CN106367378A - Glycyrrhiza glabra callus cell culture method capable of improving content of licoflavone - Google Patents
Glycyrrhiza glabra callus cell culture method capable of improving content of licoflavone Download PDFInfo
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Abstract
The invention relates to a glycyrrhiza glabra callus cell culture method being capable of improving content of licoflavone. The method is characterized by including the steps of: (1) choosing full glycyrrhiza glabra seeds, and culturing the seeds on a MS basic culture medium containing 30 g/L of saccharose and 7 g/L of agar to obtain sterile seedlings, and inoculating the sterile seedlings on a callus induction culture medium for cultivation, and moving the sterile seedlings on a subculture medium for subculture; (2) selecting slight-yellow callus after the subculture in the step (1), and transferring the callus on a liquid culture medium to perform suspended shaking cultivation; (3) performing inductive cultivation during the suspended shaking cultivation process of the callus, and increasing culturing temperature from 25 DEG C to 35-45 DEG C from the sixth to twelfth day of the subculture period, and continuously culturing the callus for 1-3 days, reducing the culturing temperature to 25 DEG C, wherein coronatine is added during the cultivation at 35-45 DEG C and p-hydroxyphenylpyruvic acid is added when the temperature is reduced to 25 DEG C, and after the suspended shaking cultivation is finished, the glycyrrhiza glabra callus cell having high content of licoflavone can be obtained. The method achieves high content of the licoflavone.
Description
Technical field
The invention belongs to biological technical field, particularly to a kind of Glycyrrhiza glabra L. wound healing group improving licoflavone content
Knit cell culture processes.
Technical background
Glycyrrhiza glabra L. is distributed mainly on the provinces and regions such as In The North of Xinjiang, Qinghai, Gansu, and main chemical compositions are flavone compound
And triterpene saponin, wherein, liquorice flavonoids compound has multiple biological activities, in addition to the effect such as antiinflammatory, antibacterial, in antioxygen
The aspects such as change, anticancer, anti-cancer also have significant biological activity, are widely used in medicine, health care of food, cosmetic industry
Prospect.
With deepening continuously that Glycyrrhiza glabra L. is developed, its demand surges.Because the licorice raw material in the world 90% is equal
There is provided by China's export, therefore, China's most area wild licorice receives destructive excavation, and some areas are even on the verge of
Exhausted.The contradiction that the scarcity of wild resource is increased with Radix Glycyrrhizae demand, becomes problem demanding prompt solution.Wild for solving
The contradiction that the scarcity in production-goods source is increased with Radix Glycyrrhizae demand, can set about: one is to utilize tissue culture technique in terms of two, to light fruit
Radix Glycyrrhizae carries out asexual Fast-propagation, and improves its quality using cell engineering or engineered means, expands improved seeds kind
Plant area；Two is the effective ingredient licoflavone producing Radix Glycyrrhizae by cell large-scale culture.But, Glycyrrhiza glabra L. is extremely
If needing less to grow 3 years to be just used as medicine that is to say, that taking the first scheme, in the period of considerably long in, Glycyrrhiza glabra L. is still not
The market demand can be met.Therefore, licoflavone is produced by cell large-scale culture extremely urgent.
Under above-mentioned background, in recent years, many scholars turn on research sight using Glycyrrhiza glabra L. stem cell
Culture, to obtain the licoflavone of high yield.This method not only can greatly speed up the growth of plant cell, improves containing of target substance
Amount, and is not limited by geography, season and weather conditions, can save wild plant and land resource, and it is short to have a growth cycle,
The advantages of remarkable in economical benefits.But easily browning occurs during Callus Tissue of Glycyrrhiza inducing culture it is difficult to obtain stable cell lines,
In addition, the research increasing secondary metabolite licoflavone to Callus Tissue of Glycyrrhiza is still at the initial stage.
