A method for separating, differentiating and culturing rehmannia stem cells

Abstract

The present invention belongs to the field of plant stem cell culture, and discloses a method for separating and differentiating Rehmannia stem cells. The method comprises the following steps: S1, cutting the tuberous root or stem of a Rehmannia plant, sterilizing and slicing, sterilizing again and then washing with sterile water to obtain an explant sheet; S2, transferring the explant sheet to a preservation solution containing plant hormones for immersion, and then transferring it to an induction culture medium for culture, inducing to obtain Rehmannia stem cells, dedifferentiated cells and adventitious roots respectively, and then culturing them separately through subculture containing plant hormones to obtain stably growing Rehmannia stem cells, dedifferentiated cells and adventitious roots, and then suspending the culture. The present invention establishes a highly efficient induction and long-term stable in vitro culture method for Rehmannia stem cells, dedifferentiated cells and adventitious roots, and at the same time establishes a method for producing catalpol, verbascoside, and rehmannia glycoside A / D cycloterpenoid compounds using the above culture system.

Description

Technical Field

[0001] The invention belongs to the technical field of plant stem cell culture, and relates to a method for separating and differentiating Rehmannia glutinosa stem cells, and specifically to a method for efficiently inducing and long-term stable in vitro culture of Rehmannia glutinosa plant stem cells, dedifferentiated cells and adventitious roots. Background Art

[0002] At present, after nearly 70 years of development, the development of plant biotechnology has made remarkable progress, mainly used in plant seed preservation, breeding, propagation and the production of active metabolites by in vitro culture of plant tissues (callus, hairy roots, adventitious buds, etc.). Because plant secondary metabolites are one of the main sources of pharmacologically active substances and drug discovery, their market application potential and value are very broad, so scientists and commercial companies have established in vitro plant tissue culture systems for more than 400 plants, and screened out stable and high-yield cell lines for the production of important active secondary metabolites.

[0003] Plant secondary metabolites are small molecule organic compounds that are not essential for plant development and have great medicinal value. Compared with microorganisms, plant genomes are more complex. In plants, the synthesis of a compound often requires the participation and transport of different types of cells. The complex spatial characteristics of plants provide the optimal environment required for the synthesis of different metabolites. In recent years, due to the annual increase in the price of medicinal plants and the high cost of raw materials, more and more researchers have turned their attention to low-cost and high-yield research based on cell biology and molecular biology. There are mainly the following methods: using plant tissues, cell culture, hairy root culture and plant cell bioreactors. Among them, using cells as "living factories" to mass-produce secondary metabolites is the best method.

[0004] There are many difficulties in the cultivation of plant cells, tissues and organs, such as the browning of cells, infection by external bacteria, the influence of external factors on cells such as the supply of oxygen and nutrients. In addition, the genes of cells usually change after the callus tissue is formed through dedifferentiation, which leads to the inability of certain enzyme-catalyzed reactions and the inability to synthesize secondary metabolites. Therefore, the key to solving the above problems is to establish genetically stable plant cells or tissues that can efficiently synthesize target secondary metabolites.

[0005] To solve the problems that arise in plant cell culture in industrial production, such as low yield of secondary metabolites caused by dedifferentiation and gene mutations that occur during the culture process. Plant stem cells mainly exist in the apical meristem and lateral meristem, and their self-valorization and differentiation pluripotency provide different types of cell sources for plant growth. In recent years, scientists have been committed to culturing stem cells from plant stem tips, root tip meristems or vascular cambium in vitro to keep plants alive and well. With the development of plant biological technology, the study of plant stem cells has become a hot topic in current research. At present, researchers use plants to separate cells from the cambium to obtain stem cells, which have stable genetic information, fast growth rate, many types of secondary metabolism and high content, and a large number of vacuoles in stem cells can make cells less sensitive to shear force and less aggregated. The above advantages make plant stem cells more suitable for large-scale bioreactor culture. At present, plant stem cell culture technology has achieved some success. For example, researchers in South Korea and the United Kingdom have invented methods for the efficient and low-cost production of paclitaxel using yew stem cells and the production of ginsenosides using ginseng stem cells. In summary, in the next few decades, humans will be able to use plant stem cell culture technology to produce active medicinal secondary metabolites and other commercial compounds on a large scale.

