CN104770304B - Plant bioreactor and its application method - Google Patents
Plant bioreactor and its application method Download PDFInfo
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- CN104770304B CN104770304B CN201510218596.7A CN201510218596A CN104770304B CN 104770304 B CN104770304 B CN 104770304B CN 201510218596 A CN201510218596 A CN 201510218596A CN 104770304 B CN104770304 B CN 104770304B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
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Abstract
The present invention relates to a kind of plant bioreactor and its application method, the plant bioreactor includes drive device, pipe-line system and at least one reacting tank body；The drive device includes control system, display system and dynamical system；The control system controls dynamical system by operation interface；The display system includes operation interface and monitoring interface；The dynamical system is connected with the pipe-line system；The pipe-line system is connected with reacting tank body sealing.The control system is programmable controller, and various forms of instructions are inputted by operation interface, then by the output of control system control dynamical system, and monitored by monitoring system；Time control by programmable controller programme-control, it is simple in construction and easy to operate so as to realize the high automaticity of reactor.
Description
Technical field
Plant cell, tissue, organ mass propgation are can be not only used for the present invention relates to one kind, it can also be used to microorganism and plant
The foundation of co-culture system is organized, it is more particularly to a kind of to obtain the culture apparatus of the natural active product with medical value
Realize the plant bioreactor of plant clearance submergence.
Background technology
At present in field of plant tissue culture, training method is broadly divided into solid and the class of Liquid Culture two, and it is each excellent
Shortcoming such as table 1.Compared with solid culture, liquid training body culture advantage is more obvious, but still suffers from shortcomings and deficiency.For
Further solve the above problems, domestic and foreign scholars have carried out lot of experiments, wherein interval immersion system cultivation effect more shows
Write.Intermittent immersed plant bioreactor huge number, many of reactor is improved training in experimenter's actual tests
More ripe in the system of supporting, these bioreactors, representative mainly has four kinds, such as table 2.
The solid culture of table 1 is compared with Liquid Culture advantage and disadvantage
2 four kinds of intermittent immersion bioreactor structure and features of table
Above-mentioned four kinds of gap immersion bioreactors represent the achievement of different times, and first 2 kinds due to pollution and space
Problem is not used widely.2 kinds employ double cylinder afterwards, and principle is the same, realize that interval is submerged using air pressure, reduction pollution
Rate, also reduces cost to a certain extent, improves efficiency, has been put into and commercially produces application, but this kind of plant life
Thing reactor still constructs complexity, maintenance difficult, can cause certain inconvenient for use.
Intermittent immersion bioreactor can not only meet plant tissue cultures growth needed nutrient matter and be supplied with oxygen
Give, also solve the Vitrification Occurred run into the culture that suspends；Such training method studies more both at home and abroad, and has applied for phase
The patent of pass, such as Chinese patent (application number 98205797.0) disclose a kind of " gas-lifting type cycle submergence illumination plant groups of cells
Knit organ culture reactor ", Chinese patent (application number 98102396.7) discloses a kind of " gas-lifting type cycle submergence illumination plant
Cell and tissue structrue method and cultivation reactor ", the reactor disclosed in above-mentioned two patent is because equipment construction is complicated, be difficult to
Scale is manufactured, and causes not being suitable for being widely popularized.Meanwhile, Chinese patent (patent announcement CN 201420083Y) is disclosed
A kind of " interval submerges the cultivation reactor of Plant Tissue Breeding organ " is although design simply, with low cost, the master of the equipment
Retort opening is smaller, and the tissue-cultured seedling after culture is not easily taken out, or even needs destruction main treatment tank could seedling taking；Further, since
Fluid reservoir and main treatment tank are two independent components, are made troubles to carrying and sterilizing, the possibility of pollution is greatly increased.China
Patent (patent announcement CN 201809355U) discloses a kind of " the open-close type plant bioreactor of interval submergence " and realized
The bioreactor of integral type, but be due to that its is cumbersome, material is heavy, and sealing is poor, cause it is seriously polluted, finally also not
It is used widely.What deserves to be explained is, above pre-existing reactors system is mainly the construction for describing bioreactor tank body,
It there is no and be related to air movement device this key element, also lack the series connection of equipment and automatically control part.
