CN113913362B - Sorbus pohuashanensis stem cell for improving anthocyanin content, culture medium and culture method - Google Patents
Sorbus pohuashanensis stem cell for improving anthocyanin content, culture medium and culture method Download PDFInfo
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Abstract
The culture medium can be applied to culture of anthocyanin synthesis of the mountain ash stem cells, and the culture method comprises the following specific steps: (1) preparation of stem cell tissue; (2) induction and accumulation of anthocyanin. The invention utilizes konjak to prepare natural culture medium, and utilizes the nutrient substances of konjak to provide major elements, trace elements and carbon source for stem cells to be cultured; substances contained in konjak have promotion effect on stem cell induction, 3-5d explants expand, and 7-10d stem cells are obviously increased; the konjak culture medium also has the effect of promoting anthocyanin synthesis, and the anthocyanin content is obviously improved; in addition, the characteristic of konjak glucomannan is utilized to ensure that the konjak glucomannan has good coagulability, the addition of agar is reduced, the culture cost is saved, and the natural culture medium also ensures that the target product of the culture is safer and more reliable.
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
Sorbus nigra (Aronia melanocarpa) belongs to Rosaceae plants, is native to the eastern North America, is later introduced into Europe and Soviet Union, and is introduced into China for cultivation in the last 90 th century. The research on the functionality of the Aronia melanocarpa polyphenol substance starts from the 80 th century, and along with the gradual discovery of the functionality of the Aronia melanocarpa polyphenol substance in the aspects of free radical removal, cancer resistance, blood pressure reduction, blood vessel softening and the like, the health care value of the Aronia melanocarpa polyphenol substance is also increasingly revealed. The Aronia melanocarpa fruits are rich in polyphenols such as anthocyanin, flavonoid, phenolic acid and procyanidine, and the effects of resisting oxidization, scavenging free radicals and affecting human metabolism are obvious for enhancing the anti-mutation, protecting the heart and reducing the blood lipid by long-term eating of the Aronia melanocarpa fruits due to the various capacities of the polyphenols in the Aronia melanocarpa fruits.
At present, the small-scale planting of the black chokeberry in some places in China can not meet the increasing demands of the market, and in the production process, the problems of the serious pest and disease damage, the climate and season limitation, the serious toxicity and side effects, pesticide residues and the like exist, the quality of fruits produced in different areas and different years is unstable, the content of active ingredients such as anthocyanin and the like is greatly changed, and the downstream processing and commercial popularization of the black chokeberry are influenced; in addition, the mountain ash fruit planted in the field has high content of tannin components, which affects the quality and taste of the deep-processed product.
Therefore, the problem that the Sorbus pohuashanensis stem cells, the culture medium and the culture method thereof, which are simple in culture method, high in anthocyanin content and short in period, are needed to be solved by the person skilled in the art, can be provided.
Disclosure of Invention
In view of the above, the invention provides a Sorbus pohuashanensis stem cell, a culture medium and a culture method for improving anthocyanin content, wherein the invention utilizes konjak to prepare a natural culture medium, and utilizes the nutrition of konjak to provide macroelements, microelements and carbon sources for stem cells to be cultured; substances contained in konjak have promotion effect on stem cell induction, 3-5d explants expand, and 7-10d stem cells are obviously increased; the konjak culture medium also has the effect of promoting anthocyanin synthesis; in addition, the characteristic of konjak glucomannan is utilized to ensure that the konjak glucomannan has good coagulability, the addition of agar is reduced, the culture cost is saved, and the natural culture medium also ensures that the target product of the culture is safer and more reliable.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
sorbus pohuashanensis stem cells for increasing anthocyanin synthesis content.
A culture medium for improving anthocyanin content of stem cells of Sorbus pohuashanensis, wherein the culture medium is a konjak culture medium; wherein each ofLifting the konjak medium comprises: 1-3g konjak flour, 0.5-3mgFeCl 2 And 0.5-5g potassium nitrate.
The preparation method of the culture medium for improving anthocyanin content of the Sorbus pohuashanensis stem cells comprises the following specific steps of:
(1) Weighing raw materials: 1g to 3g konjaku flour and 0.5mg to 3mg FeCl are weighed per liter of culture medium 2 And 0.5-5g of potassium nitrate;
(2) Adding the konjaku flour into 1L of water, and uniformly stirring to obtain a konjaku flour swelling system;
(3) Adding hydrochloric acid solution into the swelling system of rhizoma Amorphophalli powder, hydrolyzing at 40-60deg.C for 0.5-3 hr, and adding FeCl 2 Carrying out a glue coupling reaction;
(4) Adding KNO into the solution obtained in the step (3) 3 And regulating the pH to 5.0-8.0 to obtain the konjak culture medium.
