CN105145364A - Tissue culture propagation method for obtaining cibotium barometz green globular body - Google Patents
Tissue culture propagation method for obtaining cibotium barometz green globular body Download PDFInfo
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Abstract
The invention discloses a tissue culture propagation method for obtaining a cibotium barometz green globular body. The method includes the steps of obtaining an aseptic explant; inducing the green globular body, wherein the green globular body is obtained after being cultivated in two inducing pre-culture media and then cultivated in a green globular body inducing culture medium; cultivating the green globular body in two propagation culture media alternately, and conducting differentiation culture and rooting culture to obtain a tissue culture seedling. By means of the method, the rare and endangered fern type cibotium barometz green globular body is successfully induced, the propagation efficiency is high, the inducing rate of the green globular body reaches 90-94%, the propagation cycle is short, the method has great significance in increasing the population quantity of rare and endangered fern type cibotium barometz and relieving the endangered situation, and meanwhile the in-vitro conservation of germplasm resources of endangered species is achieved.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the induction of Cibotium barometz green spheroids and enrichment procedure.
Technical background
Cibotium barometz (Cibotiumbarometz), be under the jurisdiction of Dicksoniaceae Cibotium, root-like stock is flat sleeping, thick, end upwarps, and part of basseting is close by golden yellow villus, the Cibotium barometz that shape is seemingly thrown oneself on the ground, form is unique, tool ornamental value, golden fine hair can be used as medicine, and is incorporated into the Pharmacopoeia of the People's Republic of China.All kinds of Dicksoniaceae are all classified as national II Top-rated protected wild plants by " national key protected wild plants register (first) ", and CONTINENTAL AREA OF CHINA Dicksoniaceae is 1 genus a kind only, that is: the Cibotium barometz of Cibotium.
Under natural conditions, Cibotium barometz adopt spore breed, but spore germination and Development of Gametophytes comparatively strict to the requirement of environmental condition, cause its natural propagation ability extremely low, in addition environmental disruption and people are digging, and the wild stocks quantity of Cibotium barometz sharply reduces, endangered.
At present, the tissue cultures of Cibotium barometz is limited to sporogenesis approach more, its method mainly comprises: the steps such as spore germination cultivation, prothallium cultivation, sporophyte cultivation, culture of rootage, there is the defects such as the breeding cycle is grown, sporophyte inductivity is low, therefore, find a kind of breeding cycle and shortly breed the high method of efficiency simultaneously, for the expansion of Precious, Rare, Endangered fern-Cibotium barometz population quantity, and the alleviation of degree in imminent danger is significant.
Pteridophyte green spheroids refers to the orbicule be made up of numerous green particles obtained by pteridophyte explant induction, its essence is meristematic tissue aggregate, be inoculated on differential medium and can obtain a large amount of pteridophyte seedling, once induction obtains green spheroids, its reproduction coefficient will be significantly higher than sporogenesis approach, and the breeding cycle shortens.
The reason of the plant hormone screening aspect selected due to explant and be suitable for, at present, successfully induce the kind of green spheroids comparatively limited, and in green spheroids breeding, only have and select suitable plant hormone formula and training method, fast breeding could be realized while the differentiation of suppression green spheroids.Therefore, at present, there is no the relevant report that Precious, Rare, Endangered fern Cibotium barometz is studied by green spheroids approach tissue culture propagation.
Summary of the invention
The technical problem to be solved in the present invention is that to overcome Cibotium barometz natural propagation power low, longer by the sporogenesis approach tissue culture propagation cycle, and the technical problem such as reproduction coefficient is lower.