Therefore, it is badly in need of researching and developing a kind of automaticity now high, simple in construction, simple to operate, good airproof performance, and can
The plant bioreactor of standardized production.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of automaticity height, simple in construction, easy to operate, material
Gently, high leakproofness can be really realized, so as to avoid the plant bioreactor of pollution.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that the plant bioreactor includes driving
Device, pipe-line system and 1~multiple reacting tank bodies；The drive device includes control system, display system and dynamical system；Institute
Stating display system includes operation interface and monitoring interface, and the control system controls dynamical system by operation interface, described dynamic
Force system is connected with the pipe-line system, and the pipe-line system is connected with reacting tank body sealing.The control system is
Programmable controller, various forms of instructions are inputted by operation interface, then control the output of dynamical system by control system, and
Monitored by monitoring system；Time control by programmable controller programme-control；So as to realize the high automation journey of reactor
Degree, it is only necessary to carry out simple operations in operation interface, i.e., it is simple to operate and structure is also simple.
Further improvement of the present invention is that the pipe-line system includes air inlet pipeline and outlet pipe, the air inlet pipe
Road connects the dynamical system, and the outlet pipe is using isocon and is intersected in formation main pipe rail at one；The reacting tank body
Air inlet and gas outlet are provided with, the air inlet connects the air inlet pipeline, and the gas outlet connects the outlet pipe.It is logical
Pipeline air inlet pipeline and outlet pipe are crossed, it is possible to achieve the circulation of gas in reacting tank body, necessary to meeting plant growth
Condition.
Further improvement of the present invention is that the reacting tank body includes culturing room, fluid reservoir and culture plate, the reaction
Tank body is cultured disk and separates to form upper strata culturing room and lower floor's fluid reservoir two chambers, and the culture plate is provided with catheter, institute
State catheter and connect the culturing room and the fluid reservoir；The top of the catheter is provided with shunting current limliting post, the culture plate
It is transportable.
Using above-mentioned technical proposal, culture tank body is separated into by culturing room and fluid reservoir, the culture by culture plate
Building volume is more than or equal to the fluid reservoir, and culturing room accounts for the 1/2~4/5 of whole culture tank body, and fluid reservoir accounts for whole culture
The 1/5~1/2 of tank body, it is such to be designed as Plant Tissue Breeding there is provided bigger growing space；By setting catheter
Culturing room is connected with fluid reservoir, and on catheter set shunting current limliting post, can catheter then can be by the culture of fluid reservoir
Liquid imports culturing room, then the nutrition of abundance can be provided for Plant Tissue Breeding；Shunting current limliting post can control to import nutrient solution
Amount, the gap immersion culture of the plant can be better achieved；In addition, culture plate is arranged to movably, when in culture
During need to change nutrient solution or when culture medium, directly culture plate can be moved to together with the plant of culture new
Cultivate in tank body；Or after the completion of a stage is cultivated, directly can be moved to culture plate together with the plant of culture next
In the culture tank body in stage, from without influenceing the growth conditions of plant and simple to operate.
Further improvement of the present invention is that the air inlet pipeline includes the first sebific duct and the first air disinfector, described
Outlet pipe includes the second sebific duct and the second air disinfector；First air disinfector by first sebific duct with it is described
Air inlet sealing is connected；Second air disinfector is connected by second sebific duct with the gas outlet seal.By
Air disinfector is set to realize sterile culture of the plant bioreactor to plant in pipe-line system.
Further improvement of the present invention is that the dynamical system is made up of air pump, valve, valve and flow sensor,
The valve is used to control air pump；The flow sensor monitors the flow of air pump；The air pump connects institute by the valve
State pipe-line system.When air pump work, the nutrient solution in culturing room in the presence of self gravitation, pass sequentially through shunting current limliting post,
Mozzle is back in fluid reservoir；Negative pressure is now formed in whole culturing room, extraneous gas passes through air by export pipeline
Enter after the sterilization processing of sterilization device along gas outlet in culturing room, the air pressure of reacting tank body is recovered normal, so as to realize this
A circulation for the free of contamination culture plant of plant bioreactor.
Further improvement of the present invention is that the reacting tank body also includes the first sealing ring and the second sealing ring, described
The edge of reacting tank body is provided with the first sealing ring neck, for placing first sealing ring；On the edge of the culture plate
Provided with the second sealing ring neck, for placing second sealing ring.By between the fluid reservoir and culture plate of reacting tank body
One sealing ring is set, a sealing ring is set between culturing room and culture plate, the close of reacting tank body can be preferably improved
Sealing property, the sealing realized.
