CN103120124B - Method for continuously obtaining plant secondary metabolites by tissue culture and device thereof - Google Patents
Method for continuously obtaining plant secondary metabolites by tissue culture and device thereof Download PDFInfo
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Abstract
The invention discloses a method for continuously obtaining plant secondary metabolites by tissue culture. The method comprises the following steps of: (1) obtaining plant materials in batches; (2) generating and inducing the plant secondary metabolites; and (3) collecting and extracting the plant secondary metabolites. The invention further relates to a reactor device for the culture, wherein the upper part of the device uses a layered culture design of plant tissues and organs, so that cultures can be conveniently collected in a staging manner through the design of a multilayer culture rack; the lower part of the device uses a split type design, so that a liquid storage tank can be conveniently dismounted, and desired products can be obtained by collecting culture liquid; and furthermore, the inhibition on the continuous generation by the concentration of the secondary metabolites can be reduced by replacing the culture liquid, so that the secondary metabolites can be continuously obtained. The method and the device provided by the invention have the advantages of large flux, convenience in operation and low cost.
Description
Technical field
The invention belongs to plant biotechnology field; Be specifically related to a kind of device that continues to obtain the method for the active ingredients such as Secondary Metabolism of Plant product and realize this method of cultivating by tissue.
Background technology
Secondary metabolite is a class cell activities or the nonessential little molecular organic compound of the normal operation of growth and development of plants, its generation and the specificity that conventionally has kind, organ, organizes and grow period that distributes being produced by secondary metabolism.Can be divided into Phenylpropanoid Glycosides class, quinones, flavonoids, tannins, terpene, steroidal and glucoside thereof, the large class of alkaloid seven.In medicinal plant, secondary metabolite is the key component of its performance pharmacologic action.
Secondary Metabolism of Plant product is that plant adapts to the one of environment, is plant and biological and the interactional result of abiotic factor in long-term evolution process.Between the adaptation to environment-stress, plant and plant vie each other and the harm to insect of coevolution, plant, Herbivore search for food and the defence of the process such as the invasion and attack of pathogenic microorganism in play an important role.
Plant particularly contains abundant secondary metabolite in medicinal plant, and some metabolites have significant curative effect to human body diseases, and therefore the secondary metabolite of plant has been subject to very high attention.Generally, Secondary Metabolism of Plant product is to extract and obtain from the plant corpus of plantation or natural distribution, and tend to cause the utilization of certain Plants to pluck excessively for the wilderness demand of certain secondary metabolite, be exactly a good example as the utilization of taxol causes the in imminent danger of Chinese yew.
Therefore the production of secondary metabolite is carried out in the cultivation that, people start to adopt the method for Plant Tissue Breeding to carry out specified plant.But the method that traditional plant tissue is cultivated has the shortcoming of small throughput and high manpower consumption, and the application of reactor is a reasonable solution.Traditional bioreactor culture is the cultivation for plant cell, and Secondary Metabolism of Plant approach is hyperbranched approach, these approach are all not open in plant corpus or in cell, but are positioned in a certain organ, tissue, cell or organelle and independently regulated and controled.They are nonessential micromolecular compounds of cell activities or the normal operation of growth and development of plants, its generation and the specificity that conventionally has kind, organ, organizes and grow period that distributes, therefore the cultivation of specified plant histoorgan is necessary to the acquisition of secondary metabolite.
Secondary Metabolism of Plant product runs up to and tends to suppress it after certain concentration and further produce, therefore remove timely the metabolite producing in growing environment, it is controlled in certain concentration range the lasting generation of secondary metabolite is had to very important effect.In actual production process, need a kind of approach of cultivating plant organ and tissue that continues to obtain in enormous quantities, further can plant be produced and improve output by regulating and controlling the factors such as external environment, and by the concentration of certain method control secondary metabolite.The present invention provides this kind of method and discloses the device of realizing said method.
Summary of the invention
The object of the invention is to the shortcoming and defect existing for prior art, provide a kind of by the method for in enormous quantities, the lasting acquisition secondary metabolite of method for plant tissue culture.
Another object of the present invention is to provide a kind of above-mentioned device that obtains the method for secondary metabolite by method for tissue culture of realizing.
The object of the invention is to be achieved through the following technical solutions: the cultivation of plant being carried out to histoorgan level by method for plant tissue culture obtains vegetable material in batches, then make vegetable material produce required a large amount of secondary metabolites by creating suitable condition of culture, environment stress environment or adding inducible factor, thereby then by plant corpus and culture fluid are extracted and obtain needed secondary metabolite.
