CN106479951B - A method of inhibiting plant cell tissue's browning in culture - Google Patents
A method of inhibiting plant cell tissue's browning in culture Download PDFInfo
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Abstract
The invention discloses a kind of methods for inhibiting plant cell tissue's browning in culture, the method includes by plant cell tissue be placed in it is following under the conditions of cultivate, the condition makes 30% or more the synthetic quantity decline of plant cell tissue Flavonoid substances during the cultivation process.This method is for plant cell tissues such as plant callus culture, plant cell solid culture, plant cell liquid culture and plant cell liquid reactor cultures.Compared with current conventional method, this method more targetedly and controls browning significant effect, is appropriate for the industrial application of plant cell large-scale culture, wide market.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of inhibition plant cell tissue is brown in culture
The method of change.
Background technique
Plant cell and tissue culture technology is in micropropagation of plants, detoxification, acceleration breeding process, secondary metabolite production
And Germ-plasma resources protection etc. achieves huge economic benefit, social benefit and ecological benefits.Culture plant cell skill
Art is because its superiority, application field are still constantly extending, and appliable plant cell culture technology produces exogeneous animal albumen at present,
Have become the hot spot of whole world research and application.But browning problem is still prevalent in Plant cell and tissue culture field, sternly
The growth for affecting plant cell tissue again even results in its death, to reduce target metabolic product or other active matters
The accumulation of matter seriously constrains the industrial application of Plant cell and tissue culture.Plant cell and tissue culture process browning occurs
It is the quinones substance for being oxidized to toxicity by polyphenol oxidase (PPO) due to phenolic substances.
Currently, the browning during Plant cell and tissue culture relies primarily on traditional addition adsorbent, antioxidant, more
The methods of culture medium is changed to be controlled.For example, Li Chunbin etc. by into culture medium add activated carbon adsorption brown material, from
And the callus of taxus chinensis in northeast browning is made to remove browning (CN103563750A)；And Miao Chen flies to wait preparation specific shape
Silica gel adsorptive material adsorbs quinones substance, to reduce plant cell browning risk (CN103923873A)；Huang Wenmin etc. passes through
The method that PVP is added into culture medium inhibits the browning (CN102577953B) in tobacco healing tissue's squamous subculture.Bian Li Ping
Deng by adding VC into culture medium as reducing agent, to inhibit the browning (CN103125380A) of iris tissue；Xiang Yan
Deng preventing the browning (CN103749297A) of iris tissue culture by way of adding plant extraction liquid into culture medium；Liu Fuping
Deng by inventing a kind of anti-browning disinfectant 2- benzyl -4- chlorophenol, to prevent the browning of plant tissue culture explant
(CN103891713A), the action principle essence of such disinfectant or the effect of antioxidant.Li Yali etc. is by more renewing
The mode of fresh culture medium washing licorice cell prevents the browning of suspension Initial stage of culture from (CN103589679A) occurs, Yan Zhigang
Deng solid by herba fibraureae recisae callus -- the method for liquid conversion culture prevents browning from generating (CN102805032B).In addition, Qiu
Moral such as has at a kind of method by inhibiting browning after Taxus x media cells Agrobacterium tumefaciens transformation using Microrna
(CN101781650B).Although this method can play the role of preventing browning to a certain extent, cannot still hinder comprehensively
Only browning, and operating method is cumbersome.
In conclusion the method for preventing Plant cell and tissue culture browning disclosed in the above patent, carries out browning
A degree of control, but still cannot thoroughly solve the problems, such as the browning during Plant cell and tissue culture, and these methods are only
It can be used on a small scale in laboratory, implement on a large scale relatively difficult, but also there are other deficiencies, such as add antioxidant and suction
Attached dose equal to generate toxic side effect to plant cell tissue, need after incubation the phase removed from culture medium, and replace culture medium
Method it is cumbersome, production cost can be improved.Thus need to establish plant cell tissue's training effectively, convenient for implementing on a large scale
The new method of He mushroom in supporting.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of inhibition plant cell tissues to train
The method of browning fundamentally inhibits browning, thus solves by inhibiting the synthesis of plant cell flavones during the cultivation process in supporting
Certainly inhibit browning to be not thorough at present, be unable to large-scale use, the technical problem that toxic side effect and operation are numerous.