For solving the problems of the technologies described above, the invention provides a kind of tissue culture propagation method of Cibotium barometz green spheroids approach, the method comprises the following steps:
(1) acquisition of Cibotium barometz aseptic explant
By aseptic for Cibotium barometz spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is on the medium of 5.80, condition of culture is: 20 ~ 25 DEG C, the light application time of every day is 12 hours, intensity of illumination is 2000lx, obtain aseptic seedling, at superclean bench, aseptic seedling is separated stem apex with tweezers under anatomical lens, using the aseptic explant that stem apex is induced as Cibotium barometz green spheroids;
(2) induction of Cibotium barometz green spheroids
1. first time induces preculture: the stem apex that step (1) obtains is inoculated in green spheroids induction pre-culture medium I, cultivate 5 days ~ 7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium I is: 1/4MS ~ 1/2MS+6-BA1.0 ~ 1.5mg/L+NAA0.2 ~ 0.4mg/L++ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
2. second time induces preculture: the stem apex after step (2) 1. first time induction preculture is inoculated in green spheroids induction pre-culture medium II, incubation time is 5 days ~ 7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium II is: 1/4MS ~ 1/2MS+2-ip0.1 ~ 0.3mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
3. the induction of green spheroids: the stem apex after step (2) 2. second time induction preculture is inoculated in green spheroids inducing culture, condition of culture is identical with step (1), incubation time is 14 days ~ 20 days, obtain green spheroids, described green spheroids inducing culture is: 1/4MS ~ 1/2MS+TDZ0.6 ~ 0.8mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(3) propagation of Cibotium barometz green spheroids
Each proliferating cycle carries out in the following order:
1. first step Multiplying culture: the green spheroids that step (2) 3. obtains is cut into 1 ~ 3mm, be inoculated in proliferated culture medium A, cultivate 18 days ~ 21 days, condition of culture is identical with step (1), described proliferated culture medium A is: 1/4MS ~ 1/2MS+TDZ0.8 ~ 1.2mg/L+NAA0.1 ~ 0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
2. second step Multiplying culture: the green spheroids behind (3) 1. first step Multiplying culture propagation is inoculated in proliferated culture medium B, cultivate 18 days ~ 21 days, condition of culture is identical with step (1), described proliferated culture medium B is: 1/4MS ~ 1/2MS+2-ip0.1 ~ 0.2mg/L+6-BA1.0 ~ 1.2mg/L+NAA0.05 ~ 0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(4) differentiation of Cibotium barometz green spheroids
Green spheroids after step (3) 2. second step Multiplying culture propagation is divided into the orbicule of diameter 1 ~ 3mm, be inoculated in differential medium, cultivate 28 days ~ 29 days, obtain breaking up seedling, condition of culture is identical with step (1), described differential medium is: 1/6MS ~ 1/4MS+ active carbon 1 ~ 2g/L+ caseinhydrolysate 200 ~ 300mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(5) culture of rootage
Differentiation seedling inoculation step (4) obtained is in root media, Cibotium barometz plantlet in vitro is after differentiation seedling takes root, condition of culture is identical with step (1), described root media is: 1/2MS+ active carbon 1 ~ 2g/L+ caseinhydrolysate 200 ~ 300mg/L+ sucrose 30.0g/L+ agar concentration 7.0g/L, pH is 5.80.
Innovative point of the present invention:
1, successfully induced the green spheroids of Precious, Rare, Endangered pteridophyte Cibotium barometz, and the inductivity of green spheroids reaches 90 ~ 94%.
The present invention, by great many of experiments, has filtered out two green spheroids induction pre-culture mediums and a green spheroids inducing culture of suitable Precious, Rare, Endangered pteridophyte Cibotium barometz.And devise twice induction preculture and a Fiber differentiation step, successfully induce Cibotium barometz green spheroids.
2, on Multiplying culture, filter out A and B two proliferated culture mediums, and design each proliferating cycle with using A and B two proliferated culture mediums successively, both restrained effectively the differentiating phenomenon that breeding occurs, achieve again proliferation times and reach 9.5 ~ 10.2 times of significant especially technique effects, not occurring to realize high efficiently multiplying under the prerequisite of breaking up, maintain higher multiplication rate simultaneously.The Multiplying culture in several cycle need being carried out, can determine according to producing the seedling quantity needed.The present inventor finds in research process, only adopt proliferated culture medium A, can Inhibited differentiation, but proliferation times is lower, and only adopt proliferated culture medium B, proliferation times is higher but occur differentiating phenomenon, and by described in the inventive method successively with the Multiplying culture that proliferated culture medium A and proliferated culture medium B hocket, both inhibit the differentiating phenomenon that breeding occurs, the technique effect of high efficiently multiplying can have been achieved again.And be conducive to Cibotium barometz green spheroids as the propagation of middle brood body and preservation.
By innovation of the present invention, compared with prior art, beneficial effect of the present invention is as follows:
1, Precious, Rare, Endangered pteridophyte Cibotium barometz green spheroids is successfully induced; and the reproductive efficiencies such as the inductivity of green spheroids are high especially; for the expansion treasuring endangered fern Cibotium barometz group quantity that China's secondary is laid special stress on protecting, and the alleviation of degree in imminent danger is significant.
