WO2016172998A1 - 植物生物反应器及其使用方法 - Google Patents

植物生物反应器及其使用方法 Download PDF

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WO2016172998A1
WO2016172998A1 PCT/CN2015/078723 CN2015078723W WO2016172998A1 WO 2016172998 A1 WO2016172998 A1 WO 2016172998A1 CN 2015078723 W CN2015078723 W CN 2015078723W WO 2016172998 A1 WO2016172998 A1 WO 2016172998A1
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culture
plant
air
reaction tank
reactor
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French (fr)
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陈集双
张本厚
蒋海侠
陈锦周
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Nanjing Biofunction Co ltd
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Nanjing Biofunction Co ltd
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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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  • the invention relates to a culture device which can be used for large-scale cultivation of plant cells, tissues and organs, and can also be used for establishing a microbial and plant tissue co-culture system to obtain natural active products having medicinal value, in particular to realize a plant. Plant bioreactor immersed in the gap.
  • the above four gap immersion bioreactors represent the results of different periods, and the first two are not widely used due to pollution and space problems.
  • the latter two types use the double bottle type.
  • the principle is the same.
  • the use of air pressure to achieve intermittent immersion, reduce the pollution rate, and also reduce the cost to a certain extent, improve the efficiency, has been put into commercial production applications, but the plant bioreactor still The structure is complicated and the maintenance is difficult, which may cause certain inconvenience in use.
  • the intermittent immersion bioreactor can not only meet the nutrient and oxygen supply required for plant tissue culture growth, but also solve the vitrification problems encountered in suspension culture; such culture methods have been studied at home and abroad, and all have applied for relevant
  • a patent such as the Chinese patent (Application No. 98205797.0), discloses an "air-lifting cycle immersion illumination plant cell tissue culture reactor", and the Chinese patent (Application No. 98102396.7) discloses an "airlift cycle immersion illumination plant”.
  • the cell tissue culture method and the culture reactor the reactors disclosed in the above two patents are all unsuitable for widespread promotion due to complicated structure of the equipment and difficulty in large-scale manufacture. Meanwhile, the Chinese patent (Patent Publication No.
  • CN201420083Y discloses a "culture reactor for intermittently immersing plant tissue culture organs". Although the design is simple and the cost is low, the main reaction tank opening of the equipment is small, and the tissue culture seedling after cultivation is small. It is not easy to take out, and even the main reaction tank needs to be destroyed to take the seedling; in addition, since the liquid storage tank and the main reaction tank are two separate components, the carrying and sterilization are inconvenient, and the possibility of contamination is greatly increased.
  • CN201809355U discloses an "intermittently immersed open-close plant bioreactor" to realize an integrated bioreactor, but due to its cumbersome operation, bulky material, poor sealing, resulting in serious pollution, and ultimately Not widely used. It is worth noting that the above existing reactor system mainly introduces the structure of the bioreactor tank, basically does not involve the key element of the air drive device, and also lacks the series and automatic control parts of the equipment.
  • the technical problem to be solved by the present invention is to provide a plant bioreactor with high degree of automation, simple structure, convenient operation, light material, and real high sealing property to avoid pollution.
  • the plant bioreactor comprises a driving device, a pipeline system and one or more reaction tank bodies;
  • the driving device comprises a control system, a display system and a power system;
  • the display system includes an operating interface and a monitoring interface, the control system controlling a power system through an operating interface, the power system being coupled to the piping system, the piping system being sealingly coupled to the reaction tank.
  • the control system is a programmable controller, inputs various forms of instructions through an operation interface, and then the control system controls the output of the power system and is monitored by the monitoring system; the time control is controlled by the program of the programmable controller; thereby realizing the reaction
  • the high degree of automation of the device requires only a simple operation on the operation interface, that is, the operation is simple and the structure is simple.
  • a further improvement of the present invention is that the pipeline system comprises an intake line and an outlet line, the intake line is connected to the power system, and the outlet line adopts a diverter tube and meets at one place to form a main line;
  • the reaction tank body is provided with an air inlet port and an air outlet port, and the air inlet port is connected to the air inlet pipe, and the air outlet port is connected to the air outlet pipe.
  • a further improvement of the present invention is that the reaction tank body comprises a culture chamber, a liquid storage chamber and a culture tray, and the reaction tank body is separated by the culture tray to form two chambers of an upper culture chamber and a lower reservoir, the culture A liquid guiding tube is disposed on the disk, and the liquid guiding tube communicates with the culture chamber and the liquid storage chamber; a top of the liquid guiding tube is provided with a flow restricting column, and the culture plate is movable.
  • the culture tank body is separated into a culture chamber and a liquid storage chamber by a culture tray, wherein the culture chamber volume is greater than or equal to the liquid storage chamber, and the culture chamber accounts for 1/2 to 4/5 of the entire culture tank body.
  • the liquid storage chamber accounts for 1/5 to 1/2 of the entire body of the culture tank.
  • This design provides a larger growth space for plant tissue culture; the culture chamber and the liquid storage chamber are connected by a liquid guiding tube, and A flow restricting column is arranged on the liquid pipe, and the liquid guiding tube can introduce the culture liquid of the liquid storage chamber into the culture room, thereby providing sufficient nutrition for the plant tissue culture; the flow restricting column can control the amount of the introduced culture liquid, and Better to achieve interstitial immersion culture of the plant; in addition, the culture tray is set to be movable and needs to be replaced during the cultivation process When the culture medium or the culture medium is used, the culture tray can be directly transferred to the new culture tank together with the cultured plant; or after the completion of one stage of the culture, the culture tray can be directly moved along with the cultured plant to the next stage. It is cultured in the tank so as not to affect the growth state of the plant and the operation is simple and convenient.
  • a further improvement of the present invention is that the intake line includes a first hose and a first air sterilizer, the outlet line includes a second hose and a second air sterilizer; the first air sterilizer passes The first hose is sealedly connected to the air inlet; the second air sterilizer is sealedly connected to the air outlet through the second hose.
