CN112553137B - Preparation and culture method and application of stellera chamaejasme stem cells - Google Patents
Preparation and culture method and application of stellera chamaejasme stem cells Download PDFInfo
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Abstract
The invention relates to the technical field of plant stem cell preparation, in particular to a preparation and culture method and application of stellera chamaejasme stem cells. Specifically, obtaining explant material; induction of stellera chamaejasme stem cells, subculture of stellera chamaejasme stem cells, shake flask suspension culture method and the like. The method for preparing the stellera chamaejasme stem cells has the advantages that the obtained stellera chamaejasme stem cells have stable properties; the culture of the stellera chamaejasme stem cells can stably produce a large number of stellera chamaejasme stem cells by a suspension shake flask culture method, thereby providing a material for researching relevant active ingredients of stellera chamaejasme, providing a new material source for the research field of plant stem cells, and having important significance for modern development and utilization of traditional Chinese medicines. In addition, the prepared stellera chamaejasme stem cells can be applied to preparation of medicines, health-care products or cosmetics.
Description
Technical Field
The invention relates to the technical field of plant stem cell preparation, in particular to a preparation and culture method and application of stellera chamaejasme stem cells.
Background
Stellera chamaejasme L, also named Stellera chamaejasme L, gelsemium elegans, steamed bread and the like, is a Stellera chamaejasme plant of the family daphneaceae, is widely distributed in northeast and northeby, inner Mongolia, gansu, qinghai, ningxia, tibet and other places of China, is an important traditional Chinese medicine in China, and is often used as a medicine by using a dry root thereof. The stellera chamaejasme has pungent taste, and has effects of resolving hard mass and killing parasite, and can be used for treating diseases such as tuberculous lymphadenitis and tinea. The stellera chamaejasme is rich in various compounds, such as terpenoids, flavonoids, coumarins, lignins and the like. Modern pharmacological research shows that the stellera chamaejasme extract has obvious bioactivity of resisting cancer, resisting tumor, resisting virus, regulating immunity, resisting convulsion, resisting epilepsy, inhibiting enzyme, preventing pests, killing pests, etc.
However, when natural products are extracted from stellera chamaejasme, the natural products are easily influenced by factors such as the growth cycle of plants, the geographical environment, the seasonal climate and the component parts. Meanwhile, the stellera chamaejasme is toxic, and the livestock is easy to eat by mistake and die, so that the animal husbandry suffers from heavy economic loss. In addition, the breeding and planting process of the stellera chamaejasme is very complicated and difficult, and a large amount of cultivation also occupies cultivated land. These disadvantages limit the intensive research and industrial production of active natural products in stellera chamaejasme.
Plant stem cells are a class of plant cells having a multipotent differentiation potential, a self-renewal ability, which are characterized by being maintained in an undifferentiated state and a proliferative capacity for a long period of time, and have the potential to differentiate into other tissues. Compared with the callus (namely dedifferentiated cells) induced by the traditional plant tissue culture, the plant stem cells have the advantages of stable growth, stable and efficient production of plant active compounds and the like. The existing plant stem cells are mostly derived from herbaceous plants or woody plants, such as taxus chinensis, catharanthus roseus, ginseng and the like, and the preparation method of the plant stem cells of stellera chamaejasme is not reported in a public way.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a preparation method, a culture method and application of stellera chamaejasme stem cells.