[0006] Plants contain a large number of endophytes, which have formed a stable mutually beneficial symbiotic relationship with plants during the long-term co-evolution process, such as helping plants to prevent and control pests and diseases, antagonizing pathogens, increasing emergency tolerance and promoting the biosynthesis of secondary metabolites. At present, researchers have found more than 100 endophytes in Rehmannia glutinosa, mainly bacteria, such as Pseudomonas and Bacillus, followed by fungi-actinomycetes. Due to the large number of endophytes in Rehmannia glutinosa, the infection rate during explant induction is extremely high, which ultimately leads to the failure of induction to establish cell lines. Therefore, it is very difficult to use tubers as explant sources to establish stem cell, dedifferentiated cell (callus) or adventitious root culture system. In addition, the existing plant stem cell separation and culture technology cannot effectively solve the problem of infection, and cannot efficiently induce and establish in vitro cultured cambium stem cells, dedifferentiated cells and adventitious root culture systems, indicating that the existing plant cambium stem cell separation and culture technology needs to be further improved. Summary of the invention

[0007] The purpose of the present invention is to establish a highly efficient induction and long-term stable in vitro culture method for plant stem cells, dedifferentiated cells and adventitious roots derived from Rehmannia root explants in view of the above-mentioned existing problems. The three culture systems obtained can produce catalpol, verbascoside, rehmannia glycoside A / D and other iridoid terpenoid compounds. The culture system established by the present invention, especially the Rehmannia stem cell culture system, is expected to realize the large-scale culture of Rehmannia stem cells to produce catalpol and other hypoglycemic drugs.

[0008] In order to achieve the above objectives, this application adopts the following technical solutions.

[0009] A method for isolating, differentiating and culturing Rehmannia glutinosa stem cells, characterized in that it comprises the following steps:

[0010] S1. Explant sterilization: cutting tubers or stems of Rehmannia plants, washing and sterilizing to obtain yellow tuber or stem explants, slicing, sterilizing again and then washing to obtain explant slices;

[0011] S2, induction in vitro culture: the explant pieces obtained in S1 are transferred to a preservation solution containing plant hormones for immersion, and then transferred to an induction medium for culture, respectively, to induce the acquisition of Rehmannia stem cells, dedifferentiated cells and adventitious roots, and then the stably growing Rehmannia stem cells, dedifferentiated cells and adventitious roots are obtained through subculture containing plant hormones, and then suspension culture is performed;

[0012] The preservation solution contains 10-30 g / L sucrose, 100-300 mg / L citric acid, 50-500 U / mL penicillin, and 50-500 mg / mL streptomycin in MS liquid culture medium, and the plant hormone is selected from one or more of 5 μmol / L tricholoma lactone solution, 5 μmol / L brassinolide solution, and 5 mg / L NAA solution.

[0013] Preferably, in S1, the length of the cut segment is 5 to 8 cm, the sterilization treatment method is water rinsing for 1 to 3 hours, then sterilized with 8% hydrogen peroxide for 4 minutes, 75% ethanol solution for 3 minutes, 0.1% mercuric chloride solution for 10 to 20 minutes, soaked in sterile water for 1 to 4 minutes, and then rinsed with sterile water; the slices are circular slices with a thickness of 0.1 to 2 mm.

[0014] Preferably, in S2, the immersion time in the preservation solution is 1 to 5 minutes.

[0015] Preferably, in S2, the induction medium is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum, 0.1-0.5% activated carbon, and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, the plant hormones are selected from one or more of NAA, IAA, 2,4-D, and 6-BA, and the induction medium contains 2-2.6 mg / L methylchloroisothiazolinone, 0.6-0.8 mg / L methylisothiazolinone, and 15-25 mg / L sodium benzoate, a plant antibacterial agent.

[0016] Preferably, in S2, the subculture medium is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the plant hormones are selected from one or more of NAA, IAA, 2,4-D, and 6-BA.

[0017] Preferably, in S2, the culture conditions for the induction culture and subculture are: temperature 25-30°C, humidity 60-80%, and dark culture for 24 hours.

[0018] Preferably, in S2, the method for suspension culture of Rehmannia stem cells, dedifferentiated cells and adventitious roots is as follows:

[0019] Rehmannia stem cells, dedifferentiated cells and adventitious roots were inoculated into bioreactors containing suspension culture medium, respectively, and cultured continuously for 15 to 20 days in darkness. The inoculation amount of Rehmannia stem cells, dedifferentiated cells and adventitious roots was 50 to 100 g / L, respectively.

[0020] Preferably, the suspension culture medium is MS culture medium, which is an MS basal culture medium containing 10-30 g / L sucrose and 1.0-3.0 mg / L plant hormones, and the plant hormones are selected from one or more of NAA, IAA, 2,4-D, and 6-BA.

[0021] Preferably, the culture conditions of the suspension culture are: temperature of 25-30°C, rotation speed of 100-130rpm, 24h dark culture, ventilation ratio of 1:0.3-0.6, dissolved oxygen content of 0.20-0.50, and pH adjusted to 5.5-6.0.

