products, characterized in that: A health care product composition containing Dioscoreaceae plant embryonic stem cells, comprising the following components in mass ratio: 20-30% of Dioscoreaceae plant embryonic stem cells, 20-30% of lentinus edodes powder, 15-20% of pumpkin seed powder, 10-15% of barley powder, 5-10% of pea powder, 5-10% of lotus root powder, 2-5% of ginseng powder, 2-5% of soy lecithin, 2-5% of papain, and 2-5% of adzuki bean powder.

9. Use of Dioscoreaceae plant embryonic stem cells prepared as claimed in any one of claims 1-5 in food.

10. The application of Dioscoreaceae plant embryonic stem cells prepared as claimed in claim 9 in health products, characterized in that: In every 100g of flour, the addition amount of yam embryonic stem cell powder is 10-40% of the flour mass, the addition amount of carrot is 5-10% of the flour mass, the addition amount of beef is 10-15% of the flour mass, the addition amount of skim milk is 1-8% of the flour mass, and the addition amount of salt is 0.5-2% of the flour mass. After mixing evenly and pressing, a composite nutritious noodle containing yam embryonic stem cell powder is obtained.

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