Further improvement of the present invention is, the culture plate is in funnel-form, and the bottom of the culture plate is using the center of circle in
Outwards diverging is provided with the concavo-convex alternate annulus of at least two to the heart；The bottom of the culture plate is centered on shunting current limliting post to extension
Stretch provided with least 2 drainage troughs；The drainage trough connects the alternate annulus of the bumps.Concavo-convex alternate annulus is set and recessed
The drainage trough of connection is set on convex alternate annulus, the plant of culture can be made more preferably more uniformly to be cultured immersion and do not had.
Further improvement of the present invention is that the bottom of the catheter is diametrically arranged with breach；The shunting limit
Fluidization tower is hollow structure and top is closing, and the cross section of the shunting current limliting post is in " ten " font and higher than the culture plate
Outer edge, the draining hole of the side of the shunting current limliting post provided with 4 vertical-shaped openings.In shunting current limliting post is arranged to
Hollow structure, top seal and higher than the culture plate outer edge, while side have 4 vertical-shaped openings, can be preferably real
Current-limiting function is now shunted, while when nutrient solution is back to fluid reservoir, the Plant Tissue Breeding in culture plate will not be with culture
Liquid is back in fluid reservoir.
As the preferred scheme of the present invention, the reacting tank body is 1~100, and the isocon is 1~20；It is described
The quantity of concavo-convex alternate annulus is 6~9, and the drainage trough is 4.
As the preferred scheme of the present invention, the reacting tank body is 20~80.
The problem of otherwise address of the present invention, is to provide one kind and expands numerous bletilla striata seedling using afore-mentioned plants bioreactor culture
Method, comprise the following steps that：
(1) surface sterilization of bletilla striata Fruit pod and reactor tank body, the high-temperature heat sterilization of culture medium；
(2) above-mentioned sterilized fluid nutrient medium is poured into reactor tank body in super-clean bench, then by surface sterilization
Bletilla striata Fruit pod in seed sow in the culture plate of reactor tank body, inoculum density be each Fruit pod be inoculated with 3-5 react
Device tank body；
(3) the reactor tank body for being inoculated with completion is connected in drive device, is placed in culturing room and is cultivated；Set
Immersion frequency is 3min/4h.Environmental condition is：Intensity of illumination 1800lx, periodicity of illumination 10h/d, 25 ± 1 DEG C of temperature.
The plant bioreactor is applied to expand breeding seedling；It is advantageous that culture flux is big, space availability ratio is high, nothing
Culture medium need to be changed；The bletilla striata is a preferred scheme herein.
The problem of otherwise address of the present invention, which is to provide, a kind of utilizes foregoing plant bioreactor to carry out plant mutagenesis
The method of resistance screening afterwards, is comprised the following steps that：
(1) numerous plant to be mutagenic is expanded using plant bioreactor, the plant is that tissue-cultured seedling and/or plant are cured
Injured tissue, obtains a large amount of mutant materials；
(2) addition chemical mutagen makes plant produce mutation in the reactor, or by the reactor with plant
Tank body, which carries out radiation treatment, makes plant undergo mutation；
(3) the reactor tank body of above-mentioned mutagenesis is changed and adds the culture medium of predetermined substance or train under particular circumstances
Support, screen the plant of specific resistance；
(4) plant for choosing above-mentioned brush is inoculated into new plant-bioreactor, is largely expanded under given conditions
It is numerous, obtain the plant new lines of resistance.
The plant bioreactor is applied to the directed screening in gene mutation breeding, such as salt tolerant, pest-resistant etc.；Its is excellent
Gesture is that screening flux is big, environment is easily-controllable, reproducible.
