CN106479951A - A kind of method of suppression plant cell tissue browning in culture - Google Patents
A kind of method of suppression plant cell tissue browning in culture Download PDFInfo
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Abstract
The invention discloses a kind of method of suppression plant cell tissue browning in culture, methods described is cultivated under the conditions of including being placed in as follows plant cell tissue, and described condition makes the synthetic quantity of described plant cell tissue Flavonoid substances in incubation decline more than 30%.The method is directed to the plant cell tissues such as plant callus culture, plant cell solid culture, plant cell liquid culture and plant cell liquid reactor culture.Compared with current traditional method, the method is more targeted and controls browning effect is significant, is appropriate to the commercial application of plant cell large-scale culture, wide market.
Description
Technical field
The invention belongs to field of plant tissue culture technique is and in particular to a kind of suppress plant cell tissue
The method of browning in culture.
Background technology
Plant cell and tissue culture technology in micropropagation of plants, detoxification, accelerate breeding process, secondary
The aspect such as metabolite production and Germ-plasma resources protection achieves huge economic benefit, social benefit
And ecological benefits.Because of its superiority, its application still extends plant cell culture technology continuous,
Appliable plant cell culture technology produces exogeneous animal albumen at present, it has also become whole world research and application
Focus.But, browning problem is still prevalent in Plant cell and tissue culture field, has a strong impact on
The growth of plant cell tissue, even results in that it is dead, thus reduce target metabolic product or its
The accumulation of its active substance, seriously constrains the commercial application of Plant cell and tissue culture.Plant is thin
Born of the same parents' tissue culture procedures browning is because aldehydes matter is oxidized to toxicity by polyphenol oxidase (PPO)
Quinones substance.
At present, the browning during Plant cell and tissue culture rely primarily on traditional interpolation adsorbent,
Antioxidant, change the method such as culture medium to be controlled.For example, Li Chunbin etc. passes through to culture medium
Middle interpolation activated carbon adsorption brown material so that the calluss of Ramulus et folium taxi cuspidatae browning go brown
Change (CN103563750A)；And Seedling occasion fly etc. prepare given shape silica gel adsorptive material absorption quinones thing
Matter, thus reduce plant cell browning risk (CN103923873A)；Huang Wenmin etc. passes through to culture medium
The method of middle interpolation PVP suppresses the browning (CN102577953B) in tobacco healing tissue's successive transfer culture.
Bian Li Ping etc. is used as reducing agent by adding VC in culture medium, thus suppressing the browning of iris tissue
(CN103125380A)；Xiang Yan etc. prevents butterfly by way of adding plant extraction liquid in culture medium
The browning (CN103749297A) of blue tissue culture；Liu Fu equality is passed through to invent a kind of anti-browning disinfectant 2-
Benzyl -4- chlorophenol, thus preventing the browning (CN103891713A) of plant tissue culture explant, this kind of
The action principle essence of disinfectant or the effect of antioxidant.Li Yali etc. passes through to change fresh cultured
The mode that base washs Glycyrrhiza cell prevents the browning at suspension culture initial stage from occurring
(CN103589679A), Yan Zhi has just waited by Caulis Fibraureae calluss admittedly -- and the method for liquid conversion culture prevents
Browning produces (CN102805032B).Additionally, Qiu Deyou etc. presses down by using a kind of Microrna
The method (CN101781650B) of browning after Taxus x media cells Agrobacterium tumefaciens transformation processed.
Although the method can play to a certain extent and prevent the effect of browning, still can not stop comprehensively
Browning, and operational approach is loaded down with trivial details.
In sum, the method preventing Plant cell and tissue culture browning disclosed in above patent, right
Browning has carried out a certain degree of control, but during still thoroughly can not solving Plant cell and tissue culture
Browning problem, and these methods can only use in laboratory small-scale, extensive implement relatively difficult,
But also it is not enough, as interpolation antioxidant and adsorbent etc. can produce to plant cell tissue to there are other
Toxic and side effects, need the phase after incubation to remove from culture medium, and the method operation changing culture medium are numerous
Trivial, production cost can be improved.Thus need badly set up effectively, be easy to the plant cell group implemented on a large scale
Knit the new method of He mushroom in culture.
