CN103120126B - Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor - Google Patents
Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor Download PDFInfo
- Publication number
- CN103120126B CN103120126B CN201310043181.1A CN201310043181A CN103120126B CN 103120126 B CN103120126 B CN 103120126B CN 201310043181 A CN201310043181 A CN 201310043181A CN 103120126 B CN103120126 B CN 103120126B
- Authority
- CN
- China
- Prior art keywords
- reactor
- terminal bud
- roxburgh anoectochilus
- anoectochilus terminal
- stem
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 50
- 241000934230 Anoectochilus roxburghii Species 0.000 title abstract 3
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 239000000463 material Substances 0.000 claims abstract description 9
- 238000011081 inoculation Methods 0.000 claims abstract description 5
- 241000719837 Anoectochilus Species 0.000 claims description 60
- 241000196324 Embryophyta Species 0.000 claims description 29
- 238000012549 training Methods 0.000 claims description 20
- 230000008569 process Effects 0.000 claims description 19
- 239000012531 culture fluid Substances 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 14
- 238000007654 immersion Methods 0.000 claims description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims description 14
- 238000005286 illumination Methods 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- 229940088597 hormone Drugs 0.000 claims description 6
- 239000005556 hormone Substances 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 208000037824 growth disorder Diseases 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000004161 plant tissue culture Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 241000632227 Antenoron Species 0.000 description 1
- 244000247747 Coptis groenlandica Species 0.000 description 1
- 235000002991 Coptis groenlandica Nutrition 0.000 description 1
- 241000732800 Cymbidium Species 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000004017 vitrification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of a traditional Chinese medicinal material, and particularly relates to a method for carrying out tissue cultivation propagation of anoectochilus roxburghii by an intermittent submerged bioreactor. The method comprises the steps of early-stage preparation, explant selection, and aseptic treatment, inoculation, cultivation of the bioreactor, and the like. Multiplication and rooting and strong seedling cultivation are finished one time in the reactor; the method has the characteristics of being short in period, high in tissue culture seedling quality and the like; and a method with low cost and high efficiency is provided for rapid propagation of tissue cultivation of the anoectochilus roxburghii.
Description
Technical field
The invention belongs to medicinal plant tissue culture technique field, be specifically related to a kind of intermittently submergence bio-reactor that utilizes and carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation, thereby obtain the method for high quality seedling.
Background technology
Roxburgh anoectochilus terminal bud (Anoecthilus roxburghii) is opened lip Cymbidium herbaceos perennial for the orchid family, calls as Shorthairy Antenoron, rough melic herb, Shu Caolian, gold thread taiwan anetochilus herb etc.Conventionally being grown in the fertile leaf mould covering under broad leaved forest, is the traditional valuable ingredient of traditional Chinese medicine of China, has the effect of refreshing and detoxicating, nourishing Yin and falling fire, anti-inflammatory analgetic, and has no side effect, in the good reputation of among the people have " king of medicine ".Roxburgh anoectochilus terminal bud plant height 8 ~ 20cm, plant type is small and exquisite, and blade profile grace, is netted arrangement at vein golden yellow, is again the high indoor sight leaf treasure of ornamental value.Roxburgh anoectochilus terminal bud seed is small, and embryonic development is incomplete, and extremely difficulty is sprouted under field conditions (factors); If with root division or cottage propagation, length consuming time and reproduction coefficient are low, and the natural environment that roxburgh anoectochilus terminal bud is depended on for existence changes, and wild resource falls sharply, and excavates for a long time in addition, and germ plasm resource is rare, endangered, can not meet clinical demand.The highest more than 3000 yuan of the per kilogram that once increased to of wild roxburgh anoectochilus terminal bud dried food and nuts.
Appliable plant tissue culture and rapid propagation method has solved the phenomenon of roxburgh anoectochilus terminal bud seedling shortage to a certain extent, about the domestic reports that have of quick propagating technology of roxburgh anoectochilus terminal bud more.But the method for appliable plant group training is produced roxburgh anoectochilus terminal bud seedling, consumes a large amount of human and material resources, production cost is high, and cultivation cycle is long, and seedling quality is not high, still can not meet medicinal herb grower's demand.