The method that obtains secondary metabolite by method for plant tissue culture of the present invention comprises the steps: more specifically
(1) plant corpus obtains in batches, is also that plant corpus batch expands numerous: after plant corpus is processed, inoculate in device of the present invention and carry out, after culture parameters setting, carrying out scale cultivation, carry out as required next step operation;
(2) generation of Secondary Metabolism of Plant product and induction: the plant corpus of acquisition is produced under certain condition of culture or induce to obtain the secondary metabolite needing;
(3) collection of Secondary Metabolism of Plant product and extraction: according to secondary metabolite exist position, and the characteristic of secondary metabolite carry out secondary metabolite collection extract operation required to obtain;
In described (1) processing method of plant corpus be in general method for plant tissue culture by after sterilizing or the explant of germ-free condition be seeded to solid culture bottle or cultivate in reactor assembly.Inoculate different explant type according to different plants and cultivation object.Described cultivation reactor assembly is to utilize the intermittently plant bioreactor modification of submergence principle design.
The cultivation factor of described plant bioreactor device mainly contains:
Medium: carry out the selection of determining of different culture media and hormone kind and concentration according to floristic difference, comprise existing type of culture medium and pass through the type of culture medium after improvement;
Medium pH: to expand numerous pH be that the scope of plant suitable growth is 5.0 ~ 7.0 for cultivating, preferably 5.5 ~ 6.5;
Immersion frequency: according to different different Immersion frequencies, 1min/24h ~ 24h/24h, the preferably 3min/18h ~ 30min/3h of adjusting of floristics;
Inoculum density: 1/L ~ 300/L, preferably 10/L ~ 200/L.Can utilize dismountable multilayer to cultivate rack and carry out the multilayer cultivation of plant corpus, improve culture density, and can improve space utilization efficiency.
Condition of culture: intensity of illumination 1000 ~ 50000lux, preferably 1500 ~ 2000 lux, light application time 5 ~ 20 h/d, preferably 8 ~ 18 h/d, 25 ± 1 DEG C of cultivation temperature;
Incubation time: incubation time determines according to different plant growth states, general growth time is 10 ~ 90d, preferably 20 ~ 40d.Cover with culture vessel with plant corpus and be advisable, the consumption of nutrient solution during this time can be upgraded or be supplemented by the liquid storage tank structure of assembling for convenience detach in device.
It is to realize by installing dismountable multilayer cultivation support that described scale is cultivated.
In described step (2), certain condition of culture comprises the suitable condition of culture of creation, environment stress environment or adds inducible factor.
Described appropriate incubation condition is to set the different factors that is applicable to its growth for different floristics, can obtain the tissue of a large amount of plant corpus privileged sites, thereby from plant corpus or from its secretion, obtain required secondary metabolite after cultivating;
Described environment stress environment refers to the envirment factors such as high temperature, low temperature, intense light irradiation, low light level photograph, mechanical stimulus and radiation etc., make the content that produces new secondary metabolite in plant corpus or improve original secondary metabolite by environment stress environmental induction, or induction makes secondary metabolite be secreted into extracellular or improves secernment efficiency; Envirment factor can be equipped to realize by installing external environment control as the change of suitable condition of culture and environment stress environment etc., induces a large amount of plant corpus in abovementioned steps (1) to produce required secondary metabolite as temperature regulating device can improve intensity of illumination that culture environment temperature, external illumination apparatus can regulate culture environment etc.;
Described inducible factor refers to salt content, chemical substance, plant hormone, precursor substance etc., induce and make the content that produces new secondary metabolite in plant corpus or improve original secondary metabolite by inducible factor, or induction makes secondary metabolite be secreted into extracellular or improves secernment efficiency; The change of inducible factor can be by the liquid storage tank structure of assembling for convenience detach in device, realize changing in culture fluid in adding inducible factor to culture fluid as salt, the chemical substance etc. of inducing precursor substance or thering is toxic action, after interpolation, make a large amount of plant corpuss that in earlier stage obtain under the effect of inducible factor, produce required secondary metabolite by cultivations.
The position that in described step (3), secondary metabolite exists comprises: the one, there is the secondary metabolite in plant corpus; The 2nd, plant corpus is secreted into extracellular secondary metabolite; The 3rd, exist the while in plant corpus also can be secreted into the secondary metabolite outside born of the same parents;
The collection of described secondary metabolite is taked different operations according to its existing position: in secondary metabolite exists plant corpus time, the plant corpus that multilayer after induction is cultivated on support is gathered in the crops the collection of Activities of Some Plants body as required for follow-up extraction operation, and remaining part can continue to cultivate the required plant corpus of acquisition to continue;
Outside secondary metabolite is secreted into plant cell time, now secondary metabolite is washed in culture fluid, now only the culture fluid in the fluid reservoir of device bottom need to be collected to extract operation; The quantity of cultivation, the required secondary metabolite of extraction that just can continue from culture fluid are controlled in the lasting cultivation of the plant corpus by top simultaneously;
When secondary metabolite is present in plant corpus and while being secreted into extracellular simultaneously, can operate in conjunction with above-mentioned two kinds of methods, not only can in plant corpus, extract but also can from culture fluid, collect required secondary metabolite;
The concrete grammar that the extraction of described secondary metabolite adopts adopts diverse ways according to the form of secondary metabolite, as precipitation, extraction, chromatography filtration, adsorption chromatography etc.