Content of the invention
Disadvantages described above for prior art or Improvement requirement, the invention provides a kind of suppression plant is thin
The method that born of the same parents are organized in browning in culture, by suppressing the synthesis of plant cell flavone in incubation,
Fundamentally Browning control, thus solve current Browning control not thoroughly, be unable to large-scale use, have
Toxic and side effects and the numerous technical problem of operation.
For achieving the above object, the invention provides a kind of suppress plant cell tissue's browning in culture
Method, methods described include by plant cell tissue be placed in following under the conditions of cultivate, described condition makes
The synthetic quantity obtaining described plant cell tissue Flavonoid substances in incubation declines more than 30%.
Preferably, described condition includes adding stage by stage sucrose in incubation, according to consulting correlation
Document, sucrose concentration used by Plant cell and tissue culture is generally 20g/L-40g/L, but thin in plant
Born of the same parents' Initial stage of culture sucrose is too high to lead to browning degree to raise, and therefore should reduce sucrose in Initial stage of culture dense
Degree, ensures in first 3-6 days of cell culture that sucrose concentration is 10g/L-20g/L, subsequently gradually
Increase sucrose concentration to 30g/L-40g/L.
Preferably, ensure that sucrose concentration is 10g/L, is subsequently gradually increased in first 5 days of cell culture
Sucrose concentration is to 30g/L.
Preferably, described condition includes adding 10mg/L-100mg/L gibberellins in the medium.
Preferably, 30mg/L-75mg/L gibberellins are added in the medium.
Preferably, described condition includes after setting up cell line, screening minicell aggregation culture；I.e.
The cell mass of 100-800 μm of diameter of screening is cultivated.
Preferably, a diameter of 500 μm of cell mass.
Preferably, described method be applied to plant callus culture, plant cell solid culture,
The culture of plant cell liquid and the culture of plant cell liquid reactor.
In general, by the contemplated above technical scheme of the present invention compared with prior art, can
Obtain following beneficial effect：
(1) present invention based on Flavonoid substances be cause plant cell tissue in culture browning main
Establish more targetedly He mushroom method on the basis of mechanism, fundamentally prevent plant thin
Born of the same parents are organized in the generation of browning in incubation, it is to avoid the injury to plant cell tissue for the browning material.
(2) method of the present invention is free from side effects it is adaptable to different cultivation conditions to plant cell tissue
Under plant cell tissue it is not necessary to remove additive step, simple to operate it is adaptable to plant is thin
The large-scale culture of born of the same parents' tissue and commercial application.
(3) method of the present invention is simple to operate, applied widely, can effectively prevent sending out of browning
Raw；By optimizing the parameter in each method so that plant cell tissue's flavonoid thing in incubation
The content of matter is more effectively controlled, so as to preferably prevent plant cell tissue brown in culture
The generation changed.The present invention has reality to the scientific research of Plant cell and tissue culture and commercial application
Trample directive significance and commercial value.
Brief description
Fig. 1 is the cell line that in embodiment 1, Subculture Time is respectively 6 months (A) and 10 years (B)
Growth conditions.
Specific embodiment
With reference to example, the specific embodiment of the present invention is described further.Here needs explanation
, the explanation for these embodiments is used to help understand the present invention, but does not constitute to this
Bright restriction.Additionally, involved technology in each embodiment of invention described below is special
As long as levying the conflict of not constituting each other just can be mutually combined.
Present invention aim at providing effectively and be suitable for plant cell tissue's training of extensive commercial application
In supporting, the control method of browning, fundamentally prevents the generation of Plant cell and tissue culture process browning,
Avoid the injury to plant cell tissue for the browning material.
Find in research, in browning cell, general flavone content is more than 20 times of non-browning cell, leads to brown
Change browning in cell very serious, and non-browning cell does not have browning substantially because flavones content is low
Occur, and general flavone content can be reduced by adding the method specificity of gibberellins, thus avoiding
The generation of browning.This result illustrates that the synthesis of Flavonoid substances in cell is to lead to plant cell to be trained
There is the main cause of browning in foster thing.
The present invention is to provide the effective control flavonoid adopting for different plant cell tissue cultures
The synthesis of material, the method being controlled browning.The Plant cell and tissue culture of the present invention such as wound healing group
Knit culture, plant cell solid culture, plant cell liquid culture, plant cell reactor
Culture, the culture of tissue cultured seedling and other plants that brownings easily occur such as numerous, hairy root culture soon
Cell and tissue structrue.As long as Flavonoid substances synthesis can be suppressed during Plant cell and tissue culture
Method all can effectively control the browning of plant cell tissue.