The application of the culture medium for improving the anthocyanin content of the mountain ash stem cells is applied to the culture of the anthocyanin synthesis of the mountain ash stem cells.
Preferably, the final concentration of the hydrochloric acid solution is 1-2mol/L.
The culture method of the Sorbus pohuashanensis stem cells for improving the anthocyanin synthesis content comprises the following specific steps of:
(1) Preparation of stem cell tissue: pretreating root tips of aseptic seedlings of Sorbus pohuashanensis, transferring to a konjak induction culture medium for culturing for 25-30d, transferring to a konjak proliferation culture medium, and performing secondary culture for 5-10 times to obtain stem cell tissues;
(2) Induction and accumulation of anthocyanin: and transferring the stem cell tissue to an anthocyanin induction medium for 15-20d, and transferring to an anthocyanin accumulation medium for culturing for 25-30d to obtain the high anthocyanin-content Sorbus pohuashanensis stem cell.
The invention utilizes konjak to prepare natural culture medium, and utilizes the nutrient substances of konjak to provide major elements, trace elements and carbon source for stem cells to be cultured; substances contained in konjak have promotion effect on stem cell induction, 3-5d explants expand, and 7-10d stem cells are obviously increased; the konjak culture medium also has the effect of promoting anthocyanin synthesis; in addition, the characteristic of konjak glucomannan is utilized to ensure that the konjak glucomannan has good coagulability, so that the addition of agar is reduced, and the culture cost is saved.
Preferably, the pretreatment step is as follows: taking root tip of sterile seedling of Sorbus pohuashanensis 0.1mm, placing in a container containing 0.1wt% KH 2 PO 4 +0.1wt％MgCl 2 Is treated in glycerin solution for 12 hours, then washed and wiped dry, and then is sequentially transferred to dark treatment at-20 ℃ for 8-10 hours, dark treatment at 0 ℃ for 30 minutes and dark treatment at 10 ℃ for 30 minutes.
The invention uses the protective agent 0.1% KH 2 PO 4 +0.1％MgCl 2 The +glycerol protects the root tip stem cells, the stem cells are not destroyed after low-temperature treatment, and the parenchyma cells around the stem cells are destroyed, so that the root tip stem cells with high uniformity are obtained.
Preferably, the konjak induction medium in the step (1) is konjak medium added with 0.5-3 mg/L2, 4-D and 0.5-2 mg/L6-BA.
The invention adopts the culture medium to promote the growth of stem cells and obtain a large amount of stem cell seeds in a short time; the same effect is obtained at low concentration of hormone, namely, the effect of synergistic auxin is achieved, and the accumulation of exogenous hormone in cells is reduced, namely, the use amount of the hormone is reduced. By using the culture medium, 3-5d of the explant for preparing the stem cells obviously expands, and the volume of the newly added stem cell tissue of the 7-10d explant is increased by more than 30%.
Preferably, the konjak proliferation medium in the step (1) is konjak culture medium added with 0.5-1 mg/L2, 4-D.
The culture medium is adopted, anthocyanin in stem cell tissues begins to synthesize and accumulate, the stem cell tissues are gradually changed into anthocyanin synthesis state from growth state, the stem cell tissues are gradually deepened from milky white to light red, dark red and purple, and Ge-132 stimulates anthocyanin synthesis and accumulation, 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 konjak medium added with 0.01-0.05wt% of Ge-132, 0-0.5mg/L of 6-BA and 0.1-2mg/L of 2, 4-D.
By adopting the culture medium, the growth of stem cell tissues and the accumulation of anthocyanin are gradually balanced, so that a certain growth speed is maintained, the synthesis capacity of anthocyanin is maintained, the final growth stage is reached, and the accumulation of anthocyanin is maximized. The anthocyanin content reaches 1.8 percent, which is far higher than the content of cultivation and common cell culture.
Preferably, the anthocyanin accumulation medium in step (2) is konjak medium supplemented with 0.01-0.05wt% Ge-132+0-0.5 mg/L6-BA.