[0022] Preferably, the Rehmannia plants include but are not limited to Rehmannia chingii Li, Rehmannia elata NEBrown, Rehmannia glutinosa (Gaetn.) Libosch.ex Fisch.etMey., Rehmannia henryiN.E.Brown, Rehmannia piasezkii Maxim, and Rehmannia solanifolia Tsoong et Chin.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention establishes a highly efficient induction and long-term stable in vitro culture method for plant stem cells, dedifferentiated cells and adventitious roots derived from Rehmannia root tuber explants. The plant stem cells, dedifferentiated cells and adventitious root systems are all suitable for suspension culture. The stem cells exist as dispersed single cells or small cell clusters; the dedifferentiated cells grow in large cell clusters; and the adventitious roots maintain a root-like organ morphology. Experimental results show that the growth rates of Rehmannia stem cells, dedifferentiated cells and adventitious roots are 1080%, 815% and 589%, respectively. The growth rate of Rehmannia stem cells is the best, followed by dedifferentiated cells, and the lowest is adventitious roots, which are more suitable for large-scale culture in bioreactors.

[0025] (2) The present invention sterilizes the root tuber explants of Rehmannia glutinosa multiple times to effectively reduce the infection rate, and soaks them in an anti-browning preservation solution containing plant hormones to effectively increase the induction success rate of Rehmannia glutinosa stem cells, and the plant defense mechanism-related factors NO and Ca 2+ , H 2 O 2 The contents of CAT, POD in the cambium stem cells of Rehmannia glutinosa are 23, 2.8, 6.7, 2 and 1.3 times of those in the dedifferentiated cells, respectively. The three culture systems established contain relatively high contents of catalpol, verbascoside, rehmannia glutinosa A / D and other cyclopentane compounds. The culture system established by the present invention, especially the Rehmannia glutinosa stem cell culture system, is expected to realize the large-scale cultivation of Rehmannia glutinosa stem cells to produce catalpol and other hypoglycemic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the success rate of induction of Rehmannia glutinosa explants after brassinolide treatment in Example 1 of the present invention;

[0027] Figure 2 Example 1 of the present invention, the cell or tissue diagram obtained by inducing the root tubers of Rehmannia glutinosa;

[0028] Figure 3 Microscopic observation of Rehmannia glutinosa cells in Example 1 of the present invention;

[0029] Figure 4 Microscopic observation of cells after neutral red staining in Example 1 of the present invention;

[0030] Figure 5 Sensitivity diagram of Rehmannia glutinosa cells after bleomycin treatment in Example 1 of the present invention;

[0031] Figure 6 A graph showing the content of defense mechanism factors in dedifferentiated cells and stem cells of Rehmannia glutinosa according to Example 1 of the present invention;

[0032] Figure 7 A graph showing the success rate of induction of Rehmannia glutinosa explants after treatment with strigolactones in Example 2 of the present invention;

[0033] Figure 8 Graph showing the success rate of induction of cells or tissues in Rehmannia glutinosa slices after treatment with strigolactone and brassinolide in Example 3 of the present invention;

[0034] Fig. 9 A graph showing the growth rates of Rehmannia glutinosa stem cells, dedifferentiated cells and adventitious roots in suspension culture in Example 4 of the present invention;

[0035] Fig.10 Contents of catalpol, verbascoside and rehmannoside A / D in stem cells, dedifferentiated cells and adventitious roots of Rehmannia glutinosa according to Example 4 of the present invention

[0036] Fig.11 A graph showing the contents of catalpol, verbascoside and rehmannoside A / D in stem cells, dedifferentiated cells and adventitious roots of Rehmannia glutinosa in Example 5 of the present invention. DETAILED DESCRIPTION

[0037] The following will combine the data in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0039] Example 1

[0040] A method for separating, differentiating and culturing Rehmannia glutinosa stem cells comprises the following steps:

[0041] S1. Explant sterilization: In the early morning of March to May in spring, fresh wild Rehmannia plants from June to December were picked, the root tubers were cut into 5-6 cm small sections, washed with tap water for 3 hours, and then the surface water was sucked off;

[0042] The explant root tubers were placed in a sterile clean bench, sterilized with 8% hydrogen peroxide for 4 minutes, 75% ethanol solution for 2 minutes, 0.1% mercuric chloride solution for 15 minutes, then soaked in sterile distilled water for 3 minutes, and then rinsed with sterile distilled water for 6 times to obtain sterilized Rehmannia root tubers;

[0043] The sterilized Rehmannia root tubers were cut longitudinally into circular slices with a thickness of 0.5 mm, sterilized with 6% hydrogen peroxide for 2 minutes, sterilized with 75% ethanol solution for 0.5 minutes, soaked in sterilized distilled water for 4 minutes, and then rinsed with sterilized distilled water for 3 times to obtain sterilized Rehmannia root tubers circular slices;

[0044] S2, induction of in vitro culture: the explants obtained in S1 were transferred to an anti-browning preservation solution containing plant hormones and soaked for 3 min. The preservation solution was an MS liquid medium containing 30 g / L sucrose, 200 mg / L citric acid, 250 U / mL penicillin, and 250 mg / mL streptomycin. The plant hormone was a 5 μmol / L strigolactone solution.