To achieve the above object, the present invention provides a kind of method for inhibiting plant cell tissue's browning in culture, institutes
The method of stating includes cultivating under the conditions of being placed in plant cell tissue as follows, and the condition is cultivating the plant cell tissue
30% or more the synthetic quantity decline of Flavonoid substances in the process.
Preferably, the condition includes that sucrose is added stage by stage during the cultivation process, and according to pertinent literature is consulted, plant is thin
Sucrose concentration used in born of the same parents' tissue cultures is generally 20g/L-40g/L, but will lead to culture plant cell initial stage sucrose is excessively high
Browning degree increases, therefore should reduce sucrose concentration in Initial stage of culture, i.e., guarantees sucrose concentration in first 3-6 days of cell culture
For 10g/L-20g/L, sucrose concentration is then gradually increased to 30g/L-40g/L.
Preferably, guarantee that sucrose concentration is 10g/L in first 5 days of cell culture, be then gradually increased sucrose concentration extremely
30g/L。
Preferably, the condition includes adding 10mg/L-100mg/L gibberellin in the medium.
Preferably, 30mg/L-75mg/L gibberellin is added in the medium.
Preferably, the condition is included in establish cell line after, screen the culture of cellule aggregation；Screen diameter 100-
800 μm of cell mass is cultivated.
Preferably, the diameter of cell mass is 500 μm.
Preferably, the method is applied to plant callus culture, plant cell solid culture, plant cell liquid
Culture and plant cell liquid reactor culture.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect:
It (1) is the basis for causing plant cell tissue's dominant mechanism of browning in culture the present invention is based on Flavonoid substances
On establish more targeted He mushroom method, fundamentally prevent plant cell tissue's browning during the cultivation process
Occur, avoids injury of the browning substance to plant cell tissue.
(2) method of the invention is free from side effects to plant cell tissue, thin suitable for the plant under different cultivation conditions
Born of the same parents' tissue, does not need the step of removing additive, easy to operate, large-scale culture and industrialization suitable for plant cell tissue
Using.
(3) method of the invention is easy to operate, applied widely, can effectively prevent the generation of browning；Pass through optimization
Parameter in each method, so that the content of plant cell tissue's Flavonoid substances during the cultivation process is more effectively controlled,
So as to preferably prevent the generation of plant cell tissue's browning in culture.Science of the present invention to Plant cell and tissue culture
Research and industrial application all have practical advice meaning and commercial value.
Detailed description of the invention
Fig. 1 is the cell line growth state that Subculture Time is respectively 6 months (A) and 10 years (B) in embodiment 1.
Specific embodiment
Below with reference to example, specific embodiments of the present invention will be further explained.It should be noted that for
The explanation of these embodiments is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, disclosed below
The each embodiment of the present invention in involved technical characteristic can be combined with each other as long as they do not conflict with each other.
It is an object of that present invention to provide brownings in the Plant cell and tissue culture of effective and suitable extensive industrial application
Control method, fundamentally prevent the generation of Plant cell and tissue culture process browning, avoid browning substance to plant cell
The injury of tissue.
It is found in research, general flavone content is more than 20 times of non-browning cell in browning cell, is caused brown in browning cell
It is very serious to change phenomenon, and non-browning cell does not have browning since flavones content is low substantially, and can be red by adding
The method specificity of mycin reduces general flavone content, to avoid the generation of browning.The result illustrates flavonoids in cell
The synthesis of substance is the main reason for causing plant cell cultures that browning occurs.