Another technical problem that the present invention will solve is to provide the cultivation reactor assembly of realizing by the method for the lasting acquisition secondary metabolite of method for tissue culture, this device comprises air disinfector, the first tube connector, reactor tank body lid, bioreactor culture tank body, the second tube connector, fluid reservoir, fluid reservoir lid, multilayer is cultivated rack, inflator pump, time controller, servicing unit.
Described air disinfector is arranged on reactor tank body lid by the first tube connector; Multilayer is cultivated rack and is placed into and in bioreactor culture tank body, carries out layering cultivation, and described multilayer is cultivated rack, for rack is cultivated in layering that can disassembly and assembly; The second tube connector is connected with bioreactor culture tank body, and its length can touch the bottom of fluid reservoir, and described fluid reservoir upper end is also provided with fluid reservoir lid; Also include the inflator pump and the time controller that are connected with bioreactor culture tank body;
Described air disinfector, for filtered air, makes the air of cultivating turnover in reactor in germ-free condition; Described the first tube connector, for can high temperature high voltage resistant sterile material, for coupled reaction device tank body lid, bioreactor culture tank body and air disinfector; Described reactor tank body lid, has screw thread or other modes and is connected with reactor tank body, and the top of reactor tank body can be connected with air disinfector by the first tube connector, realizes the discharge of air pressure in tank body.Reactor tank body lid is can high temperature high voltage resistant (121 DEG C, 30 min) sterile material, and material is transparent material, be beneficial to cultivate passing through of time, thus the supply of illumination when plant is cultivated.
Described bioreactor culture tank body, for cultivating the part of plant tissue, tank body middle part arranges baffle plate, baffle plate centre position has outstanding osculum downwards, be used for connecting the second tube connector, and this outstanding osculum and baffle plate link position have screen pack, prevent that the explant of cultivating from falling into; The bottom of bioreactor culture tank body has screw thread or other modes can be tightly connected with fluid reservoir; The outside of bioreactor culture tank body, baffle plate bottom has laterally projecting air inlet with the centre position that is connected top, can be connected with air disinfector by the first tube connector, the object that realizes filtrated air and enter reactor; The material of bioreactor culture tank body can high temperature high voltage resistant (121 DEG C, 30 min) sterile material, and material is transparent material, thus the supply of illumination while ensureing that plant is cultivated.
Described the second tube connector can be connected to bioreactor culture tank body, length is the bottom position that can touch fluid reservoir, to make culture fluid under the effect of air pressure, arrive cultivation tank body from the second tube connector and carry out the submergence of plant, and in the time that air pressure disappears, make culture fluid get back in liquid storage tank body by it.
Described layering cultivate rack be can disassembly and assembly layering cultivate rack, according to the difference of cultivating plant variety, can be placed into and in bioreactor culture tank body, carry out layering cultivation, improve the culture efficiency of unit reactor, it is can high temperature high voltage resistant (121 DEG C that described multilayer is cultivated rack, 30 min) sterile material, mesh size 0.5mm ~ 3cm left and right is to support plant bulk material and to make culture fluid smoothly by being as the criterion, between rack, utilizing can assembly and disassembly shore supports, can be according to the number of plies of cultivating object adjustment rack, the rack number of plies is 2 ~ 10 layers, preferably 3 ~ 8 layers; Rack spacing is 0.25 ~ 25.0cm, preferably 3.0 ~ 10cm.
Described fluid reservoir lid, having the screw thread or other modes that match with fluid reservoir can seal combination with fluid reservoir, bioreactor culture process change liquid step in application when separately culture fluid being carried out to sterilizing.
Described fluid reservoir, for storing culture fluid, top has screw thread or other modes can seal combination with bioreactor culture tank body lower part, and fluid reservoir tank body material can high temperature high voltage resistant (121 DEG C) sterile material.
Above-mentioned parts are the main part of device, and apparatus main body diameter is 5 ~ 50cm, preferably 10 ~ 30cm.
Apparatus main body height 10 ~ 50cm, preferably 20 ~ 40cm.
Described inflator pump, the Pneumatic pressure power when work is provided, the atmospheric pressure of described air pump need coordinate the amount of liquid in fluid reservoir, so that culture fluid pump in 2min is advisable in culturing room.