[0045] The preparation method of the anti-browning preservation solution is as follows: dissolve penicillin, streptomycin, L-ascorbic acid, citric acid and strigolactone respectively with appropriate amount of sterile water and mix well, then filter and sterilize with a 0.22 μm organic filter membrane, and then add to the sterile MS liquid culture medium;

[0046] The basic formula of MS culture medium is: macroelements KNO 3 1900mg / L, NH 4 NO 3 1650mg / L, KH 2 PO 4 170mg / L, MgSO 4 ﹒ 7H 2 O 370mg / L, CaCl 2 ﹒ 2H 2 O 440mg / L; trace elements KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO 4 ﹒ 4H 2 O 22.3mg / L, ZnSO 4 ﹒ 7H 2 O 8.6mg / L, Na 2 MoO 4 ﹒ 2H 2 O 0.25mg / L, CuSO 4 ﹒ 5H 2 O 0.025mg / L, CoCl 2 0.025mg / L; Iron salt: Na 2 EDTA 37.3mg / L, FeSO 4 ﹒ 7H 2 O 27.8mg / L; organic ingredients: inositol 100mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L;

[0047] When preparing MS liquid medium, dissolve and mix with distilled water according to the above formula, adjust the volume, adjust pH to 5.75, sterilize with high pressure steam at 121℃ for 20min, and then cool to obtain MS liquid medium; add 4.0g / L gellan gum to MS liquid medium, sterilize, and cool to obtain MS solid medium (all the following MS medium formulas are the same). The following induction medium, separation medium and subculture medium are solid medium, while the suspension medium uses liquid medium;

[0048] After soaking in the preservation solution, the cells were transferred to three induction culture media A, B, and C and cultured for 20 days. The culture conditions were: temperature 25°C, humidity 70%, and dark culture for 24 hours to obtain Rehmannia stem cells, dedifferentiated cell groups, and adventitious roots.

[0049] The cambium stem cells in the root tubers in the induction medium A divide and grow rapidly, and the cells continue to proliferate to form soft and loose cell clusters. The cell clusters and explant residues are gradually peeled off naturally, and the cambium stem cells cultured in vitro are obtained, which are light yellow-green in color.

[0050] The cambium stem cells in the tuberous roots and the surrounding cells in the induction medium B rapidly divide and differentiate, and as the cells proliferate, pale white granular cell masses can be observed to form, which are dedifferentiated cells (callus);

[0051] The cambium stem cells and the surrounding cells in the root tubers in the induction medium C rapidly divide and differentiate, and as the cells proliferate, adventitious roots can be observed to form, and cell clusters are associated with the roots;

[0052] Induction medium A is MS medium containing 30 g / L sucrose, 4.0 g / L gellan gum, and 0.3% activated carbon, with a medium pH of 5.80, and the added plant hormones are naphthylacetic acid NAA (3.0 mg / L) and 6-benzylaminopurine 6-BA (0.5 mg / L);

[0053] Induction medium B was MS medium containing 30 g / L sucrose, 4.0 g / L gellan gum, and 0.3% activated carbon, with a medium pH of 5.80, and the added plant hormones were NAA (3.0 mg / L) and 2,4-dichlorophenoxyacetic acid 2,4-D (3 mg / L);

[0054] Induction medium C is MS medium containing 30 g / L sucrose, 4.0 g / L gellan gum, and 0.3% activated carbon, with a medium pH of 5.80, and added plant hormones NAA (1.0 mg / L) and 6-BA (0.5 mg / L);

[0055] S3, stabilization and subculture: the cell clusters, tissues or organs obtained in S2 are transferred to a subculture medium containing plant hormones for culture. The subculture conditions are: temperature 25°C, humidity 60%, 24h dark culture, subculture to the subculture medium every 14d for continued culture, and this is repeated for 6 months until stably growing Rehmannia stem cells, dedifferentiated cells and adventitious roots are obtained;

[0056] The subculture medium of Rehmannia stem cells is MS medium containing 30g / L sucrose, 4.0g / L gellan gum and 3.0mg / L plant hormones. The pH value of the medium is 5.80, and the plant hormones are NAA 2mg / L and 6-BA 0.5mg / L.

