CN113913362A - Sorbus commixta stem cell capable of increasing anthocyanin content, culture medium and culture method - Google Patents
Sorbus commixta stem cell capable of increasing anthocyanin content, culture medium and culture method Download PDFInfo
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- 229930002877 anthocyanin Natural products 0.000 title claims abstract description 71
- 235000010208 anthocyanin Nutrition 0.000 title claims abstract description 71
- 239000004410 anthocyanin Substances 0.000 title claims abstract description 71
- 150000004636 anthocyanins Chemical class 0.000 title claims abstract description 71
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 61
- 239000001963 growth medium Substances 0.000 title claims abstract description 60
- 238000012136 culture method Methods 0.000 title claims abstract description 18
- 230000001965 increasing effect Effects 0.000 title claims abstract description 13
- 235000004489 Sorbus commixta Nutrition 0.000 title claims description 11
- 241001115347 Sorbus commixta Species 0.000 title claims description 11
- 229920002752 Konjac Polymers 0.000 claims abstract description 68
- 235000010485 konjac Nutrition 0.000 claims abstract description 66
- 230000006698 induction Effects 0.000 claims abstract description 23
- 241001655175 Sorbus pohuashanensis Species 0.000 claims abstract description 20
- 235000012072 hua qui shu Nutrition 0.000 claims abstract description 20
- 238000009825 accumulation Methods 0.000 claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 18
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 18
- 239000002609 medium Substances 0.000 claims description 21
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- 238000012258 culturing Methods 0.000 claims description 12
- 235000013312 flour Nutrition 0.000 claims description 9
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- 238000002360 preparation method Methods 0.000 claims description 6
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- 150000002215 flavonoids Chemical class 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 description 2
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- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
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- C12N5/04—Plant cells or tissues
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Abstract
A sorbus pohuashanensis stem cell for improving the anthocyanin content, a culture medium and a culture method are provided, the culture medium can be applied to the sorbus pohuashanensis stem cell synthesis anthocyanin culture, and the culture method comprises the following specific steps: (1) preparing stem cell tissue; (2) induction and accumulation of anthocyanins. The invention uses konjak to prepare a natural culture medium, and uses the nutrient substances of the konjak to provide macroelements, microelements and carbon sources for stem cells to be cultured; substances contained in the konjac have a promoting effect on stem cell induction, explants are expanded within 3-5d, and stem cells are obviously increased within 7-10 d; the konjak culture medium also has the function of promoting the synthesis of anthocyanin, and the content of anthocyanin is obviously improved; in addition, the konjac glucomannan has good coagulability by utilizing the characteristics of the konjac glucomannan, the addition of agar is reduced, the culture cost is saved, and the target product cultured by the konjac glucomannan is safer and more reliable due to the natural culture medium.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a sorbus pohuashanensis stem cell capable of improving anthocyanin content, a culture medium and a culture method.
Background
Aronia melanocarpa (Aronia melanocarpa) belongs to Rosaceae, is native to eastern North America, is introduced into Europe and Soviet Union later, and is introduced into China for 90 years in the last century. The research on the functionality of aronia melanocarpa polyphenol substances starts in the 80 th century, and the health care value of the aronia melanocarpa polyphenol substances is increasingly shown along with the gradual discovery of the functionality of the aronia melanocarpa polyphenol substances in the aspects of free radical removal, cancer resistance, blood pressure reduction, blood vessel softening and the like. The aronia melanocarpa fruit is rich in polyphenols such as anthocyanin, flavonoid, phenolic acid, procyanidine and the like, and has obvious effects of enhancing mutation resistance, protecting heart and reducing blood fat after long-term eating due to the capabilities of resisting oxidation, removing free radicals and influencing human metabolism of the polyphenols in the aronia melanocarpa fruit.
At present, the mountain ash trees are planted in some places in China on a small scale, the increasing market demands can not be met, in addition, in the production process, the prominent problems of pest and disease damage and yield reduction, climate and season limitation, serious toxicity and side effects, pesticide residue and the like exist, moreover, the quality of the fruits produced in different regions and different years is unstable, the content of active ingredients such as anthocyanin and the like is greatly changed, and the downstream processing and the commercial popularization of the mountain ash trees are influenced; in addition, the content of tannin in the mountain ash fruit planted in the field is high, which affects the quality and taste of the deep-processed product.