The konjak culture medium adopted by the invention has the advantages of good water absorption, expansion rate and physical elasticity of the product after polysaccharide hydrolysis, good water absorption and expansion rate obtained by low dosage, good water retention effect and promotion of cell growth. Under the use amount lower than 3g/L, the culture medium has good elasticity and water absorption rate reaching 1:500, and the culture medium is not easy to volatilize and lose the conservation 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 anthocyanin synthesis is related to the growth state and the environmental condition, and the relationship between the anthocyanin synthesis and the cells of the Sorbus pohuashanensis belongs to the intermediate type and is between the growth coupling type and the non-growth coupling type. Thus, a balance point is found between the 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 culturing conditions in step (2) are: culturing at 26-28deg.C under light with light intensity of 1500-2000 LX.
The anthocyanin synthesis of the Sorbus pohuashanensis has a certain dependence on the illumination time, but the illumination time is not longer than a certain period, otherwise, the imbalance of anthocyanin accumulation and decomposition is affected.
Preferably, the illumination time is 14h/24h.
The anthocyanin extraction rate and yield have great dependence on extraction conditions, including polarity of extraction solvent, pH value, extraction time, feed-liquid ratio and temperature. These factors all affect the efficiency of anthocyanin elution from stem cell tissue.
Preferably, the anthocyanin extraction method comprises the following steps: extracting the obtained high anthocyanin content Sorbus pohuashanensis stem cells with 3wt% hydrochloric acid-methanol as extractant at a feed liquid ratio of 1:15 for 18h at 35 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention utilizes konjak to prepare natural culture medium, and utilizes the nutrient substances of konjak to provide major elements, trace elements and carbon source for stem cells to be cultured; substances contained in konjak have promotion effect on stem cell induction, 3-5d explants expand, and 7-10d stem cells are obviously increased; the konjak culture medium also has the effect of promoting anthocyanin synthesis; the konjak glucomannan hydrolysate has an inducer effect and has an induction effect on secondary metabolism such as anthocyanin and the like; in addition, the characteristic of konjak glucomannan is utilized to ensure that the konjak glucomannan has good coagulability, the addition of agar is reduced, the culture cost is saved, and the natural culture medium also ensures that the target product of the culture is safer and more reliable.
(2) The invention uses the characteristics of strong stem cell division capability and quick growth to reduce the complicated preparation process of culturing cell seeds, so as to obtain more anthocyanin yield in a short period and replace the mountain ash cultivated in a field.
(3) The anthocyanin obtained by the invention is a natural pigment biosynthesized by stem cells of Aronia melanocarpa, 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 antioxidant activity and anti-mutagenicity, has physiological functions of reducing blood fat, protecting liver function, improving eyesight and the like for human bodies, can be used in industries such as food, medicine and the like, and can meet the demands of people on the natural pigment.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A culture method for improving anthocyanin content of Sorbus pohuashanensis stem cells comprises the following specific steps:
(1) Preparation of konjak culture medium
1) 1g of konjaku flour is weighed, 1L of water is added, and the mixture is stirred uniformly until swelling of the konjaku flour is stable, so that a konjaku flour swelling system is obtained;
2) Heating the swelling system of konjak powder in a constant temperature water bath at 55 ℃, adding 1mol/L hydrochloric acid solution, stirring at 40 ℃ for reaction for 2 hours, and then adding FeCl with the final concentration of 0.15mg 2 Adding the mixture into a culture medium to carry out a crosslinking reaction;
3) After the crosslinking reaction is completed, 1.5g of potassium nitrate is added, and the pH is adjusted to 7.5, thus obtaining the konjak culture medium.
(2) Preparation of stem cell tissue:
taking root tip of Aronia melanocarpa aseptic seedling 0.1mm, placing in sterilized container containing 0.1% KH 2 PO 4 +0.1％MgCl 2 The glycerol solution of (2) is treated for 12h, the sterile water is quickly washed for 3 times, the surface moisture is absorbed by sterile paper, the surface moisture is sequentially transferred to the dark treatment at-20 ℃ for 8h, the dark treatment at 0 ℃ for 30min and the dark treatment at 10 ℃ for 30min, the surface moisture is transferred to a konjak induction culture medium, the surface moisture is subjected to dark culture at 25 ℃ for 5d, the swelling is generated, the generated stem cell tissue is transferred to a konjak proliferation culture medium after 25d, and a large amount of stem cell tissue is obtained after 5 times of secondary dark culture at 25 ℃, and the induction rate is 100%; wherein the konjak induction culture medium is konjak culture medium added with 1 mg/L2, 4-D and 0.5 mg/L6-BA, and the konjak proliferation culture medium is konjak culture medium added with 0.5 mg/L2, 4-D;
(3) Induction and accumulation of anthocyanin:
transferring the obtained stem cell tissue to an anthocyanin induction culture medium, culturing for 15d under the conditions of 26 ℃ and 2500LX light intensity and 14h/24h illumination time, and transferring to an anthocyanin accumulation culture medium for culturing for 30d under the same conditions to obtain the high anthocyanin content mountain ash stem cell; wherein the anthocyanin induction culture medium is konjak culture medium added with 0.01% of Ge-132, 0.5mg/L of 6-BA and 0.1mg/L of 2,4-D, and the anthocyanin accumulation culture medium is konjak culture medium added with 0.01% of Ge-132+0.5mg/L of 6-BA.