The preferably following three kinds of simple He mushroom methods of the present invention：Sucrose feeding culture, i.e. sugarcane
The sugar stage adds in culture medium；Minicell aggregation is cultivated；Gibberellins applied culture, at different groups
Knit being used alone or in combination of culture.
The present invention solves the problems, such as that the browning of Plant cell and tissue culture preferably employs below scheme：
(1) add sucrose in incubation stage by stage, ensure in first 3-6 days of cell culture
Sucrose concentration is 10g/L-20g/L, is subsequently gradually increased sucrose concentration to 30g/L-40g/L.Preferably
Ground, ensures that in first 5 days of cell culture sucrose concentration is 10g/L, is subsequently gradually increased sucrose concentration
To 30g/L.
(2) 10mg/L-100mg/L gibberellins are added in the medium.Preferably, in the medium
Add 30mg/L-75mg/L gibberellins.It is further preferred that the addition of gibberellins is 50mg/L.
Wherein, gibberellins add operation the simplest it is adaptable to whole culture plant cell amplification process,
It is applied to plant tissue and tissue cultured seedling culture large-scale production.
(3) after setting up cell line, screening minicell aggregation culture；Screen 100-800 μm of diameter
Cell mass cultivated.Preferably, a diameter of 500 μm of cell mass.Minicell aggregation
Culture can suppress the synthesis of Flavonoid substances, osmotic pressure and fluid shearing can be greatly lowered to thin
The injury of born of the same parents, it is to avoid the generation of browning.
Method of the present invention is applied to plant callus culture, plant cell solid culture, plant
The culture of thing cellular liquid and the culture of plant cell liquid reactor.
(1) He mushroom of plant callus culture.The calluss growing newly are induced to be easy to
Browning, leads to calluss dead.Using the sucrose feeding culture of the present invention, (i.e. sucrose adds stage by stage
Enter in culture medium), minicell aggregation cultivate, gibberellins applied culture, or more two methods phase knot
The method closed, can effectively suppress its browning.More preferably using sucrose feeding culture and gibberellins application training
Support, different according to floristics, it is 10-20g/L that the initial stage adds sucrose concentration scope, adds gibberellins
Concentration range is 10-100mg/L, it is possible to decrease the accumulation of total flavones in cell, thus avoiding calluss
The generation of culture browning.
(2) He mushroom of plant cell solid culture.Plant cell solid culture includes plant
Calluss are tamed culture in solid medium and are obtained cell line, and cell line long-term subculture and guarantor
Culture during Tibetan, browning is more serious.The He mushroom of plant cell solid culture
The method more preferably being combined using initial stage cane sugar content in reduction culture medium and interpolation gibberellins.According to
Plant cell species is different, and adding sucrose concentration scope in plant cell solid culture in the early stage is
10-20g/L, interpolation gibberellins concentration range is 10-100mg/L, it is possible to decrease total flavones in cell
Synthesis, thus obtain the plant cell of no browning.
(3) He mushroom of plant cell liquid culture.Plant cell is transferred to from solid medium
Fluid medium carries out suspension culture, and osmotic pressure and fluid shearing cause to cell to damage, can inducible enzyme
Promote browning to occur, cause plant cell liquid culture browning to be particularly acute.Plant cell liquid is trained
The He mushroom of foster thing uses three kinds of methods of the present invention, being applied in combination of more preferably three kinds of methods：Training
Adding gibberellins concentration range in foster base is 10-100mg/L, can stop the generation of enzymatic browning；Little thin
Born of the same parents' aggregation is cultivated, and the cell mass that screening diameter is less than 500 μm is cultivated, can be significantly
Reduce the injury to cell of osmotic pressure and fluid shearing, it is to avoid the generation of enzymatic browning.Sucrose stream
Plus cultivate, suspension culture initial stage sucrose concentration is not higher than 10g/L, and controllable browning occurs, subsequently gradually
Increase sucrose concentration to 30g/L.