The research that utilizes submergence cultivation at intermittence reactor successfully to carry out other plant cultivation has been reported.In Chinese patent literature CN101720668B, disclose and a kind ofly utilized intermittent immersed submergence at intermittence culture systems to carry out the method for tissue culturing and quick propagation of sugarcane, it utilizes intermittently submergence to cultivate reactor the sugarcane of three kinds is cultivated and introduced.In Chinese patent literature CN101213942A, disclosed a kind of medicinal roxburgh anoectochilus terminal bud tissue and cultivated forming seedling through one step culture method for quickly breeding, but incubation time is long, inefficiency.
Due to the intermittently method of operating difference of submergence culture apparatus of difference, the culture apparatus using in above-mentioned patent documentation is different with this patent on the one hand; Due to the difference on various plant physiologies, biochemistry, identical cultural method is cultivated different to different plants, need different condition of culture and processing mode on the other hand, can not obtain by simple limited number of time experiment reasoning.The step of mentioning in above-mentioned document patent and the concrete training method wherein relating to and condition of culture can not be used for utilizing intermittently submergence bio-reactor to cultivate roxburgh anoectochilus terminal bud completely.
The application production that intermittently submergence bio-reactor carries out roxburgh anoectochilus terminal bud seedling can make up the defect of produced in conventional processes, for producing cheaply high-quality roxburgh anoectochilus terminal bud seedling, provides effective method.
In view of the production that utilizes intermittent immersed cultural method to carry out roxburgh anoectochilus terminal bud seedling has no report and open application, therefore, be necessary to work out a kind of method of utilizing submergence at intermittence bio-reactor to carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation.
Summary of the invention
The technical problem to be solved in the present invention is to provide the roxburgh anoectochilus terminal bud seedling production method that a kind of cycle is short, cost is low, simple to operate.
For solving the problems of the technologies described above, the technical solution used in the present invention is: the method for utilizing submergence at intermittence bio-reactor to carry out roxburgh anoectochilus terminal bud tissue culture quick breeding comprises preparation, explant selection and aseptic process, inoculation, bioreactor culture, particularly, the method comprises the following steps:
(1) early-stage preparations
Culture fluid preparation: MS liquid is basic culture solution, adds hormone NAA0.2 ~ 1.5mg/L; Hormone 6-BA 0.5 ~ 2.5mg/L; Sucrose concentration 30g/L; Roxburgh anoectochilus terminal bud 0.1 ~ 1.0g/L; After culture fluid has been prepared, put into triangular flask, standby after HTHP moist heat sterilization;
Reactor sterilizing: by reactor wrapping, standby after HTHP moist heat sterilization;
(2) explant is selected and aseptic process
When plant that explant is self-sow, need to be to stem aseptic process;
When explant is aseptic group of training seedling, directly inoculate;
(3) inoculation
In gnotobasis, sterilized culture fluid is poured in sterilized reactor;
When plant that explant is self-sow, the stem that aseptic process is crossed is cut into each some stem sections that contain a stipes after removing blade, is first inoculated in MS solid culture medium, through the observations of 3 ~ 5 days, cultivates, and is inoculated in above-mentioned reactor after aseptic;
When explant is aseptic group of training seedling, remove blade and equally stem is cut into each some stem sections that contain a stipes, be directly inoculated in above-mentioned reactor;
35 ~ 80 stem section/L of reactor inoculum density;
(4) bioreactor culture
Set the intermittently parameter of submergence reactor: Immersion frequency: 3 ~ 15min/6 ~ 12h, preferably 5 ~ 10 min/8 ~ 10h; Incubation time is 60 ~ 120d.
Setting culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12 h/d, 25 ± 1 ℃ of temperature.