The present invention is to provide the conjunctions of the effective control Flavonoid substances used for different plant cell tissue cultures
At the method for carrying out control browning.Plant cell and tissue culture of the invention such as callus tissue culture, plant cell solid are trained
Support object, plant cell liquid culture, plant cell bioreactor culture object, the culture of tissue-cultured seedling and fast numerous, hairy culture etc.
Other easy Plant cell and tissue cultures that browning occurs.As long as flavonoids can be inhibited during Plant cell and tissue culture
Substance synthetic method can effectively control the browning of plant cell tissue.
The preferably following three kinds of simple and easy He mushroom methods of the present invention: sucrose fed-batch cultivation, i.e. sucrose add stage by stage
Enter in culture medium；Cellule aggregation culture；Gibberellin applied culture, in being used alone or in combination for different tissues culture.
The present invention solves the problems, such as that the browning of Plant cell and tissue culture preferably uses following scheme:
(1) sucrose is added stage by stage during the cultivation process, i.e., guarantees that sucrose concentration is in first 3-6 days of cell culture
10g/L-20g/L is then gradually increased sucrose concentration to 30g/L-40g/L.Preferably, guarantee in first 5 days of cell culture
Sucrose concentration is 10g/L, is then gradually increased sucrose concentration to 30g/L.
(2) 10mg/L-100mg/L gibberellin is added in the medium.Preferably, 30mg/L- is added in the medium
75mg/L gibberellin.It is further preferred that the additive amount of gibberellin is 50mg/L.Wherein, gibberellin addition operation is the simplest,
Suitable for entire culture plant cell amplification process, it is also applied for plant tissue and tissue-cultured seedling culture large-scale production.
(3) after establishing cell line, the culture of cellule aggregation is screened；Screen 100-800 μm of diameter of cell mass
It is cultivated.Preferably, the diameter of cell mass is 500 μm.The culture of cellule aggregation can inhibit the conjunction of Flavonoid substances
At the injury of osmotic pressure and fluid shearing to cell can be greatly lowered, avoid the generation of browning.
Method of the present invention is applied to plant callus culture, plant cell solid culture, plant cell liquid
Culture and plant cell liquid reactor culture.
(1) He mushroom of plant callus culture.It newly induces the callus grown to be easy to browning, causes to be cured
Injured tissue is dead.Using sucrose fed-batch cultivation (i.e. sucrose is added in culture medium stage by stage) of the invention, the training of cellule aggregation
It supports, its browning can be effectively suppressed in the method that gibberellin applied culture or both the above method combine.More preferably use sucrose
Fed-batch cultivation and gibberellin applied culture, according to floristics difference, it is 10-20g/L, addition that initial stage, which adds sucrose concentration range,
Gibberellin concentration range is 10-100mg/L, the accumulation of general flavone in cell can be reduced, to avoid callus tissue culture brown
The generation of change.
(2) He mushroom of plant cell solid culture.Plant cell solid culture includes that plant callus exists
Domestication culture obtains the culture in cell line and cell line long-term subculture and preserving process in solid medium, and browning is existing
As more serious.The He mushroom of plant cell solid culture more preferably using initial stage cane sugar content in reduction culture medium and adds
Add the method that gibberellin combines.It is different according to plant cell type, sucrose is added in the early stage in plant cell solid culture
Concentration range is 10-20g/L, and addition gibberellin concentration range is 10-100mg/L, can reduce the synthesis of general flavone in cell, from
And obtain the plant cell of no browning.