  • Sterile cultivation of plants by plant bioreactors can be achieved by providing an air sterilizer on the piping system.
  • a further improvement of the present invention is that the power system is composed of a gas pump, a gas nozzle, a valve and a flow sensor, the valve is for controlling a gas pump; the flow sensor is for monitoring a flow rate of the air pump; and the air pump is connected through the gas nozzle The piping system.
  • the air pump works, the culture liquid in the culture chamber is returned to the liquid storage chamber through the flow restricting column and the draft tube under the action of its own gravity; at this time, a negative pressure is formed in the entire culture chamber, and the outside gas passes through the outlet line. After being sterilized by the air sterilizer, the air enters the culture chamber along the air outlet, and the air pressure of the reaction tank returns to normal, thereby achieving a cycle of the plant bioreactor without pollution.
  • the reaction tank further includes a first sealing ring and a second sealing ring, and a first sealing ring groove is disposed on the edge of the reaction can body for placing the first sealing ring a second sealing ring groove is disposed on the edge of the culture tray for placing the second sealing ring.
  • the culture tray has a funnel shape, and the bottom of the culture tray is outwardly dispersed with at least two annular rings at the center of the center; the bottom of the culture tray is divided by a flow restricting column. There are at least two drainage grooves extending outward from the center; the drainage grooves communicate with the annular rings.
  • a further improvement of the present invention is that the bottom of the liquid guiding tube is symmetrically provided with a notch; the shunting restricting column is a hollow structure and the top is closed, and the cross-section of the shunting restricting column has a "ten" shape And above the outer edge of the culture tray, the side of the flow restricting column is provided with four vertical opening drainage ports.
  • the split restrictor column By arranging the split restrictor column as a hollow structure, the top is closed and higher than the outer edge of the culture plate At the same time, there are 4 vertical openings on the side, which can better realize the shunt current limiting function.
  • the plant tissue culture in the culture tray will not return to the liquid storage chamber with the culture liquid. .
  • the number of the reaction tanks is 1 to 100
  • the number of the shunt tubes is 1 to 20
  • the number of the rings of the unevenness is 6 to 9
  • the number of the drainage grooves is 4 .
  • the reaction tank body is 20 to 80.
  • Another problem to be solved by the present invention is to provide a method for cultivating and expanding white peony seedlings by using the aforementioned plant bioreactor, and the specific steps are as follows:
  • the inoculated reactor tank was connected to a driving device, placed in a culture chamber for cultivation; and the immersion frequency was set to 3 min/4 h.
  • the environmental conditions are: light intensity 1800lx, photoperiod 10h/d, temperature 25 ⁇ 1°C.
  • the plant bioreactor is applied to the expanded seedling; the advantage is that the culture flux is large, the space utilization rate is high, and the medium is not required to be replaced; here, the chalk is only a preferred solution.
  • Another problem to be solved by the present invention is to provide a method for screening for resistance after plant mutagenesis using the aforementioned plant bioreactor, and the specific steps are as follows:
  • the plant bioreactor is applied to the targeted screening in gene mutation breeding, such as salt tolerance and insect resistance; the advantages are large screening flux, easy environment control and good repeatability.
  • Another technical problem solved by the present invention is to provide a plant bioreactor using the aforementioned plant bioreactor A method for producing a plant secondary metabolite, the plant being comfrey, the specific steps are as follows:
  • the culture parameters mainly include: inoculating the comfrey callus into the plant bioreactor of the invention, the seeding density is 120/L, and the medium formula is: B5+1.0 mg/L 6-BA+0.1 mg/ L NAA+30g/L sucrose; medium pH: 6.0; immersion frequency: 1min/6h; culture conditions: light intensity 1000lux, illumination time 18h/d, culture temperature 25 ⁇ 1°C, culture time: 60d;
  • the replaced culture solution was concentrated, and acetone was extracted from shikonin, and the yield of comfrey was calculated by a spectrophotometer at 520 nm.
  • the plant bioreactor is used to produce plant secondary metabolites for the production and preparation of natural products, such as crocin, comfrey and the like, which have the advantages of continuous enrichment through environmental control including continuous collection and addition of the culture solution.
  • Natural product here comfrey is only a preferred solution.
  • the beneficial effects of the invention are: the plant bioreactor has high degree of automation, simple structure and operation, no pollution, sealing property, light material, good light transmission, more reasonable design and better culture effect. Good, and low cost, easy to scale production and popularization.
  • Figure 1 is a general flow chart of the plant bioreactor of the present invention
  • Figure 2 is a schematic structural view of a driving device of the present invention
  • Figure 3 is a schematic structural view of a reaction can body of the present invention.
  • Figure 4 is a schematic structural view of a culture tray of the present invention.
  • Figure 5 is a schematic structural view of a shunt current limiting column of the present invention.
  • FIG. 6 is a culture cycle diagram of cultivating white peony seedlings using the plant bioreactor according to the third embodiment of the present invention.
  • Figure 7 is a technical roadmap for utilizing the plant bioreactor resistance selection of the present invention.
  • Figure 8 is a technical roadmap for extracting secondary metabolites using the plant bioreactor of the present invention.
  • 1-drive device 101-control system; 102-display system; 1021-operation interface; 1022-monitoring interface; 103-power system; 1031-air pump; 1032-gas nozzle; 1033-valve; 201-intake line; 2011-first hose; 2012-first air sterilizer; 202-exhaust line; 2021-second hose; 2022-second air sterilizer; 3-reaction tank; Intake port; 302-outlet port; 303-tank cover; 3031-inverted tapered handle; 304-foot pad; 4-culture plate; 401-catheter; 4011-notch; 402-shunt current-limiting column; - second seal ring card slot; 404-drainage slot; 405-drainage port; 5-culture chamber; 6-reservoir chamber; 7-first one-way air valve; 8-second one-way air valve; Three one-way air valve; 10-fourth one-way air valve;
  • A culture for 20 days
  • B culture for 40 days
  • C culture for 60 days.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the plant bioreactor comprises a driving device 1, a piping system and four reaction tanks 3.