The invention relates to a technical scheme of a preparation method of stellera chamaejasme stem cells, which specifically comprises the following steps:
step one, obtaining explant materials: taking roots and leaves of fresh stellera chamaejasme as explants, cleaning, soaking for 5min by using a soaking solution, then washing the explants by using clear water, putting the explants into a super clean bench, respectively placing the roots and the leaves on a culture dish padded with sterile filter paper after sterilization treatment, and then cutting the roots and the leaves into small pieces under the aseptic condition, wherein the pulp parts of the roots are stripped off to obtain root tissue blocks and leaf tissue blocks containing cambium cells;
step two, induction of stellera chamaejasme stem cells: placing the tissue block containing cambium cells obtained in the step one on induction culture media of different systems for induction, and carrying out dark culture for 7-15 days under the conditions that the temperature is 24-26 ℃ and the humidity is 55-75%; wherein the formula of the induction culture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 0-2 mg/mL phytohormone and 8.5-9.5 g/L agar, and its pH value is 5.75-5.85; the phytohormone is the combination of any two of 6-benzylamino adenine (6-BA), naphthylacetic acid (NAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and indole-3-butyric acid (IBA);
step three, subculturing the stellera chamaejasme stem cells: inoculating the cambium cells obtained in the step two into a subculture medium, and performing dark culture for 9-21 days at the temperature of 24-26 ℃ and the humidity of 55-75%; wherein the formula of the subculture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 0-2 mg/mL phytohormone and 8.5-9.5 g/L agar, and its pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), indole-3-butyric acid (IBA), naphthalene Acetic Acid (NAA), and 2, 4-dichlorophenoxy acetic acid (2, 4-D).
Further, the explant material in the first step is derived from stellera chamaejasme which is a daphne plant, including but not limited to roots and leaves thereof.
Further, the soaking solution in the first step is a laundry powder solution.
Further, in the step one, forceps and a knife are adopted for cutting the roots and the leaves, wherein the size of the leaf tissue block is 0.6 multiplied by 0.6cm; the root tissue block size was 0.3X 0.8cm.
Further, the sterilization treatment in the first step comprises the following specific steps: soaking in 75% ethanol for 3min for sterilization, washing with sterile water for 3 times, and adding 0.1% HgCl 2 Soaking the solution for 6min, washing with sterile water for 6 times, carefully wiping off with sterile filter paper, and soaking in 150mg/L citric acid solution.
Further, the optimal combination of the subculture medium in the third step is a B system and an H system, wherein:
and a system B: MS + NAA 1mg/L +2, 4-D1 mg/L + sucrose 30g/L, pH5.8;
h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L, pH5.8;
the invention also protects the stellera chamaejasme stem cells prepared by the method.
The invention also protects the application of the stellera chamaejasme stem cells in preparing medicines, health-care products or cosmetics.
The invention also provides a culture method of the stellera chamaejasme stem cells, which is a shake flask suspension culture method and specifically comprises the following steps:
inoculating the stellera chamaejasme stem cells into a liquid subculture medium, wherein the inoculation amount is 50g/L of wet weight of the cells, the culture temperature is 24-26 ℃, the rotating speed is 110-130 rpm/min, and dark culture is carried out for 10-25 days; wherein the formula of the liquid subculture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 1mg/mL phytohormone and 8.5-9.5 g/L agar, and the pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D).
Compared with the prior art, the invention has the following beneficial effects: the method for preparing the stellera chamaejasme stem cells has the advantages that the obtained stellera chamaejasme stem cells have stable properties; the culture of the stellera chamaejasme stem cells can stably produce a large number of stellera chamaejasme stem cells by a suspension shake flask culture method, thereby providing a material for researching relevant active ingredients of stellera chamaejasme, providing a new material source for the research field of plant stem cells, and having important significance for modern development and utilization of traditional Chinese medicines. In addition, the prepared stellera chamaejasme stem cells can be applied to preparation of medicines, health-care products or cosmetics.
