CN103120126A - 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
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 241000934230 Anoectochilus roxburghii Species 0.000 title abstract 3
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 239000000463 material Substances 0.000 claims abstract description 9
- 238000011081 inoculation Methods 0.000 claims abstract description 6
- 241000719837 Anoectochilus Species 0.000 claims description 62
- 241000196324 Embryophyta Species 0.000 claims description 29
- 238000012549 training Methods 0.000 claims description 20
- 239000012531 culture fluid Substances 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 17
- 230000001954 sterilising effect Effects 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 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 12
- 238000011534 incubation Methods 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 8
- 229940088597 hormone Drugs 0.000 claims description 8
- 239000005556 hormone Substances 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 239000001963 growth medium 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
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 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
- 238000004519 manufacturing process Methods 0.000 description 6
- 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
- 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
- 230000008901 benefit Effects 0.000 description 2
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- 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
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- 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 utilization intermittently the submergence bio-reactor carry out the 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 to be Shorthairy Antenoron, rough melic herb, Shu Caolian, gold thread taiwan anetochilus herb etc.Usually being grown in the fertile leaf mould that covers under broad leaved forest, is the traditional valuable ingredient of traditional Chinese medicine of China, the effect of refreshing and detoxicating, nourishing Yin and falling fire, anti-inflammatory analgetic is arranged, and have 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 is graceful, and vein is golden yellow, be netted arrangement, is again the high indoor sight leaf treasure of ornamental value.The 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, and be endangered, can not satisfy 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.
The appliable plant tissue culture and rapid propagation method has solved the phenomenon of roxburgh anoectochilus terminal bud seedling shortage to a certain extent, the domestic reports that have of the quick propagating technology of relevant roxburgh anoectochilus terminal bud more.But the method for appliable plant group training is produced the roxburgh anoectochilus terminal bud seedling, consumes a large amount of human and material resources, and production cost is high, and cultivation cycle is long, and seedling quality is not high, still can not satisfy 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 and the sugarcane of three kinds is cultivated is introduced.Disclosed a kind of medicinal roxburgh anoectochilus terminal bud tissue and cultivate the forming seedling through one step culture method for quickly breeding in Chinese patent literature CN101213942A, but incubation time is long, inefficiency.
Method of operating due to difference submergence at intermittence culture apparatus is different on the one hand, and the culture apparatus that uses in above-mentioned patent documentation is different with this patent; 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 that wherein relates to and condition of culture can not be used for utilizing intermittently submergence bio-reactor cultivation roxburgh anoectochilus terminal bud fully.
Use the defective that production that submergence bio-reactor intermittently carries out the roxburgh anoectochilus terminal bud seedling can make up produced in conventional processes, provide effective method for producing cheaply high-quality roxburgh anoectochilus terminal bud seedling.
In view of the production that utilizes intermittent immersed cultural method to carry out the roxburgh anoectochilus terminal bud seedling has no report and open the application, therefore, be necessary to work out a kind of method of utilizing submergence at intermittence bio-reactor to carry out the 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 the 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
The 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 the culture fluid preparation is completed, put into triangular flask, standby after the HTHP moist heat sterilization;
Reactor sterilization: with the reactor wrapping, standby after the HTHP moist heat sterilization;
(2) explant is selected and aseptic process
When explant is the plant of self-sow, need to be to the 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 explant is the plant of self-sow, after removing blade, the stem that aseptic process is crossed is cut into some stem sections that each contains a stipes, at first be inoculated in the MS solid culture medium, cultivate through the observations of 3 ~ 5 days, be inoculated in above-mentioned reactor after aseptic;
When explant is aseptic group of training seedling, remove blade and equally stem is cut into some stem sections that each contains a stipes, directly be 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, preferred 5 ~ 10 min/8 ~ 10h; Incubation time is 60 ~ 120d.
Setting the culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12 h/d, 25 ± 1 ℃ of temperature.
Further improve being as the present invention, 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 experiment confirms, in culture fluid, suitable roxburgh anoectochilus terminal bud addition is 0.2 ~ 0.5 g/L, when in culture fluid, the 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 that obtains and common cultivation obtain does not have significant difference, the group that group training seedling leaf length, plant height, stem are thick etc. obtains when 0.2 ~ 0.5 g/L less than addition is trained seedling and significant difference; When the roxburgh anoectochilus terminal bud addition 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 during lower than 45 stem sections/L, group training seedling is not full of whole reactor when inoculum concentration, and space availability ratio is low; During higher than 60 stem sections/L, in reactor, group training seedling is crowded, lopsided seedling occurred when inoculum concentration.