(3) He mushroom of plant cell liquid culture.Plant cell is transferred to fluid nutrient medium from solid medium
Suspension culture is carried out, osmotic pressure and fluid shearing cause to damage to cell, can induce enzymatic browning generation, cause plant cell liquor
Body culture browning is particularly acute.The He mushroom of plant cell liquid culture uses three kinds of methods of the invention, more excellent
Select being applied in combination for three kinds of methods: it is 10-100mg/L that gibberellin concentration range is added in culture medium, can prevent enzymatic browning
Occur；Cellule aggregation culture, screening cell mass of the diameter less than 500 μm are cultivated, and infiltration can be greatly lowered
Pressure and injury of the fluid shearing to cell, avoid the generation of enzymatic browning.Sucrose fed-batch cultivation, suspension Initial stage of culture sucrose
Concentration is not higher than 10g/L, can control browning, then gradually increases sucrose concentration to 30g/L.
(4) He mushroom of plant cell liquid reactor culture.Plant cell bioreactor culture object includes that plant is thin
Born of the same parents are amplified to bioreactor culture from flask suspension culture, then the culture of commercial scale reactor is amplified to from small-scale reactor,
Due to the acute variation of culture environment, the influence of fluid shearing, browning is easy to happen.In plant cell bioreactor culture object
He mushroom in, the present invention listed by method have good effect.
The common type of culture medium in Plant cell and tissue culture field has MS, B5, N6 culture medium, three kinds of culture mediums compositions at
Divide unanimously, only there is some difference for the height of certain or several content of material.Wherein the most commonly used is MS culture medium.
The present invention is with xylophyta Chinese yew and herbaceous plant Radix Glycyrrhizae, cotton with very high medicinal valence and application prospect
For flower, fragrant coke, induction and squamous subculture from callus are outstanding to the liquid suspension culture of plant cell, then to reactor
Under the different culture states of floating culture, He mushroom is carried out.
The following are embodiments
Embodiment 1
Chinese Chinese yew browning cell line and non-browning cell line enzymatic browning correlated characteristic comparative analysis
(1) there are two types of cell lines for laboratory preservation, and one is the cell line of subculture half a year (NA), the cell line is either solid
Body or liquid suspension culture all show browning very serious, and another kind is 10 years cell line of subculture (CA), should
Cell line not generation of browning substantially in solid Subculture.Two kinds of cell lines are seeded in MS solid culture
On base, sucrose 30g/L, controlled at 25 DEG C, dark culturing.NA cells show goes out dark brown, 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 of, the flavonoids of both cell lines of comparative analysis
Content of material and PPO activity.The study found that in 5d there is maximum difference in intracellular Flavonoid substances content in two kinds of cell lines
Value is 17 times, and extracellular Flavonoid substances content difference is 5 times in 5d.And the PPO activity difference very little of two kinds of cell line,
In 3d, 6 months intracellular PPO activity are only 1.4 times of 10 years cells, and extracellular 2 times of PPO activity maximum difference or so.Two
The Flavonoid substances content and PPO expression activitiy explanation, the greatest differences of exactly Flavonoid substances content of kind cell line have caused
Complete different cell browning degree, also further illustrates compared with PPO activity, the height of Flavonoid substances content more can be anti-
Answer the height of browning degree.
The He mushroom of the Chinese Chinese yew browning cell line of embodiment 2
It is 10mg/L, 50mg/L containing gibberellin that the cell line (NA) of subculture half a year in embodiment 1, which is inoculated in accordingly,
In the MS solid subculture medium of 100mg/L, sucrose 30g/L, controlled at 25 DEG C, dark culturing, every 25 days for 1 after
For the period, fresh above-mentioned subculture medium is replaced.By the culture in two periods, the general flavone content in cell declines respectively
41%, 77% and 86%, to obtain occurring without browning on the culture medium of addition 50mg/L and 100mg/L gibberellin
Solid taxus callus and yew cell solid culture.
Embodiment 3
Cotton healing tissue induces He mushroom
Cotton healing tissue's induction uses MS culture medium, and addition gibberellin concentration is 50mg/L.It is good to choose upgrowth situation
Cotton aseptic seedling hypocotyl be cut into 1cm or so segment, cotyledon is cut into 1cm × 1cm or so fritter, be placed in red mould containing 50mg/L
The MS solid culture primary surface of plain concentration.Cultivation temperature is 28 DEG C, and daily illumination 12 hours is cultivated 30 days.Add in MS culture medium
Adding 50mg/L gibberellin finally makes hypocotyl and cotyledon general flavone content have dropped 58% and 67% respectively, to obtain no browning
The cotton healing tissue of generation.