Another technical problem that the present invention is solved is to provide a kind of produced using foregoing plant bioreactor and planted
The method of thing secondary metabolite, the plant is Asian puccoon, is comprised the following steps that：
(1) batch of Asian puccoon callus is obtained：
The culture parameters mainly have：In the plant bioreactor that Asian puccoon callus is inoculated with to the present invention, inoculation
Density：120/L, culture medium prescription is：B5+1.0mg/L 6-BA+0.1mg/L NAA+30g/L sucrose；Medium pH：6.0；
Immersion frequency：1min/6h；Condition of culture：Intensity of illumination 1000lux, light application time 18h/d, 25 ± 1 DEG C of cultivation temperature, culture
Time：60d；
(2) induction of Secondary metabolites：While culture medium is changed without, CuSO is added in nutrient solution4·
5H2O, makes the CuSO in nutrient solution4Concentration is 0.075mg/L；Continuation is cultivated Asian puccoon callus, described to continue to cultivate
Parameter synchronization rapid (1) described in, except that incubation time is 20d；After culture 20d, fluid nutrient medium is taken out, simultaneously
Change CuSO4Concentration is 0.075mg/L fresh culture；The receipts of old nutrient solution are carried out while fresh medium is changed
Collect to extract required alkannin, and so on；
(3) collection and extraction of Secondary metabolites：
The nutrient solution changed is subjected to acetone extraction alkannin after concentration, using spectrophotometer by
Detect to calculate the yield of Asian puccoon Soviet Union under 520nm.
Production and preparation that Secondary metabolites are applied to natural products are produced using the plant bioreactor, than
Such as crocin, Asian puccoon plant, it is advantageous that by the control of environment include the continuous collection of nutrient solution and addition so as to
Lasting enrichment natural products；Asian puccoon is a preferred scheme herein.
Compared with prior art, the beneficial effects of the invention are as follows：The plant bioreactor automaticity is high, structure and
Simple to operate, pollution-free, sealing, material is light, and translucency is good, designs more reasonable, culture effect more preferably, and cost is low, just
In large-scale production and promote the use of.
Brief description of the drawings
In order that present disclosure is easier to be clearly understood, it is right below according to specific embodiment and with reference to accompanying drawing
The present invention is described in further detail：
Fig. 1 is the general flow chart of plant bioreactor of the present invention；
Fig. 2 is the structural representation of inventive drive means；
Fig. 3 is the structural representation of the reacting tank body of the present invention；
Fig. 4 is the structural representation of the culture plate of the present invention；
Fig. 5 is the structural representation of the shunting current limliting post of the present invention；
Fig. 6 is cultivation cycle figure of the embodiment of the present invention three using the plant bioreactor culture bletilla striata seedling；
Fig. 7 is Technology Roadmap of the present invention using plant bioreactor resistance general's choosing of the present invention；
Fig. 8 is to extract secondary metabolite Technology Roadmap using the plant bioreactor of the present invention；
Wherein：1- drive devices；101- control systems；102- shows system；1021- operation interfaces；1022- monitors boundary
Face；103- dynamical systems；1031- air pumps；1032- valves；1033- valves；1034- flow sensors；201- air inlet pipelines；
The sebific ducts of 2011- first；The air disinfectors of 2012- first；202- outlet pipes；The sebific ducts of 2021- second；The air of 2022- second is removed
Bacterium device；3- reacting tank bodies；301- air inlets；302- gas outlets；303- Cap for tin body；3031- back taper handles；304- foots pad；4-
Culture plate；401- catheters；4011- breach；402- shunts current limliting post；403- the second sealing ring necks；404- drainage troughs；405-
Draining hole；5- culturing room；6- fluid reservoirs；The Non-return air valves of 7- first；The Non-return air valves of 8- second；The Non-return air valves of 9- the 3rd；10- the 4th
Non-return air valve；
Wherein, A：Culture 20 days, B：Culture 40 days, C：Culture 60 days.