(4) He mushroom of plant cell liquid reactor culture.Plant cell bioreactor culture thing
It is amplified to bioreactor culture including plant cell from flask suspension culture, then amplify from small-scale reactor
To the culture of commercial scale reactor, due to the acute variation of culture environment, the impact of fluid shearing,
Browning is susceptible to.In the He mushroom of plant cell bioreactor culture thing, listed by the present invention
The method going out all has good effect.
The type of culture medium that Plant cell and tissue culture field is commonly used has MS, B5, N6 culture medium, three kinds
Culture medium constituent is consistent, is that there is some difference for the height of certain or several content of material.Its
In the most commonly used be MS culture medium.
The present invention is to have xylophyta Ramulus et folium taxi cuspidatae and the herbaceous plant of very high medicinal valency and application prospect
As a example Radix Glycyrrhizae, Cotton Gossypii, perfume are burnt, from induction and the successive transfer culture of calluss, to the liquid of plant cell
Body suspension culture, then under the different culture states of reactor suspension culture, carry out He mushroom.
It is below embodiment
Embodiment 1
Chinese Ramulus et folium taxi cuspidatae browning cell line and non-browning cell line enzymatic browning correlated characteristic comparative analysiss
(1) laboratory preserves two kinds of cell lines, a kind of be subculture half a year cell line (NA), should
Cell line either solid or fluid suspension culture all shows very serious browning, another
Planting is the subculture cell line of 10 years (CA), and this cell line does not have in solid Subculture substantially
The generation of browning.Two kinds of cell lines are seeded on MS solid medium, sucrose 30g/L, control
Temperature is 25 DEG C, dark culturing.NA cells show goes out pitchy, and cell growth state is not good enough,
CA cells show goes out ecru, and grows vigorous (Fig. 1).
(2) in order to probe into two kinds of cell line browning degree differences the reason, comparative analysiss both cells
The Flavonoid substances content of system and PPO activity.Research finds, intracellular flavonoid in two kinds of cell lines
Maximum different value in 5d and is 17 times in content of material, and extracellular Flavonoid substances content difference exists
5d is 5 times.And the PPO activity difference very little of two kinds of cell lines, in 3d, 6 months are thin
Intracellular PPO activity is only 1.4 times of 10 years cells, extracellular PPO activity 2 times of left sides of maximum difference
Right.The Flavonoid substances content of two kinds of cell lines and the explanation of PPO expression activitiy, exactly Flavonoid substances
The greatest differences of content cause diverse cell browning degree, also further illustrate and live with PPO
Property is compared, and the height of Flavonoid substances content more can react the height of browning degree.
The He mushroom of the Chinese Ramulus et folium taxi cuspidatae browning cell line of embodiment 2
The cell line (NA) of subculture half a year in embodiment 1 is inoculated in and contains gibberellins accordingly for 10
In the MS solid subculture medium of mg/L, 50mg/L and 100mg/L, sucrose 30g/L, control temperature
Spend for 25 DEG C, dark culturing, it is within every 25 days 1 subculture cycle, change fresh above-mentioned successive transfer culture
Base.Through the culture in two cycles, the general flavone content in cell have dropped 41% respectively, 77% He
86%, thus all having obtained no browning in the culture medium adding 50mg/L and 100mg/L gibberellins
The solid taxus callus occurring and yew cell solid culture.
Embodiment 3
Cotton healing tissue induces He mushroom
Cotton healing tissue's induction adopts MS culture medium, and interpolation gibberellins concentration is 50mg/L.Choose
The good Cotton Gossypii aseptic seedling hypocotyls of upgrowth situation be cut into 1cm about segment, cotyledon be cut into 1cm × 1cm
Left and right fritter, is placed in the MS solid culture primary surface containing 50mg/L gibberellins concentration.Cultivation temperature
For 28 DEG C, daily illumination 12 hours, cultivate 30 days.Add 50mg/L gibberellins in MS culture medium
Hypocotyls and cotyledon general flavone content is finally made to have dropped 58% and 67% respectively, thus obtaining no browning
The cotton healing tissue occurring.
Embodiment 4
Fructus Musae callus induction He mushroom
Fructus Musae callus induction adopts MS culture medium, and interpolation gibberellins concentration is 75mg/L.Choose
The good Fructus Musae shoot apical meristem of upgrowth situation be cut into 1cm about segment, be placed in containing 75mg/L
The MS solid culture primary surface of gibberellins concentration.Cultivation temperature is 25 DEG C, dark culturing 30 days.MS
Adding 75mg/L gibberellins in culture medium finally makes calluss general flavone content decline 63% respectively,
Thus obtaining the Fructus Musae calluss that no browning occurs.