It is high all especially that the inventive method makes green spheroids inductivity, green spheroids multiplication rate, green spheroids differentiation rate, single green spherical differentiation seedling number and differentiation seedling be trained the efficiency of plantlet in vitro, the inductivity of its green spheroids reaches 90 ~ 94%, the proliferation times of green spheroids reaches 9.5 ~ 10.2 times, the differentiation rate 100% of green spheroids, rooting rate 100%, therefore, the reproductive efficiency such as inductivity of green spheroids of the present invention is high especially; And the sporophyte inductivity of existing sporogenesis approach is only 28.30%, compared with breeding with existing spore approach, reproductive efficiency of the present invention creates unforeseeable technique effect.
2, the breeding cycle is short
After the present invention induces green spheroids, namely can green spheroids as middle propagating materials, high effect culture goes out a large amount of plantlet in vitro, without the need to repeat again from spore cultivate, therefore, the breeding cycle is short.With green spheroids for middle propagating materials calculates, breeding cycle is 92 days ~ 98 days, and the existing spore approach breeding cycle needs about 224 days ~ 284 days, and (wherein spore germination forms prothallium 60-90 days, prothallium inductive formation sporophyte 80-110 days, sporophyte breeds 42 days, take root 42 days), the present invention is compared with the propagation method of conventional sporogenesis approach, and the breeding cycle shortens 132 ~ 186 days.
3, the Plantlet in vitro of endangered species Cibotium barometz germ plasm resource is achieved
The Cibotium barometz green spheroids obtained by method provided by the invention and Cibotium barometz plantlet in vitro be can be used as germ plasm resource and preserve, and for realizing, Precious, Rare, Endangered fern Cibotium barometz germ plasm resource Plantlet in vitro is significant.
Accompanying drawing explanation
Fig. 1 is Cibotium barometz green spheroids.Line segment in figure is engineer's scale, and the length of expression is 1mm.
Fig. 2 is the Cibotium barometz green spheroids starting to break up.Line segment in figure is engineer's scale, and the length of expression is 1mm.
Specific implementation method
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment is commercially available.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
The preparation method of following embodiment medium, for conventional method is namely by after component each described in culture medium prescription and content mixing thereof, namely makes by the pH value that pH meter tune pH is this medium requirement.
Embodiment 1 the inventive method
(1) acquisition of Cibotium barometz aseptic explant
By aseptic for Cibotium barometz spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is on the medium of 5.80, and condition of culture is: 20 ~ 25 DEG C, the light application time of every day is 12 hours, and intensity of illumination is 2000lx, obtains the aseptic seedling that children is tender.In superclean bench, the aseptic seedling obtained is separated stem apex with tweezers under anatomical lens, using the aseptic explant that stem apex is induced as Cibotium barometz green spheroids.
The preparation method of the aseptic spore of Cibotium barometz is: 10mg spore is placed in 1.5ml centrifuge tube, soak the centrifugal 2min of 1-2 hour, 6000r/min with sterile water, obtain spore sediment, adding volume fraction is 5%NaClO solution sterilization 4min, and namely sterile water wash obtains the aseptic spore of Cibotium barometz for 3-5 time.
(2) induction of Cibotium barometz green spheroids
1. first time induces preculture: be inoculated in by the stem apex that step (1) obtains in green spheroids induction pre-culture medium I, cultivate 6 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium I is: 1/4MS+6-BA1.5mg/L+NAA0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80; Inoculate several 50 stem apexs.
2. second time induces preculture: be inoculated in green spheroids induction pre-culture medium II by the stem apex after step (2) 1. first time induction preculture, incubation time is 6 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium II is: 1/4MS+2-ip0.3mg/L+NAA0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
3. the induction of green spheroids: the stem apex after step (2) 2. second time induction preculture is inoculated in green spheroids inducing culture, incubation time is 14 days, condition of culture is identical with step (1), obtain green spheroids, described green spheroids inducing culture is: 1/4MS+TDZ0.8mg/L+NAA0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
After induction preculture and Fiber differentiation, the stem apex of Cibotium barometz expands formation green spheroids, and the inductivity of green spheroids is 94.00%.
Inductivity=(producing the aseptic explant number of the aseptic seedling number/inoculation of green spheroids) × 100%.