Described time controller, controls the work of inflator pump by controlling the break-make of electric current, carry out the Immersion frequency of realization response device by controlling the break-make frequency of electric current.
Described servicing unit comprises: heater, in order to the temperature control of culture fluid to be provided; Illumination apparatus, with provide plant cultivate time illumination; CO
2input unit, adjusts the constituent of air that passes into reactor can ventilate in conjunction with air pump time, make plant growth more healthy and stronger; Pickup probe, the PH in can Real-Time Monitoring culture fluid and the variation of various compositions are to instruct concrete operation.In addition, servicing unit can also provide different environment stresses to reach different cultivation objects.
Principle of the present invention is:
General to cultivate to produce secondary metabolite for plant be to utilize fermentation tank etc. to obtain by the cultivation of carrying out cellular level, but most secondary metabolites need to can generate in histoorgan level.What top of the present invention adopted is that the cultivation of carrying out plant tissue organ is cultivated in dismountable layering, can carry out stage by stage easily the extraction operation of gathering to carry out secondary metabolite of culture, and the design of multilayer cultivation rack can be cultivated the plant corpus of sufficient amount simultaneously, thereby the concentration that provides enough raising plant corpuss to be secreted into the utility in culture fluid, is convenient to the enrichment of secondary metabolite and follow-up concentrated collection of extraction.Bottom of the present invention adopts split type design, the fluid reservoir of bottom can be dismantled easily, after cultivation after a while, the secondary metabolite of some exocytosiss can be secreted in culture fluid, collection by culture fluid can obtain required material, and replaces with the secondary metabolite in the acquisition culture fluid that can continue after cultivation after a while after fresh culture.And because culture fluid is changed conveniently, can in culture fluid, add certain precursor substance and stress factors etc. and carry out the enrichment operation of required metabolite by the regulation and control of metabolic pathway.Heating, supplementary illumination and auxiliary CO
2the necessary condition of plant corpus growth can be provided in device, can improve the state of plant corpus growth, and can provide this necessary adverse circumstance environment that raw metabolite produces by adjustment.
The relative prior art of the present invention has following advantage and effect:
(1) save cost.Compared with organizing training method with tradition, the consumption that adopts device and method of the present invention to carry out extensive Plant Tissue Breeding can to save a large amount of materials that can not reuse (as agar etc.), and the raising of the automaticity of the increase of culture vessel volume and the simplification of seeded process and incubation also greatly reduces the consumption of manpower.Therefore there is the effect that reduces Plant Tissue Breeding cost.
(2) can realize numerously soon on a large scale, improve unit quantum of output.The present invention and traditional tissue culture room can well coordinate carry out numerous soon on a large scale.And can greatly improve the numerous efficiency of expansion by the adjustment of tissue being cultivated to microenvironment, and can make the grow metabolism state of plant in the best, quality and the quality of raising group training seedling.And device of the present invention can amplify according to production scale.And be equipped with layering and cultivated rack, can carry out layering cultivation to the plant tissue of cultivating as required.So just improve unit quantum of output.
(3) production of the secondary metabolite of convenience and high-efficiency.The density of cultivating by raising can output more contains the plant corpus of metabolite, can therefrom extract and be secreted into extracellular secondary metabolite by the collection of the culture fluid to after cultivation a period of time in addition, and can regulate and control by adjusting the formation of nutrient solution the generation of desired substance.Reduce secondary metabolite concentration by the replacing of culture fluid it is continued to the inhibition producing, thereby improve the output of product.
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further details:
Fig. 1 cultivates by tissue the operational flowchart that continues the method that obtains Secondary Metabolism of Plant product;
Fig. 2 cultivates to use reactor assembly exploded view.
Embodiment
Easy for what compose a piece of writing, below relating to is consistent with the orientation in accompanying drawing up and down.
embodiment 1
As shown in Figure 1, 2, a kind of related device each several part of method that obtains Secondary Metabolism of Plant product by Plant Tissue Breeding carries out the assembling of device.Reactor tank body lid 3, has screw thread, is connected with reactor tank body 4, and fluid reservoir 8 tops have screw thread is combined with the lower seal of bioreactor culture tank body 4, and fluid reservoir lid 7 has the screw thread matching with fluid reservoir 8 can seal combination with fluid reservoir 8.
Apparatus main body diameter is 20cm, apparatus main body height 30cm; It is can high temperature high voltage resistant (121 DEG C, 30min) sterile material that multilayer is cultivated rack 6, and mesh size 0.5cm left and right is with support plant bulk material and make culture fluid smoothly by being as the criterion.Multilayer is cultivated each layer rack utilization of rack 6 can assembly and disassembly shore supports, can be according to the number of plies of cultivating object adjustment rack, and the rack number of plies in Fig. 2 is 3 layers; Rack spacing is 5cm.