Further improve being as the present invention, in described step (1) early-stage preparations, add roxburgh anoectochilus terminal bud juice in culture fluid.Contain in roxburgh anoectochilus terminal bud juice in the roxburgh anoectochilus terminal bud process of growth the indispensable and trace element that can't manually synthesize, that the culture fluid that adds roxburgh anoectochilus terminal bud juice cultivates than common culture fluid the roxburgh anoectochilus terminal bud group training seedling that obtains is more healthy and stronger through a large amount of experiment showed.Take common culture fluid as contrast, cultivate and compared afterwards roxburgh anoectochilus terminal bud group training seedling growing way situation such as following table in 30 days:
| 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 |
Further improve being as the present invention, the preparation method of roxburgh anoectochilus terminal bud juice is that fresh material is ground and/or squeezes the juice.Fresh material is ground and/or is squeezed the juice and obtains the purpose that roxburgh anoectochilus terminal bud juice can satisfy disorganize and cell acquisition cell inclusion, can not destroy again macromolecular structure in cell.
Further improve being as the present invention, use blade and/or root to prepare roxburgh anoectochilus terminal bud juice.The explant that the present invention uses is the roxburgh anoectochilus terminal bud stem, need to remove blade and root when obtaining explant, and blade and root are used as preparation roxburgh anoectochilus terminal bud juice, has namely saved material and has avoided again waste.
Further improve being as the present invention, when explant was the plant of self-sow, the method for aseptic process is divided into cleaned and sterilization.
Further improve being as the present invention, the method for described cleaning is: select the disease-free plant of healthy growth, remove root, soak 5 ~ 10min with liquor potassic permanganate, then flowing water cleans 30 ~ 60min; The method of described sterilization is: in gnotobasis, the plant after above-mentioned cleaning is removed blade, and the alcohol-pickled stem 15 ~ 30s with 75%, after sterile water wash 2 times, then with mercuric chloride immersion 10 ~ 20min of 0.1%, sterile water wash 5 ~ 7 times.
The technical scheme that adopts the present invention to produce the roxburgh anoectochilus terminal bud seedling, the beneficial effect of its generation is that significant technique effect is:
(1) rate of increase is high.Intermittently the 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 takes full advantage 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 the immersion liquid culture systems be semi-automatic control, whole incubation only needs once to inoculate, and once changes culture fluid in same reactor, has saved the operation of the can 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.Compare with traditional solid culture and reduced subculture number, reduced aberration rate, and carried out sufficient gas exchange in incubation, reduce and even avoided Vitrification, adaptive capacity to environment is strong, transplanting survival rate is high.
Description of drawings
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 the bioreactor culture situation;
Wherein:
Cultivated for 3 weeks; 2. cultivated for 6 weeks; 3. cultivated for 9 weeks; 4. cultivated for 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 wrapping reactor, standby after the HTHP moist heat sterilization;
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select the disease-free plant of healthy growth, remove root, soak 10min with liquor potassic permanganate, then flowing water cleans 60min;
Sterilization: in gnotobasis, the plant after above-mentioned cleaning is removed blade, the alcohol-pickled stem 30s with 75%, after sterile water wash 2 times, then with 0.1% mercuric chloride immersion 20min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into some stem sections that each contains a stipes after removing blade, at first is inoculated in the MS solid culture medium, cultivates through the observations of 3 ~ 5 days, 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; The culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12 h/d, 25 ± 1 ℃ of temperature.
The growth situation that has shown roxburgh anoectochilus terminal bud group training seedling each incubation time cycle in reactor in Fig. 1.
It is 26.1 that roxburgh anoectochilus terminal bud is cultivated growth coefficient, will organize the training transplantation of seedlings after 3 months, survival rate of plant 96.0%, and plant strain growth is healthy.
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 wrapping reactor, standby after the HTHP moist heat sterilization.
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select the disease-free plant of healthy growth, remove root, soak 7min with liquor potassic permanganate, then flowing water cleans 45min;
Sterilization: in gnotobasis, the plant after above-mentioned cleaning is removed blade, the alcohol-pickled stem 20s with 75%, after sterile water wash 2 times, then with 0.1% mercuric chloride immersion 15min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into some stem sections that each contains a stipes after removing blade, at first is inoculated in the MS solid culture medium, cultivates through the observations of 3 ~ 5 days, 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.The 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 the training transplantation of seedlings after 3 months, survival rate of plant 98.2%, and plant strain growth is healthy.
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 wrapping reactor, standby after the HTHP moist heat sterilization.
(2) take wild roxburgh anoectochilus terminal bud stem as explant, aseptic process:
Clean: select the disease-free plant of healthy growth, remove root, soak 5min with liquor potassic permanganate, then flowing water cleans 30min;
Sterilization: in gnotobasis, the plant after above-mentioned cleaning is removed blade, the alcohol-pickled stem 15s with 75%, after sterile water wash 2 times, then with 0.1% mercuric chloride immersion 10min, sterile water wash 5 ~ 7 times.