As the present invention, further improve and be, in described step (1) early-stage preparations, described hormone NAA0.5 ~ 1.0mg/L, hormone 6-BA 1.0 ~ 1.5 mg/L, roxburgh anoectochilus terminal bud 0.2 ~ 0.5 g/L, adjusts pH value 5.8 ~ 6.2; In described step (3) inoculation, 45 ~ 60 stem section/L of reactor inoculum density; Incubation time in described step (4) bioreactor culture is 80 ~ 90d.
By a large amount of experiments, confirm, in culture fluid, suitable roxburgh anoectochilus terminal bud addition is 0.2 ~ 0.5 g/L, when in culture fluid, roxburgh anoectochilus terminal bud addition is lower than 0.2 g/L, the group training seedling growing way that the roxburgh anoectochilus terminal bud group training seedling obtaining and common cultivation obtain does not have significant difference, group training seedling leaf length, plant height, stem are thick etc. is less than the group that addition obtains when 0.2 ~ 0.5 g/L and trains seedling and significant difference; When roxburgh anoectochilus terminal bud addition is during higher than 0.5 g/L, along with the increase group training seedling of addition does not change significantly.By one group of experiment, confirm, the suitable inoculum density of bioreactor culture roxburgh anoectochilus terminal bud is 45 ~ 60 stem section/L, and when inoculum concentration is during lower than 45 stem section/L, group training seedling is not full of whole reactor, and space availability ratio is low; When inoculum concentration is during higher than 60 stem section/L, in reactor, group training seedling is crowded, has occurred lopsided seedling.
As the present invention, further improve and be, in described step (1) early-stage preparations, in culture fluid, add roxburgh anoectochilus terminal bud juice.In roxburgh anoectochilus terminal bud juice, contain in roxburgh anoectochilus terminal bud process of growth indispensable and trace element that cannot be manually synthetic, it is more healthy and stronger that the culture fluid that adds roxburgh anoectochilus terminal bud juice through a large amount of experiment showed, cultivates than common culture fluid the roxburgh anoectochilus terminal bud group training seedling obtaining.Take common culture fluid as contrast, cultivate 30 days afterwards relatively roxburgh anoectochilus terminal bud group training seedling growing way situation as following table:
| Culture fluid | Blade long (cm) | Plant height (cm) | Stem thick (mm) |
| Roxburgh anoectochilus terminal bud juice (0.2 g/L) | 2.36 | 5.24 | 3.72 |
| Ordinary culture medium | 1.88 | 4.32 | 3.28 |
As the present invention, further improve and be, the preparation method of roxburgh anoectochilus terminal bud juice is that fresh material is ground and/or squeezes the juice.Fresh material grinding and/or the acquisition roxburgh anoectochilus terminal bud juice of squeezing the juice can meet the object of disorganize and cell acquisition cell inclusion, can not destroy again macromolecular structure in cell.
As the present invention, further improve and be, use blade and/or root to prepare roxburgh anoectochilus terminal bud juice.The explant that the present invention uses is roxburgh anoectochilus terminal bud stem, need to remove blade and root obtaining during explant, and by blade and root as preparing roxburgh anoectochilus terminal bud juice, saved material and avoided again waste.
As the present invention, further improve and be, when plant that explant is self-sow, the method for aseptic process is divided into cleans and sterilization.
As the present invention, further improve and be, the method for described cleaning is: select the disease-free plant of healthy growth, remove root, with liquor potassic permanganate immersion 5 ~ 10min, then flowing water cleans 30 ~ 60min; The method of described sterilization is: in gnotobasis, plant after above-mentioned cleaning is removed to blade, and with alcohol-pickled stem 15 ~ 30s of 75%, after sterile water wash 2 times, then by mercuric chloride immersion 10 ~ 20min of 0.1%, sterile water wash 5 ~ 7 times.