It is as follows that use device is realized concrete operation step of the present invention:
(1) plant corpus obtains in batches, is also that plant corpus batch expands numerous:
In fluid reservoir 8, inject after a certain amount of culture fluid, after being connected in turn according to all parts shown in Fig. 2, by its high temperature and high pressure steam sterilizing 20min at 121 DEG C.Take out cooling for subsequent use.Reactor body part good sterilizing, under aseptic condition, after bioreactor culture tank body lid 3 is opened, is inoculated into needing scale to expand numerous explant, and rear sealing completes inoculation; After time controller 10 is set to different break-make frequencies as required, connect inflator pump 9, and then be connected with reactor body partial reaction device cultivation tank body 4.
The cultivation connecting is switched on power to be placed on reactor assembly and in normal tissue culturing room, carry out illumination cultivation.Incubation as shown in Figure 1.In the time that inflator pump 9 is in running order, the air disinfector 2 that gas is connected by the air inlet smallmouth laterally projecting with bioreactor culture tank body 4 middle parts carries out degerming processing, then enters fluid reservoir 8 by the first tube connector 1; Culture fluid in fluid reservoir 8 enters in bioreactor culture tank body 4 by the second tube connector 5 under the effect of air pressure, make in bioreactor culture tank body 4 plant tissue organ be immersed in culture fluid.The first tube connector 2 and air disinfector 1 that gas in bioreactor culture tank body 4 connects by bioreactor culture tank body lid 3 central exit positions are discharged from.
In the time that inflator pump 9 quits work, the culture fluid in bioreactor culture tank body 4, under the effect of self gravitation, is back in fluid reservoir 8 by downward outstanding smallmouth and the second tube connector 5 of bioreactor culture tank body 4 middle part baffle plates; Because the baffle plate center at bioreactor culture tank body 4 middle parts is provided with screen pack in outstanding smallmouth position downwards, therefore, the plant tissue organ in bioreactor culture tank 4 can be along with culture fluid is back in fluid reservoir 8.Now, the interior formation negative pressure of whole bioreactor culture tank body 4, the filtration sterilization of the air disinfector 1 that extraneous gas is connected through the smallmouth projecting upwards with bioreactor culture tank body lid 3 enters in bioreactor culture tank body 4 along the first tube connector 2 after processing.Like this, the plant tissue organ in bioreactor culture tank body 4 has just completed one by the intermittently circulation of submergence.Can be by regulating time controller 10 to regulate the time of above-mentioned circulation.
When cultivating after a period of time, in culture fluid, composition has certain consumption, needs more to bring the growth that ensures plant to culture fluid according to cultivating.Changing the step of culture fluid is: first fresh culture fluid is injected to fluid reservoir for subsequent use 8, utilize after 7 sealings of fluid reservoir lid, and autoclave sterilization 20min at 121 DEG C, cooling for subsequent use.Then by its with cultivate after the reactor body part ultraviolet sterilization of plant, the fluid reservoir 8 that fresh medium is housed is replaced to the fluid reservoir 8 that the culture fluid that nutrition exhausts is housed under aseptic condition, as shown in Figure 2, the fluid reservoir 8 on the right is exactly for replacing the fluid reservoir 8 on the left side.Complete the culture fluid process of changing.
Concrete cultivation factor is carried out different settings according to different plants.Carry out scale cultivation, carry out as required next step operation;
(2) generation of Secondary Metabolism of Plant product and induction: utilize envirment factor or inducible factor to carry out the induction of plant corpus, thereby carry out the induction of secondary metabolite.
Envirment factor can be equipped to realize by installing external environment control as the change of suitable condition of culture and environment stress environment etc., induces a large amount of plant corpus in abovementioned steps (1) to produce required secondary metabolite as temperature regulating device can improve intensity of illumination that culture environment temperature, external illumination apparatus can regulate culture environment etc.;
The change of inducible factor can be by the liquid storage tank structure of assembling for convenience detach in device, realize changing in adding inducible factor to culture fluid as salt, induction precursor substance or chemical substance etc. in culture fluid, after interpolation, make a large amount of plant corpuss that in earlier stage obtain under the effect of inducible factor, produce required secondary metabolite by cultivations.