Embodiment
Embodiment one：
As Figure 1-5, the plant bioreactor includes drive device 1, pipe-line system and 4 reacting tank bodies 3, driving
Device 1 includes control system 101, display system 102 and dynamical system 103；Dynamical system 103 by air pump 1031, valve 1032,
Valve 1033 and flow sensor 1034 are constituted, and valve 1033 is used to control air pump 1031；Display system 102 includes operation interface
1021 and monitoring interface 1022, control system 101 passes through operation interface 1021 and controls dynamical system 103, flow sensor 1034
Monitor the flow of air pump 1031；Air pump 1031 seals phase by the connecting line system of valve 1032, pipe-line system with reacting tank body 3
Even；Pipe-line system includes air inlet pipeline 201 and outlet pipe 202, the connection dynamical system 103 of air inlet pipeline 201, air inlet pipeline
201 use isocon and are intersected in formation main pipe rail at one；Pass through the first Non-return air valve 7, the second Non-return air valve the 8, the 3rd respectively
The regulation control of the Non-return air valve 10 of Non-return air valve 9 and the 4th is passed through the flow of the gas of 4 reacting tank bodies；Reacting tank body 3 is integrated
Shaping is set；Reacting tank body 3 includes air inlet 301, gas outlet 302, Cap for tin body 303, the sealing ring neck of foot pad 304 and first,
Air inlet pipeline 201 includes the first sebific duct 2011 and the first air disinfector 2012, and outlet pipe 202 includes the He of the second sebific duct 2021
Second air disinfector 2022；First air disinfector 2012 is connected by the first sebific duct 2011 with the sealing of air inlet 301；Second
Air disinfector 2021 is connected by the second sebific duct 2022 with the sealing of gas outlet 302；The inside of reacting tank body 3 is provided with culture plate 4,
Reacting tank body 3 is cultured disk 4 and separates to form culturing room 5 and the two chambers of fluid reservoir 6, and culture plate 4 is provided with catheter 401, leads
Liquid pipe 401 connects culturing room 5 and fluid reservoir 6；The top of catheter 401 is provided with shunting current limliting post 402.Reacting tank body 3 also includes
First sealing ring and the second sealing ring, the edge of reacting tank body 3 are provided with the first sealing ring neck, close for placing described first
Seal；The edge of culture plate 4 is provided with the second sealing ring neck 403, for placing second sealing ring；Culture plate 4 is in leakage
Bucket-shaped, outwards diverging is provided with the alternate annulus of 7 bumps centered on the center of circle for the bottom of culture plate 4；The bottom of culture plate 4 with point
Stretch out centered on ductility limit fluidization tower provided with 4 drainage troughs 404；Drainage trough 404 connects concavo-convex alternate annulus；Catheter 401
Bottom be diametrically arranged with breach 4011；Shunting current limliting post 402 is hollow structure and top is closing, shunts current limliting post
402 cross section is in " ten " font and higher than the outer edge of culture plate 4, and the side of shunting current limliting post 402 is provided with 4 vertical-shaped openings
Draining hole 405.
Specific work process：First, by it as the high pressure moist heat sterilization that 25~35min is carried out at 121 DEG C, then super
Net workbench opens Cap for tin body 303, appropriate nutrient solution is put into fluid reservoir 6, nutrient solution is by shunting current limliting post 402, water conservancy diversion
Pipe 4 enters in fluid reservoir 6, covers Cap for tin body 303；Aseptically, Cap for tin body 303 is opened, by plant to be cultivated (as carefully
Born of the same parents, tissue, organ), microorganism and both co-culture systems are put into the culture plate 4 of culturing room 5, close Cap for tin body 303, are protected
Hold whole main reactor sealing；Control air pump 1031 to carry out discontinuous operation by control system 101, pass through flow sensor
The flow of 1034 monitoring air pumps 1031；When air pump 1034 works, the nutrient solution in culturing room 5 in the presence of self gravitation, according to
It is secondary to be back to by shunting current limliting post 402, mozzle 4 in fluid reservoir 6；Due to its upper end closed and there are 4 vertical-shaped openings sideways
Draining hole 405, therefore Plant Tissue Breeding in culturing room 5 will not be back in fluid reservoir 6 with nutrient solution.It is now whole
Form negative pressure in culturing room 5, extraneous gas is by export pipeline system after the sterilization processing of the second air disinfector 308
Enter along gas outlet 302 in culturing room 5, the air pressure of reacting tank body 3 is recovered normal, so as to realize that free of contamination culture is planted
Thing.So, the plant tissue organ in culturing room 5 just completes the circulation that a quilt is intermittently submerged；Can be by adjusting control system
System 101 adjusts time of above-mentioned circulation.Culture terminates after under aseptic condition, discharges culture medium, then opens lid, takes out again
Raw plant.