The technical scheme that adopts the present invention to produce roxburgh anoectochilus terminal bud seedling, the beneficial effect of its generation is that significant technique effect is:
(1) rate of increase is high.Intermittently immersion liquid culture systems is to utilize pressure that filtrated air produces contacting liquid nutrient medium and plant tissue culture seedling intermittence, combine solid culture and maximize the advantage that gas exchanges and liquid culture nutrition makes full use of, the rate of increase that roxburgh anoectochilus terminal bud is cultivated is more than 10 times of common solid culture, 5 ~ 7 times of liquid culture.
(2) automaticity is high, the amount of labour is little.Due to intermittence immersion liquid culture systems be semi-automatic control, whole cultivation process only need to once be inoculated, and once in same reactor, changes culture fluid, has saved the operation of the filling cleaning etc. of frequent switching and tissue culture bottle in enormous quantities, reduce greatly the amount of labour, reduced production cost.
(3) group training seedling quality is good, strong adaptability.With traditional solid culture, compare and reduced subculture number, reduced aberration rate, and in cultivation process, carried out sufficient gas exchange, reduce and even avoided Vitrification, adaptive capacity to environment is strong, transplanting survival rate is high.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further details:
Fig. 1 is that roxburgh anoectochilus terminal bud group training seedling is in bioreactor culture situation;
Wherein:
cultivate 3 weeks; 2. cultivate 6 weeks; 3. cultivate 9 weeks; 4. cultivate 12 weeks.
Embodiment
Embodiment 1:
(1) according to formula MS+ NAA1.0mg/L+6-BA1.5mg/L+ sucrose 30g/L+ roxburgh anoectochilus terminal bud fresh material 0.5g/L, adjust pH value 5.8, preparation culture fluid, and wrap up reactor, standby after HTHP moist heat sterilization;
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select healthy growth disease-free plant, remove root, soak 10min with liquor potassic permanganate, then flowing water cleans 60min;
Sterilization: in gnotobasis, plant after above-mentioned cleaning is removed to blade, with 75% alcohol-pickled stem 30s, after sterile water wash 2 times, then by 0.1% mercuric chloride immersion 20min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into each some stem sections that contain a stipes after removing blade, is first inoculated in MS solid culture medium, through the observations of 3 ~ 5 days, cultivates, and is inoculated in reactor 60 stem section/L of inoculum density after aseptic.
(4) set the intermittently parameter of submergence reactor: Immersion frequency: 10min/10h; Incubation time is 90d; Culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12 h/d, 25 ± 1 ℃ of temperature.
In Fig. 1, shown the growth situation in roxburgh anoectochilus terminal bud group training seedling each incubation time cycle in reactor.
It is 26.1 that roxburgh anoectochilus terminal bud is cultivated growth coefficient, will organize training transplantation of seedlings after 3 months, survival rate of plant 96.0%, and plant strain growth health.
Embodiment 2:
(1) according to formula MS+NAA0.7mg/L+6-BA1.2mg/L+ sucrose 30g/L+ roxburgh anoectochilus terminal bud fresh material 0.3g/L, adjust pH value 6.0, preparation culture fluid, and wrap up reactor, standby after HTHP moist heat sterilization.
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select healthy growth disease-free plant, remove root, soak 7min with liquor potassic permanganate, then flowing water cleans 45min;
Sterilization: in gnotobasis, plant after above-mentioned cleaning is removed to blade, with 75% alcohol-pickled stem 20s, after sterile water wash 2 times, then by 0.1% mercuric chloride immersion 15min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into each some stem sections that contain a stipes after removing blade, is first inoculated in MS solid culture medium, through the observations of 3 ~ 5 days, cultivates, and is inoculated in reactor 50 stem section/L of inoculum density after aseptic.
(4) set the intermittently parameter of submergence reactor: Immersion frequency: 7 min/9h; Incubation time is 85d.Culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12h/d, 25 ± 1 ℃ of temperature.
It is 29.4 that roxburgh anoectochilus terminal bud is cultivated growth coefficient, will organize training transplantation of seedlings after 3 months, survival rate of plant 98.2%, and plant strain growth health.