Drawings
FIG. 1 shows stem cells of stellera chamaejasme induced from stellera chamaejasme leaves;
FIG. 2 shows stem cells of stellera chamaejasme induced from stellera chamaejasme roots;
FIG. 3 is the suspension culture growth curve of stellera chamaejasme stem cells;
FIG. 4 is a microstructure of plant cells obtained from stellera chamaejasme roots induced with H system medium;
FIG. 5 is a microstructure of plant cells obtained from stellera chamaejasme roots induced with the B system medium;
FIG. 6 is a microstructure of plant cells induced from stellera chamaejasme leaves with the B system medium.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Obtaining of stellera chamaejasme stem cell induced explant material
Collecting fresh radix Euphorbiae Fischerianae, cleaning its root and leaf, soaking in washing powder solution for 5min, washing with clear water, placing into super clean bench, soaking in 75% ethanol for 3min for sterilization, washing with sterile water for 3 times, and adding 0.1% HgCl 2 Soaking the solution for 6min, washing with sterile water for 6 times, carefully wiping the solution with sterile filter paper, and soaking in 150mg/L citric acid solution to prevent tissue oxidation and browning; placing the root and leaf on a culture dish filled with sterile filter paper, carefully cutting into small pieces with sterile forceps and a knife, and removing medulla of the root to obtain root tissue block and leaf tissue block containing cambium cells, wherein the size of the leaf tissue block is 0.6 × 0.6cm; the root tissue block size was 0.3X 0.8cm.
2. Screening of stellera chamaejasme stem cell induction culture medium
MS culture medium containing 20-30 g/L of cane sugar, 0-2 mg/mL of plant hormone and 8.5-9.5 g/L of agar is selected as an induction culture medium, and the pH value is 5.75-5.85. Wherein the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), naphthalene Acetic Acid (NAA), 2, 4-dichlorophenoxy acetic acid (2, 4-D) and indole-3-butyric acid (IBA).
Preparing an induction culture medium, weighing 4.43g/L of MS powder, adding 30g/L of sucrose to prepare the MS culture medium, adding phytohormone according to different formulas, adjusting the pH to 5.75-5.85 by using HCl and NaOH solutions, weighing 50mL by using a measuring cylinder, subpackaging the weighed 50mL into 150mL conical bottles filled with 0.45g of agar powder, sealing the bottle openings by using 8 layers of medical cotton yarns and newspapers, placing the bottles into a high-pressure steam sterilization pot for sterilization at 121 ℃ for 20min; and (3) after the sterilized culture medium is shaken lightly and mixed uniformly, the mixture is placed in a shade place to be kept stand until the mixture is naturally cooled and solidified, and an induction culture medium (see table 1 for details) is obtained.
TABLE 1 ingredient table of induction medium for stellera chamaejasme L
In the application, the formula of the subculture medium is the same as that of the induction medium, the subculture medium also adopts an MS culture medium containing 20-30 g/L of sucrose, 0-2 mg/mL of phytohormone and 8.5-9.5 g/L of agar as the induction medium, and the pH of the MS culture medium is 5.75-5.85. Wherein the plant hormone is any two of 6-benzylamino adenine (6-BA), indole-3-butyric acid (IBA), naphthalene Acetic Acid (NAA) and 2, 4-dichlorophenoxy acetic acid (2, 4-D) (see Table 1 for details).
Preferred subculture media are a B system and an H system, wherein:
and B system: MS + NAA 1mg/L +2, 4-D1 mg/L + sucrose 30g/L, pH5.8;
h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L, pH5.8.
Example 2
A preparation method of stellera chamaejasme stem cells specifically comprises the following steps:
step one, obtaining explant materials: collecting fresh stellera chamaejasme L, cleaning its root and leaf, soaking in washing powder solution for 5min, washing with clear water, placing in super clean bench, soaking in 75% ethanol for 3min for sterilization, washing with sterile water for 3 times, and adding 0.1% HgCl 2 Soaking the solution for 6min, washing with sterile water for 6 times, carefully wiping the solution with sterile filter paper, and soaking in 150mg/L citric acid solution to prevent tissue oxidation and browning; placing the root and leaf on a culture dish filled with sterile filter paper, cutting into small pieces with sterile forceps and knife, removing medulla of the root to obtain root tissue block and leaf tissue block containing cambium cells, wherein the size of the leaf tissue block is 0.6 × 0.6cm; the root tissue block size is 0.3 × 0.3 × 0.8cm;
step two, induction of stellera chamaejasme stem cells: placing the tissue block containing the cambium cells obtained in the step one on induction culture media of different systems for induction, carefully and lightly pressing with forceps to ensure that one surface containing the cambium cells is fully contacted with the induction culture media, and culturing for 7-15 days in dark under the conditions of the temperature of 24-26 ℃ and the humidity of 55-75%; wherein the formula of the induction culture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 0-2 mg/mL phytohormone and 8.5-9.5 g/L agar, and its pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), naphthylacetic acid (NAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and indole-3-butyric acid (IBA);
inducing 10 bottles of each culture medium of the two explants, putting 4-6 explants in each bottle, and observing regularly every day;
and B system: MS + NAA 1mg/L +2, 4-D1 mg/L + sucrose 30g/L, pH5.8;
h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L, pH5.8.
