CN112293250B

CN112293250B - Culture method of nardostachys chinensis callus cells

Info

Publication number: CN112293250B
Application number: CN201910710851.8A
Authority: CN
Inventors: 李惠玲; 蒋耀权; 章漳
Original assignee: Jala Group Co
Current assignee: Shanghai Natural Hall Group Co ltd
Priority date: 2019-08-02
Filing date: 2019-08-02
Publication date: 2023-02-17
Anticipated expiration: 2039-08-02
Also published as: CN112293250A

Abstract

The invention belongs to the field of tissue culture, and particularly relates to a culture method of nardostachys chinensis callus cells, which comprises the following steps: A. washing and disinfecting petioles and leaves of wild nardostachys chinensis to obtain base tissues; B. cutting the base tissue prepared in the step A, and inoculating the cut base tissue into a callus induction culture medium for culture to obtain callus; C. b, inoculating the callus prepared in the step B into a callus subculture medium for subculture to obtain subculture callus cells; the callus cells of the leaves and the petioles of the nardostachys clavipes plant used in the invention are induced, and the callus cells with high secondary metabolite (chlorogenic acid and polyphenol) content are obtained under low-temperature stimulation induction; the method can obtain large amount of callus cells of radix Et rhizoma Nardostachyos embryo containing active substances in short time, and has the advantages of safety, no toxicity, short period, rapid propagation, simple operation and low cost, and provides convenience for application of radix Et rhizoma Nardostachyos extract in practical product.

Description

Culture method of nardostachys chinensis callus cells

Technical Field

The invention belongs to the field of tissue culture, and particularly relates to a culture method of nardostachys chinensis callus cells.

Background

Plant cell and tissue culture (plant cell and tissue culture) refers to a technique of separating one or several somatic cells or a part of a plant body under aseptic conditions to undergo processes of dedifferentiation, callus formation, redifferentiation, and the like. By using the plant cell tissue culture method, a large amount of secondary metabolites can be extracted from the cultured plant cells or callus under the controllable and repeated conditions, and enzyme preparations, medicinal products and additives can be provided for human beings. The method for producing the plant secondary metabolite by utilizing the plant cell large-scale culture technology is widely applied, and on one hand, the method can overcome the defect that the quality of the medicinal material is directly influenced due to large difference in the composition and the content of the plant chemical components caused by different growing conditions such as geographical environment, climate, soil and the like and different time for collecting the medicinal material; on the other hand, rare wild plant resources can be protected from being damaged, and reasonable utilization and sustainable development of the resources are promoted.

Nardostachys chinensis (Nardostachys jatamansi DC.) is a perennial herb of Nardostachys of Valerianaceae (Valerianaceae), grows on alpine grassland, shrubs or gravelly land with the elevation of 2500-5000 m, is distributed in areas of Gansu, qinghai, sichuan, tibet, yunnan and the like, and is a typical plant of Himalayan, namely Indian, nipol and Plumbum preparatium. Nardostachys chinensis is not only a famous spice plant in Tibet, but also an important medicinal source plant of Chinese herbs, tibetan herbs and Indian herbs. The root and rhizome of the Chinese medicinal composition are used as medicines, have warm property, pungent and sweet taste, contain terpenes, flavonoids, polysaccharides, polyphenols, lignin, volatile oil and the like as chemical components, have the functions of regulating qi to alleviate pain, relieving stagnation and activating spleen, and are used for treating abdominal distension and pain, vomiting and inappetence and treating toothache and foot pain by external application. The research at home and abroad reports that the nardostachys chinensis batal has the effects of reducing blood pressure, calming, resisting depression, tumors, oxidation, inflammation, cardiovascular injury, protecting human neuroblastoma cell injury and the like. The nardostachys plants are mainly bred by rhizomes or seeds, are mostly wild, and are occasionally cultivated in India and China. The nardostachys chinensis bunge has great medicinal value and aromatic value, so that the resources of the nardostachys chinensis bunge are seriously damaged due to over-development of people, and the nardostachys chinensis bunge is listed in the name list of endangered plants in the world.