Embodiment 5
Chinese Ramulus et folium taxi cuspidatae callus tissue culture and the He mushroom of cell line solid culture
(1) explant sterilization：Take raw China Ramulus et folium taxi cuspidatae tender stem then, detergent water soaks 30 minutes,
Flowing water rushes 4 hours, and 75% ethanol soaks 10 seconds, 0.1%HgCl2Soak 10 minutes, and with a large number
Aseptic water washing 6 times, is cut into the segment of 1 cm, thus it is standby to obtain aseptic explant.
(2) induction of callus：Aseptic explant obtained by step (1) is inoculated in respectively
It is respectively 10mg/L, the MS solid inducing culture of 50mg/L and 100mg/L containing gibberellins concentration
Base, sucrose 20g/L, control temperature to be 25 DEG C, dark culturing, treat that callus induction rate reaches 90%
During left and right, the calluss of no browning are peeled off standby.
(3) calluss successive transfer culture：The calluss that step (2) is peeled off, are inoculated in corresponding
Be 10mg/L containing gibberellins, in the MS solid subculture medium of 50mg/L and 100mg/L,
Sucrose 30g/L, controls temperature to be 25 DEG C, and dark culturing every 25 days is 1 subculture cycle, changes
Fresh above-mentioned subculture medium.Adding variable concentrations gibberellins in MS culture medium finally makes total flavones contain
Amount have dropped 36%, 71% and 89%, thus in the training adding 50mg/L and 100mg/L gibberellins
Solid taxus callus and yew cell cell solid that no browning occurs all have been obtained on foster base
Culture.
Embodiment 6
The He mushroom of taxus chinensis cell liquid culture
3 kinds of He mushroom methods are available, final control taxus chinensis cell liquid culture browning
The generation of phenomenon：
(1) the solid culture cell line of long term subculture no browning is pressed 10% inoculum concentration (m/V)
It is inoculated in and contain gibberellins 10mg/L respectively, in the MS fluid medium of 50mg/L and 100mg/L,
Sucrose 30g/L, controls temperature to be 25 DEG C, by shaking flask as dark culturing on shaking table, controls shaking table to turn
Speed is 110-130RPM.Adding gibberellins 10mg/L makes general flavone content in cell decline 32%, carefully
Born of the same parents' browning decreases；Add gibberellins 50mg/L and 100mg/L and make yew cell total flavones
Content have dropped 78% and 92%, thus obtaining the yew cell liquid culture that no browning occurs.
(2) by the solid culture cell line MS fluid medium of long term subculture no browning, put
Shake scattered on shaking table, make cell mass separately.Under aseptic conditions, sieve collection diameter using steel to be less than
500 μm, 500-800 μm of diameter and cell mass with diameter greater than 800 μm (as comparison).
Cell mass is re-seeded into MS fluid medium respectively by 5% inoculum concentration (m/V), sucrose is dense
Spend for 30g/L, control temperature to be 25 DEG C, by shaking flask as dark culturing on shaking table, control shaking table to turn
Speed is 110-130RPM.The yew cell general flavone content that diameter is less than 500 μm relatively compares decline
57%, thus obtaining the yew cell liquid culture that no browning occurs, 500-800 μm of diameter
Culture in general flavone content relatively compare and have dropped 46%, cell browning degree decreases.
(3) the solid culture cell line of long term subculture no browning is pressed 10% inoculum concentration (m/V)
It is inoculated in MS fluid medium, Initial stage of culture setting sucrose concentration is 10g/L and 20g/L within first 5 days,
In cell, the content of total flavones declines 71% and 52% respectively, adds within the 5th day corresponding addition 20 respectively
G/L and 10g/L sucrose.Control temperature to be 25 DEG C, by shaking flask as dark culturing on shaking table, control
Shaking speed is 110-130RPM, thus obtaining the yew cell liquid culture that no browning occurs.