Embodiment 4
Banana callus induction He mushroom
Banana callus induction uses MS culture medium, and addition gibberellin concentration is 75mg/L.It is good to choose upgrowth situation
Banana shoot apical meristem be cut into 1cm or so segment, be placed in the MS solid culture primary surface containing 75mg/L gibberellin concentration.
Cultivation temperature is 25 DEG C, dark culturing 30 days.75mg/L gibberellin is added in MS culture medium finally contains callus general flavone
Amount declines 63% respectively, to obtain the banana callus that no browning occurs.
Embodiment 5
The He mushroom of Chinese Chinese yew callus tissue culture and cell line solid culture
(1) explant sterilizes: current year raw Chinese Chinese yew tender stem is taken, washing powder water impregnates 30 minutes, and flowing water rushes 4 hours,
75% ethyl alcohol impregnates 10 seconds, 0.1%HgCl2It impregnates 10 minutes, and with a large amount of aseptic water washings 6 times, is cut into 1 centimetre or so
Segment, so that it is spare to obtain aseptic explant.
(2) induction of callus: step (1) obtained aseptic explant is inoculated in dense containing gibberellin respectively
Degree is respectively the MS solid induced medium of 10mg/L, 50mg/L and 100mg/L, and sucrose 20g/L is black controlled at 25 DEG C
Dark culture, it is when callus induction rate reaches 90% or so, the callus removing of no browning is spare.
(3) callus squamous subculture: the callus that step (2) is removed is inoculated in and contains gibberellin accordingly
For in the MS solid subculture medium of 10mg/L, 50mg/L and 100mg/L, sucrose 30g/L, controlled at 25 DEG C, dark is trained
It supports, every 25 days are 1 subculture cycle, replace fresh above-mentioned subculture medium.Various concentration gibberellin is added in MS culture medium
Finally general flavone content is made to have dropped 36%, 71% and 89%, thus in the culture medium of addition 50mg/L and 100mg/L gibberellin
On obtained without browning occur solid taxus callus and yew cell cell solid culture.
Embodiment 6
The He mushroom of taxus chinensis cell liquid culture
3 kinds of He mushroom methods are available, the final hair for controlling taxus chinensis cell liquid culture browning
It is raw:
(1) the solid culture cell line by long term subculture without browning is inoculated in difference by 10% inoculum concentration (m/V)
In MS fluid nutrient medium containing gibberellin 10mg/L, 50mg/L and 100mg/L, sucrose 30g/L will controlled at 25 DEG C
For shaking flask as dark culturing on shaking table, control shaking speed is 110-130RPM.Addition gibberellin 10mg/L makes total yellow in cell
Ketone content decline 32%, cell browning decreases；Addition gibberellin 50mg/L and 100mg/L make yew cell general flavone
Content has dropped 78% and 92%, to obtain the yew cell liquid culture that no browning occurs.
(2) by long term subculture without in the solid culture cell line MS fluid nutrient medium of browning, be placed on shaking table shake it is scattered,
Separate cell mass.Under aseptic conditions, it is sieved using steel and collects diameter less than 500 μm, 500-800 μm of diameter and diameter are greater than
800 μm of cell mass (as control).Cell mass is reinoculated on the training of MS liquid by 5% inoculum concentration (m/V) respectively
Base is supported, sucrose concentration 30g/L, controlled at 25 DEG C, by shaking flask as dark culturing on shaking table, controlling shaking speed is
110-130RPM.Yew cell general flavone content of the diameter less than 500 μm has dropped 57% compared with control, to obtain no browning
The yew cell liquid culture of generation, general flavone content has dropped 46% compared with control in 500-800 μm of diameter of culture,
Cell browning degree decreases.