Document shows, during calluss suspension amplification culture, suitable condition of culture can be obviously improved secondary generation
Thank to the yield of product, such as illumination, temperature, fungal biodegradation etc., the change of these conditions can induce secondary metabolite route of synthesis
The generation of middle relevant enzyme and expression, thus promote the synthesis of target product.Secondly, the addition of the coherent signal factor also can induce plant
Thing chemical defence, the expression of activated plant defense-related gene, thus promote secondary metabolite to generate.He Feng et al. is in Radix Glycyrrhizae
Add 10 mg/l salicylic acid, up to 4.3mg/g, other is verified effective signal to licoflavone content in suspension culture system
The factor also includes (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and MDJ.Additionally, being also that a kind of raising is secondary by adding precursor substance
The effective ways of metabolite, this is because can there is rate-limiting enzyme in route of synthesis, usually make the yield of end-product be limited by
The quantity of some substrates in metabolic pathway, therefore, target product metabolic pathway substantially clearly under the conditions of, add its biology close
The precursor substance becoming is to release this restriction it is likely that improving the yield of end-product.Yang Ying et al. is in Glycyrrhiza cell suspended substance
Add Phenylalanine, tyrosine, cinnamic acid and sodium acetate that to study precursor, Glycyrrhiza cell is produced with the impact of licoflavone in system,
Result shows, 4 kinds of precursors all can promote the biosynthesiss of intracellular licoflavone, adds under concentration and time optimum, Radix Glycyrrhizae
Flavones content all can reach 6.5mg/g.Although researchers are to licoflavone content in raising Callus Tissue of Glycyrrhiza cell
Make related endeavors, but in the related ends of report, licoflavone has only accounted for the 0.4% ~ 0.7% of callus cell dry weight
Left and right, still cannot meet the market demand.Therefore, find and a kind of improve the thin of the accumulation of licoflavone in Glycyrrhiza glabra L. calluss
Born of the same parents' cultural method becomes problem demanding prompt solution.
Content of the invention
It is an object of the invention to provide a kind of Glycyrrhiza glabra L. callus cell culture improving licoflavone content
Method.
For solving above-mentioned technical problem, the present invention employs the following technical solutions: a kind of light improving licoflavone content
Fruit Callus Tissue of Glycyrrhiza cell culture processes are it is characterised in that adopt following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, the upper culture of ms basal medium (ms basal medium is purchased from sigma, m 5524) of 7g/l agar obtain aseptic seedling；Take no
Vaccine plumular axis is explant, accesses dark culturing 10 days in callus inducing medium, induces calluss, afterwards in light
It is 1500 according to intensitylxUnder conditions of continue culture 14 days, daily illumination 12h；Then calluss are proceeded to subculture medium
In, once, successive transfer culture 2 times, to obtain stable Glycyrrhiza glabra L. calluss, successive transfer culture temperature 25 for every 28 days subculturesoC,
Subculture medium ph 5.8；Described callus inducing medium is containing 1 ~ 2mg/l naphthalene acetic acid, 0.1 ~ 0.5mg/l 6- benzyl ammonia
Base purine, 20 ~ 30g/l sucrose, the ms basal medium of 5 ~ 8g/l agar；Described subculture medium is by induction of callus
Add methionine (cas 59-51-8, be purchased from sigma) in base to form, concentration in subculture medium for the methionine is