Therefore, providing the sorbus pohuashanensis stem cells with simple culture method, high anthocyanin content and short period, the culture medium and the culture method thereof are the problems that need to be solved by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a sorbus pohuashanensis stem cell with increased anthocyanin content, a culture medium and a culture method, the invention utilizes konjak to prepare a natural culture medium, and utilizes nutrient substances of the konjak to provide macroelements, microelements and carbon sources for the stem cell to be cultured; substances contained in the konjac have a promoting effect on stem cell induction, explants are expanded within 3-5d, and stem cells are obviously increased within 7-10 d; the konjak culture medium also has the function of promoting anthocyanin synthesis; in addition, the konjac glucomannan has good coagulability by utilizing the characteristics of the konjac glucomannan, the addition of agar is reduced, the culture cost is saved, and the target product cultured by the konjac glucomannan is safer and more reliable due to the natural culture medium.
In order to achieve the purpose, the invention adopts the following technical scheme:
a Sorbus commixta stem cell for increasing anthocyanin synthesis content.
Increase of anthocyanin content in sorbus pohuashanensis stem cellsThe culture medium is a konjak culture medium; wherein, each liter of the konjak culture medium comprises: 1-3g of konjaku flour and 0.5-3mg of FeCl2And 0.5-5g potassium nitrate.
The preparation method of the culture medium for improving the anthocyanin content of the sorbus pohuashanensis stem cells comprises the following specific steps:
(1) weighing raw materials: weighing 1-3g rhizoma Amorphophalli powder and 0.5-3mg FeCl per liter of culture medium2And 0.5-5g potassium nitrate;
(2) adding the konjac flour into 1L of water, and uniformly stirring to obtain a konjac flour swelling system;
(3) adding hydrochloric acid solution into the konjac flour swelling system, hydrolyzing at 40-60 deg.C for 0.5-3h, adding FeCl2Carrying out a cross-linking reaction;
(4) KNO is added into the solution obtained in the step (3)3Adjusting pH to 5.0-8.0 to obtain rhizoma Amorphophalli culture medium.
The application of the culture medium for improving the anthocyanin content of the sorbus pohuashanensis stem cells is the application of the culture medium in the synthesis of anthocyanin by sorbus pohuashanensis stem cells.
Preferably, the final concentration of the hydrochloric acid solution is 1-2 mol/L.
The culture method of the sorbus pohuashanensis stem cells for improving the synthesis content of anthocyanin comprises the following specific steps:
(1) preparation of stem cell tissue: pretreating the root tip of the aseptic seedling of the Sorbus commixta, transferring to a konjak induction culture medium for culture for 25-30d, transferring to a konjak multiplication culture medium for subculture for 5-10 times to obtain a stem cell tissue;
(2) induction and accumulation of anthocyanins: and transferring the stem cell tissue to an anthocyanin induction culture medium for culturing for 15-20d, and then transferring to an anthocyanin accumulation culture medium for culturing for 25-30d to obtain the sorbus pohuashanensis stem cell with high anthocyanin content.
The invention uses konjak to prepare a natural culture medium, and uses the nutrient substances of the konjak to provide macroelements, microelements and carbon sources for stem cells to be cultured; substances contained in the konjac have a promoting effect on stem cell induction, explants are expanded within 3-5d, and stem cells are obviously increased within 7-10 d; the konjak culture medium also has the function of promoting anthocyanin synthesis; in addition, the konjac glucomannan has good coagulability by utilizing the characteristics of the konjac glucomannan, so that the addition of agar is reduced, and the culture cost is saved.
Preferably, the pretreatment step is: taking 0.1mm root tip of aseptic seedling of Sorbus pohuashanensis, placing in a container containing 0.1 wt% KH2PO4+0.1wt％MgCl2Treating in glycerol solution for 12h, washing, wiping, and sequentially transferring to-20 deg.C dark treatment for 8-10h, 0 deg.C dark treatment for 30min, and 10 deg.C dark treatment for 30 min.