  • the driving device 1 comprises a control system 101, a display system 102 and a power system 103;
  • the power system 103 is composed of a gas pump 1031, a gas nozzle 1032, a valve 1033 and a flow sensor 1034, the valve 1033 is used to control the air pump 1031;
  • the display system 102 includes an operation interface 1021 and a monitoring interface 1022, and the control system 101 controls the power system 103 through the operation interface 1021, and the flow sensor 1034 monitors
  • the air pump 1031 is connected to the pipeline system through the air nozzle 1032, and the pipeline system is sealedly connected with the reaction tank body 3;
  • the pipeline system includes an intake pipeline 201 and an outlet conduit 202, and the intake pipeline 201 is connected to the power system 103,
  • the intake pipe 201 adopts a diverter pipe and meets at one place to form a main pipe; is
  • the reaction tank body 3 is integrally formed; the reaction tank body 3 includes the air inlet 301, the air outlet 302, the tank cover 303, the foot pad 304, and the first seal ring card groove,
  • the gas line 201 includes a first hose 2011 and a first air sterilizer 2012
  • the outlet line 202 includes a second hose 2021 and a second air sterilizer 2022; the first air sterilizer 2012 passes the first hose 2011 and the air inlet
  • the mouth 301 is sealed and connected; the second air sterilizer 2021 is sealedly connected to the air outlet 302 through the second hose 2022; the inside of the reaction tank 3 is provided with a culture tray 4, and the reaction tank 3 is separated by the culture tray 4.
  • the culture tray 4 is provided with a liquid guiding tube 401, the liquid guiding tube 401 is connected to the culture chamber 5 and the liquid storage chamber 6; the top of the liquid guiding tube 401 is provided with a shunting restricting column 402.
  • the reaction tank body 3 further includes a first sealing ring and a second sealing ring, and a first sealing ring groove is disposed on the edge of the reaction can body 3 for placing the first sealing ring; the edge of the culture plate 4 is provided a second sealing ring slot 403 for placing the second sealing ring; the culture disk 4 is in the shape of a funnel, and the bottom of the culture disk 4 is outwardly dispersed with seven concave and convex rings as a center of the center; the culture disk 4
  • the bottom portion of the bottom of the liquid guiding tube 401 is symmetrically provided with a notch 4011;
  • the shunting restricting column 402 is The hollow structure and the top portion are closed.
  • the cross-section of the flow-limiting restricting column 402 has a "ten" shape and is higher than the outer edge of the culture tray 4.
  • the side of the flow-limiting restricting column 402 is provided with four vertical-shaped openings 405.
  • the restrictor column 402 and the draft tube 4 enter the liquid storage chamber 6 and are covered with the can body cover 303; under aseptic conditions, the can body cover 303 is opened, the plants to be cultured (such as cells, tissues, organs), microorganisms And the co-culture system of the two are placed in the culture tray 4 of the culture chamber 5, the can lid 303 is closed, and the entire main reactor is sealed; the air pump 1031 is controlled by the control system 101 to perform intermittent operation, and the air pump is monitored by the flow sensor 1034.
  • the culture liquid in the culture chamber 5 is returned to the liquid storage chamber 6 through the flow restriction column 402 and the flow guiding tube 4 under the action of its own gravity; since the upper end is closed and the side surface has Four vertically open drainage ports 405, so that the plant tissue culture in the culture chamber 5 does not flow back into the reservoir 6 with the culture solution.
  • a negative pressure is formed in the entire culture chamber 5, and the outside air is sterilized by the second air sterilizer 308 through the outlet piping system, and then enters the culturing chamber 5 along the gas outlet 302 to restore the gas pressure of the reaction tank 3. Normal, thus achieving a pollution-free culture of plants.
  • the plant tissue organ in the culture chamber 5 completes a cycle of intermittent immersion; the time of the above cycle can be adjusted by adjusting the control system 101.
  • the medium was drained under aseptic conditions, and the lid was opened, and the regenerated plants were taken out.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the difference from the embodiment 1 is that the number of the reaction tank bodies 3 is different.
  • the number of the reaction tank bodies 3 is 20, and the gas is supplied to the diverter valve through two.
  • the driving device 1 includes a control system 101, a display system 102 and a power system 103.
  • the power system 103 is composed of a gas pump 1031 and a gas nozzle 103. 2.
  • the valve 1033 and the flow sensor 1034 are composed.
  • the valve 1033 is used to control the air pump 1031.
  • the display system 102 includes an operation interface 1021 and a monitoring interface 1022.
  • the control system 101 controls the power system 103 through the operation interface 1021, and the flow sensor 1034 monitors the flow of the air pump 1031.
  • the air pump 1031 is connected to the pipeline system through the gas nozzle 1032, and the pipeline system is sealedly connected with the reaction tank body 3;
  • the pipeline system includes an intake pipeline 201 and an outlet pipeline 202, and the intake pipeline 201 is connected to the power system 103, and the intake pipeline 201
  • the shunt tube is used and meets at one place to form a main pipeline;
  • the control inlet 4 is adjusted by the first one-way air valve 7, the second one-way air valve 8, the third one-way air valve 9, and the fourth one-way air valve 10, respectively.
  • the flow rate of the gas of the reaction tank body; the reaction tank body 3 is integrally formed; the reaction tank body 3 includes the air inlet 301, the air outlet 302, the tank cover 303, the foot pad 304 and the first sealing ring card slot, the intake pipe
  • the road 201 includes a first hose 2011 and a first air sterilizer 2012
  • the outlet pipe 202 includes a second hose 2021 and a second air sterilizer 2022; the first air sterilizer 2012 passes through the first hose 2011 and the air inlet 301 is sealed and connected;
  • the second air sterilizer 2021 is sealedly connected to the air outlet 302 through the second hose 2022; the inside of the reaction tank 3 is provided with a culture tray 4, and the reaction tank 3 is separated by the culture tray 4 to form the cultivating chamber 5 and the liquid storage chamber 6.