(3) the solid culture cell line by long term subculture without browning is inoculated in MS liquid by 10% inoculum concentration (m/V)
In body culture medium, preceding 5 days Initial stage of culture setting sucrose concentration is 10g/L and 20g/L, and the content of general flavone declines respectively in cell
71% and 52%, the corresponding addition 20g/L of the difference of addition in the 5th day and 10g/L sucrose.Controlled at 25 DEG C, by shaking flask as
Dark culturing on shaking table, control shaking speed are 110-130RPM, to obtain the yew cell liquid training that no browning occurs
Support object.
Embodiment 7
The He mushroom of taxus chinensis cell liquid reactor culture
3 kinds of He mushroom methods are available, final control taxus chinensis cell bioreactor culture object browning
Occur:
(1) the solid culture cell line by long term subculture without browning is inoculated in difference by 10% inoculum concentration (m/V)
In MS fluid nutrient medium containing gibberellin 10mg/L, 50mg/L and 100mg/L, sucrose 30g/L.Using 7.5L bioreactor
(NBS, BioFlo 115), working volume 5L, screw mixing paddle revolving speed are 60RPM, ventilatory capacity 0.2VVM, temperature 25
DEG C, dark culturing.Addition various concentration gibberellin makes yew cell general flavone content have dropped 35% respectively, 75% He
87%, the yew cell reactor that no browning occurs is obtained on the culture medium of addition 50mg/L and 100mg/L gibberellin
Liquid culture.
(2) by long term subculture without in the solid culture cell line MS fluid nutrient medium of browning, be placed on shaking table shake it is scattered,
Separate cell mass.Under aseptic conditions, using steel screening diameter less than 500 μm, 500-800 μm of diameter and diameter are greater than
800 μm of cell mass (as control).Cell mass is reinoculated on reactor MS by 5% inoculum concentration (m/V) respectively
Fluid nutrient medium, sucrose concentration 30g/L.Using 7.5L bioreactor (NBS, BioFlo 115), working volume 5L, spiral shell
Rotating rotating speed of agitator is 60RPM, and ventilatory capacity 0.2VVM, temperature is 25 DEG C, dark culturing.Red bean of the diameter less than 500 μm
General flavone content has dropped 63% in China fir cell, so that the yew cell liquid reactor culture that no browning occurs is obtained,
General flavone content has dropped 48% in 500-800 μm of diameter of yew cell, and browning degree decreases.
(3) the solid culture cell line by long term subculture without browning is inoculated in reaction by 10% inoculum concentration (m/V)
In device MS fluid nutrient medium, preceding 5 days Initial stage of culture setting sucrose concentration is 10g/L and 20g/L, can effectively reduce the product of general flavone
Tired, yew cell general flavone content decline 71% and 58%, addition in the 5th day is separately added into corresponding 20g/L and 10g/L sugarcane
Sugar.Using 7.5L bioreactor (NBS, BioFlo 115), working volume 5L, screw mixing paddle revolving speed is 60RPM, is led to
Tolerance is 0.2VVM, and temperature is 25 DEG C, dark culturing.Above-mentioned culture obtains the yew cell reactor liquid that no browning occurs
Body culture.
Embodiment 8
The He mushroom of licorice cell liquid culture
3 kinds of He mushroom methods are available, the final generation for controlling licorice cell liquid culture browning:
(1) the solid culture licorice cell system by long term subculture without browning is inoculated in point by 6% inoculum concentration (m/V)
Not in the shaking flask MS fluid nutrient medium containing gibberellin 30mg/L, sucrose 30g/L, by shaking flask as on shaking table, control shaking table turns
Speed is 110-130RPM, and temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Addition gibberellin 50mg/L makes sweet
Careless cell general flavone content has dropped 69%, to obtain the licorice cell liquid culture that no browning occurs.