(3) collection of Secondary Metabolism of Plant product and extraction: according to secondary metabolite exist position, and the characteristic of secondary metabolite carry out secondary metabolite collection extract operation required to obtain; In secondary metabolite exists plant corpus time, the plant corpus that the multilayer after induction is cultivated on rack is gathered in the crops the collection of Activities of Some Plants body as required for follow-up extraction operation, and remaining part can continue to cultivate the required plant corpus of acquisition to continue;
Outside secondary metabolite is secreted into plant cell time, now secondary metabolite is washed in culture fluid, now only the culture fluid in the fluid reservoir of device bottom need to be collected to extract operation; The quantity of cultivation, the required secondary metabolite of extraction that just can continue from culture fluid are controlled in the lasting cultivation of the plant corpus by top simultaneously;
When secondary metabolite is present in plant corpus and while being secreted into extracellular simultaneously, can operate in conjunction with above-mentioned two kinds of methods, not only can in plant corpus, extract but also can from culture fluid, collect required secondary metabolite.
embodiment 2
Carry out the assembling of device according to a kind of related device each several part of method that obtains Secondary Metabolism of Plant product by Plant Tissue Breeding shown in Fig. 2, reactor tank body lid 3, having draw-in groove mode is connected with reactor tank body 4, fluid reservoir 8 tops have draw-in groove mode can be combined with bioreactor culture tank body 4 lower seal, and fluid reservoir lid 7 has draw-in groove mode can seal combination with fluid reservoir 8;
Apparatus main body diameter is 30 cm.Apparatus main body height 40cm;
It is can high temperature high voltage resistant (121 DEG C, 30 min) sterile material that described multilayer is cultivated rack, and mesh size 1mm left and right to be to support plant bulk material and to make culture fluid smoothly by being as the criterion, and between rack, utilization can assembly and disassembly shore supports, and the rack number of plies is 5 layers; Rack spacing is 4cm;
All the other concrete operation steps are consistent with embodiment 1.
embodiment 3
Carry out the assembling of device according to a kind of related device each several part of method that obtains Secondary Metabolism of Plant product by Plant Tissue Breeding shown in Fig. 2, described in embodiment 1, difference is that the rack number of plies is 2 layers; Rack spacing is 10cm.
embodiment 4
Carry out the assembling of device according to a kind of related device each several part of method that obtains Secondary Metabolism of Plant product by Plant Tissue Breeding shown in Fig. 2, described in embodiment 1, difference is that the rack number of plies is 10 layers; Rack spacing is 2cm.
embodiment 5
According to the device described in embodiment 1, the method for carrying out the acquisition of tuber of pinellia Ephedrine by Plant Tissue Breeding, concrete operation step is as follows:
(1) batch of tuber of pinellia seedling obtains:
Described culture parameters mainly contains: tuber of pinellia Multiple Buds is inoculated in the device described in embodiment 1 to inoculum density: 60/L, 3 layers of separate inoculations,, cultivate after adjusting medium by 20 every layer.Wherein: culture medium prescription is: Ms+1.5 mg/L 6-BA+0.2mg/L NAA+30 g/L sucrose; Medium pH: 5.8; Immersion frequency: 5min/12h.Condition of culture: intensity of illumination 2000 lux, light application time 12 h/d, 25 ± 1 DEG C of cultivation temperature, incubation time: after 40d, plant corpus covers with culture vessel.
(2) induction of Secondary Metabolism of Plant product: under the condition that needn't change at culture fluid, the enough plant corpuss that obtain and device are reached to 35 DEG C by the additional auxiliary equipment temperature that controls environment, after processing times 15 d, after the tuber of pinellia falls seedling, can obtain extracting the tuber of pinellia stem tuber of secondary metabolite.Utilize dismountable multilayer to cultivate the feature of rack, take out part tuber of pinellia stem tuber and carry out the extraction of secondary metabolite, remainder continues to cultivate to obtain more extraction material after changing fresh medium, described in the parameter synchronization rapid (1) that described continuation is cultivated.
(3) collection of Secondary Metabolism of Plant product and extraction: after tuber of pinellia stem tuber is taken out, by grinding, the steps such as ultrasonic extraction, vacuum drying obtain ephedrine extraction thing, detect by HPLC method, and the content that obtains obtained tuber of pinellia stem tuber Ephedrine can reach 160.30ug/g.
embodiment 6
According to the device described in embodiment 3, the method for carrying out the acquisition of tuber of pinellia Ephedrine by Plant Tissue Breeding, concrete operation step is with described in embodiment 5, and the content of the tuber of pinellia stem tuber Ephedrine finally obtaining reaches 190.70 ug/g.
embodiment 7
According to the device described in embodiment 4, the method for carrying out the acquisition of tuber of pinellia Ephedrine by Plant Tissue Breeding, concrete operation step is with described in embodiment 5, and the content of the tuber of pinellia stem tuber Ephedrine finally obtaining reaches 120.50ug/g.
embodiment 8
According to the device described in embodiment 1, the method for carrying out the acquisition of tuber of pinellia Ephedrine by Plant Tissue Breeding, concrete operation step is as follows:
(1) batch of tuber of pinellia seedling obtains:
Described culture parameters is with in embodiment 3 described in (1), and difference is that incubation time is 30d.