Embodiment two：
Difference with embodiment 1 is that the quantity of reacting tank body 3 is different, and the quantity of the embodiment reacting tank body 3 is 20
It is individual, supplied by two to flow divider.It is specific as follows：Including drive device 1, pipe-line system and 4 reacting tank bodies 3, drive device 1
Including control system 101, display system 102 and dynamical system 103；Dynamical system 103 is by air pump 1031, valve 1032, valve
1033 and flow sensor 1034 constitute, valve 1033 be used for control air pump 1031；Display system 102 includes operation interface 1021
With monitoring interface 1022, control system 101 controls dynamical system 103 by operation interface 1021, and flow sensor 1034 is monitored
The flow of air pump 1031；Air pump 1031 is connected by the connecting line system of valve 1032, pipe-line system with the sealing of reacting tank body 3；
Pipe-line system includes air inlet pipeline 201 and outlet pipe 202, and the connection dynamical system 103 of air inlet pipeline 201, air inlet pipeline 201 is adopted
With isocon and it is intersected at one formation main pipe rail；Pass through the first Non-return air valve 7, the second Non-return air valve 8, the 3rd unidirectional gas respectively
The regulation control of the Non-return air valve 10 of valve 9 and the 4th is passed through the flow of the gas of 4 reacting tank bodies；Reacting tank body 3, which is formed in one, to be set
Put；Reacting tank body 3 includes air inlet 301, gas outlet 302, Cap for tin body 303, the sealing ring neck of foot pad 304 and first, air inlet pipe
Road 201 includes the first sebific duct 2011 and the first air disinfector 2012, and it is empty that outlet pipe 202 includes the second sebific duct 2021 and second
Gas sterilization device 2022；First air disinfector 2012 is connected by the first sebific duct 2011 with the sealing of air inlet 301；Second air is removed
Bacterium device 2021 is connected by the second sebific duct 2022 with the sealing of gas outlet 302；The inside of reacting tank body 3 is provided with culture plate 4, retort
Body 3 is cultured disk 4 and separates to form culturing room 5 and the two chambers of fluid reservoir 6, and culture plate 4 is provided with catheter 401, catheter 401
Connect culturing room 5 and fluid reservoir 6；The top of catheter 401 is provided with shunting current limliting post 402.Reacting tank body 3 also includes the first sealing
Circle and the second sealing ring, the edge of reacting tank body 3 are provided with the first sealing ring neck, for placing first sealing ring；Training
The edge for supporting disk 4 is provided with the second sealing ring neck 403, for placing second sealing ring；Culture plate 4 is in funnel-form, training
The bottom for supporting disk 4 is outwards dissipated provided with the alternate annulus of 7 bumps centered on the center of circle；The bottom of culture plate 4 is to shunt current limliting
Stretch out centered on post provided with 4 drainage troughs 404；Drainage trough 404 connects concavo-convex alternate annulus；The bottom of catheter 401
Diametrically it is arranged with breach 4011；Shunting current limliting post 402 is hollow structure and top is closing, shunting current limliting post 402
Cross section is in " ten " font and higher than the outer edge of culture plate 4, the side of shunting current limliting post 402 drawing provided with 4 vertical-shaped openings
Head piece 405.
Embodiment three：Expand numerous bletilla striata
As shown in fig. 6, expanding the method for numerous bletilla striata seedling using the plant bioreactor of the present invention：
(1) surface sterilization of bletilla striata Fruit pod and reactor tank body, the high-temperature heat sterilization of culture medium；
(2) above-mentioned sterilized fluid nutrient medium is poured into reactor tank body in super-clean bench, then by surface sterilization
Bletilla striata Fruit pod in seed sow in the culture plate of reactor tank body, inoculum density be each Fruit pod be inoculated with 3-5 react
Device tank body；
(3) the reactor tank body for being inoculated with completion is connected in drive device, is placed in culturing room and is cultivated；Set
Immersion frequency is 3min/4h.Environmental condition is：Intensity of illumination 1800lx, periodicity of illumination 10h/d, 25 ± 1 DEG C of temperature.
Example IV：Resistance screening
As shown in fig. 7, the method for carrying out the resistance screening after plant mutagenesis using the plant bioreactor of the present invention：
(1) expand numerous plant (tissue-cultured seedling and/or plant callus) to be mutagenic using plant bioreactor, obtain
A large amount of mutant materials；
(2) addition chemical mutagen makes plant produce mutation in the reactor, or by the reactor with plant
Tank body, which carries out radiation treatment, makes plant undergo mutation；
(3) the reactor tank body of above-mentioned mutagenesis is changed and adds the culture medium of predetermined substance or train under particular circumstances
Support, screen the plant of specific resistance；
(4) plant for choosing above-mentioned brush is inoculated into new plant-bioreactor, is largely expanded under given conditions
It is numerous, obtain the plant new lines (kind) of resistance.