[0057] The subculture medium of dedifferentiated Rehmannia glutinosa cells is MS medium containing 30 g / L sucrose, 4.0 g / L gellan gum and 3.0 mg / L plant hormones. The pH value of the medium is 5.80. The plant hormones are NAA 1 mg / L and 2,4-D 1 mg / L.

[0058] The subculture medium for Rehmannia glutinosa adventitious roots is MS medium containing 30 g / L sucrose, 4.0 g / L gellan gum and 3.0 mg / L plant hormones. The pH value of the medium is 5.80, and the plant hormones are NAA 1 mg / L and 6-BA 0.5 mg / L.

[0059] Taking the Rehmannia glutinosa stem cell obtained by the method provided in the above Example 1 as an example, its characteristics were identified as follows:

[0060] 1. The following study on the sterilization of Rehmannia root explants by using mercuric chloride

[0061] (1) The explant root tubers were placed in a sterile clean bench and sterilized with 8% hydrogen peroxide for 4 minutes and 75% ethanol solution for 2 minutes. Then, the explant root tubers were sterilized with 0.01%, 0.05%, and 0.10% mercuric chloride solution for 10, 15, or 20 minutes. The root tubers treated with the above different concentrations of mercuric chloride were cut into circular slices (when slicing, the sterile blade was replaced once for each slice). There were 9 groups in total, with 100 slices in each group, and they were placed in the induction culture medium. The infection rate and stem cell induction rate were calculated. The results are shown in Table 1. When 0.1% mercuric chloride was used for sterilization for 15 minutes, the infection rate reached the lowest, which was 60%, and the success rate of stem cell induction reached 15%.

[0062] Table 1 Results of the investigation on the sterilization time of Rehmannia glutinosa root explants by mercuric chloride (n=3)

[0063]

[0064]

[0065] (2) The sterilized Rehmannia root tubers were cut longitudinally into circular slices with a thickness of 0.5 mm (when slicing, the sterile blade was replaced once for each slice), and treated with 3%, 6%, and 9% hydrogen peroxide for 1, 2, and 3 minutes, respectively, and then treated with 75% ethanol for 0.5 minutes. The Rehmannia slices obtained by the above different treatment methods were divided into 9 groups, each with 100 slices, and placed in the induction culture medium, and the infection rate and stem cell induction rate were calculated. The results of the investigation are shown in Table 2. Using 6% hydrogen peroxide, sterilization for 2 minutes, treatment with 75% ethanol for 0.5 minutes, and 0.1% mercuric chloride for 15 minutes, the infection rate was 25%, and the stem cell induction success rate reached 68%.

[0066] Table 2. Results of the study on the sterilization time of Rehmannia glutinosa slice explants by hydrogen peroxide and ethanol (n=3)

[0067]

[0068] (3) Sterilized Rehmannia root tubers were cut longitudinally into circular slices with a thickness of 0.5 mm (the sterile blade was replaced every time a slice was cut), and the slices were treated with 6% hydrogen peroxide for 2 min, 75% ethanol for 0.5 min, and mercuric chloride for 15 min. The Rehmannia slices obtained by the above-mentioned different treatment methods were divided into 4 groups, each with 100 slices, and placed in an induction medium to which methylchloroisothiazolinone, methylisothiazolinone, and benzyl alcohol were added. Sodium benzoate was added and divided into the following groups: 1) blank group, no addition; 2) 2mg / L methylchloroisothiazolinone + 0.6mg / L methylisothiazolinone + 15mg / L sodium benzoate; 3) 2.3mg / L methylchloroisothiazolinone + 0.7mg / L methylisothiazolinone + 20mg / L sodium benzoate; 4) 2.6mg / L methylchloroisothiazolinone + 0.8mg / L methylisothiazolinone + 25mg / L sodium benzoate, and the infection rate and stem cell induction rate were calculated. The results of the investigation are shown in Table 3. Using 2.3mg / L methylchloroisothiazolinone, 0.7mg / L methylisothiazolinone, and 20mg / L sodium benzoate, the infection rate was 15%, and the success rate of stem cell induction reached 87%.