The invention utilizes 0.1% KH protective agent2PO4+0.1％MgCl2The glycerol protects the root tip stem cells, the stem cells are not damaged after low-temperature treatment, and the parenchyma cells around the stem cells are damaged, so that the root tip stem cells with high uniformity are obtained.
Preferably, the konjak induction medium of step (1) is a konjak medium supplemented with 0.5-3 mg/L2, 4-D and 0.5-2 mg/L6-BA.
The culture medium adopted by the invention can promote the growth of the stem cells and obtain a large amount of stem cell seeds in a short time; the same effect is obtained under the condition of low concentration hormone, namely the effect of the auxin is synergistic, the accumulation of exogenous hormone in cells is reduced, and the using amount of the hormone is reduced. By using the culture medium, the explant for preparing the stem cells is obviously expanded in 3-5 days, and the volume of the stem cell tissue newly increased by the explant in 7-10 days is increased by more than 30%.
Preferably, the konjak propagation medium in the step (1) is a konjak medium supplemented with 0.5-1 mg/L2, 4-D.
The anthocyanin in the stem cell tissue begins to be synthesized and accumulated by adopting the culture medium, the stem cell tissue is gradually changed into the anthocyanin synthesis state from the growth state by using the culture medium, the stem cell tissue is gradually deepened from milk white to light red, deep red and purple red, and the synthesis and accumulation of the anthocyanin are stimulated by Ge-132, so that the anthocyanin synthesis and accumulation can be improved by more than 1.2 times.
Preferably, the anthocyanin induction medium in the step (2) is a konjak medium supplemented with 0.01-0.05 wt% Ge-132, 0-0.5 mg/L6-BA and 0.1-2 mg/L2, 4-D.
By adopting the culture medium, the stem cell tissue growth and anthocyanin accumulation are gradually balanced, namely a certain growth speed is kept, the synthesis capacity of the anthocyanin is also kept, and the anthocyanin accumulation reaches the maximum at the final growth stage. The content of the anthocyanin reaches 1.8 percent, which is far higher than the content of the anthocyanin in cultivation and common cell culture.
Preferably, the anthocyanin accumulation medium in the step (2) is a konjak medium supplemented with 0.01-0.05 wt% Ge-132+ 0-0.5 mg/L6-BA.
The konjak culture medium has the advantages that the water absorption rate, the expansion rate and the physical elasticity of the product after polysaccharide hydrolysis are good, and the lower using amount can obtain the better water absorption rate and the expansion rate, so that the better water retention effect is achieved, and the cell growth is promoted. Under the use amount of less than 3g/L, the culture medium has good elasticity and water absorption rate of 1:500, and the culture medium contains less volatile and lost water, thereby being beneficial to the growth of cell tissues.
Preferably, the conditions of said culturing and said subculture in step (1) are dark culturing at 21-25 ℃.
The synthesis of anthocyanin is related to growth state and environmental condition, and the synthesis of anthocyanin of Sorbus pohuashanensis belongs to an intermediate type, which is between a growth coupling type and a non-growth coupling type. Therefore, a balance point is found between the requirement of biomass and anthocyanin accumulation, and the temperature interval and dark culture of the invention have promotion effects on cell growth division and anthocyanin accumulation.
Preferably, the conditions of the culturing in step (2) are: culturing under the irradiation condition of 26-28 ℃ and the light intensity of 1500-.
The synthesis of anthocyanin of the sorbus pohuashanensis has certain dependence on illumination time, but the illumination time is not suitable to exceed a certain time, otherwise the accumulation and decomposition imbalance of the anthocyanin is influenced.
Preferably, the illumination time is 14h/24 h.
The anthocyanin extraction rate and yield have great dependence on extraction conditions, including extraction solvent polarity, pH value, extraction time, feed-liquid ratio and temperature. These factors all affect the efficiency of anthocyanin dissolution from stem cell tissue.