Embodiment 3:
(1) according to formula MS+NAA0.5mg/L+6-BA1.0mg/L+ sucrose 30g/L+ roxburgh anoectochilus terminal bud fresh material 0.2g/L, adjust pH value 6.2, preparation culture fluid, and wrap up reactor, standby after HTHP moist heat sterilization.
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select healthy growth disease-free plant, remove root, soak 5min with liquor potassic permanganate, then flowing water cleans 30min;
Sterilization: in gnotobasis, plant after above-mentioned cleaning is removed to blade, with 75% alcohol-pickled stem 15s, after sterile water wash 2 times, then by 0.1% mercuric chloride immersion 10min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into each some stem sections that contain a stipes after removing blade, is first inoculated in MS solid culture medium, through the observations of 3 ~ 5 days, cultivates, and is inoculated in reactor 45 stem section/L of inoculum density after aseptic.
(4) set the intermittently parameter of submergence reactor: Immersion frequency 5min/8h; Incubation time is 80d.Culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12h/d, 25 ± 1 ℃ of temperature.
It is 30.0 that roxburgh anoectochilus terminal bud is cultivated growth coefficient, will organize training transplantation of seedlings after 3 months, survival rate of plant 100%, and plant strain growth health.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments 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 (5)
1. utilize submergence at intermittence bio-reactor to carry out a method for roxburgh anoectochilus terminal bud tissue-culturing quick-propagation, comprise the steps:
(1) early-stage preparations
Culture fluid preparation: MS liquid is basic culture solution, adds hormone NAA0.5～1.0mg/L; Hormone 6-BA1.0～1.5mg/L; Sucrose concentration 30g/L; Roxburgh anoectochilus terminal bud 0.2～0.5g/L; Adjust pH value 5.8～6.2, after culture fluid has been prepared, put into triangular flask, standby after HTHP moist heat sterilization;
Reactor sterilizing: by reactor wrapping, standby after HTHP moist heat sterilization;
(2) explant is selected and aseptic process
When plant that explant is self-sow, need to be to stem aseptic process;
When explant is aseptic group of training seedling, directly inoculate;
(3) inoculation
In gnotobasis, sterilized culture fluid is poured in sterilized reactor;
When plant that explant is self-sow, the stem that aseptic process is crossed is cut into each some stem sections that contain a stipes after removing blade, is first inoculated in MS solid culture medium, through the observations of 3～5 days, cultivates, and is inoculated in above-mentioned reactor after aseptic;
When explant is aseptic group of training seedling, remove blade and equally stem is cut into each some stem sections that contain a stipes, be directly inoculated in above-mentioned reactor;
45～60 stem section/L of reactor inoculum density;
(4) bioreactor culture
Set the intermittently parameter of submergence reactor: Immersion frequency: 5～10min/8～10h; Incubation time is 80～90d;
Setting culture environment condition is: intensity of illumination 1500～1800lux, illumination 12h/d, 25 ± 1 ℃ of temperature;
In described step (1) early-stage preparations, the roxburgh anoectochilus terminal bud adding in culture fluid is roxburgh anoectochilus terminal bud juice.
2. the method for utilizing submergence at intermittence bio-reactor to carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 1, is characterized in that, the preparation method of roxburgh anoectochilus terminal bud juice is that fresh material is ground and/or squeezes the juice.
3. the method for utilizing submergence at intermittence bio-reactor to carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 2, is characterized in that, uses blade and/or root to prepare roxburgh anoectochilus terminal bud juice.
4. the method for utilizing submergence bio-reactor intermittently to carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 1 and 2, is characterized in that, when plant that explant is self-sow, the method for aseptic process is divided into cleans and sterilization.