  • the chamber 4 is provided with a liquid guiding tube 401.
  • the liquid guiding tube 401 is connected to the culture chamber 5 and the liquid storage chamber 6.
  • the top of the liquid guiding tube 401 is provided with a flow restricting flow column 402.
  • the reaction tank body 3 further includes a first sealing ring and a second sealing ring, and a first sealing ring groove is disposed on the edge of the reaction can body 3 for placing the first sealing ring; the edge of the culture plate 4 is provided a second sealing ring slot 403 for placing the second sealing ring;
  • the culture disk 4 is in the shape of a funnel, and the bottom of the culture disk 4 is outwardly dispersed with seven concave and convex rings as a center of the center; the culture disk 4
  • the bottom portion of the bottom of the liquid guiding tube 401 is symmetrically provided with a notch 4011; the shunting restricting column 402 is The hollow structure and the top portion are closed.
  • Embodiment 3 Expanding the white
  • a method for expanding a white peony seedling using the plant bioreactor of the present invention As shown in Fig. 6, a method for expanding a white peony seedling using the plant bioreactor of the present invention:
  • the inoculated reactor tank was connected to a driving device, placed in a culture chamber for cultivation; and the immersion frequency was set to 3 min/4 h.
  • the environmental conditions are: light intensity 1800lx, light cycle 10h/d, Temperature 25 ⁇ 1 °C.
  • a method for screening for resistance after plant mutagenesis using the plant bioreactor of the present invention is as follows:
  • the culture parameters mainly include: inoculating the comfrey callus into the plant bioreactor of the invention, the seeding density is 120/L, and the medium formula is: B5+1.0 mg/L 6-BA+0.1 mg/ L NAA + 30 g / L sucrose; medium pH: 6.0; immersion frequency: 1 min / 6 h.
  • Culture conditions light intensity 1000 lux, illumination time 18 h / d, culture temperature 25 ⁇ 1 ° C, culture time: 60d;
  • the replaced culture solution was concentrated, and acetone was extracted from shikonin, and the yield of the comfrey was calculated by a spectrophotometer at 520 nm to obtain shikonin in the replaced culture solution.
  • the content can reach 2.3mg/L.

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Abstract

本申请提供了一种植物生物反应器及其使用方法，所述植物生物反应器包括驱动装置、管路系统和至少一个反应罐体；所述驱动装置包括控制系统、显示系统和动力系统；所述控制系统通过操作界面输入指令，控制动力系统的输出，并通过监控系统监控，从而提高了反应器的自动化程度。

Description

植物生物反应器及其使用方法 技术领域
本发明涉及一种既可用于植物细胞、组织、器官大量培养，也可用于微生物与植物组织共培养体系的建立，来获取具有药用价值的天然活性产物的培养装置，特别涉及一种实现植物间隙浸没的植物生物反应器。
背景技术