(3) propagation of Cibotium barometz green spheroids
Each proliferating cycle carries out according to the following steps:
1. first step Multiplying culture: the green spheroids that step (2) 3. obtains is cut into 2mm, be inoculated on proliferated culture medium A, cultivate 18 days, condition of culture is identical with step (1), described proliferated culture medium A is: 1/2MS+TDZ0.8mg/L+NAA0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80; The fresh weight of this 2mm green spheroids is 8mg, inoculates several 50 green spheroids.
2. second step Multiplying culture: the green spheroids behind (3) 1. first step Multiplying culture propagation is inoculated on proliferated culture medium B, cultivate 18 days, condition of culture is identical with step (1), described proliferated culture medium B is: 1/2MS+2-ip0.2mg/L+6-BA1.0mg/L+NAA0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
The green spheroids mean fresh recruitment of every 18 days is 76mg, average 9.5 times of proliferation times, and does not occur green spheroids differentiating phenomenon in breeding.Each Multiplying culture cycle is undertaken by said sequence, need carry out the Multiplying culture in several cycle, determines according to producing the plantlet in vitro quantity needed.
(4) differentiation of Cibotium barometz green spheroids
Green spheroids after step (3) 2. second step Multiplying culture propagation is divided into the orbicule of diameter 2mm, is inoculated on differential medium, cultivates 28 days, obtain breaking up seedling, condition of culture is identical with step (1); Described differential medium is: 1/6MS+ active carbon 1g/L+ caseinhydrolysate 200mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
Inoculate number 50 green spheroids, average mark rate is 100%.
Differentiation rate=(quantity of the green spheroids of the green spheroids quantity/inoculation of differentiation occurs) × 100%.
(5) culture of rootage
The differentiation seedling inoculation that step (4) obtains is cultivated on root media, Cibotium barometz plantlet in vitro is after differentiation seedling takes root, condition of culture is identical with step (1), described root media is: 1/2MS+ active carbon 1g/L+ caseinhydrolysate 200mg/L+ sucrose 30.0g/L+ agar concentration 7.0g/L, pH is 5.80.
Cultivate 4 weeks, the average height of Cibotium barometz plantlet in vitro reaches 3.15cm.Differentiation seedling all takes root and survives formation plantlet in vitro, and the production rate of plantlet in vitro reaches 100%.
Embodiment 2 and embodiment 3 are the inventive method
Embodiment 2 and embodiment 3 are except the concentration of each composition in medium each listed by table 1 is different, and all the other measures are all identical with embodiment 1, repeat no more.
The difference of each step of table 1 embodiment 2-embodiment 3 and embodiment 1
The Breeding results of embodiment 1: the inductivity of green spheroids is 94%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 3.15cm of plantlet in vitro.Diameter is the green spheroids fresh weight 8mg of 2mm, and green spheroids every 18 days mean freshs increase 76mg, average 9.5 times of proliferation times.With the green spheroids obtained for middle brood body, through two step Multiplying culture for 36 days, differentiation cultivation 28 days, culture of rootage form plantlet in vitro 28 days, repoductive time for 92 days.
The Breeding results of embodiment 2: the inductivity of green spheroids is 90.00%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 3.08cm of plantlet in vitro.Diameter is the green spheroids fresh weight 4.5mg of 1mm, and green spheroids every 20 days mean freshs increase 45.9mg, average 10.2 times of proliferation times.With the green spheroids obtained for middle brood body, through two step Multiplying culture for 40 days, differentiation cultivation 29 days, culture of rootage form plantlet in vitro 28 days, repoductive time for 97 days.
The Breeding results of embodiment 3: the inductivity of green spheroids is 92%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 3.30cm of plantlet in vitro.Diameter is the green spheroids fresh weight 8mg of 2mm, and green spheroids every 21 days mean freshs increase 77.6mg, average 9.7 times of proliferation times.With the green spheroids obtained for middle brood body, through two step Multiplying culture for 42 days, differentiation cultivation 28 days, culture of rootage form plantlet in vitro 28 days, repoductive time for 98 days.
The green spheroids of a 1 ~ 3mm is once broken up can obtain 20 ~ 25 strain plantlet in vitro.
Above-described embodiment 1 to embodiment 3 shows: the inductivity of green spheroids of the present invention is 90 ~ 94%, and the proliferation times of green spheroids reaches 9.5 ~ 10.2 times, the differentiation rate 100% of green spheroids, rooting rate 100%, the formation rate 100% of plantlet in vitro, therefore, reproductive efficiency is high especially.After the present invention induces green spheroids, namely can green spheroids as middle propagating materials, high effect culture goes out a large amount of plantlet in vitro, without the need to repeat again from spore cultivate, therefore, the breeding cycle is short.With green spheroids for middle propagating materials calculates, the breeding cycle is 92 days ~ 98 days.