[0069] Table 3. Effects of plant antimicrobial agents on the infection rate and induction rate of explants during induction (n=3)

[0070]

[0071] 2. Investigate the induction success rate of Rehmannia glutinosa slices after soaking in plant hormones

[0072] The sterilized circular slices of Rehmannia glutinosa were placed in a sterile anti-browning preservation solution for 3 minutes. Preservation solution 1 contained 0, 5, and 10 μmol / L (μM) of strigolactone, and preservation solution 2 contained 0, 5, and 10 μmol / L of brassinolide solution. The Rehmannia glutinosa slices were divided into 9 groups, 100 slices in each group, and placed in the induction medium respectively to calculate the induction rate. Figure 1 As shown, the success rate of stem cell induction was the highest after treatment with 5 μM brassinolide, which was 62%.

[0073] 3. Comparative observation of stem cells, dedifferentiated cells and adventitious roots of Rehmannia glutinosa cambium using an optical microscope

[0074] When cultured in complete darkness on solid culture medium, it can be observed that Figure 2 A is the cambium stem cell of Rehmannia glutinosa, which is light yellow, soft in texture, and the cells are adhered together; Figure 2 B is dedifferentiated cells of Rehmannia glutinosa, which are light green, hard, and loosely distributed in granules; Figure 2 C is an adventitious root which is light green tissue surrounded by hard cell clusters.

[0075] When observed using an optical microscope, Figure 3 As shown in A, Rehmannia stem cells are dispersed single cells or small cell clusters; Figure 3 As shown in B, the dedifferentiated cells of Rehmannia glutinosa are large cell clusters formed by multi-cell aggregation.

[0076] Vacuole staining experiment: Take 0.1g fresh weight of Rehmannia stem cells and dedifferentiated cells respectively, add 5mL Ringer solution (Ringer solution formula: NaCl 8.5g / L, CaCl 2 0.12g / L, NaHCO 3 0.2g / L, KCl 0.14g / L, Na 2 HPO 4 0.01g / L, glucose 2.0g / L), blow evenly, take a drop of cell suspension and drop it on a slide, add two drops of 0.25% neutral red-Ringer solution, stain for 8 minutes, dry with filter paper, wash with Ringer solution 3 times, then drop a drop of Ringer solution on the cells, cover with a coverslip, and observe under a microscope. Figure 4 As shown in A, Rehmannia stem cells have a large number of small and numerous vacuoles; Figure 4 As shown in B, the dedifferentiated cell has only a single large vacuole.

[0077] 4. Bleomycin sensitivity test

[0078] Dedifferentiated cells and stem cells of Rehmannia glutinosa were cultured in suspension for 12 days, 1 mL of the cell suspension was placed in a 12-well plate, 250 μg / mL of bleomycin solution was added, and the culture was shaken for 24 h and 48 h. 250 ng / mL of fluorescein diethyl ester was added, and the mixture was shaken and mixed. The cell death rate (%) was observed under a fluorescence microscope. (Total number of cells - number of live cells) / total number of cells * 100%. Figure 5 As shown, after treatment with 250 μg / mL bleomycin, the mortality rate of Rehmannia glutinosa stem cells was significantly higher than that of dedifferentiated cells, and they were more sensitive to bleomycin and possessed the typical characteristics of plant stem cells.

[0079] 5. Defense factors NO, Ca 2+ , H 2 O 2 , CAT, POD content analysis

[0080] When plants are stimulated by external stimuli, they will trigger their defense responses, such as the accumulation of NO, Ca 2+ Influx, burst of reactive oxygen species, (O 2- , H 2 O 2 OH - , O 2 ), and the plant's defense response is closely related to the synthesis of alkaloids. Figure 6 As shown, plant defense mechanism-related factors NO, Ca 2+ , H 2 O 2 The contents of CAT and POD in Rehmannia glutinosa cambium stem cells were 23, 2.8, 6.7, 2 and 1.3 times that in dedifferentiated cells, respectively.

[0081] The above results show that disinfecting the root explants of Rehmannia glutinosa twice can effectively reduce the infection rate, and soaking them with plant hormones strigolactone and brassinolide can effectively increase the induction success rate of Rehmannia glutinosa stem cells; in addition, the cell growth rate, defense response factors (NO, Ca 2+ , H 2 O 2 , CAT, POD), and iridoid ether terpenoids (catalpol, verbascoside, rehmannia glycoside A and D) in Rehmannia stem cells were also higher than those in dedifferentiated cells.

[0082] Example 2

[0083] A method for isolating, differentiating and culturing Rehmannia glutinosa stem cells is basically the same as that in Example 1, except that the plant hormone in the preservation solution in S2 is a 5 μmol / L strigolactone solution.

[0084] The induction success rate of Rehmannia glutinosa slices treated with plant hormones in Example 2 was investigated

[0085] The sterilized circular slices of Rehmannia glutinosa were placed in a sterile anti-browning preservation solution for 3 minutes, and 0, 5, and 10 μmol / L brassinolide solution was added to the preservation solution. The Rehmannia glutinosa slices were divided into 9 groups, 100 slices in each group, and placed in the induction medium respectively, and the induction rate was calculated. Figure 7 As shown, the success rate of stem cell induction was the highest after treatment with 5 μM strigolactone, which was 68%.