At present, chemical components, pharmacological effects, quality standards and the like of nardostachys chinensis benth are researched more at home and abroad, but research reports about tissue culture of nardostachys chinensis benth plants are not seen for a while. The 2017 reported project of Tibetan medicine-Nardostachys chinensis in Nacizi Linke institute of Sichuan province is based on molecular laboratories of Sichuan university of agriculture, and a Nardostachys chinensis tissue culture sterile system is preliminarily established through multiple experimental exploration.

The key point of establishing the nardostachys chinensis bunge tissue culture sterile system lies in the selection of an explant part of the nardostachys chinensis bunge. In the early stage experiment, the root (root bud, root bud stem and rhizome), stem (stem base and stem segment) and bud of the nardostachys chinensis bunge are respectively selected for tissue culture, and the result shows that the germination rate of the stem base of the nardostachys chinensis bunge is the highest, so that the stem base of the nardostachys chinensis bunge is selected as an explant. The explants were cut into sections and placed in 45 ℃ warm water Tween 10 minutes, and then washed with running water for 1 hour. The washed explants were then placed on a clean bench and washed with sterile water 1 time, then soaked in 75% alcohol for 15 seconds, and then soaked in 0.1% mercuric chloride for 8-10 minutes. Then, the mixture was washed with sterile water 1 time and inoculated into a blank MS medium for culture. However, because the rhizoma nardostachyos is an international endangered plant, the project only uses rhizomes and buds of the rhizoma nardostachyos as explants, the rhizoma nardostachyos plant is easily damaged, the disinfection and sterilization steps are complicated, and the successful culture of the whole aseptic seedling of the rhizoma nardostachyos is not reported so far.

Disclosure of Invention

In order to solve the technical problems, the invention provides a culture method of callus cells of nardostachys chinensis bunge, and the embryonic callus cells of nardostachys chinensis bunge with active substances obtained by culture are extracted and active substance polyphenol and chlorogenic acid are measured.

A culture method of nardostachys chinensis bunge callus cells comprises the following steps:

A. washing and disinfecting petioles and leaves of wild nardostachys chinensis to obtain base tissues;

B. cutting the base tissue prepared in the step A, and inoculating the cut base tissue into a callus induction culture medium for culture to obtain callus;

C. and C, inoculating the callus prepared in the step B into a callus subculture medium for subculture to obtain a subculture callus cell.

In the process of induction culture and subculture, the culture conditions are that the temperature is 20 +/-1 ℃, the humidity is 60-70%, the illumination is carried out for 12 hours every day, and the illumination intensity is 1500 Lx-2000 Lx.

In the step A, the washing and disinfecting steps are as follows: washing with running water under natural water for 1 to 2 hours, and then disinfecting with 70% ethanol for 30 s in an aseptic environment; washing with sterile water for 3 times, and sterilizing with 0.1% HgCl2 dripping 1-2 drops of Tween20 for 6 min; after washing with sterile water for 3 to 5 times, the surface water was blotted with sterile filter paper.

In the step B, cutting the sterilized petioles and leaves into explants of 0.5 to 0.8 cm in length by using an aseptic scalpel with the base tissue in a super clean bench; the callus induction culture medium comprises the following components: MS + NAA 0.3-0.8 mg/L +6-BA 0.6-1.3 mg/L + sucrose 30g/L + agar 7g/L. (ii) a In step B, the induction culture period is 20 days.

In the step C, the callus subculture medium comprises the following components: MS +6-BA 0.5-2.0 mg/L + NAA 0.2-1.0 mg/L + sucrose 30g/L + agar 7g/L; the subculture period is 25-40 days.

And C, carrying out low-temperature treatment on the subcultured callus cells obtained in the step C to obtain the nardostachys chinensis embryonic callus cells with the active substances.

The low-temperature treatment comprises the following steps: the callus cells of Nardostachys chinensis were treated at 4 ℃ at low temperature 1d,3d,5d,7d, three times each.