The results show that for leaf-derived explants, in system B: MS + NAA 1mg/L + 2, 4-D1 mg/L + sucrose 30g/L, can obtain the stem cell of stellera chamaejasme leaf in good state, and takes place the stratification phenomenon with dedifferentiated cell in the culture medium of pH5.8; for root-derived explants, in system B: MS + NAA 1mg/L + 2, 4-D1 mg/L + sucrose 30g/L, pH5.8 and H system: the stellera chamaejasme root stem cells with good state and delamination phenomenon with dedifferentiated cells can be obtained under the conditions of MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L and pH5.8.
Step three, subculturing the stellera chamaejasme stem cells: carefully peeling the cambium cells obtained in the step two from the explants by using long tweezers, inoculating the cambium cells into a subculture medium, and performing dark culture for 9 to 21 days under the conditions that the temperature is 24 to 26 ℃ and the humidity is 55 to 75 percent; wherein the formula of the subculture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 0-2 mg/mL phytohormone and 8.5-9.5 g/L agar, and the pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), indole-3-butyric acid (IBA), naphthalene Acetic Acid (NAA), and 2, 4-dichlorophenoxy acetic acid (2, 4-D).
Subculturing once every 2-3 weeks to obtain stellera chamaejasme stem cells with stable performance;
the results show that: for stem cells derived from stellera chamaejasme leaves, a B system is selected: MS + NAA 1mg/L + 2, 4-D1 mg/L + sucrose 30g/L, pH5.8 culture medium as subculture medium; for stem cells derived from stellera chamaejasme roots, an H system is selected: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L, pH5.8 culture medium as subculture medium, and its cell morphology is shown in figures 1 and 2 respectively.
Example 3
A culture method of stellera chamaejasme stem cells is a shake flask suspension culture method and specifically comprises the following steps:
inoculating the stellera chamaejasme stem cells into a liquid subculture medium, wherein the inoculation amount is 50g/L of wet weight of the cells, the culture temperature is 24-26 ℃, the rotating speed is 110-130 rpm/min, and dark culture is carried out for 10-25 days; wherein the formula of the liquid subculture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 1mg/mL phytohormone and 8.5-9.5 g/L agar, and the pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine (6-BA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D).
And (3) measuring a stellera chamaejasme stem cell suspension cell growth curve: h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L, suspension culture 30 bottles of stellera chamaejasme root-derived stem cells in culture medium with pH5.8, taking 3 bottles of cells every 5 days, filtering and separating the cells and liquid culture medium by using a cloth type funnel, placing the separated cells in a culture dish, freeze-drying, weighing the dry weight of the cells after complete drying, and recording the growth curve as shown in figure 3.