Embodiment 7
The He mushroom of taxus chinensis cell liquid reactor culture
3 kinds of He mushroom methods are available, and final control taxus chinensis cell bioreactor culture thing is brown
Change the generation of phenomenon：
(1) the solid culture cell line of long term subculture no browning is pressed 10% inoculum concentration (m/V)
It is inoculated in and contain gibberellins 10mg/L respectively, in the MS fluid medium of 50mg/L and 100mg/L,
Sucrose 30g/L.Using 7.5L bioreactor (NBS, BioFlo 115), working volume is 5L,
Screw mixing oar rotating speed is 60RPM, and ventilation is 0.2VVM, and temperature is 25 DEG C, dark culturing.
Adding variable concentrations gibberellins makes yew cell general flavone content have dropped 35% respectively, 75% He
87%, the culture medium adding 50mg/L and 100mg/L gibberellins all obtains what no browning occurred
Yew cell liquid reactor culture.
(2) by the solid culture cell line MS fluid medium of long term subculture no browning, put
Shake scattered on shaking table, make cell mass separately.Under aseptic conditions, it is less than using steel screening diameter
500 μm, 500-800 μm of diameter and cell mass with diameter greater than 800 μm (as comparison).
Cell mass is re-seeded into reactor MS fluid medium by 5% inoculum concentration (m/V) respectively,
Sucrose concentration is 30g/L.Using 7.5L bioreactor (NBS, BioFlo 115), working body
Amass as 5L, screw mixing oar rotating speed is 60RPM, ventilation is 0.2VVM, and temperature is 25 DEG C, black
Light culture.Diameter is less than general flavone content in 500 μm of yew cell and have dropped 63%, thus
Obtain the yew cell liquid reactor culture that no browning occurs, the Semen Phaseoli of 500-800 μm of diameter
In China fir cell, general flavone content have dropped 48%, and browning degree decreases.
(3) the solid culture cell line of long term subculture no browning is pressed 10% inoculum concentration (m/V)
It is inoculated in reactor MS fluid medium, Initial stage of culture setting sucrose concentration is 10g/L within first 5 days
And 20g/L, can effectively reduce the accumulation of total flavones, yew cell general flavone content declines 71% He
58%, addition in the 5th day is separately added into corresponding 20g/L and 10g/L sucrose.Biological using 7.5L
Reactor (NBS, BioFlo 115), working volume is 5L, and screw mixing oar rotating speed is 60RPM,
Ventilation is 0.2VVM, and temperature is 25 DEG C, dark culturing.Above-mentioned culture all obtains what no browning occurred
Yew cell liquid reactor culture.
Embodiment 8
The He mushroom of Glycyrrhiza cell liquid culture
3 kinds of He mushroom methods are available, final control Glycyrrhiza cell liquid culture browning
Occur：
(1) the solid culture Glycyrrhiza cell system of long term subculture no browning is pressed 6% inoculum concentration
(m/V) it is inoculated in respectively in the shaking flask MS fluid medium containing gibberellins 30mg/L, sucrose 30g/L,
By shaking flask as on shaking table, control shaking speed is 110-130RPM, and temperature is 25 DEG C, 12 hours
Dark and 12 hours illumination alternate cultures.Adding gibberellins 50mg/L makes Glycyrrhiza cell general flavone content
Have dropped 69%, thus obtaining the Glycyrrhiza cell liquid culture that no browning occurs.
(2) by the Glycyrrhiza cell inoculation MS fluid medium of long term subculture no browning, it is placed in
Shake scattered on shaking table, make cell mass separately.Under aseptic conditions, it is less than 500 using steel screening diameter
μm cell mass.This diameter cells agglomerate is re-seeded into MS by 4% inoculum concentration (m/V)
Fluid medium, sucrose concentration is 30g/L, by shaking flask as on shaking table, controls the shaking speed to be
110-130RPM, temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Diameter is little
Have dropped 59% in 500 μm of Glycyrrhiza cell general flavone contents, thus obtaining the sweet of no browning generation
Careless cellular liquid culture.
(3) the solid culture cell line of long term subculture no browning is pressed 10% inoculum concentration (m/V)
It is inoculated in MS fluid medium, Initial stage of culture setting sucrose concentration is 10g/L within first 5 days, can be effective
Reduce the accumulation of total flavones, add other 20g/L sucrose within the 5th day.Control shaking speed is 110-130
RPM, temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Diameter is less than 500 μ
The Glycyrrhiza cell general flavone content of m have dropped 59%, thus obtaining the Glycyrrhiza cell liquid that no browning occurs
Culture.