[0086] Example 3

[0087] The isolation, differentiation and culture methods of Rehmannia glutinosa stem cells are the same as those in Example 1, except that the plant hormones in the preservation solution in S2 are 5 μmol / L strigolactone solution and 5 μM brassinolide.

[0088] The induction success rate of Rehmannia glutinosa slices treated with plant hormones in Example 3 was investigated

[0089] The sterilized circular slices of Rehmannia glutinosa were placed in a sterile anti-browning preservation solution for 3 minutes, and 0, 5, and 10 μmol / L of brassinolide solution and brassinolide were added to the preservation solution. The Rehmannia glutinosa slices were divided into 9 groups, 100 slices in each group, and placed in the induction medium respectively, and the induction rate was calculated. Figure 8 As shown, the success rate of stem cell induction was the highest, 80%, after treatment with 5 μM brassinolide solution and 5 μM strigolactone.

[0090] Example 4

[0091] Example 1 Bioreactor suspension culture method for Rehmannia stem cells, dedifferentiated cells and adventitious roots

[0092] Rehmannia stem cells, dedifferentiated cells and adventitious roots were inoculated into a 5.0L stirred airlift bioreactor at an inoculation rate of 50.0g / L, cultured in complete darkness at 25°C and harvested after 15 days of culture. The culture conditions for suspension culture were: temperature of 25°C, rotation speed of 100rpm, 24h dark culture, ventilation ratio of 1:0.5, and pH adjusted to 6.0; the suspension culture medium was MS medium, MS basal medium containing 10-30g / L sucrose and 1.0-3.0mg / L plant hormones, and plant hormones: 2mg / L NAA and 1mg / L 6-BA.

[0093] like Fig. 9 As shown, the growth rates of Rehmannia stem cells, dedifferentiated cells and adventitious roots were 943%, 723% and 508% respectively, and the growth rate of Rehmannia stem cells was the highest.

[0094] like Fig.10 As shown, analysis of secondary metabolites in cells revealed that the contents of catalpol, verbascoside, rehmannoside A and D in Rehmannia stem cells were higher than those in dedifferentiated cells and adventitious roots.

[0095] Example 5

[0096] Example 2 Bioreactor suspension culture method for Rehmannia stem cells, dedifferentiated cells and adventitious roots

[0097] Rehmannia stem cells, dedifferentiated cells and adventitious roots were inoculated into a 5.0L stirred airlift bioreactor at an inoculation rate of 60.0g / L, cultured in complete darkness at 25°C and harvested after 20 days of culture. The culture conditions for suspension culture were: temperature of 25°C, rotation speed of 100rpm, 24h dark culture, ventilation ratio of 1:0.5, and pH adjusted to 6.0; the suspension culture medium was MS medium, MS basal medium containing 30g / L sucrose and 2.0mg / L plant hormones, and plant hormones: 2mg / L NAA and 1mg / L 6-BA.

[0098] The experimental results showed that the growth rates of Rehmannia stem cells, dedifferentiated cells, and adventitious roots were 1080%, 815%, and 589%, respectively, with Rehmannia stem cells having the highest growth rate. Fig.11 As shown, analysis of secondary metabolites in cells revealed that the contents of catalpol, verbascoside, rehmannoside A and D in Rehmannia stem cells were higher than those in dedifferentiated cells and adventitious roots.

[0099] Furthermore, we also used other Rehmannia plants as raw materials, including Rehmannia chingii Li, Rehmannia elata NEBrown, Rehmannia glutinosa (Gaetn.) Libosch. ex Fisch. et Mey., Rehmannia henryi N.E. Brown, Rehmannia solanifolia Tsoong et Chin, to conduct experiments on the differentiation and culture method of Rehmannia stem cells provided in Example 1 and the bioreactor culture method provided in Example 3, and confirmed that the method of the present invention can be used to prepare cambium stem cells derived from tuberous roots of Rehmannia plants, dedifferentiated cells, and the preparation and culture of adventitious root culture systems.