0.1~1.0 mmol/l；
2) calluss suspension shaken cultivation: selecting step 1) in growth is vigorous, faint yellow calluss that quality is loose, transfer
The suspension shaken cultivation to fluid medium, inoculum concentration 40 ~ 100g/l, g/l=calluss fresh weight/fluid medium volume, shake
Bed rotating speed 60 ~ 150 rpm, intensity of illumination 1500 ~ 2000lx, light application time 8 ~ 16 h/day, fluid medium ph 5.6 ~
6.8；Every 21 days successive transfer culture 1 time, successive transfer culture 2 ~ 5 times；Described fluid medium is to be added with 6-benzyl aminopurine, naphthalene second
Acid, the ms basal medium of 2,4- dichlorphenoxyacetic acid, sucrose, the concentration of described 6-benzyl aminopurine is 1.0 ~ 2.0mg/l, naphthalene
The concentration of acetic acid is 0.5 ~ 1.0mg/l, the concentration of 2,4- dichlorphenoxyacetic acids is 0.5 ~ 1.0mg/l, the concentration of sucrose be 20 ~
30g/l；
3) the induction accumulation of licoflavone: in step 2) during described calluss suspension shaken cultivation, also it is lured
Lead culture, to stimulate the accumulation of secondary metabolite licoflavone, described inductive condition is: in step 2) described successive transfer culture week
The 6th ~ 12 day of phase starts, by cultivation temperature from 25oC improves to 35 ~ 45oC, after maintaining this temperature to continue culture 1 ~ 3 day, recovers
Cultivation temperature is to 25oC, until this cultivation cycle terminates；35 ~ 45oDuring c culture, add through filtration sterilization toward in fluid medium
Hat toxin (coronatine) afterwards, is preced with the final concentration of 0.8 ~ 2.2mg/l of toxin；Recover to 25oAfter c, toward in fluid medium
Add the Single-chip microcomputer (cas 156-39-8, be purchased from aldrich) after filtration sterilization, Single-chip microcomputer is eventually
Concentration is 60 ~ 150mg/l；Suspension shaken cultivation obtains the high Glycyrrhiza glabra L. callus cell of licoflavone content after terminating.
Under condition of culture of the present invention, the content of licoflavone increases to 28 ~ 48mg/g, accounts for callus cell dry weight
2.8% ~ 4.8%, compared with the cell culture mode of report, the yield of target product licoflavone lifts 4 ~ 16 times with present.
The present invention, in the induction of Glycyrrhiza glabra L. calluss and Subculture, adds anti-browning material first sulfur ammonia
Acid, can increase calluss healing rate, and in identical cultivation cycle, cellular biomass (do by the cell of per unit volume culture medium
Weight, g/l) increase by 56% ~ 83%, improve licoflavone yield.
The present invention passes through during calluss suspension shaken cultivation, periodically by cultivation temperature from 25oC rises paramount
In 35oC, builds high temperature stress environment, to start Glycyrrhiza glabra L. chemical defense mechanisms；It is simultaneously introduced signal factor hat toxin, ginseng
The signals transmission related to plant defense, promotes the generation of key enzyme in licoflavone biosynthetic process, realizes secondary
The Fast back-projection algorithm of metabolite.
The present invention, after terminating high temperature stress, adds precursor substance Single-chip microcomputer in culture fluid, can improve sweet
The reaction rate of straw colour ketone biosynthetic process, thus greatly improve the yield improving secondary metabolite.
In sum, the present invention has the beneficial effect that can improve licoflavone content in Glycyrrhiza glabra L. callus cell
Really.
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the invention is not limited in following examples.