Embodiment 9
The He mushroom of Glycyrrhiza cell liquid reactor culture
3 kinds of He mushroom methods are available, final control Glycyrrhiza cell bioreactor culture thing browning
Generation：
(1) the solid culture Glycyrrhiza cell system of long term subculture no browning is pressed 6% inoculum concentration
(m/V) it is inoculated in respectively in the MS fluid medium containing gibberellins 50mg/L, sucrose 30g/L.
Using 7.5L bioreactor (NBS, BioFlo 115), working volume is 5L, rotating speed of agitator
For 60RPM, ventilation is 0.2VVM, and temperature is 25 DEG C, dark and illumination in 12 hours friendship in 12 hours
For culture.Adding gibberellins 50mg/L makes Glycyrrhiza cell general flavone content have dropped 72%, thus
Obtain the Glycyrrhiza cell liquid reactor culture that no browning occurs.
(2) by the solid culture Glycyrrhiza cell system MS fluid medium of long term subculture no browning,
It is placed on shaking table and shakes scattered, make cell mass separately.Under aseptic conditions, little using steel screening diameter
In 500 μm of cell mass.This diameter cells agglomerate is connect again by 4% inoculum concentration (m/V)
Plant in reactor MS fluid medium, sucrose concentration is 30g/L.Using 7.5L bioreactor (NBS,
BioFlo 115), working volume is 5L, and rotating speed of agitator is 60RPM, and ventilation is 0.2VVM, temperature
Spend for 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Diameter is sweet less than 500 μm
Careless cell general flavone content have dropped 61%, thus obtaining the Glycyrrhiza cell reactor liquid that no browning occurs
Body culture.
(3) the solid culture Glycyrrhiza cell system of long term subculture no browning is pressed 6% inoculum concentration
(m/V) it is inoculated in reactor MS fluid medium, Initial stage of culture setting sucrose concentration is within first 5 days
10g/L, can effectively reduce the accumulation of total flavones, add other 20g/L sucrose within the 5th day.Using 7.5
L bioreactor (NBS, BioFlo 115), working volume is 5L, and rotating speed of agitator is 60RPM,
Ventilation is 0.2VVM, and temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Directly
The Glycyrrhiza cell general flavone content that footpath is less than 500 μm have dropped 64%, thus obtain no browning occurring
Glycyrrhiza cell liquid reactor culture.
The He mushroom method of the present invention, based on culture plant cell browning mechanism, is cultivated by suppression
During the synthesis of Flavonoid substances carry out Browning control, with strong points, can fundamentally prevent browning
Occur.
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention,
Not in order to limit the present invention, all any modifications made within the spirit and principles in the present invention, etc.
With replacement and improvement etc., should be included within the scope of the present invention.
Claims (8)
1. the method for a kind of suppression plant cell tissue browning in culture, methods described is included plant
Cell tissue is cultivated under the conditions of being placed in as follows, and described condition makes described plant cell tissue cultivate
In journey, the synthetic quantity of Flavonoid substances declines more than 30%.
2. the method for claim 1 is it is characterised in that described condition is included in incubation
In add sucrose stage by stage, that is, in first 3-6 days of cell culture ensure sucrose concentration be 10g/L-20
G/L, is subsequently gradually increased sucrose concentration to 30g/L-40g/L.
3. the method for claim 1 is it is characterised in that protect in first 5 days of cell culture
Card sucrose concentration is 10g/L, is subsequently gradually increased sucrose concentration to 30g/L.
4. method as described in any of claims 1 is it is characterised in that described condition includes
Add 10mg/L-100mg/L gibberellins in culture medium.
5. the method for claim 1 is it is characterised in that add in the medium
30mg/L-75mg/L gibberellins.
6. the method for claim 1 is it is characterised in that described condition includes setting up cell
After system, screening minicell aggregation culture；I.e. the cell mass of 100-800 μm of diameter of screening is trained
Support.
7. the method for claim 1 is it is characterised in that a diameter of 500 μm of cell mass.
8. method as described in any of claims 1 is it is characterised in that described method is applied
Thin in plant callus culture, the culture of plant cell solid culture, plant cell liquid and plant
Born of the same parents' liquid reactor is cultivated.
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