(3) stem of aseptic process being crossed is cut into some stem sections that each contains a stipes after removing blade, at first is inoculated in the MS solid culture medium, cultivates through the observations of 3 ~ 5 days, 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.The 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 the training transplantation of seedlings after 3 months, survival rate of plant 100%, and plant strain growth is healthy.
At last, 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, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (7)
1. a method of utilizing submergence at intermittence bio-reactor to carry out the roxburgh anoectochilus terminal bud tissue-culturing quick-propagation, comprise the steps:
(1) early-stage preparations
The 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; Adjust pH value 5.8 ~ 6.2, after the culture fluid preparation is completed, put into triangular flask, standby after the HTHP moist heat sterilization;
Reactor sterilization: with the reactor wrapping, standby after the HTHP moist heat sterilization;
(2) explant is selected and aseptic process
When explant is the plant of self-sow, need to be to the 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 explant is the plant of self-sow, after removing blade, the stem that aseptic process is crossed is cut into some stem sections that each contains a stipes, at first be inoculated in the MS solid culture medium, cultivate through the observations of 3 ~ 5 days, be inoculated in above-mentioned reactor after aseptic;
When explant is aseptic group of training seedling, remove blade and equally stem is cut into some stem sections that each contains a stipes, directly be 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, preferred 5 ~ 10 min/8 ~ 10h; Incubation time is 60 ~ 120d;
Setting the culture environment condition is: intensity of illumination 1500 ~ 1800lux, illumination 12h/d, 25 ± 1 ℃ of temperature.
2. utilization submergence at intermittence bio-reactor according to claim 1 carries out the method for roxburgh anoectochilus terminal bud tissue-culturing quick-propagation, it is characterized in that, in described step (1) early-stage preparations, described hormone NAA0.5 ~ 1.0mg/L, hormone 6-BA1.0 ~ 1.5mg/L, roxburgh anoectochilus terminal bud 0.2 ~ 0.5g/L; 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.
3. the method for utilizing submergence at intermittence bio-reactor to carry out the roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 1 and 2, is characterized in that, in described step (1) early-stage preparations, adds roxburgh anoectochilus terminal bud juice in culture fluid.
4. the method for utilizing submergence at intermittence bio-reactor to carry out the roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 3, is characterized in that, the preparation method of roxburgh anoectochilus terminal bud juice is that fresh material is ground and/or squeezes the juice.
5. the method for utilizing submergence at intermittence bio-reactor to carry out the roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 3, is characterized in that, uses blade and/or root to prepare roxburgh anoectochilus terminal bud juice.
6. the method for utilizing submergence at intermittence bio-reactor to carry out the roxburgh anoectochilus terminal bud tissue-culturing quick-propagation according to claim 1 and 2, is characterized in that, when explant was the plant of self-sow, the method for aseptic process is divided into cleaned and sterilization.
7. utilization submergence at intermittence bio-reactor according to claim 6 carries out the method for roxburgh anoectochilus terminal bud tissue-culturing quick-propagation, it is characterized in that, the method of described cleaning is: select the disease-free plant of healthy growth, remove root, soak 5 ~ 10min with liquor potassic permanganate, then flowing water cleans 30 ~ 60min; The method of described sterilization is: in gnotobasis, the plant after above-mentioned cleaning is removed blade, and the alcohol-pickled stem 15 ~ 30s with 75%, after sterile water wash 2 times, then with mercuric chloride immersion 10 ~ 20min of 0.1%, sterile water wash 5 ~ 7 times.
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| 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 |
| CN104186314A (en) * | 2014-08-06 | 2014-12-10 | 宁明县红枫中药材种植专业合作社 | Anoectochilus formosanus seedling culturing method |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 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 |
| CN104719168A (en) * | 2015-04-03 | 2015-06-24 | 南京工业大学 | Method for culturing bletilla striata seedling by virtue of intermittent immersion type bioreactor |
| CN104782484A (en) * | 2015-04-13 | 2015-07-22 | 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 |
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| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 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 |
| CN104719168A (en) * | 2015-04-03 | 2015-06-24 | 南京工业大学 | Method for culturing bletilla striata seedling by virtue of intermittent immersion type bioreactor |
| CN104782484A (en) * | 2015-04-13 | 2015-07-22 | 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 |
| WO2021179527A1 (en) * | 2020-03-13 | 2021-09-16 | 广西壮族自治区农业科学院 | Method for culturing pueraria thomsonii by proliferation of single-bud stem segment and one-step seedling formation |
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