5. the method for utilizing submergence at intermittence bio-reactor to carry out roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 4, it is characterized in that, the method of described cleaning is: select the disease-free plant of healthy growth, remove root, with liquor potassic permanganate immersion 5～10min, then flowing water cleans 30～60min; The method of described sterilization is: in gnotobasis, plant after above-mentioned cleaning is removed to blade, and with alcohol-pickled stem 15～30s of 75%, after sterile water wash 2 times, then by mercuric chloride immersion 10～20min of 0.1%, sterile water wash 5～7 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310043181.1A CN103120126B (en) | 2013-02-04 | 2013-02-04 | Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310043181.1A CN103120126B (en) | 2013-02-04 | 2013-02-04 | Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103120126A CN103120126A (en) | 2013-05-29 |
| CN103120126B true CN103120126B (en) | 2014-05-07 |
Family
ID=48451426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310043181.1A Expired - Fee Related CN103120126B (en) | 2013-02-04 | 2013-02-04 | Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103120126B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104106464B (en) * | 2014-06-12 | 2016-03-30 | 南京工业大学大丰海洋产业研究院 | The method of explant sterilization in a kind of Plant Tissue Breeding |
| CN104106465A (en) * | 2014-06-12 | 2014-10-22 | 南京工业大学大丰海洋产业研究院 | Explant disinfection method in plant tissue culture |
| CN104186314B (en) * | 2014-08-06 | 2016-08-24 | 宁明县红枫中药材种植专业合作社 | A kind of method for culturing seedlings of Herba Anoectochili roxburghii |
| 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 |
| CN104719168B (en) * | 2015-04-03 | 2018-01-12 | 南京工业大学 | The method for cultivating bletilla striata seedling using interval submergence bioreactor |
| CN104782484B (en) * | 2015-04-13 | 2017-02-01 | 云南省农业科学院生物技术与种质资源研究所 | Method for obtaining tissue culture plantlets fast by utilizing homogenate of stems and leaves of hydrilla verticillata |
| CN107581069A (en) * | 2017-11-02 | 2018-01-16 | 张玉华 | The method that bioreactor fast-propagation passion fruit tissue-cultured seedling is submerged using interval |
| CN108812306A (en) * | 2018-04-28 | 2018-11-16 | 河南师范大学 | Chinese medicine glutinous rehmannia tissue cultures expanding propagation method is carried out using interval submergence bioreactor |
| CN108849531A (en) * | 2018-09-06 | 2018-11-23 | 河南师范大学 | RHIIZOMA DIOSCOREAE from Henan of China tissue cultures expanding propagation method is carried out using interval submergence bioreactor |
| CN111149704B (en) * | 2020-03-13 | 2023-04-14 | 广西壮族自治区农业科学院 | Proliferation and one-step seedling culture method for single-bud stem of pachyrhizua angulatus |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213942A (en) * | 2008-01-10 | 2008-07-09 | 浙江省中药研究所有限公司 | Medicinal anoectochilus Formosan tissue culture one-step seedling establishment fast replication method |
| CN101300958A (en) * | 2007-05-11 | 2008-11-12 | 中国科学院沈阳应用生态研究所 | Intermittent immersed plant cultivation device and control method thereof |
| EP2067401A1 (en) * | 2007-12-04 | 2009-06-10 | Nestec S.A. | Cocoa somatic embryogenesis |
| CN101720668A (en) * | 2009-09-18 | 2010-06-09 | 广西壮族自治区农业科学院 | Method for tissue culturing and quick propagation of sugarcane with intermittent immersion bioreactor |
| CN102144566A (en) * | 2011-03-30 | 2011-08-10 | 贵州省亚热带作物研究所 | Method for culturing test tube plantlet of Xingren Anoectochilus |
| CN102150624A (en) * | 2011-04-29 | 2011-08-17 | 南京工业大学 | Tissue culture and rapid propagation method for pinellia tuber plant |
| WO2012044239A1 (en) * | 2010-09-30 | 2012-04-05 | Plant Form Ab | Bioreactor for in vitro culture of plants |
| CN202211050U (en) * | 2011-07-20 | 2012-05-09 | 江苏农林职业技术学院 | Split temporary immersion type cultivation device |
-
2013