(2) induction of Secondary Metabolism of Plant product:
By the design of detachable in device, easily culture fluid is changed, the formula of metabolite induction culture fluid is: Ms+0.5 mg/L 6-BA+2.0 mg/L 2,4-D+30 g/L sucrose, after changing, continue double summer and cultivate, described in the parameter synchronization rapid (1) that described continuation is cultivated.
Cultivating after certain hour, changing fresh metabolite induction culture fluid and proceed to cultivate.Described in the parameter synchronization rapid (1) that described continuation is cultivated.
Utilize dismountable multilayer to cultivate the feature of rack, according to the growing state of culture, take out part tuber of pinellia stem tuber and carry out the extraction of secondary metabolite, remainder continues to cultivate to obtain more extraction material changing after fresh medium.
(3) collection of Secondary Metabolism of Plant product and extraction:
The culture fluid changing is passed through centrifugal, precipitation is directly carried out ultrasonic extraction vacuum drying, it is dry that supernatant extracts final vacuum after concentrated, the ephedrine product obtaining is extracted in rear merging, detect and obtain by HPLC, can reach 12.56ug/L at the content of the culture fluid Ephedrine changing.
By the part tuber of pinellia callus tuber of pinellia taking out, by grinding, the steps such as ultrasonic extraction, vacuum drying obtain ephedrine extraction thing, detect by HPLC method, and the content that obtains obtained tuber of pinellia stem tuber Ephedrine can reach 92.50 ug/g.
embodiment 9
According to the device described in embodiment 2, the method for carrying out the acquisition of alkannin in Asian puccoon by Plant Tissue Breeding, concrete operation step is as follows:
(1) batch of Asian puccoon callus obtains:
Described culture parameters mainly contains: Asian puccoon callus is inoculated in the device described in embodiment 2 to inoculum density: 120/L, 6 layers of separate inoculations,, cultivate after adjusting medium by 20 every layer.Wherein: culture medium prescription is: B5+1.0 mg/L 6-BA+0.1mg/L NAA+30 g/L sucrose; Medium pH: 6.0; Immersion frequency: 1min/6h.Condition of culture: intensity of illumination 1000 lux, light application time 18 h/d, 25 ± 1 DEG C of cultivation temperature, incubation time: 60d.
(2) induction of Secondary Metabolism of Plant product: by the design of detachable in device, easily culture fluid is changed, the formula of metabolite induction culture fluid is: B5+1.0 mg/L 6-BA+0.1mg/L NAA+30 g/L sucrose, and in culture fluid, add CuSO simultaneously
45H
2o, makes the CuSO in culture fluid
4concentration is 0.075mg/L.After changing, continue Asian puccoon callus to cultivate, described in the parameter synchronization rapid (1) that described continuation is cultivated, difference is that incubation time is 20 d.Cultivating after 20 d, again changing fresh metabolite induction culture fluid and proceed to cultivate.In changing fresh medium, carry out the collection of old culture fluid and extract required alkannin, and so forth.The quantity of simultaneously cultivating the structural adjustment Asian puccoon callus of rack by multilayer makes it keep suitable vigor.
(3) collection of Secondary Metabolism of Plant product and extraction:
The culture fluid changing is carried out to acetone extraction alkannin after concentrated, utilize spectrophotometer by detect the output of calculating Asian puccoon Soviet Union under 520nm, the content that obtains alkannin in the culture fluid changing can reach 2.3mg/L.
embodiment 10
According to the device described in embodiment 3, the method for carrying out the acquisition of alkannin in Asian puccoon by Plant Tissue Breeding, concrete operation step is with described in embodiment 9, and the content that finally obtains alkannin in the culture fluid changing can reach 1.6mg/L.
embodiment 11
According to the device described in embodiment 4, the method for carrying out the acquisition of alkannin in Asian puccoon by Plant Tissue Breeding, concrete operation step is with described in embodiment 9, and the content that finally obtains alkannin in the culture fluid changing can reach 4.2mg/L.
embodiment 12
According to the device described in embodiment 1, cultivate the preparation method that carries out panaxoside in Panax ginseng hairy by tissue, concrete operation step is as follows:
(1) batch of Panax ginseng hairy obtains:
Described culture parameters mainly contains: Panax ginseng hairy is inoculated in the device described in embodiment 1 to inoculum density: 120/L, 3 layers of separate inoculations,, cultivate after adjusting medium by 40 every layer.Wherein: culture medium prescription is: Ms+0.1 mg/L IBA+30 g/L sucrose; Medium pH: 5.8; Immersion frequency: 3min/4h.Condition of culture: intensity of illumination 2000 lux, light application time 8 h/d, 25 ± 1 DEG C of cultivation temperature, incubation time: 10d.