目前在植物组织培养领域，培养方式大致可分为固体和液体培养两类，其各自优缺点如表1。与固体培养相比较，液体培体培养优点较为明显，但仍存在诸多缺点与不足。为了进一步解决上述问题，国内外学者进行了大量试验，其中间歇浸没系统增殖效果较为显著。间歇浸没式植物生物反应器种类众多，其中多种反应器是实验者实际试验中改进的培养系统，这些生物反应器中较成熟，具有代表性的主要有四种，如表2。
表1固体培养与液体培养优缺点比较
Figure PCTCN2015078723-appb-000001
表2四种间歇浸没式生物反应器结构与特点
Figure PCTCN2015078723-appb-000002
Figure PCTCN2015078723-appb-000003
上述四种间隙浸没式生物反应器代表了不同时期的成果，前2种由于污染和空间问题没有得到广泛应用。后2种采用了双瓶式，原理一样，利用气压实现间歇浸没，降低污染率，也在一定程度上降低了成本，提高了效率，已经投入商业化生产应用，但是该种植物生物反应器依然构造复杂，维修困难，会造成一定的使用不便。
间歇浸没式生物反应器不仅能满足植物组织培养物生长所需营养物质与氧气供给，还解决了悬浮培养中遇到的玻璃化问题；此类培养方式国内外研究较多，且均申请了相关的专利，如中国专利(申请号98205797.0)公开了一种“气升式周期浸没光照植物细胞组织器官培养反应器”，中国专利(申请号98102396.7)公开了一种“气升式周期浸没光照植物细胞组织培养方法及培养反应器”，上述两个专利所公开的反应器均因设备构造复杂、难以规模化制造，导致不适用于广泛推广。同时，中国专利(专利公告号CN201420083Y)公开了一种“间歇浸没植物组织培养器官的培养反应器”虽然设计简单，成本低廉，但该设备的主反应罐开口较小，培养后的组培苗不易取出，甚至需要破坏主反应罐才能取苗；此外，由于储液罐和主反应罐为两个独立元件，给携带和灭菌带来不便，污染的可能性大大增加。中国专利(专利公告号CN201809355U)公开了一种“间歇浸没的开合式植物生物反应器”实现了一体式的生物反应器，但是由于其操作繁琐，材质笨重，密封性差，导致污染严重，最终也未得到广泛应用。值得说明的是，以上现有反应器系统主要是介绍了生物反应器罐体的构造，基本上没有涉及空气驱动装置这一关键要素，也缺少设备的串联和自动控制部分。
因此，现在急需要研发一种自动化程度高，结构简单，操作简单，密封性好，且能够标准化生产的植物生物反应器。
发明内容
本发明要解决的技术问题是，提供一种自动化程度高，结构简单，操作方便，材质轻，能够真正实现高密封性，从而避免污染的植物生物反应器。
为了解决上述技术问题，本发明采用的技术方案是，该植物生物反应器包括驱动装置、管路系统和1～多个反应罐体；所述驱动装置包括控制系统、显示系统和动力系统；所述显示系统包括操作界面和监控界面，所述控制系统通过操作界面控制动力系统，所述动力系统与所述管路系统相连接，所述管路系统与所述反应罐体密封相连。所述控制系统为可编程控制器，通过操作界面输入各种形式的指令，再由控制系统控制动力系统的输出，并通过监控系统监控；时间控制由可编程控制器的程序控制；从而实现反应器的高自动化程度，只需要在操作界面进行简单操作即可，即操作简单且结构也简单。
本发明的进一步改进在于，所述管路系统包括进气管路和出气管路，所述进气管路连接所述动力系统，所述出气管路采用分流管且交汇于一处形成总管路；所述反应罐体上设有进气口与出气口，所述进气口连接所述进气管路，所述出气口连接所述出气管路。通过管路进气管路和出气管路，可以实现反应罐体的里的气体流通，满足植物生长所必需的条件。
本发明的进一步改进在于，所述反应罐体包括培养室、储液室和培养盘，所述反应罐体被培养盘隔开形成上层培养室和下层储液室两个腔室，所述培养盘上设有导液管，所述导液管连通所述培养室和所述储液室；所述导液管的顶部设有分流限流柱，所述培养盘是可以移动的。
采用上述技术方案，通过培养盘将培养罐本体分隔成培养室和储液室，所述培养室体积大于或等于所述储液室，培养室占整个培养罐本体的1/2～4/5，储液室占整个培养罐本体的1/5～1/2，这样的设计为植物组织培养提供了更大的生长空间；通过设置导液管将培养室和储液室连通，并在导液管上设置分流限流柱，可导液管则可以将储液室的培养液导入培养室，则可以为植物组织培养提供充足的营养；分流限流柱可以控制导入培养液的量，可以更好地实现该植物的间隙浸没式培养；此外，培养盘设置成可移动的，当在培养过程中需要更换 培养液或培养基的时候，可以直接将培养盘连同培养的植物一起移至新的培养罐体中；或者在培养一个阶段完成后，可以直接将培养盘连同培养的植物移动到下一阶段的培养罐体中，从而不影响植物的生长状态且操作简单方便。
本发明的进一步改进在于，所述进气管路包括第一胶管和第一空气除菌器，所述出气管路包括第二胶管和第二空气除菌器；所述第一空气除菌器通过所述第一胶管与所述进气口密封相连；所述第二空气除菌器通过所述第二胶管与所述出气口密封相连。通过在管路系统上设置空气除菌器可以实现植物生物反应器对植物的无菌培养。
本发明的进一步改进在于，所述动力系统由气泵、气嘴、阀门和流量传感器组成，所述阀门用于控制气泵；所述流量传感器监控气泵的流量；所述气泵通过所述气嘴连接所述管路系统。当气泵工作，培养室里的培养液在自身重力的作用下，依次通过分流限流柱、导流管回流至储液室中；此时整个培养室内形成负压，外界的气体通过出口管路经过空气除菌器的杀菌处理后沿着出气口进入培养室内，使反应罐体的气压恢复正常，从而实现该植物生物反应器无污染的培养植物的一个循环。
本发明的进一步改进在于，所述反应罐体还包括第一密封圈和第二密封圈，所述反应罐体的边沿上设有第一密封圈卡槽，用于放置所述第一密封圈；所述培养盘的边沿上设有第二密封圈卡槽，用于放置所述第二密封圈。通过在反应罐体的储液室与培养盘之间设置一个密封圈，在培养室和培养盘之间设置一个密封圈，可以更好地提高反应罐体的密封性能，实现好的密封性。
本发明的进一步改进在于，所述培养盘呈漏斗状，所述培养盘的底部以圆心为中心向外发散设有至少2个凹凸相间的圆环；所述培养盘的底部以分流限流柱为中心向外延伸设有至少2条引流槽；所述引流槽连通所述凹凸相间的圆环。设置凹凸相间的圆环及在凹凸相间的圆环上设置连通的引流槽，可以使培养的植物更好更均匀地被培养液浸没。