Embodiment five：Secondary metabolite
The method for producing Secondary metabolites using the plant bioreactor of the present invention：
As shown in figure 8, exemplified by producing alkannin, concrete operation step is as follows：
(1) batch of Asian puccoon callus is obtained：
The culture parameters mainly have：In the plant bioreactor that Asian puccoon callus is inoculated with to the present invention, inoculation
Density：120/L, culture medium prescription is：B5+1.0mg/L 6-BA+0.1mg/L NAA+30g/L sucrose；Medium pH：6.0；
Immersion frequency：1min/6h.Condition of culture：Intensity of illumination 1000lux, light application time 18h/d, 25 ± 1 DEG C of cultivation temperature, culture
Time：60d；
(2) induction of Secondary metabolites：While culture medium is changed without, CuSO is added in nutrient solution4·
5H2O, makes the CuSO in nutrient solution4Concentration is 0.075mg/L.Continuation is cultivated Asian puccoon callus, described to continue to cultivate
Parameter synchronization rapid (1) described in, except that incubation time is 20d.After culture 20d, fluid nutrient medium is taken out, simultaneously
Change CuSO4Concentration is 0.075mg/L fresh culture.The receipts of old nutrient solution are carried out while fresh medium is changed
Collect to extract required alkannin, and so on；
(3) collection and extraction of Secondary metabolites：
The nutrient solution changed is subjected to acetone extraction alkannin after concentration, using spectrophotometer by
Detect to calculate the yield of Asian puccoon Soviet Union under 520nm, obtaining the content of the alkannin in the nutrient solution changed can reach
2.3mg/L。
Finally, in addition it is also necessary to it is noted that listed above is only a specific embodiment of the invention.Obviously, it is of the invention
Above example is not limited to, there can also be many deformations, for example, change the plant of culture, Asian puccoon is changed to safflower etc..Ability
All deformations that the those of ordinary skill in domain directly can export or associate from present disclosure, are considered as this hair
Bright protection domain.
Claims (7)
1. a kind of plant bioreactor, it is characterised in that：Including drive device, pipe-line system and at least one reacting tank body；
The drive device includes control system, display system and dynamical system；The display system includes operation interface and monitoring circle
Face, the control system controls dynamical system by operation interface, and the dynamical system is connected with the pipe-line system, described
Pipe-line system is connected with reacting tank body sealing, and the reacting tank body includes culturing room, fluid reservoir and culture plate, the reaction
Tank body is cultured disk and separates to form upper strata culturing room and lower floor's fluid reservoir two chambers, and the culture plate is provided with catheter, institute
State catheter and connect the culturing room and the fluid reservoir；The top of the catheter is provided with shunting current limliting post；The culture plate
It is transportable；The culture plate is in funnel-form, and outwards diverging is provided with least 2 centered on the center of circle for the bottom of the culture plate
The alternate annulus of individual bumps；The bottom of the culture plate is stretched out centered on shunting current limliting post provided with least 2 drainage troughs；
The drainage trough connects the alternate annulus of the bumps；The bottom of the catheter is diametrically arranged with breach；The shunting
Current limliting post is hollow structure and top is closing, and the cross section of the shunting current limliting post is in " ten " font and higher than the culture
The outer edge of disk, draining hole of the side provided with 4 vertical-shaped openings of the shunting current limliting post.
2. plant bioreactor according to claim 1, it is characterised in that the pipe-line system include air inlet pipeline and
Outlet pipe, the air inlet pipeline connects the dynamical system, and the air inlet pipeline uses isocon and is intersected at one and formed
Main pipe rail；The reacting tank body is provided with air inlet and gas outlet, and the air inlet connects the air inlet pipeline, the gas outlet
Connect the outlet pipe.
3. plant bioreactor according to claim 2, it is characterised in that the air inlet pipeline include the first sebific duct and
First air disinfector, the outlet pipe includes the second sebific duct and the second air disinfector；First air disinfector leads to
First sebific duct is crossed with air inlet sealing to be connected；Second air disinfector by second sebific duct with it is described go out
Gas port sealing is connected.