[0100] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0101] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing and culturing Rehmannia glutinosa stem cells, characterized in that: The following steps are involved: S1. Explant sterilization: cutting the tuberous roots of Rehmannia plants, washing and sterilizing to obtain Rehmannia root explants, slicing, sterilizing again and then washing to obtain explant slices; S2, induction in vitro culture: the explant pieces obtained in S1 are transferred to a preservation solution containing plant hormones for immersion, and then transferred to an induction culture medium for culture, respectively, to induce the acquisition of Rehmannia stem cells, dedifferentiated cells and adventitious roots, and then the stably growing Rehmannia stem cells, dedifferentiated cells and adventitious roots are obtained through subculture containing plant hormones, and then suspension culture is performed; the preservation solution contains 10-30 g / L sucrose, 100-300 mg / L citric acid, 50-500 U / mL penicillin, and 50-500 mg / mL streptomycin in an MS liquid culture medium, and the plant hormones are selected from 5 μmol / L strigolactone solution and / or 5 μmol / L brassinolide solution; The induction medium of Rehmannia stem cells is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum, 0.1-0.5% activated carbon, and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the added plant hormones are 3.0 mg / L NAA+0.5 mg / L 6-BA; the induction medium contains 2-2.6 mg / L methylchloroisothiazolinone, 0.6-0.8 mg / L methylisothiazolinone, and 15-25 mg / L sodium benzoate; The induction medium of dedifferentiated cells is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum, 0.1-0.5% activated carbon, and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the added plant hormones are 3.0 mg / L NAA+3.0 mg / L 2,4-D; the induction medium contains 2-2.6 mg / L methylchloroisothiazolinone, 0.6-0.8 mg / L methylisothiazolinone, and 15-25 mg / L sodium benzoate; The adventitious root induction medium is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum, 0.1-0.5% activated carbon, and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the added plant hormones are 1.0 mg / L NAA+0.5 mg / L 6-BA; the induction medium contains 2-2.6 mg / L methylchloroisothiazolinone, 0.6-0.8 mg / L methylisothiazolinone, and 15-25 mg / L sodium benzoate; The subculture medium of Rehmannia stem cells is an MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the added plant hormones are 1.0 mg / L NAA + 0.5 mg / L 6-BA; The subculture medium of dedifferentiated cells of Rehmannia glutinosa is MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum and 0.5-5.0 mg / L plant hormones, the pH value of the medium is 5.5-6.0, and the added plant hormones are 1 mg / L NAA + 1 mg / L 2,4-D; The subculture medium of Rehmannia glutinosa adventitious roots is MS medium containing 10-30 g / L sucrose, 3.0-4.0 g / L gellan gum and 0.5-5.0 mg / L plant hormones. The pH value of the medium is 5.5-6.0, and the added plant hormones are 1 mg / L NAA + 0.5 mg / L 6-BA.

2. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 1, characterized in that: In S1, the length of the cut section is 5 to 8 cm, the sterilization treatment method is water washing for 1 to 3 hours, then sterilized with 8% hydrogen peroxide for 4 minutes, 75% ethanol solution for 2 minutes, 0.1% mercuric chloride solution for 10 to 20 minutes, soaked in sterile water for 1 to 4 minutes, and then rinsed with sterile water; the slices are circular slices with a thickness of 0.1 to 2 mm.

3. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 1, characterized in that: In S2, the immersion time of the preservation solution is 1 to 5 minutes.

4. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 1, characterized in that: In S2, the culture conditions for the induction culture and subculture are: temperature 25-30° C., humidity 60-80%, and dark culture for 24 hours.

5. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 1, characterized in that: In S2, the method for suspension culture of Rehmannia stem cells, dedifferentiated cells and adventitious roots is as follows: Rehmannia stem cells, dedifferentiated cells and adventitious roots were inoculated into bioreactors containing suspension culture medium, respectively, and cultured continuously for 15 to 20 days in darkness. The inoculation amount of Rehmannia stem cells, dedifferentiated cells and adventitious roots was 50 to 100 g / L, respectively.

6. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 5, characterized in that: The suspension culture medium is an MS basic culture medium containing 10-30 g / L sucrose and 1.0-3.0 mg / L plant hormones, and the plant hormones are selected from one or more of NAA, IAA, 2,4-D, and 6-BA.

7. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 5, characterized in that: The culture conditions of the suspension culture are: temperature of 25-30° C., rotation speed of 100-130 rpm, 24-hour dark culture, ventilation ratio of 1:0.3-0.6, dissolved oxygen content of 0.20-0.50, and pH adjusted to 5.5-6.

0.

8. The method for preparing and culturing Rehmannia glutinosa stem cells according to claim 1, characterized in that: The Rehmannia plants include but are not limited to Rehmannia chingii Li, Rehmannia elata NEBrown, Rehmannia glutinosa (Gaetn.) Libosch.ex Fisch.et Mey., Rehmannia henryiN.E.Brown, Rehmannia piasezkii Maxim, and Rehmannia solanifoliaTsoong et Chin.

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