Preferably, the extraction method of the anthocyanin is as follows: extracting the obtained Sorbus commixta stem cells with high anthocyanin content with 3 wt% hydrochloric acid-methanol as extractant at a material-liquid ratio of 1:15, an extraction time of 18h and an extraction temperature of 35 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention uses konjak to prepare a natural culture medium, and uses the nutrient substances of the konjak to provide macroelements, microelements and carbon sources for stem cells to be cultured; substances contained in the konjac have a promoting effect on stem cell induction, explants are expanded within 3-5d, and stem cells are obviously increased within 7-10 d; the konjak culture medium also has the function of promoting anthocyanin synthesis; the konjac glucomannan hydrolysate has the effect of an inducer and has an induction effect on secondary metabolism such as anthocyanin; in addition, the konjac glucomannan has good coagulability by utilizing the characteristics of the konjac glucomannan, the addition of agar is reduced, the culture cost is saved, and the target product cultured by the konjac glucomannan is safer and more reliable due to the natural culture medium.
(2) The invention utilizes the characteristics of strong division capability and rapid growth of stem cells to reduce the complicated preparation process of cultured cell seeds, so as to obtain more anthocyanin yield in a short time and replace the wild ash cultivated in the field.
(3) The anthocyanin obtained by the invention is a natural pigment biosynthesized by the aronia melanocarpa stem cells, is a flavonoid substance, can be directly and stably absorbed by tissues as a natural edible pigment, has the advantages of no toxicity, no harm and high safety compared with a synthetic pigment, has the physiological functions of resisting oxidation activity and mutagenicity, reducing blood fat, protecting liver function, improving vision and the like for a human body, can be used in the industries of food, medicine and the like, and can meet the requirements of people on the natural pigment.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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
A culture method for improving the anthocyanin content of sorbus pohuashanensis stem cells comprises the following specific steps:
(1) preparation of konjak Medium
1) Weighing 1g of konjac flour, adding 1L of water, uniformly stirring, and waiting until the swelling of the konjac flour is stable to obtain a konjac flour swelling system;
2) heating the konjac flour swelling system in a constant-temperature water bath at 55 ℃, adding 1mol/L hydrochloric acid solution, stirring at 40 ℃, reacting for 2 hours, and then adding FeCl with the final concentration of 0.15mg2Adding the mixture into a culture medium for crosslinking reaction;
3) after the crosslinking reaction was completed, 1.5g of potassium nitrate was added, and the pH thereof was adjusted to 7.5, to obtain a konjak medium.
(2) Preparation of stem cell tissue:
placing root tip of aseptic seedling of Aronia melanocarpa 0.1mm in sterilized container containing 0.1% KH2PO4+0.1％MgCl2Treating in glycerol solution for 12h, rapidly washing with sterile water for 3 times, absorbing surface water by sterile paper, transferring to-20 deg.C dark treatment for 8h, 0 deg.C dark treatment for 30min and 10 deg.C dark treatment for 30min, transferring to rhizoma Amorphophalli induction culture medium, dark culturing at 25 deg.C for 5d to obtain swelling, transferring to rhizoma Amorphophalli proliferation culture medium after 25d, and performing 5 subcultures at 25 deg.C to obtain a large amount of stem cell tissue with induction rate of 100%; wherein the konjak induction culture medium is a konjak culture medium added with 1 mg/L2, 4-D and 0.5 mg/L6-BA, and the konjak propagation culture medium is a konjak culture medium added with 0.5 mg/L2, 4-D;
(3) induction and accumulation of anthocyanins:
transferring the obtained stem cell tissue to an anthocyanin induction culture medium, culturing for 15d under the conditions of 26 ℃, light intensity of 2500LX and illumination time of 14h/24h, and then transferring to an anthocyanin accumulation culture medium to culture for 30d under the same conditions to obtain the sorbus pohuashanensis stem cell with high anthocyanin content; wherein the anthocyanin induction culture medium is a konjak culture medium added with 0.01% Ge-132, 0.5 mg/L6-BA and 0.1 mg/L2, 4-D, and the anthocyanin accumulation culture medium is a konjak culture medium added with 0.01% Ge-132+0.5 mg/L6-BA.