- 2013-02-04 CN CN201310043181.1A patent/CN103120126B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101300958A (en) * | 2007-05-11 | 2008-11-12 | 中国科学院沈阳应用生态研究所 | Intermittent immersed plant cultivation device and control method thereof |
| EP2067401A1 (en) * | 2007-12-04 | 2009-06-10 | Nestec S.A. | Cocoa somatic embryogenesis |
| CN101213942A (en) * | 2008-01-10 | 2008-07-09 | 浙江省中药研究所有限公司 | Medicinal anoectochilus Formosan tissue culture one-step seedling establishment fast replication method |
| CN101720668A (en) * | 2009-09-18 | 2010-06-09 | 广西壮族自治区农业科学院 | Method for tissue culturing and quick propagation of sugarcane with intermittent immersion bioreactor |
| WO2012044239A1 (en) * | 2010-09-30 | 2012-04-05 | Plant Form Ab | Bioreactor for in vitro culture of plants |
| CN102144566A (en) * | 2011-03-30 | 2011-08-10 | 贵州省亚热带作物研究所 | Method for culturing test tube plantlet of Xingren Anoectochilus |
| CN102150624A (en) * | 2011-04-29 | 2011-08-17 | 南京工业大学 | Tissue culture and rapid propagation method for pinellia tuber plant |
| CN202211050U (en) * | 2011-07-20 | 2012-05-09 | 江苏农林职业技术学院 | Split temporary immersion type cultivation device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103120126A (en) | 2013-05-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103120126B (en) | Method for carrying out tissue cultivation propagation of anoectochilus roxburghii by intermittent submerged bioreactor | |
| CN102150624B (en) | Tissue culture and rapid propagation method for pinellia tuber plant | |
| CN101720668B (en) | Method for tissue culturing and quick propagation of sugarcane with intermittent immersion bioreactor | |
| CN102119655B (en) | Natural light rapid breeding method for dendrobium officinale | |
| CN103598098B (en) | Pachyrhizua angulatus tissue culturing fast seedling-cultivating method | |
| CN102499088B (en) | Method for quickly breeding seedlings of Guangxi anoectochilus roxburghii capsules by utilizing Guangxi anoectochilus roxburghii capsules | |
| CN103931492A (en) | Tissue-culture rapid seedling growing method for apple rootstock M9 | |
| CN104585033B (en) | Blood aspidistra quick breeding method for tissue culture | |
| CN102845308B (en) | Method for dendrobium officinale tissue culture one-step seedling | |
| CN103004599B (en) | Method for obtaining crowtoe regeneration plantlet by anther culture | |
| CN101574055A (en) | Lonicera edulis tissue culturing method | |
| CN104705188A (en) | Method for culturing dendrobium candidum protocorms | |
| CN105104190A (en) | Anoectochilus roxburghii tissue culture seedling method | |
| CN102090327A (en) | Method for quickly breeding Akebia trifoliata Koidz fruit seedling in test tube | |
| CN103270946B (en) | A kind of seedlings of Dendrobium officinale is produced and hardening synchronous method | |
| CN104137779A (en) | Method for regenerating sapium japonicum plant by inducing sapium japonicum stem rapidly | |
| CN104041408A (en) | Potting domestication method of traditional Chinese medicinal material mint | |
| CN105532473B (en) | A kind of tissue culture method of HERBA DENDROBII seedling | |
| CN103548691B (en) | The method of tea-tree tissue culture seedling culture of rootage | |
| CN105075858A (en) | Liquid rapid-propagation method for rhizoma bletillae seeds | |
| CN106922536A (en) | A kind of method that fast energy-saving cultivates bletilla seedling | |
| CN100394845C (en) | In-bottle production method of detoxified small seed ball of east lily | |
| CN104813931A (en) | Tissue culture and rapid propagation method for Dendrobium officinale | |
| CN104719168A (en) | Method for culturing bletilla striata seedling by virtue of intermittent immersion type bioreactor | |
| CN108243959A (en) | It is a kind of using yellow fine strain of millet wood stem section as the highly efficient regeneration method of explant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140507 Termination date: 20150204 |
|
| EXPY | Termination of patent right or utility model |