Embodiment 1:
A kind of Glycyrrhiza glabra L. callus cell cultural method improving licoflavone content, using following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, the ms base culture base of 7g/l agar obtain aseptic seedling；Take aseptic seedling plumular axis to be explant, access and contain 1.0mg/l
In naphthalene acetic acid, 0.4mg/l 6-benzyl aminopurine, 22g/l sucrose, the ms basal medium of 6g/l agar, dark culturing 10 days,
Induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture 14 days, daily illumination 12h；Then will
Calluss proceed to containing 1.0mg/l naphthalene acetic acid, 0.4mg/l 6-benzyl aminopurine, 0.1mmol/l methionine, 22g/l sugarcane
In sugar, the ms basal medium of 6g/l agar, successive transfer culture temperature 25oC, subculture medium ph 5.8, every 28 days subcultures once,
Successive transfer culture 2 times, obtains stable Glycyrrhiza glabra L. calluss, and described ms basal medium is purchased from sigma, trade mark m 5524；
2) the induction accumulation of calluss suspension shaken cultivation and licoflavone: selecting step 1) in growth is vigorous, quality is loose
Faint yellow calluss, be transferred to containing 2.0mg/l 6-benzyl aminopurine, 0.5mg/l naphthalene acetic acid, 0.8mg/l 2,4- bis-
Suspension shaken cultivation in chlorophenoxyacetic acid, the fluid medium of sucrose 22g/l, inoculum concentration 40g/l, shaking speed 120 rpm, light
According to intensity 1500lx, light application time 10h/day, fluid medium ph 5.6；Every 21 days successive transfer culture 1 time, and in successive transfer culture
The 10th day of cycle starts, by cultivation temperature from 25oC improves to 35oC, is simultaneously introduced hat toxin, makes hat toxin in liquid culture
Final concentration of 2.2mg/l in base, maintains 35oAfter c cultivates 1 day, renewal cultivation temperature to 25oC, adds Single-chip microcomputer,
Final concentration of 60mg/l in Single-chip microcomputer liquid medium within；Licoflavone content is obtained high after successive transfer culture 2 times
Glycyrrhiza glabra L. callus cell.
After culture terminates, measure licoflavone content, result shows that licoflavone content accounts for the 3.12% of dry cell weight.
Embodiment 2:
A kind of Glycyrrhiza glabra L. callus cell cultural method improving licoflavone content, using following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, culture on the ms basal medium of 7g/l agar obtains aseptic seedling；Take aseptic seedling plumular axis to be explant, access and contain 1.2mg/
In l naphthalene acetic acid, 0.3mg/l 6-benzyl aminopurine, 20g/l sucrose, the ms basal medium of 8g/l agar, dark culturing 10 days,
Induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture 14 days, daily illumination 12h；Then will
Calluss proceed to containing 1.2mg/l naphthalene acetic acid, 0.3mg/l 6-benzyl aminopurine, 0.3mmol/l methionine, 20g/l sugarcane
In sugar, the ms basal medium of 8g/l agar, successive transfer culture temperature 25oC, subculture medium ph 5.8, every 28 days subcultures once,
Successive transfer culture 2 times, obtains stable Glycyrrhiza glabra L. calluss；
2) the induction accumulation of calluss suspension shaken cultivation and licoflavone: selecting step 1) in growth is vigorous, quality is loose
Faint yellow calluss, be transferred to containing 1.5mg/l 6-benzyl aminopurine, 0.7mg/l naphthalene acetic acid, 0.9mg/l 2,4- bis-
Suspension shaken cultivation in chlorophenoxyacetic acid, the fluid medium of sucrose 20g/l, inoculum concentration 60g/l, shaking speed 100 rpm, light
According to intensity 1700lx, light application time 14h/day, fluid medium ph 6.0；Every 21 days successive transfer culture 1 time, and in successive transfer culture
The 6th day of cycle starts, by cultivation temperature from 25oC improves to 42oC, is simultaneously introduced hat toxin, makes hat toxin liquid medium within
In final concentration of 1.8mg/l, maintain 42oAfter c cultivates 2 days, renewal cultivation temperature to 25oC, adds Single-chip microcomputer, right
Final concentration of 100mg/l in hydroxyphenylphruvic acid liquid medium within；Licoflavone content is obtained high after successive transfer culture 4 times
Glycyrrhiza glabra L. callus cell.
After culture terminates, measure licoflavone content, result shows that licoflavone content accounts for the 4.66% of dry cell weight.