The obtained Sorbus commixta stem cells with high anthocyanin content are extracted under the conditions of 3% hydrochloric acid-methanol as an extracting agent, the material-liquid ratio of 1:15, the extraction time of 18h and the extraction temperature of 35 ℃, the light absorption value is measured at 565nm to obtain the anthocyanin content, wherein, the Sorbus commixta fruit and common callus (young and tender leaves, the same culture method as that of example 1) are used as a contrast, and the results are shown in Table 2.
TABLE 2 Induction Effect of anthocyanins in Sorbus commixta Stem cells
Example 2 Effect of konjak Medium on anthocyanin content in Sorbus commixta
The stem cells and the common callus tissues adopted by the invention are respectively cultivated in different culture media and fields in the prior art for anthocyanin synthesis comparison, and the results are shown in table 3.
TABLE 3 comparison of the anthocyanin content of the konjak Medium with that of the conventional Medium
The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments can be referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A Sorbus commixta stem cell for increasing anthocyanin synthesis content.
2. A culture medium for improving the anthocyanin content of sorbus pohuashanensis stem cells is characterized in that the culture medium is a konjak culture medium; wherein, each liter of the konjak culture medium comprises: 1-3g of konjaku flour and 0.5-3mg of FeCl2And 0.5-5g potassium nitrate.
3. A method for culturing sorbus pohuashanensis stem cells with increased anthocyanin synthesis content is characterized by comprising the following specific steps:
(1) preparation of stem cell tissue: pretreating the root tip of the aseptic seedling of the Sorbus commixta, transferring to a konjak induction culture medium for culture for 25-30d, transferring to a konjak multiplication culture medium for subculture for 5-10 times to obtain a stem cell tissue;
(2) induction and accumulation of anthocyanins: and transferring the stem cell tissue to an anthocyanin induction culture medium for culturing for 15-20d, and then transferring to an anthocyanin accumulation culture medium for culturing for 25-30d to obtain the sorbus pohuashanensis stem cell with high anthocyanin content.
4. The culture method according to claim 3, wherein the pretreatment step is: taking 0.1mm root tip of aseptic seedling of Sorbus pohuashanensis, placing in a container containing 0.1% KH2PO4+0.1％MgCl2Treating in glycerol solution for 12h, washing, wiping, and sequentially transferring to-20 deg.C dark treatment for 8-10h, 0 deg.C dark treatment for 30min, and 10 deg.C dark treatment for 30 min.
5. The culture method according to claim 3, wherein the konjak induction medium of the step (1) is a konjak medium supplemented with 0.5 to 3mg/L of 2,4-D and 0.5 to 2mg/L of 6-BA.
6. The culture method according to claim 3, wherein the konjak propagation medium in the step (1) is a konjak medium supplemented with 0.5 to 1mg/L of 2, 4-D.
7. The culture method according to claim 3, wherein the anthocyanin induction medium of the step (2) is konjak medium supplemented with 0.01 to 0.05% Ge-132, 0.1 to 0.5 mg/L6-BA and 0.5 to 2 mg/L2, 4-D.
8. The culture method according to claim 3, wherein the anthocyanin accumulation medium in the step (2) is konjak medium supplemented with 0.01 to 0.05% Ge-132+0.1 to 0.5mg/L of 6-BA.
9. The culture method according to claim 3, wherein the culture conditions in step (2) are: culturing under the irradiation condition of 26-28 ℃ and the light intensity of 1500-.
10. The culture method according to claim 9, wherein the irradiation time is 14h/24 h.
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| CN116889197A (en) * | 2023-08-08 | 2023-10-17 | 大连工业大学 | Method for inducing adventitious roots of Aronia melanocarpa to produce anthocyanin |
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| CN116889197A (en) * | 2023-08-08 | 2023-10-17 | 大连工业大学 | Method for inducing adventitious roots of Aronia melanocarpa to produce anthocyanin |
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