(2) long term subculture is placed on shaking table without in the licorice cell inoculation MS fluid nutrient medium of browning and shakes scattered, made
Cell mass separates.Under aseptic conditions, using steel screening cell mass of the diameter less than 500 μm.By the diameter cells group
Block is reinoculated on MS fluid nutrient medium by 4% inoculum concentration (m/V), sucrose concentration 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.Diameter is less than
500 μm of licorice cell general flavone content has dropped 59%, to obtain the licorice cell liquid culture that no browning occurs.
(3) the solid culture cell line by long term subculture without browning is inoculated in MS liquid by 10% inoculum concentration (m/V)
In body culture medium, preceding 5 days Initial stage of culture setting sucrose concentration is 10g/L, can effectively reduce the accumulation of general flavone, is added within the 5th day
In addition 20g/L sucrose.Control shaking speed is 110-130RPM, and temperature is 25 DEG C, 12 hours dark and alternatings of illumination in 12 hours
Culture.Licorice cell general flavone content of the diameter less than 500 μm has dropped 59%, to obtain the licorice cell that no browning occurs
Liquid culture.
Embodiment 9
The He mushroom of licorice cell liquid reactor culture
3 kinds of He mushroom methods are available, the final generation for controlling licorice cell bioreactor culture object browning:
(1) the solid culture licorice cell system by long term subculture without browning is inoculated in point by 6% inoculum concentration (m/V)
Not in the MS fluid nutrient medium containing gibberellin 50mg/L, sucrose 30g/L.Using 7.5L bioreactor (NBS, BioFlo
115), working volume 5L, rotating speed of agitator 60RPM, ventilatory capacity 0.2VVM, temperature are 25 DEG C, 12 hours dark and 12
Hour illumination alternate culture.Addition gibberellin 50mg/L makes licorice cell general flavone content have dropped 72%, to obtain nothing
The licorice cell liquid reactor culture that browning occurs.
(2) long term subculture is placed on shaking table without in the solid culture licorice cell system MS fluid nutrient medium of browning
It shakes scattered, separates cell mass.Under aseptic conditions, using steel screening cell mass of the diameter less than 500 μm.By the diameter
Cell mass is reinoculated on reactor MS fluid nutrient medium, sucrose concentration 30g/L by 4% inoculum concentration (m/V).Using
7.5L bioreactor (NBS, BioFlo 115), working volume 5L, rotating speed of agitator 60RPM, ventilatory capacity 0.2VVM,
Temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Under licorice cell general flavone content of the diameter less than 500 μm
61% is dropped, to obtain the licorice cell liquid reactor culture that no browning occurs.
(3) the solid culture licorice cell system by long term subculture without browning is inoculated in instead by 6% inoculum concentration (m/V)
It answers in device MS fluid nutrient medium, preceding 5 days Initial stage of culture setting sucrose concentration is 10g/L, it can effectively reduce the accumulation of general flavone, the
Other 20g/L sucrose is added within 5 days.Using 7.5L bioreactor (NBS, BioFlo 115), working volume 5L, agitating paddle turns
Speed is 60RPM, and ventilatory capacity 0.2VVM, temperature is 25 DEG C, 12 hours dark and 12 hours illumination alternate cultures.Diameter is less than
500 μm of licorice cell general flavone content has dropped 64%, to obtain the licorice cell liquid reactor training that no browning occurs
Support object.
He mushroom method of the invention is based on culture plant cell browning mechanism, by inhibiting flavones in incubation
The synthesis of substance inhibits browning, with strong points, can fundamentally prevent the generation of browning.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (7)
1. it is a kind of inhibit plant cell tissue's browning in culture method, the method includes by plant cell tissue be placed in as
It is cultivated under the conditions of lower, the condition makes the synthetic quantity decline of plant cell tissue Flavonoid substances during the cultivation process
30% or more；The condition is that sucrose is added stage by stage during the cultivation process, i.e., guarantees sucrose in first 3-6 days of cell culture
Concentration is 10g/L-20g/L, is then gradually increased sucrose concentration to 30g/L-40g/L.