The Nardostachys jatamansi DC plant used in the method is collected from Asian county of Japan, autonomous region, japan, and fresh Nardostachys jatamansi leaf and petiole of the Nardostachys jatamansi are taken as explants through experiments, callus cells are induced and proliferated under certain conditions, and finally, the callus cells are subjected to low-temperature treatment to obtain cells with high secondary metabolite content. The method can obtain a large amount of the embryonic callus cells of the nardostachys chinensis which contain active substances in a short time, has the characteristics of safety, no toxicity, short period, quick propagation, simple operation and low cost, and compared with the only known nardostachys chinensis project at present, the leaves and the petioles are simple in aseptic operation compared with the use of roots and stems, and the aseptic system is easier to establish; the invention uses the leaf and the petiole of the nardostachys chinensis bunge plant to induce the callus cells, and obtains the callus cells with high secondary metabolite (chlorogenic acid and polyphenol) content under the low-temperature stimulation induction. The technical scheme of the invention provides convenience for the application of the nardostachys chinensis bunge extract in actual products.

Drawings

FIG. 1 is a comparison of the results of subculture of the experimental nardostachys chinensis benth callus.

FIG. 1-A shows callus with adventitious roots, FIG. 1-B shows greenish-yellow embryogenic callus, and FIG. 1-C shows callus with browned edges.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Preparation of the experiment:

taking one of culture medium MS,1/2MS, WPM, B5 and N6, matching with different plant hormones alpha-naphthylacetic acid (alpha-NAA), 6-benzylamino adenine (6-BA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D), preparing a fresh culture medium according to a certain proportion, adding 30g/L of cane sugar, 7g/L of agar, and 5.7-5.8 of pH, and sterilizing for 20min at 121 ℃ in a sterilizing pot for later use.

The culture conditions of all plant explants are 20 +/-1 ℃, the humidity is 60% -70%, the illumination is carried out for 12 hours every day, and the illumination intensity is 1500 Lx-2000 Lx.

Callus induction rate = number of induced explants/number of inoculated explants × 100%

Proliferation coefficient = weight of callus after proliferation/weight of callus before proliferation

Experiment one:

explant disinfection: taking petioles and leaves of wild nard plants, washing the petioles and leaves with running water in natural water for 1 to 2 hours, then disinfecting the petioles and leaves with 70 percent ethanol for 30 s under an aseptic condition, washing the petioles and leaves with sterile water for 3 times, then disinfecting the petioles and leaves with 0.1 percent HgCl2 dripped with 1 to 2 drops of Tween20 for 6 min, washing the petioles and the leaves with the sterile water for 3 to 5 times, and then sucking the surface water with sterile filter paper for later use.

Induction of nard callus: cutting the petioles and leaves of the nardostachys chinensis subjected to sterilization treatment into explants with the length of 0.5-0.8 cm in a super clean bench by using an aseptic scalpel, and inoculating the explants to MS culture media with different hormone ratios. After 20 days of culture, the induction was observed and the callus induction rate was counted, and the results are shown in Table 1.

As shown in Table 1, MS + NAA 0.3-0.8 mg/L +6-BA 0.6-1.3 mg/L + sucrose 30g/L + agar 7g/L can successfully induce callus; wherein the nardostachys chinensis benth explant is inoculated in a No. 4 culture medium for about 7 days, the two ends of the explant begin to swell and form a faint yellow loose callus, and the callus cells gradually turn into yellow green along with the prolonging of the culture time. The formula of the optimal induction medium for the nardostachys chinensis bunge explant is MS + NAA 0.5 mg/L +6-BA 1.0mg/L + sucrose 30g/L + agar 7g/L, and the induction rate is 61.5%.

TABLE 1 Induction of Nardostachys chinensis callus

Subculturing callus of rhizoma nardostachyos:

the induced callus with good growth and uniform shape is taken and inoculated into MS,1/2MS, WPM, B5 and N6 culture mediums respectively, each culture medium contains 1 mg/L NAA and 1.0 mg/L6-BA, each treatment is inoculated into 3 bottles, and the cell state is observed and the proliferation coefficient is calculated after 28 days of culture. The results are shown in Table 2.

As can be seen from the table 2, the callus of nardostachys chinensis has the highest proliferation coefficient in WPM medium, which is 6.201; followed by 5.995 in MS medium. The lowest proliferation factor was 3.457 in N6 medium. The callus cultured by the WPM is in a sphere-like shape, the center of the callus is provided with a brown small sphere and more adventitious roots (figure 1-A), while the callus cultured by the MS culture medium is granular, loose in texture and yellow-green (figure 1-B), so the optimal basic culture medium for callus subculture of the nardostachys is the MS culture medium. The callus cultured on the N6 medium has hard texture and browning phenomenon (figure 1-C), and is not suitable for a subculture medium.