本发明的进一步改进在于，所述导液管的底部沿直径对称设有缺口；所述分流限流柱是中空结构且顶部是封闭的，所述分流限流柱的横截面呈“十”字形且高于所述培养盘的外边沿，所述分流限流柱的侧面设有4条竖形开口的引流口。通过将分流限流柱设置成中空结构，顶部封闭的且高于所述培养盘的外边沿 ，同时侧面有4条竖形开口，可以更好地实现分流限流功能，同时在培养液回流至储液室时，培养盘内的植物组织培养不会随着培养液回流至储液室中。
作为本发明的优选方案，所述反应罐体为1～100个，所述分流管为1～20根；所述凹凸相间的圆环的数量为6～9个，所述引流槽为4条。
作为本发明的优选方案，所述反应罐体为20～80个。
本发明另外解决的问题是提供一种利用前述植物生物反应器培养扩繁白芨种苗的方法，具体步骤如下：
(1)白芨果荚的表面消毒与反应器罐体、培养基的高温湿热灭菌；
(2)在超净台中将上述已灭菌的液体培养基倒入反应器罐体中，再将已表面消毒的白芨果荚内的种子播撒到反应器罐体的培养盘中，接种密度为每个果荚接种3-5个反应器罐体；
(3)将接种完成的反应器罐体连接到驱动装置上，放置在培养室中进行培养；设置浸没频率为3min/4h。环境条件为：光照强度1800lx，光照周期10h/d，温度25±1℃。
将该植物生物反应器应用于扩繁种苗；其优势在于培养通量大、空间利用率高、无需更换培养基；此处白芨只是一个优选方案。
本发明另外解决的问题是提供一种利用前述的植物生物反应器进行植物体诱变后的抗性筛选的方法，具体步骤如下：
(1)利用植物生物反应器扩繁待诱变的植物体，所述植物体为组培苗和/或植物愈伤组织，获得大量诱变材料；
(2)在反应器中添加化学诱变剂使植物体产生突变，或者将带有植物体的反应器罐体进行辐射处理使植物体发生突变等；
(3)将上述诱变的反应器罐体更换添加特定物质的培养基或在特定的环境下培养，筛选特定抗性的植物体；
(4)将上述刷选到的植物体接种到新的植物反应器中，在特定条件下进行大量扩繁，获得抗性稳定的植物新品系。
将该植物生物反应器应用于基因突变育种中的定向筛选，比如耐盐、抗虫等；其优势在于筛选通量大、环境易控、重复性好。
本发明解决的另外一个技术问题是提供一种利用前述的植物生物反应器生 产植物次生代谢产物的方法，所述植物为紫草，具体步骤如下：
(1)紫草愈伤的批量获得：
所述培养参数主要有：将紫草愈伤组织接种入本发明的植物生物反应器中，接种密度：120个/L，培养基配方为：B5+1.0mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖；培养基pH：6.0；浸没频率：1min/6h；培养条件：光照强度1000lux，光照时间18h/d，培养温度25±1℃，培养时间：60d；
(2)植物次生代谢产物的诱导：在不更换培养基的同时，在培养液中添加CuSO4·5H2O，使培养液中的CuSO4浓度为0.075mg/L；继续对紫草愈伤组织进行培养，所述继续培养的参数同步骤(1)中所述，所不同的是培养时间为20d；在培养20d后，取出液体培养基，同时更换CuSO4浓度为0.075mg/L的新鲜培养基；在更换新鲜培养液的同时进行旧的培养液的收集来提取所需紫草素，如此往复；
(3)植物次生代谢产物的收集和提取：
将更换下来的培养液经过浓缩后进行丙酮提取紫草素，利用分光光度计通过在520nm下检测来计算紫草苏的产量。
采用该植物生物反应器生产植物次生代谢产物应用于天然产物的生产与制备，比如藏红花素、紫草等植物，其优势在于通过环境的控制包括培养液的不断收集与添加从而持续的富集天然产物；此处紫草只是一个优选方案。
与现有技术相比，本发明的有益效果是：该植物生物反应器自动化程度高，结构和操作简单，无污染，密封性，材质轻，透光性好，设计更为合理、培养效果更好，且成本低，便于规模化生产和推广使用。
附图说明
为了使本发明的内容更容易被清楚地理解，下面根据具体实施例并结合附图，对本发明作进一步详细的说明：
图1是本发明植物生物反应器的总流程图；
图2是本发明的驱动装置的结构示意图；
图3是本发明的反应罐体的结构示意图；
图4是本发明的培养盘的结构示意图；
图5是本发明的分流限流柱的结构示意图；
图6是本发明实施例三使用该植物生物反应器培养白芨种苗的培养周期图；
图7是本发明利用本发明的植物生物反应器抗性帅选的技术路线图；
图8是利用本发明的植物生物反应器提取次生代谢产物技术路线图；
其中：1-驱动装置；101-控制系统；102-显示系统；1021-操作界面；1022-监控界面；103-动力系统；1031-气泵；1032-气嘴；1033-阀门；1034-流量传感器；201-进气管路；2011-第一胶管；2012-第一空气除菌器；202-出气管路；2021-第二胶管；2022-第二空气除菌器；3-反应罐体；301-进气口；302-出气口；303-罐体盖；3031-倒锥形手柄；304-脚垫；4-培养盘；401-导液管；4011-缺口；402-分流限流柱；403-第二密封圈卡槽；404-引流槽；405-引流口；5-培养室；6-储液室；7-第一单向气阀；8-第二单向气阀；9-第三单向气阀；10-第四单向气阀；
其中，A：培养20天，B：培养40天，C：培养60天。
具体实施方式
实施例一：
如图1-5所示，该植物生物反应器包括驱动装置1、管路系统和4个反应罐体3，驱动装置1包括控制系统101，显示系统102和动力系统103；动力系统103由气泵1031、气嘴1032、阀门1033和流量传感器1034组成，阀门1033用于控制气泵1031；显示系统102包括操作界面1021和监控界面1022，控制系统101通过操作界面1021控制动力系统103，流量传感器1034监控气泵1031的流量；气泵1031通过气嘴1032连接管路系统，管路系统与反应罐体3密封相连；管路系统包括进气管路201和出气管路202，进气管路201连接动力系统103，进气管路201采用分流管且交汇于一处形成总管路；分别通过第一单向气阀7、第二单向气阀8、第三单向气阀9和第四单向气阀10调节控制通入4个反应罐体的气体的流量；反应罐体3为一体成型设置；反应罐体3包括进气口301、出气口302、罐体盖303、脚垫304和第一密封圈卡槽，进气管路201包括第一胶管2011和第一空气除菌器2012，出气管路202包括第二胶管2021和第二空气除菌器2022；第一空气除菌器2012通过第一胶管2011与进气口301密封相连；第二空气除菌器2021通过第二胶管2022与出气口302密封相连；反应罐体3的内部设有培养盘4，反应罐体3被培养盘4隔开形成 培养室5和储液室6两个腔室，培养盘4上设有导液管401，导液管401连通培养室5和储液室6；导液管401的顶部设有分流限流柱402。