Embodiment 3:
A kind of Glycyrrhiza glabra L. callus cell cultural method improving licoflavone content, using following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, culture on the ms basal medium of 7g/l agar obtains aseptic seedling；Take aseptic seedling plumular axis to be explant, access and contain 1.4mg/
In l naphthalene acetic acid, 0.1mg/l 6-benzyl aminopurine, 26g/l sucrose, the ms basal medium of 5g/l agar, dark culturing 10 days,
Induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture 14 days, daily illumination 12h；Then will
Calluss proceed to containing 1.4mg/l naphthalene acetic acid, 0.1mg/l 6-benzyl aminopurine, 0.6mmol/l methionine, 26g/l sugarcane
In sugar, the ms basal medium of 5g/l agar, successive transfer culture temperature 25oC, subculture medium ph 5.8, every 28 days subcultures once,
Successive transfer culture 2 times, obtains stable Glycyrrhiza glabra L. calluss；
2) the induction accumulation of calluss suspension shaken cultivation and licoflavone: selecting step 1) in growth is vigorous, quality is loose
Faint yellow calluss, be transferred to containing 1.4mg/l 6-benzyl aminopurine, 0.6mg/l naphthalene acetic acid, 0.7mg/l 2,4- bis-
Suspension shaken cultivation in chlorophenoxyacetic acid, the fluid medium of sucrose 26g/l, inoculum concentration 80g/l, shaking speed 60 rpm, light
According to intensity 1800lx, light application time 8h/day, fluid medium ph 5.8；Every 21 days successive transfer culture 1 time, and in successive transfer culture
The 12nd day of cycle starts, by cultivation temperature from 25oC improves to 38oC, is simultaneously introduced hat toxin, makes hat toxin in liquid culture
Final concentration of 1.2mg/l in base, maintains 38oAfter c cultivates 3 days, renewal cultivation temperature to 25oC, adds Single-chip microcomputer,
Final concentration of 80mg/l in Single-chip microcomputer liquid medium within；Licoflavone content is obtained high after successive transfer culture 5 times
Glycyrrhiza glabra L. callus cell.
After culture terminates, measure licoflavone content, result shows that licoflavone content accounts for the 3.83% of dry cell weight.
Embodiment 4:
A kind of Glycyrrhiza glabra L. callus cell cultural method improving licoflavone content, using following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, culture on the ms basal medium of 7g/l agar obtains aseptic seedling；Take aseptic seedling plumular axis to be explant, access and contain 1.8mg/
In l naphthalene acetic acid, 0.5mg/l 6-benzyl aminopurine, 24g/l sucrose, the ms basal medium of 7g/l agar, dark culturing 10 days,
Induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture 14 days, daily illumination 12h；Then will
Calluss proceed to containing 1.8mg/l naphthalene acetic acid, 0.5mg/l 6-benzyl aminopurine, 0.9mmol/l methionine, 24g/l sugarcane
In sugar, the ms basal medium of 7g/l agar, successive transfer culture temperature 25oC, subculture medium ph 5.8, every 28 days subcultures once,
Successive transfer culture 2 times, obtains stable Glycyrrhiza glabra L. calluss.
2) calluss suspension shaken cultivation and the induction of licoflavone accumulate: selecting step 1) the vigorous, quality of middle growth
Loose faint yellow calluss, are transferred to containing 1.8mg/l 6-benzyl aminopurine, 0.9mg/l naphthalene acetic acid, 1.0mg/l 2,
Suspension shaken cultivation in 4- dichlorphenoxyacetic acid, the fluid medium of sucrose 24g/l, inoculum concentration 100g/l, shaking speed 150
Rpm, intensity of illumination 1600lx, light application time 16h/day, fluid medium ph 6.4；Every 21 days successive transfer culture 1 time, and in continuing
The 8th day of the culture cycle starts, by cultivation temperature from 25oC improves to 45oC, is simultaneously introduced hat toxin, makes hat toxin in liquid
Final concentration of 1.5mg/l in culture medium, maintains 45oAfter c cultivates 2 days, renewal cultivation temperature to 25oC, adds para hydroxybenzene third
Keto acid, the final concentration of 130mg/l in Single-chip microcomputer liquid medium within；Licoflavone is obtained after successive transfer culture 3 times
The high Glycyrrhiza glabra L. callus cell of content.