2. the method as described in claim 1, which is characterized in that guarantee that sucrose concentration is 10g/ in first 5 days of cell culture
L is then gradually increased sucrose concentration to 30g/L.
3. method as described in claim 1, which is characterized in that the condition further includes adding 10mg/L- in the medium
100mg/L gibberellin.
4. the method as described in claim 1, which is characterized in that the condition further includes adding 30mg/L- in the medium
75mg/L gibberellin.
5. it is a kind of inhibit plant cell tissue's browning in culture method, the method includes by plant cell tissue be placed in as
It is cultivated under the conditions of lower, the condition makes the synthetic quantity decline of plant cell tissue Flavonoid substances during the cultivation process
30% or more, which is characterized in that the condition is to screen the culture of cellule aggregation after establishing cell line；Screen diameter
100-800 μm of cell mass is cultivated.
6. method as claimed in claim 5, which is characterized in that the cell mass of 500 μm of diameter of screening is cultivated.
7. the method as described in claim 1 or 5, which is characterized in that the method be applied to plant callus culture,
Plant cell solid culture, the culture of plant cell liquid and plant cell liquid reactor culture.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737124A (en) * | 2005-08-08 | 2006-02-22 | 中国林业科学研究院林业研究所 | Method for preventing plant callus browning |
| CN102577953A (en) * | 2012-02-13 | 2012-07-18 | 中国科学院水生生物研究所 | Method for restraining browning in subculture of tobacco callus |
| CN102805032A (en) * | 2012-08-20 | 2012-12-05 | 广西壮族自治区药用植物园 | Method for preventing daemonorops margaritae callus browning phenomena from occurring |
| CN103202233A (en) * | 2013-04-28 | 2013-07-17 | 安徽农业大学 | Butterfly orchid anti-browning tissue culture method and anti-browning culture medium |
| CN104604677A (en) * | 2015-01-04 | 2015-05-13 | 黑龙江省科学院自然与生态研究所 | Tissue culture propagation method for reducing tissue culture browning rate of traxacum koksaghyz |
-
2015
- 2015-08-25 CN CN201510527002.0A patent/CN106479951B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737124A (en) * | 2005-08-08 | 2006-02-22 | 中国林业科学研究院林业研究所 | Method for preventing plant callus browning |
| CN102577953A (en) * | 2012-02-13 | 2012-07-18 | 中国科学院水生生物研究所 | Method for restraining browning in subculture of tobacco callus |
| CN102805032A (en) * | 2012-08-20 | 2012-12-05 | 广西壮族自治区药用植物园 | Method for preventing daemonorops margaritae callus browning phenomena from occurring |
| CN103202233A (en) * | 2013-04-28 | 2013-07-17 | 安徽农业大学 | Butterfly orchid anti-browning tissue culture method and anti-browning culture medium |
| CN104604677A (en) * | 2015-01-04 | 2015-05-13 | 黑龙江省科学院自然与生态研究所 | Tissue culture propagation method for reducing tissue culture browning rate of traxacum koksaghyz |
Non-Patent Citations (5)
| Title |
|---|
| 岩白菜组培中活性的研究;彭红卫等;《西华师范大学学报（自然科学版）》;20110930;227-230 |
| 植物组织培养中褐化问题的研究;司晶星等;《科技信息》;20091231;30 |
| 红蔑愈伤组织中黄酮代谢途径的探讨;张缨等;《甘肃农业人学学报》;20000331;63-65 |
| 红豆杉细胞培养过程中褐化机理及其控制;董艳山;《中国博士学位论文全文数据库》;20160715;A006-56 |
| 银杏组织培养中控制褐化的研究;张明文等;《中国南方果树》;20031231;51-52 |
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