(2) induction of Secondary Metabolism of Plant product: utilize dismountable design to be convenient for changing culture fluid to be: Ms+30 g/L sucrose; Medium pH: 5.8; Immersion frequency: 10min/2h.Condition of culture: intensity of illumination 1000 lux, light application time 5 h/d, 25 ± 1 DEG C of cultivation temperature, incubation time: 90d.
(3) collection of Secondary Metabolism of Plant product and extraction: the content of measuring TSPG and ginsenoside Re+Rg after Panax ginseng hairy is taken out according to Chinese people's pharmacopeia version in 2010, in the Panax ginseng hairy that obtained after testing, ginsenoside content can reach 6.182%, and the content of ginsenoside Re+Rg can reach 0.3752%.
embodiment 13
According to the device described in embodiment 2, cultivate the preparation method that carries out panaxoside in Panax ginseng hairy by tissue.Concrete operation step, with described in embodiment 12, finally obtains ginsenoside content in Panax ginseng hairy and can reach 4.8912%, and the content of ginsenoside Re+Rg can reach 0.2527%.
embodiment 14
According to the device described in embodiment 3, cultivate the preparation method that carries out panaxoside in Panax ginseng hairy by tissue, concrete operation step is with described in embodiment 12, finally obtain ginsenoside content in Panax ginseng hairy and can reach 6.532%, the content of ginsenoside Re+Rg can reach 0.3852%.
embodiment 15
According to the device described in embodiment 4, cultivate the preparation method that carries out panaxoside in Panax ginseng hairy by tissue, concrete operation step is with described in embodiment 12, finally obtain ginsenoside content in Panax ginseng hairy and can reach 4.6752%, the content of ginsenoside Re+Rg can reach 0.2319%.
Finally, it is also to be noted that, what more than enumerate is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (1)
1. cultivate by tissue a method that continues to obtain Asian puccoon secondary metabolite, comprise the following steps:
(1) batch of Asian puccoon callus obtains:
Culture parameters mainly contains: Asian puccoon callus is inoculated to cultivation with in reactor assembly, inoculum density: 120/L, 6 layers of separate inoculations, 20 every layer, after adjusting medium, cultivate, wherein: culture medium prescription is: B5+1.0 mg/L 6-BA+0.1mg/L NAA+30 g/L sucrose; Medium pH: 6.0; Immersion frequency: 1min/6h, condition of culture: intensity of illumination 1000 lux, light application time 18h/d, 25 ± 1 DEG C of cultivation temperature, incubation time: 60d;
(2) induction of Asian puccoon secondary metabolite: by the design of detachable in device, easily culture fluid is changed, the formula of metabolite induction culture fluid is: B5+1.0 mg/L 6-BA+0.1mg/L NAA+30 g/L sucrose, and in culture fluid, add CuSO simultaneously
45H
2o, makes the CuSO in culture fluid
4concentration is 0.075mg/L; After changing metabolite induction culture fluid, continue Asian puccoon callus to cultivate, described in the parameter synchronization rapid (1) that described continuation is cultivated, difference is that incubation time is 20d; Cultivating after 20d, again changing fresh metabolite induction culture fluid and proceed to cultivate; In changing fresh medium, carry out the collection of old culture fluid and extract required alkannin, and so forth; The quantity of simultaneously cultivating the structural adjustment Asian puccoon callus of rack by multilayer makes it keep suitable vigor;
(3) collection of Asian puccoon secondary metabolite and extraction: the culture fluid changing is carried out to acetone extraction alkannin after concentrated, utilize spectrophotometer by detect the output of calculating alkannin under 520nm, the content that obtains alkannin in the culture fluid changing reaches 2.3mg/L;
Cultivation in described step (1) comprises reactor tank body lid (3) with reactor assembly, having draw-in groove mode is connected with reactor tank body (4), fluid reservoir (8) top has draw-in groove mode is combined with bioreactor culture tank body (4) lower seal, and fluid reservoir lid (7) has draw-in groove mode and fluid reservoir (8) sealing combination;
Cultivation is 30 cm with reactor assembly main diameter, apparatus main body height 40cm;
It is can high temperature high voltage resistant sterile material that multilayer in described step (2) is cultivated rack (6), and mesh size 1mm left and right to be to support plant bulk material and to make culture fluid smoothly by being as the criterion, and between rack, utilization can assembly and disassembly shore supports; Rack spacing is 4cm.
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| CN201410264732.1A CN103999777B (en) | 2013-02-04 | 2013-02-04 | The method that Rhizoma Pinelliae seedling secondary metabolite is persistently obtained by tissue culture |
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