After culture terminates, measure licoflavone content, result shows that licoflavone content accounts for the 4.05% of dry cell weight
Embodiment 5:
A kind of Glycyrrhiza glabra L. callus cell cultural method improving licoflavone content, using following steps:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
Sugar, culture on the ms basal medium of 7g/l agar obtains aseptic seedling；Take aseptic seedling plumular axis to be explant, access and contain 2.0mg/
In l naphthalene acetic acid, 0.2mg/l 6-benzyl aminopurine, 30g/l sucrose, the ms basal medium of 8g/l agar.Dark culturing 10 days,
Induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture 14 days, daily illumination 12h；Then will
Calluss proceed to containing 2.0mg/l naphthalene acetic acid, 0.2mg/l 6-benzyl aminopurine, 1.0mmol/l methionine, 30g/l sugarcane
In sugar, the ms basal medium of 8g/l agar, successive transfer culture temperature 25oC, subculture medium ph 5.8, every 28 days subcultures once,
Successive transfer culture 2 times, obtains stable Glycyrrhiza glabra L. calluss.
2) calluss suspension shaken cultivation and the induction of licoflavone accumulate: selecting step 1) the vigorous, quality of middle growth
Loose faint yellow calluss, are transferred to containing 1.0mg/l 6-benzyl aminopurine, 1.0mg/l naphthalene acetic acid, 0.5mg/l 2,
Suspension shaken cultivation in 4- dichlorphenoxyacetic acid, the fluid medium of sucrose 30g/l, inoculum concentration 50g/l, shaking speed 80
Rpm, intensity of illumination 2000lx, light application time 12h/day, fluid medium ph 6.8；Every 21 days successive transfer culture 1 time, and in continuing
The 9th day of the culture cycle starts, by cultivation temperature from 25oC improves to 40oC, is simultaneously introduced a certain amount of hat toxin, makes hat toxin
Final concentration of 0.8mg/l in liquid medium within, maintains 40oAfter c cultivates 1 day, renewal cultivation temperature to 25oC, adds to hydroxyl
Base phenylpyruvic acid, the final concentration of 150mg/l in Single-chip microcomputer liquid medium within；Obtain sweet after successive transfer culture 2 times
The high Glycyrrhiza glabra L. callus cell of careless flavones content.
After culture terminates, measure licoflavone content, result shows that licoflavone content accounts for the 3.42% of dry cell weight.
The foregoing is only embodiments of the invention, not thereby limit the present invention the scope of the claims, every using this
Equivalent structure or equivalent flow conversion that bright description is done, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
Claims (1)
1. the Glycyrrhiza glabra L. callus cell cultural method of licoflavone content can be improved it is characterised in that adopting following step
Rapid:
1) induction of Glycyrrhiza glabra L. calluss and successive transfer culture: select full Glycyrrhiza glabra L. seed, containing 30g/l sugarcane
On sugar, the ms basal medium of 7g/l agar, culture obtains aseptic seedling；Take aseptic seedling plumular axis to be explant, access calluss and lure
Lead dark culturing 10 days in culture medium, induce calluss, afterwards intensity of illumination be 1500lxUnder conditions of continue culture
14 days, daily illumination 12h；Then calluss are proceeded in subculture medium, every 28 days subcultures once, successive transfer culture 2 times, with
Obtain stable Glycyrrhiza glabra L. calluss, successive transfer culture temperature 25oC, subculture medium ph 5.8；Described callus induction
Culture medium is containing 1 ~ 2mg/l naphthalene acetic acid, 0.1 ~ 0.5mg/l 6-benzyl aminopurine, 20 ~ 30g/l sucrose, 5 ~ 8g/l agar
Ms basal medium；Described subculture medium is formed by interpolation methionine in callus inducing medium, and methionine exists
Concentration in subculture medium is 0.1 ~ 1.0 mmol/l；