TABLE 2 proliferation of callus cells from Nardostachys chinensis by different basal media

Note that: "0D" indicates the mass of cells at day 0 of seeding and "4W" indicates the mass of cells at day 28 of seeding (4 weeks).

After determining that the MS culture medium is the optimal culture medium, inoculating the induced callus with good growth and uniform shape into MS subculture multiplication culture media with different hormone ratios, inoculating each culture medium into 3 bottles, and observing cell growth vigor and calculating multiplication coefficients after culturing for 35 days. The experiment uses an orthogonal design method, and the hormone proportion design and the results of the proliferation medium are shown in Table 3.

As shown in Table 3, the callus cells of Nardostachys chinensis can be successfully proliferated within the range of 0.5-2.0 mg/L of MS +6-BA, 0.2-1.0 mg/L of NAA, 30g/L of sucrose and 7g/L of agar. The callus of Nardostachys chinensis is the best No. 6 and No. 9, the callus is quick to proliferate, granular on the surface, loose in texture, yellow green and embryonic callus. Therefore, the optimal formulation of the subculture multiplication medium is No. 6, namely MS +6-BA 1.0mg/L + NAA1.0 mg/L + sucrose 30g/L + agar 7g/L, and the multiplication coefficient reaches up to 9.8. And secondly, 9, namely MS +6-BA 2.0mg/L + NAA1.0 mg/L + sucrose 30g/L + agar 7g/L, and the increment coefficient reaches 9.4.

TABLE 3 proliferation of callus cells from Nardostachys chinensis by different phytohormone combinations

After confirming the growth effect of the culture mediums No. 6 and No. 9, in order to further confirm the growth cultivation period, the above-mentioned callus with good growth and uniform morphology was taken and inoculated into MS subculture medium containing 1.0mg/L of 6-BA + 1.0mg/L of NAA (No. 6) and 2.0mg/L of 6-BA + 1.0mg/L of NAA (No. 9), 3 flasks were inoculated into each of the culture mediums, and after culturing for 40 days, the growth vigor of the cells was observed and the growth coefficient was calculated. The experiment uses an orthogonal design method, the proliferation cycle, the hormone proportion of a corresponding proliferation culture medium and the results are shown in a table 4.

Referring to table 4, the subculture cycle of this experiment was 25-40 days, wherein the period of fastest proliferation was around 30 days, and thus this period was the most efficient period.

TABLE 4 Effect of different incubation times on the proliferation factor of Nardostachys chinensis Franch cells

Experiment two:

extracting callus cells of nardostachys chinensis:

to enrich the activity of the callus cells of Nardostachys jatamansi, the well-grown callus cells of Nardostachys jatamansi after the secondary culture according to the experiment were treated at a low temperature of 4 ℃ in steps 1d,3d,5d,7d, three times each. The treated cells are taken out, dried and ground into powder, and the powder has special fragrance of the nardostachys chinensis bunge.

Weighing a certain amount of dried rhizoma nardostachyos histiocyte powder, transferring the dried rhizoma nardostachyos histiocyte powder to a 10mL bottle, weighing a certain amount of methanol (the material-liquid ratio is about 1.

And (3) determining polyphenol by a color development method:

the total polyphenol content is detected by Folin-Ciocalteu method and gallic acid as reference substance. Taking 0.1 mL gallic acid standard solution and sample solution to be tested, paralleling for 3 times, adding 0.5 mL 10% Folin-Ciocalteu reagent, mixing, standing at room temperature for 5min, adding 0.4 mL 7.5% Na2CO3 solution, mixing, standing at room temperature for 60 min, and determining 765nm OD value. A standard curve was drawn from OD765 of each tube of the standard solution for gallic acid content, the polyphenol content in the sample was calculated from the standard curve, and the percentage of total polyphenol in Nardostachys chinensis Franch cells was calculated from the standard curve, and the results are shown in Table 5.

As shown in Table 5, the total polyphenol content of the callus cells of Nardostachys chinensis was 0.649% after the callus cells of Nardostachys chinensis were treated at 4 ℃, which is 0.221% higher than that of the cells (0.428%) which were not treated at low temperature.