反应罐体3还包括第一密封圈和第二密封圈，反应罐体3的边沿上设有第一密封圈卡槽，用于放置所述第一密封圈；培养盘4的边沿上设有第二密封圈卡槽403，用于放置所述第二密封圈；培养盘4呈漏斗状，培养盘4的底部以圆心为中心向外发散设有7个凹凸相间的圆环；培养盘4的底部以分流限流柱为中心向外延伸设有4条引流槽404；引流槽404连通凹凸相间的圆环；导液管401的底部沿直径对称设有缺口4011；分流限流柱402是中空结构且顶部是封闭的，分流限流柱402的横截面呈“十”字形且高于培养盘4的外边沿，分流限流柱402的侧面设有4条竖形开口的引流口405。
具体工作过程：首先，将其至于121℃下进行25～35min的高压湿热灭菌，然后在超净工作台打开罐体盖303，将适量培养液放入储液室6内，培养液通过分流限流柱402、导流管4进入储液室6内，盖上罐体盖303；在无菌条件下，打开罐体盖303，将待培养的植物(如细胞、组织、器官)，微生物及其两者共培养体系放入培养室5的培养盘4内，合上罐体盖303，保持整个主反应器密封性；通过控制系统101控制气泵1031进行间歇工作，通过流量传感器1034监控气泵1031的流量；当气泵1034工作，培养室5里的培养液在自身重力的作用下，依次通过分流限流柱402、导流管4回流至储液室6中；由于其上端封闭且侧面有4条竖形开口的引流口405，因此培养室5内的植物组织培养不会随着培养液回流至储液室6中。此时整个培养室5内形成负压，外界的气体通过出口管路系统经过第二空气除菌器308的杀菌处理后沿着出气口302进入培养室5内，使反应罐体3的气压恢复正常，从而实现无污染的培养植物。这样，培养室5内的植物组织器官就完成了一个被间歇浸没的循环；可通过调节控制系统101来调节上述循环的时间。培养结束后于无菌条件下，排出培养基，再打开盖子，取出再生植株。
实施例二：
与实施例1的区别在于，反应罐体3的数量不同，该实施例反应罐体3的数量为20个，通过二给分流阀供气。具体如下：包括驱动装置1、管路系统和4个反应罐体3，驱动装置1包括控制系统101，显示系统102和动力系统103；动力系统103由气泵1031、气嘴103 2、阀门1033和流量传感器1034组成，阀门1033用于控制气泵1031；显示系统102包括操作界面1021和监控界面1022，控制系统101通过操作界面1021控制动力系统103，流量传感器1034监控气泵1031的流量；气泵1031通过气嘴1032连接管路系统，管路系统与反应罐体3密封相连；管路系统包括进气管路201和出气管路202，进气管路201连接动力系统103，进气管路201采用分流管且交汇于一处形成总管路；分别通过第一单向气阀7、第二单向气阀8、第三单向气阀9和第四单向气阀10调节控制通入4个反应罐体的气体的流量；反应罐体3为一体成型设置；反应罐体3包括进气口301、出气口302、罐体盖303、脚垫304和第一密封圈卡槽，进气管路201包括第一胶管2011和第一空气除菌器2012，出气管路202包括第二胶管2021和第二空气除菌器2022；第一空气除菌器2012通过第一胶管2011与进气口301密封相连；第二空气除菌器2021通过第二胶管2022与出气口302密封相连；反应罐体3的内部设有培养盘4，反应罐体3被培养盘4隔开形成培养室5和储液室6两个腔室，培养盘4上设有导液管401，导液管401连通培养室5和储液室6；导液管401的顶部设有分流限流柱402。反应罐体3还包括第一密封圈和第二密封圈，反应罐体3的边沿上设有第一密封圈卡槽，用于放置所述第一密封圈；培养盘4的边沿上设有第二密封圈卡槽403，用于放置所述第二密封圈；培养盘4呈漏斗状，培养盘4的底部以圆心为中心向外发散设有7个凹凸相间的圆环；培养盘4的底部以分流限流柱为中心向外延伸设有4条引流槽404；引流槽404连通凹凸相间的圆环；导液管401的底部沿直径对称设有缺口4011；分流限流柱402是中空结构且顶部是封闭的，分流限流柱402的横截面呈“十”字形且高于培养盘4的外边沿，分流限流柱402的侧面设有4条竖形开口的引流口405。
实施例三：扩繁白芨
如图6所示，利用本发明的植物生物反应器扩繁白芨种苗的方法：
(1)白芨果荚的表面消毒与反应器罐体、培养基的高温湿热灭菌；
(2)在超净台中将上述已灭菌的液体培养基倒入反应器罐体中，再将已表面消毒的白芨果荚内的种子播撒到反应器罐体的培养盘中，接种密度为每个果荚接种3-5个反应器罐体；
(3)将接种完成的反应器罐体连接到驱动装置上，放置在培养室中进行培养；设置浸没频率为3min/4h。环境条件为：光照强度1800lx，光照周期10h/d， 温度25±1℃。
实施例四：抗性筛选
如图7所示，利用本发明的植物生物反应器进行植物体诱变后的抗性筛选的方法：
(1)利用植物生物反应器扩繁待诱变的植物体(组培苗和或植物愈伤组织)，获得大量诱变材料；
(2)在反应器中添加化学诱变剂使植物体产生突变，或者将带有植物体的反应器罐体进行辐射处理使植物体发生突变等；
(3)将上述诱变的反应器罐体更换添加特定物质的培养基或在特定的环境下培养，筛选特定抗性的植物体；
(4)将上述刷选到的植物体接种到新的植物反应器中，在特定条件下进行大量扩繁，获得抗性稳定的植物新品系(种)。
实施例五：次生代谢产物
利用本发明的植物生物反应器生产植物次生代谢产物的方法：
如图8所示，以生产紫草素为例，具体操作步骤如下：
(1)紫草愈伤的批量获得：
所述培养参数主要有：将紫草愈伤组织接种入本发明的植物生物反应器中，接种密度：120个/L，培养基配方为：B5+1.0mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖；培养基pH：6.0；浸没频率：1min/6h。培养条件：光照强度1000lux，光照时间18h/d，培养温度25±1℃，培养时间：60d；