Claims (1)
1. a tissue culture propagation method for Cibotium barometz green spheroids approach, its feature is comprising the following steps:
(1) acquisition of Cibotium barometz aseptic explant
By aseptic for Cibotium barometz spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is on the medium of 5.80, condition of culture is: 20 ~ 25 DEG C, the light application time of every day is 12 hours, intensity of illumination is 2000lx, obtain aseptic seedling, at superclean bench, aseptic seedling is separated stem apex with tweezers under anatomical lens, using the aseptic explant that stem apex is induced as Cibotium barometz green spheroids;
(2) induction of Cibotium barometz green spheroids
1. first time induces preculture: the stem apex that step (1) obtains is inoculated in green spheroids induction pre-culture medium I, cultivate 5 days ~ 7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium I is: 1/4MS ~ 1/2MS+6-BA1.0 ~ 1.5mg/L+NAA0.2 ~ 0.4mg/L++ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
2. second time induces preculture: the stem apex after step (2) 1. first time induction preculture is inoculated in green spheroids induction pre-culture medium II, incubation time is 5 days ~ 7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture medium II is: 1/4MS ~ 1/2MS+2-ip0.1 ~ 0.3mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
3. the induction of green spheroids: the stem apex after step (2) 2. second time induction preculture is inoculated in green spheroids inducing culture, condition of culture is identical with step (1), incubation time is 14 days ~ 20 days, obtain green spheroids, described green spheroids inducing culture is: 1/4MS ~ 1/2MS+TDZ0.6 ~ 0.8mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(3) propagation of Cibotium barometz green spheroids
Each proliferating cycle carries out in the following order:
1. first step Multiplying culture: the green spheroids that step (2) 3. obtains is cut into 1 ~ 3mm, be inoculated in proliferated culture medium A, cultivate 18 days ~ 21 days, condition of culture is identical with step (1), described proliferated culture medium A is: 1/4MS ~ 1/2MS+TDZ0.8 ~ 1.2mg/L+NAA0.1 ~ 0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
2. second step Multiplying culture: the green spheroids behind (3) 1. first step Multiplying culture propagation is inoculated in proliferated culture medium B, cultivate 18 days ~ 21 days, condition of culture is identical with step (1), described proliferated culture medium B is: 1/4MS ~ 1/2MS+2-ip0.1 ~ 0.2mg/L+6-BA1.0 ~ 1.2mg/L+NAA0.05 ~ 0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(4) differentiation of Cibotium barometz green spheroids
Green spheroids after step (3) 2. second step Multiplying culture propagation is divided into the orbicule of diameter 1 ~ 3mm, be inoculated in differential medium, cultivate 28 days ~ 29 days, obtain breaking up seedling, condition of culture is identical with step (1), described differential medium is: 1/6MS ~ 1/4MS+ active carbon 1 ~ 2g/L+ caseinhydrolysate 200 ~ 300mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(5) culture of rootage
Differentiation seedling inoculation step (4) obtained is in root media, Cibotium barometz plantlet in vitro is after differentiation seedling takes root, condition of culture is identical with step (1), described root media is: 1/2MS+ active carbon 1 ~ 2g/L+ caseinhydrolysate 200 ~ 300mg/L+ sucrose 30.0g/L+ agar concentration 7.0g/L, pH is 5.80.
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| CN109699495A (en) * | 2019-02-12 | 2019-05-03 | 云南农业大学 | A method of improving scythian lamb rhizome spore germination rate |
| CN117089510A (en) * | 2023-08-30 | 2023-11-21 | 毕节市中药研究所 | Culture medium combination for inducing germination and proliferation of Cibotium barometz spores, and method and application for inducing protophylls |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109699495A (en) * | 2019-02-12 | 2019-05-03 | 云南农业大学 | A method of improving scythian lamb rhizome spore germination rate |
| CN109699495B (en) * | 2019-02-12 | 2022-04-08 | 云南农业大学 | Method for improving germination rate of cibotium barometz spores |
| CN117089510A (en) * | 2023-08-30 | 2023-11-21 | 毕节市中药研究所 | Culture medium combination for inducing germination and proliferation of Cibotium barometz spores, and method and application for inducing protophylls |
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