TABLE 5 Effect of different cryopreservation times on the total intracellular Polyphenol content of Nardostachys chinensis (dry weight per 100 g)

Detecting chlorogenic acid by HPLC:

an Agilent 1260-type high performance liquid chromatograph is adopted, the chromatographic column is Agilent Eclipse C18 XDB (150 mm multiplied by 4.6 mm,5 mu m), the fluidity is acetonitrile-0.1 percent phosphoric acid water solution, gradient elution is carried out, the flow rate is 1 mL/min, the column temperature is 30 ℃, and the detection wavelength is 327 nm. The chlorogenic acid standards were used as controls, the callus cell extracts of Nardostachys jatamansi were analyzed qualitatively for retention time and quantitatively by external standard method, and the results are shown in Table 6.

As shown in Table 6, the chlorogenic acid content of the callus cells of Nardostachys chinensis treated at 4 ℃ can reach 0.266%, which is 0.091% higher than that of the cells (0.175%) which are not treated at low temperature

TABLE 6 Effect of different cryogenic treatment times on the intracellular chlorogenic acid content of Nardostachys chinensis (dry weight per 100 g)

The experimental data prove that the induced nardostachys chinensis bunge callus cells can be cultured by the technical scheme of the invention, and the cells with high secondary metabolite content can be obtained after the low-temperature treatment of the nardostachys chinensis bunge callus cells. The method can obtain large amount of callus cells containing active substances in short time, and has the advantages of safety, no toxicity, short period, and rapid propagation. The reagents MS, WPM, B5 and N6 culture media, 6-BA, NAA and 2,4-D involved in the first and second experiments were purchased from Phototechnology, and agar and sucrose were purchased from national drug group.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A culture method of nardostachys chinensis bunge callus cells is characterized by comprising the following steps:

a, washing and disinfecting leafstalks and leaves of wild nardostachys chinensis to obtain base tissues;

b, cutting the base tissue prepared in the step A, and inoculating the cut base tissue into a callus induction culture medium for culture to obtain callus;

c, inoculating the callus prepared in the step B into a callus subculture medium for subculture to obtain subculture callus cells;

c, carrying out low-temperature treatment on the secondary callus cells obtained in the step C to obtain the embryonic callus cells of the nardostachys chinensis with the active substances, wherein the low-temperature treatment step comprises the following steps: the callus cells of the nardostachys chinensis batal are respectively treated at the low temperature of 4 ℃ for 1d,3d,5d,7d;

wherein the callus induction culture medium in the step B comprises the following components: MS, 0.3-0.8 mg/L NAA, 0.6-1.3 mg/L6-BA, 30g/L sucrose and 7g/L agar,

the callus subculture medium in the step C comprises the following components: 0.5-2.0 mg/L of MS +6-BA + 0.2-1.0 mg/L of NAA + 30g/L of sucrose + 7g/L of agar.

2. The method for culturing the callus cells of nardostachys chinensis as claimed in claim 1, wherein in the process of the induction culture and the subculture, the culture conditions are that the temperature is 20 +/-1 ℃, the humidity is 60% -70%, the illumination is 12 hours per day, and the illumination intensity is 1500 Lx-2000 Lx.

3. The method for culturing callus cells of nardostachys chinensis as claimed in claim 2, wherein in step a, the washing and disinfecting steps are as follows: washing with running water under natural water for 1 to 2 hours, and then disinfecting with 70% ethanol for 30 s in an aseptic environment; washing with sterile water for 3 times, and sterilizing with 0.1% HgCl2 dripping 1-2 drops of Tween20 for 6 min; and (5) washing with sterile water for 3-5 times, and then sucking surface water by using sterile filter paper.

4. The method for culturing callus cells of nardostachys chinensis benth as claimed in claim 2, wherein in step B, the sterilized petioles and leaves are cut into 0.5-0.8 cm long explants by using a sterile scalpel in a super clean bench.

5. The method for culturing Nardostachys chinensis callus cells according to claim 4, wherein in the step B, the induction culture period is 20 days.

6. The method for culturing callus cells of nardostachys chinensis as claimed in claim 1, wherein in step C, the period of subculture is 25-40 days.