Example 4
Microscopic shape inspection of stellera chamaejasme stem cells
Respectively taking a small amount of plant cells induced by an H system culture medium from stellera chamaejasme roots; plant cells induced from stellera chamaejasme roots with a B system medium; plant cells induced from stellera chamaejasme leaves by using a B system culture medium are placed on a glass slide, smashed by using a blade, dropwise added with a drop of neutral red dye, dyed for 30-60s, covered with a cover glass, washed by clear water, placed under a microscope for observation, observed by using a 100X objective lens, and the microstructure of the plant cells is shown as an attached figure 3-6.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A preparation method of stellera chamaejasme stem cells is characterized by comprising the following steps:
step one, obtaining explant materials: taking roots and leaves of fresh stellera chamaejasme as explants, cleaning, soaking for 5min by using a soaking solution, then washing the explants by using clear water, putting the explants into a super clean bench, respectively placing the roots and the leaves on a culture dish padded with sterile filter paper after sterilization treatment, and then cutting the roots and the leaves into small pieces under the aseptic condition, wherein the pulp parts of the roots are stripped off to obtain root tissue blocks and leaf tissue blocks containing cambium cells; the soaking solution is a laundry powder solution; the sterilization treatment comprises the following specific steps: soaking in 75% ethanol for 3min for sterilization, washing with sterile water for 3 times, and adding 0.1% of HgCl 2 Soaking the solution for 6min, washing with sterile water for 6 times, carefully wiping with sterile filter paper, and soaking in 150mg/L citric acid solution;
step two, induction of stellera chamaejasme stem cells: placing the tissue block containing cambium cells obtained in the step one on induction culture media of different systems for induction, and culturing in the dark for 7-15 days under the conditions that the temperature is 24-26 ℃ and the humidity is 55-75%; wherein the formula of the induction culture medium is as follows: b system or H system, wherein, B system: MS + NAA 1mg/L +2, 4-D1 mg/L + sucrose 30g/L + 8.5-9.5 g/L agar, pH5.8; h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L + 8.5-9.5 g/L agar, pH5.8;
step three, subculturing stellera chamaejasme stem cells: inoculating the cambium cells obtained in the step two into a subculture medium, and performing dark culture for 9-21 days at the temperature of 24-26 ℃ and the humidity of 55-75%; the formula of the subculture medium is as follows: b system or H system, wherein, B system: MS + NAA 1mg/L +2, 4-D1 mg/L + sucrose 30g/L + 8.5-9.5 g/L agar, pH5.8; h system: MS +6-BA 1mg/L + IBA 1mg/L + sucrose 30g/L + 8.5-9.5 g/L agar, pH5.8.
2. The method for preparing stellera chamaejasme stem cells as claimed in claim 1, wherein the cutting of the roots and leaves in the first step is performed by using forceps and knife, wherein the size of the tissue block of the leaves is 0.6 x 0.6cm; the root tissue block size was 0.3X 0.8cm.
3. The method for preparing stellera chamaejasme stem cells according to claim 1 or 2, wherein the stellera chamaejasme stem cells are obtained by the method.
4. The method for preparing stellera chamaejasme stem cells according to claim 3, further comprising a culture method of stellera chamaejasme stem cells, which is a shake flask suspension culture method, and specifically comprises the following steps:
inoculating the stellera chamaejasme stem cells into a liquid subculture medium, wherein the inoculation amount is 50g/L of wet weight of the cells, the culture temperature is 24-26 ℃, the rotation speed is 110-130 rpm, and dark culture is carried out for 10-25 days; wherein the formula of the liquid subculture medium is as follows: MS culture medium containing 20-30 g/L sucrose, 1mg/mL phytohormone and 8.5-9.5 g/L agar, and the pH value is 5.75-5.85; the plant hormone is the combination of any two of 6-benzylamino adenine, indole-3-butyric acid, naphthylacetic acid and 2, 4-dichlorophenoxyacetic acid.
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| CN102870672A (en) * | 2011-07-12 | 2013-01-16 | 中国林业科学研究院林业研究所 | Method of induction and tissue culture for adventitious roots of Euphorbia fischeriana |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102870672A (en) * | 2011-07-12 | 2013-01-16 | 中国林业科学研究院林业研究所 | Method of induction and tissue culture for adventitious roots of Euphorbia fischeriana |
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