(2)植物次生代谢产物的诱导：在不更换培养基的同时，在培养液中添加CuSO4·5H2O，使培养液中的CuSO4浓度为0.075mg/L。继续对紫草愈伤组织进行培养，所述继续培养的参数同步骤(1)中所述，所不同的是培养时间为20d。在培养20d后，取出液体培养基，同时更换CuSO4浓度为0.075mg/L的新鲜培养基。在更换新鲜培养液的同时进行旧的培养液的收集来提取所需紫草素，如此往复；
(3)植物次生代谢产物的收集和提取：
将更换下来的培养液经过浓缩后进行丙酮提取紫草素，利用分光光度计通过在520nm下检测来计算紫草苏的产量，得到在更换下来的培养液中紫草素的 含量可以达到2.3mg/L。
最后，还需要注意的是，以上列举的仅是本发明的一个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形，例如改变培养的植物，将紫草改为藏红花等。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (13)

  1. 一种植物生物反应器，其特征在于：包括驱动装置、管路系统和至少一个反应罐体；所述驱动装置包括控制系统、显示系统和动力系统；所述显示系统包括操作界面和监控界面，所述控制系统通过操作界面控制动力系统，所述动力系统与所述管路系统相连接，所述管路系统与所述反应罐体密封相连。
  2. 根据权利要求1所述的植物生物反应器，其特征在于，所述管路系统包括进气管路和出气管路，所述进气管路连接所述动力系统，所述进气管路采用分流管且交汇于一处形成总管路；所述反应罐体上设有进气口与出气口，所述进气口连接所述进气管路，所述出气口连接所述出气管路。
  3. 根据权利要求1所述的植物生物反应器，其特征在于，所述反应罐体包括培养室、储液室和培养盘，所述反应罐体被培养盘隔开形成上层培养室和下层储液室两个腔室，所述培养盘上设有导液管，所述导液管连通所述培养室和所述储液室；所述导液管的顶部设有分流限流柱；所述培养盘是可以移动的。
  4. 根据权利要求2所述的植物生物反应器，其特征在于，所述进气管路包括第一胶管和第一空气除菌器，所述出气管路包括第二胶管和第二空气除菌器；所述第一空气除菌器通过所述第一胶管与所述进气口密封相连；所述第二空气除菌器通过所述第二胶管与所述出气口密封相连。
  5. 根据权利要求3所述的植物生物反应器，其特征在于，所述动力系统由气泵、气嘴、阀门和流量传感器组成，所述阀门用于控制气泵；所述流量传感器监控气泵的流量；所述气泵通过所述气嘴连接所述管路系统。
  6. 根据权利要求3所述的植物生物反应器，其特征在于，所述反应罐体还包括第一密封圈和第二密封圈，所述反应罐体的边沿上设有第一密封圈卡槽，用于放置所述第一密封圈；所述培养盘的边沿上设有第二密封圈卡槽，用于放置所述第二密封圈。
  7. 根据权利要求6所述的植物生物反应器，其特征在于，所述培养盘呈漏斗状，所述培养盘的底部以圆心为中心向外发散设有至少2个凹凸相间的圆环；所述培养盘的底部以分流限流柱为中心向外延伸设有至少2条引流槽；所述引流槽连通所述凹凸相间的圆环。
  8. 根据权利要求7所述的植物生物反应器，其特征在于，所述导液管的底部沿 直径对称设有缺口；所述分流限流柱是中空结构且顶部是封闭的，所述分流限流柱的横截面呈“十”字形且高于所述培养盘的外边沿，所述分流限流柱的侧面设有4条竖形开口的引流口。
  9. 根据权利要求1-8任一项所述的植物生物反应器，其特征在于，所述反应罐体为1～100个，所述分流管为1～20根；所述凹凸相间的圆环的数量为6～9个，所述引流槽为4条。
  10. 根据权利要求9所述的植物生物反应器，其特征在于，所述反应罐体为20～80个。
  11. 一种利用如权利要求1-10任一项所述的植物生物反应器培养扩繁白芨种苗的方法，其特征在于，具体步骤如下：
    (1)白芨果荚的表面消毒与反应器罐体、培养基的高温湿热灭菌；
    (2)在超净台中将上述已灭菌的液体培养基倒入反应器罐体中，再将已表面消毒的白芨果荚内的种子播撒到反应器罐体的培养盘中，接种密度为每个果荚接种3-5个反应器罐体；
    (3)将接种完成的反应器罐体连接到驱动装置上，放置在培养室中进行培养；设置浸没频率为3min/4h。环境条件为：光照强度1800lx，光照周期10h/d，温度25±1℃。
  12. 一种利用如权利要求1-10任一项所述的植物生物反应器进行植物体诱变后的抗性筛选的方法，其特征在于，具体步骤如下：
    (1)利用植物生物反应器扩繁待诱变的植物体，所述植物体为组培苗和/或植物愈伤组织，获得大量诱变材料；
    (2)在反应器中添加化学诱变剂使植物体产生突变，或者将带有植物体的反应器罐体进行辐射处理使植物体发生突变等；
    (3)将上述诱变的反应器罐体更换添加特定物质的培养基或在特定的环境下培养，筛选特定抗性的植物体；
    (4)将上述刷选到的植物体接种到新的植物反应器中，在特定条件下进行大量扩繁，获得抗性稳定的植物新品系。
  13. 一种利用如权利要求1-10任一项所述的植物生物反应器生产植物次生代谢产物的方法，其特征在于，所述植物为紫草，具体步骤如下：
    (1)紫草愈伤的批量获得：
    所述培养参数主要有：将紫草愈伤组织接种入本发明的植物生物反应器中，接种密度：120个/L，培养基配方为：B5+1.0mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖；培养基pH：6.0；浸没频率：1min/6h；培养条件：光照强度1000lux，光照时间18h/d，培养温度25±1℃，培养时间：60d；
    (2)植物次生代谢产物的诱导：在不更换培养基的同时，在培养液中添加CuSO4·5H2O，使培养液中的CuSO4浓度为0.075mg/L；继续对紫草愈伤组织进行培养，所述继续培养的参数同步骤(1)中所述，所不同的是培养时间为20d；在培养20d后，取出液体培养基，同时更换CuSO4浓度为0.075mg/L的新鲜培养基；在更换新鲜培养液的同时进行旧的培养液的收集来提取所需紫草素，如此往复；
    (3)植物次生代谢产物的收集和提取：
    将更换下来的培养液经过浓缩后进行丙酮提取紫草素，利用分光光度计通过在520nm下检测来计算紫草苏的产量。
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