4. plant bioreactor according to claim 3, it is characterised in that the dynamical system is by air pump, valve, valve
Door and flow sensor composition, the valve are used to control air pump；The flow sensor monitors the flow of air pump；The air pump
The pipe-line system is connected by the valve.
5. plant bioreactor according to claim 3, it is characterised in that the reacting tank body also includes the first sealing
Circle and the second sealing ring, the edge of the reacting tank body are provided with the first sealing ring neck, for placing first sealing ring；
The edge of the culture plate is provided with the second sealing ring neck, for placing second sealing ring.
6. the plant bioreactor according to claim any one of 1-5, it is characterised in that the reacting tank body is 1 ~
100, the isocon is 1 ~ 20；The quantity of the alternate annulus of the bumps is 6 ~ 9, and the drainage trough is 4.
7. plant bioreactor according to claim 6, it is characterised in that the reacting tank body is 20 ~ 80.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510218596.7A CN104770304B (en) | 2015-04-30 | 2015-04-30 | Plant bioreactor and its application method |
| PCT/CN2015/078723 WO2016172998A1 (en) | 2015-04-30 | 2015-05-12 | Plant bioreactor and use method therefor |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510218596.7A CN104770304B (en) | 2015-04-30 | 2015-04-30 | Plant bioreactor and its application method |
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| CN104770304A CN104770304A (en) | 2015-07-15 |
| CN104770304B true CN104770304B (en) | 2017-08-25 |
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| WO (1) | WO2016172998A1 (en) |
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| CN108076726B (en) * | 2017-12-28 | 2023-08-25 | 江苏哈工药机科技股份有限公司 | Sterile culture system for ginseng seeds |
| CN108271458B (en) * | 2017-12-28 | 2021-06-22 | 江苏哈工药机科技股份有限公司 | Ginseng seed sterile culture method |
| CN111280063B (en) * | 2020-04-02 | 2023-09-05 | 安赛搏(重庆)生物技术有限公司 | Rapid desert rose callus induction and large-scale suspension culture method |
| CN112970482B (en) * | 2021-02-04 | 2023-07-04 | 福建荣冠环境建设集团有限公司 | Saline-alkali resistance screening experimental device for vine |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101300958B (en) * | 2007-05-11 | 2012-01-11 | 中国科学院沈阳应用生态研究所 | Intermittent immersed plant cultivation device and control method thereof |
| CN201420083Y (en) * | 2009-03-24 | 2010-03-10 | 浙江理工大学 | Cultivation reaction vessel for intermittent plant tissue and organ immersion |
| CN101768566A (en) * | 2010-01-25 | 2010-07-07 | 大连普瑞康生物技术有限公司 | Method for culturing cendrobium officinale protocorm by intermittent fermentation |
| CN201809355U (en) * | 2010-08-10 | 2011-04-27 | 南京工业大学 | Intermittent-immersion openable plant bioreactor |
| CN103525696B (en) * | 2012-07-04 | 2015-08-26 | 神华集团有限责任公司 | Monitoring system and monitoring method for multi-stage biomass fermentation reaction system |
| CN103999777B (en) * | 2013-02-04 | 2017-03-08 | 南京工业大学大丰海洋产业研究院 | The method that Rhizoma Pinelliae seedling secondary metabolite is persistently obtained by tissue culture |
| CN104026019A (en) * | 2013-02-04 | 2014-09-10 | 南京工业大学大丰海洋产业研究院 | Method for continuously obtaining secondary ginseng hairy root metabolites through tissue culture |
| CN203333668U (en) * | 2013-07-04 | 2013-12-11 | 南京博方生物科技有限公司 | One-piece intermittent immersion plant bioreactor |
| CN203482715U (en) * | 2013-09-02 | 2014-03-19 | 南京博方生物科技有限公司 | Combined plant seedling production device |
| CN104412901B (en) * | 2013-09-03 | 2017-07-04 | 南京博方生物科技有限公司 | One kind automation plant seedling culture apparatus |
| CN104542277B (en) * | 2014-12-19 | 2017-01-11 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
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| CN104770304A (en) | 2015-07-15 |
| WO2016172998A1 (en) | 2016-11-03 |
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