The obtained high anthocyanin content Sorbus pohuashanensis stem cells are extracted by using 3% hydrochloric acid-methanol as an extractant, the feed liquid ratio is 1:15, the extraction time is 18h and the extraction temperature is 35 ℃, the light absorption value is measured at 565nm, and the anthocyanin content is obtained, wherein Sorbus pohuashanensis fruits and common callus (tender leaves, adopting the same culture method as in example 1) are used as controls, and the results are shown in Table 2.
TABLE 2 Induction of anthocyanin in Sorbus nigra Stem cells
EXAMPLE 2 Effect of konjak Medium on the anthocyanin content of Sorbus pohuashanensis
The stem cells adopted by the invention and common callus are respectively subjected to anthocyanin synthesis comparison by adopting different culture media and field cultivation in the prior art, and the results are shown in Table 3.
TABLE 3 comparison of anthocyanin content of konjak Medium and conventional Medium
The various embodiments are described in a progressive manner, each embodiment focusing on differences from the other embodiments, and identical and similar parts between the various embodiments are sufficient to be seen with 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 (1)
1. A culture method of mountain ash stem cells for improving anthocyanin synthesis content is characterized by comprising the following specific steps:
(1) Preparation of stem cell tissue: pretreating root tips of aseptic seedlings of Sorbus pohuashanensis, transferring to a konjak induction culture medium for culturing for 25-30d, transferring to a konjak proliferation culture medium, and performing secondary culture for 5-10 times to obtain stem cell tissues;
the pretreatment steps are as follows: taking root tip of aseptic seedling of Sorbus pohuashanensis 0.1mm, placing in a container containing 0.1% KH 2 PO 4 +0.1%MgCl 2 Is treated in glycerol solution for 12 hours, then washed and wiped dry, and then sequentially transferred to dark treatment at-20 ℃ for 8-10 hours, dark treatment at 0 ℃ for 30 minutes and dark treatment at 10 ℃ for 30 minutes;
the konjak induction culture medium is konjak culture medium added with 0.5-3 mg/L2, 4-D and 0.5-2 mg/L6-BA; the konjak proliferation culture medium is konjak culture medium added with 0.5-1 mg/L2, 4-D;
(2) Induction and accumulation of anthocyanin: transferring the stem cell tissue to an anthocyanin induction culture medium for 15-20d, and transferring to an anthocyanin accumulation culture medium for 25-30d to obtain the anthocyanin-high-content Sorbus pohuashanensis stem cell;
the anthocyanin induction culture medium is konjak culture medium added with 0.01-0.05% of Ge-132, 0.1-0.5mg/L of 6-BA and 0.5-2mg/L of 2, 4-D; the anthocyanin accumulation culture medium is konjak culture medium added with 0.01-0.05% of Ge-132+0.1-0.5 mg/L6-BA;
the culture conditions are as follows: culturing at 26-28deg.C under light with light intensity of 1500-2000 LX; the illumination time is 14h/24h;
wherein each liter of the konjak culture medium comprises: 1-3g konjak flour, 0.5-3mgFeCl 2 And 0.5-5g of potassium nitrate;
the preparation method of the konjak culture medium comprises the following specific steps:
1) Weighing raw materials: 1g to 3g konjaku flour and 0.5mg to 3mg FeCl are weighed per liter of culture medium 2 And 0.5-5g of potassium nitrate;
2) Adding the konjaku flour into 1L of water, and uniformly stirring to obtain a konjaku flour swelling system;
3) Adding hydrochloric acid solution into the swelling system of rhizoma Amorphophalli powder, hydrolyzing at 40-60deg.C for 0.5-3 hr, and adding FeCl 2 Carrying out a glue coupling reaction;
4) Adding KNO into the solution obtained in the step (3) 3 And regulating the pH to 5.0-8.0 to obtain the konjak culture medium.
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