2) calluss suspension shaken cultivation: selecting step 1) in growth is vigorous, faint yellow calluss that quality is loose, transfer
The suspension shaken cultivation to fluid medium, inoculum concentration 40 ~ 100g/l, g/l=calluss fresh weight/fluid medium volume, shake
Bed rotating speed 60 ~ 150 rpm, intensity of illumination 1500 ~ 2000lx, light application time 8 ~ 16 h/day, fluid medium ph 5.6 ~
6.8；Every 21 days successive transfer culture 1 time, successive transfer culture 2 ~ 5 times；Described fluid medium is to be added with 6-benzyl aminopurine, naphthalene second
Acid, the ms basal medium of 2,4- dichlorphenoxyacetic acid, sucrose, the concentration of described 6-benzyl aminopurine is 1.0 ~ 2.0mg/l, naphthalene
The concentration of acetic acid is 0.5 ~ 1.0mg/l, the concentration of 2,4- dichlorphenoxyacetic acids is 0.5 ~ 1.0mg/l, the concentration of sucrose be 20 ~
30g/l；
3) the induction accumulation of licoflavone: in step 2) during described calluss suspension shaken cultivation, also it is lured
Lead culture, to stimulate the accumulation of secondary metabolite licoflavone, described inductive condition is: in step 2) described successive transfer culture week
The 6th ~ 12 day of phase starts, by cultivation temperature from 25oC improves to 35 ~ 45oC, after maintaining this temperature to continue culture 1 ~ 3 day, recovers
Cultivation temperature is to 25oC, until this cultivation cycle terminates；35 ~ 45oDuring c culture, add through filtration sterilization toward in fluid medium
Hat toxin afterwards, is preced with the final concentration of 0.8 ~ 2.2mg/l of toxin；Recover to 25oAfter c, add toward in fluid medium and go out through filtration
Single-chip microcomputer after bacterium, the final concentration of 60 ~ 150mg/l of Single-chip microcomputer；Suspension shaken cultivation obtains after terminating
The high Glycyrrhiza glabra L. callus cell of licoflavone content.
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| CN106754633A (en) * | 2017-02-14 | 2017-05-31 | 天津艾赛博生物技术有限公司 | A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones |
| CN109122323A (en) * | 2018-10-22 | 2019-01-04 | 覃家日 | The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum |
| CN111718888A (en) * | 2020-06-23 | 2020-09-29 | 中国医学科学院药用植物研究所 | Culture method for improving glycyrrhizic acid content in suspension culture cells of liquorice |
| CN112005883A (en) * | 2020-08-22 | 2020-12-01 | 延安大学 | Method for establishing liquorice tissue culture rapid propagation system |
| CN114793906A (en) * | 2022-06-02 | 2022-07-29 | 珀莱雅化妆品股份有限公司 | Method for increasing forsythiaside content in calli of marrubium vulgare |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754633A (en) * | 2017-02-14 | 2017-05-31 | 天津艾赛博生物技术有限公司 | A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones |
| CN109122323A (en) * | 2018-10-22 | 2019-01-04 | 覃家日 | The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum |
| CN111718888A (en) * | 2020-06-23 | 2020-09-29 | 中国医学科学院药用植物研究所 | Culture method for improving glycyrrhizic acid content in suspension culture cells of liquorice |
| CN111718888B (en) * | 2020-06-23 | 2022-08-19 | 中国医学科学院药用植物研究所 | Culture method for improving glycyrrhizic acid content in suspension culture cells of liquorice |
| CN112005883A (en) * | 2020-08-22 | 2020-12-01 | 延安大学 | Method for establishing liquorice tissue culture rapid propagation system |
| CN114793906A (en) * | 2022-06-02 | 2022-07-29 | 珀莱雅化妆品股份有限公司 | Method for increasing forsythiaside content in calli of marrubium vulgare |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN116574668B (en) * | 2023-06-13 | 2023-12-05 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
| CN117448254B (en) * | 2023-10-23 | 2024-04-09 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
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