CN105145365A - Green globular body tissue culture propagation method for Alsophila costularis - Google Patents
Green globular body tissue culture propagation method for Alsophila costularis Download PDFInfo
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Abstract
The invention discloses a green globular body tissue culture propagation method for Alsophila costularis. The method includes the steps of obtaining aseptic explants and inducing green globular bodies. In the inducing process, culture is performed through two inducing pre-culture media, then culture is performed through a green globular body inducing culture medium, and the green globular bodies are obtained; then, alternate culture is performed through two proliferation culture media, differentiation culture and rooting culture are performed, and tissue culture seedlings are obtained. Through the method, the dendritic woody fern green globular bodies are successfully induced, the propagation efficiency is high, the green globular body inducing rate reaches 82%-88%, the proliferation multiple reaches 7.5-9.3, the differentiation rate of the green globoids reaches 100%, the rooting rate reaches 100%, and the propagation period is short; the method has great significance in increasing the population quantity of the rare endangered fern Alsophila costularis and relieving the endangered situation of the rare endangered fern Alsophila costularis, and meanwhile in-vitro preservation 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 Chinese spinulose tree fern green spheroids and enrichment procedure.
Technical background
China spinulose tree fern (Alsophilacostularis) is under the jurisdiction of Cyatheaceae spinulose tree fern and belongs to (wooden spinulose tree fern belongs to).Terrestrial stem is upright, tree-shaped, can, up to 10m, be woody pteridophyte few in number.
Cyatheaceae plant comes across Mesozoic Jurassic Period morning or Triassic period in evening the earliest, is the coeval species of dinosaur.Afterwards due to geology transition and climatic variation, a large amount of kind extinction, finally existed only in " sanctuary " that in Perenniporia martius, some weather is suitable especially, was therefore called as botanic " Relict Plant ", " living fossil ".All kinds of China spinulose tree fern and Cyatheaceae are all classified as national secondary Top-rated protected wild plants " national key protected wild plants register (first) ".
Under natural conditions, China spinulose tree fern adopts spore to breed, but spore germination and Development of Gametophytes comparatively strict to the requirement of environmental condition, cause the natural propagation ability of Chinese spinulose tree fern extremely low, in addition environmental disruption and people are digging, the wild stocks quantity of China spinulose tree fern sharply reduces, endangered.
At present, the tissue cultures of China spinulose tree fern is only confined to sporogenesis approach, its method mainly comprises: the steps such as the cultivation of Spore cultivation, prothallium, prothallium propagation, sporophyte cultivation, there is breeding cycle long, the defect such as reproductive efficiency is low, therefore, find a kind of breeding cycle short and breed the high method of efficiency, for the expansion treasuring endangered fern-Chinese Alsophila spinulosa 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 the large-scale tree-shaped woody pteridophytes such as Chinese spinulose tree fern are 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 Chinese spinulose tree fern 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 Chinese spinulose tree fern green spheroids approach, the method comprises the following steps:
(1) acquisition of Chinese spinulose tree fern aseptic explant
By aseptic for Chinese spinulose tree fern 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 Chinese spinulose tree fern green spheroids;
(2) induction of Chinese spinulose tree fern 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 ~ 2.0mg/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+2-ip0.5 ~ 0.8mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(3) propagation of Chinese spinulose tree fern 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 1 ~ 3mm, be inoculated in proliferated culture medium A, cultivate 21 days ~ 22 days, condition of culture is identical with step (1), described proliferated culture medium A is: 1/4MS ~ 1/2MS+TDZ0.8 ~ 1.0mg/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 21 days ~ 22 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 Chinese spinulose tree fern 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, Chinese spinulose tree fern 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 large-scale tree-shaped woody pteridophyte China spinulose tree fern, the inductivity of green spheroids reaches 82 ~ 88%.
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 large-scale tree-shaped woody pteridophyte China spinulose tree fern.And devise twice induction preculture and a Fiber differentiation step, successfully induced Chinese spinulose tree fern green spheroids, and the inductivity of green spheroids reaches 82 ~ 88%.
2, on Multiplying culture, filter out A and B two proliferated culture mediums, and design and use A and B two proliferated culture mediums each proliferating cycle successively, both restrained effectively the differentiating phenomenon that breeding occurs, achieve again proliferation times and reach 7.5 ~ 9.3 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 invention 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 Chinese spinulose tree fern green spheroids as the propagation of middle brood body and preservation.
By innovation of the present invention, compared with prior art, the present invention has following beneficial effect:
1, successfully induce tree-shaped woody pteridophyte green spheroids, and reproductive efficiency is high especially, for the expansion treasuring endangered fern-Chinese Alsophila spinulosa population 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 is 82 ~ 88%, the proliferation times of green spheroids reaches 7.5 ~ 9.3 times, the differentiation rate 100% of green spheroids, rooting rate 100%, the formation rate 100% of plantlet in vitro, therefore, reproductive efficiency of the present invention is high especially.And the spore germination rate of existing sporogenesis approach is 34.33%, prothallium bunch propagation 2 ~ 3 times, sporophyte seedling planting percent is 24.00%.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 98 days ~ 101 days, and existing spore approach breeding, each breeding all need be bred from spore germination, its breeding cycle needs 217 days ~ 252 days, the present invention shortens 119 ~ 151 days compared with the propagation method of conventional sporogenesis approach, and the breeding cycle shortens 54.8 ~ 59.9%.
3, the Plantlet in vitro of endangered species germ plasm resource is achieved
The Chinese spinulose tree fern green spheroids obtained by method provided by the invention and Chinese spinulose tree fern plantlet in vitro be can be used as germ plasm resource and preserve, and for realizing, Precious, Rare, Endangered fern-Chinese spinulose tree fern germ plasm resource Plantlet in vitro is significant.
Accompanying drawing explanation
Fig. 1 is Chinese spinulose tree fern green spheroids.Line segment in figure is engineer's scale, and the length of expression is 1mm.
Fig. 2 is the Chinese spinulose tree fern 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 Chinese spinulose tree fern aseptic explant
By aseptic for Chinese spinulose tree fern 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 Chinese spinulose tree fern green spheroids.
The preparation method of the aseptic spore of China spinulose tree fern is: 10mg spore is placed in 1.5ml centrifuge tube, soak 1 ~ 2 hour with sterile water, the centrifugal 2min of 6000r/min, obtain spore sediment, adding volume fraction is 5%NaClO solution sterilization 4min, and namely sterile water wash obtains the aseptic spore of Chinese spinulose tree fern for 3-5 time.
(2) induction of Chinese spinulose tree fern 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 7 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 7 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+2-ip0.8mg/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 Chinese spinulose tree fern expands formation green spheroids, and the inductivity of green spheroids is 88.00%.
Inductivity=(producing the aseptic explant number of the aseptic seedling number/inoculation of green spheroids) × 100%.
(3) propagation of Chinese spinulose tree fern green spheroids
Each proliferating cycle carries out Multiplying culture 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 21 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, pH is 5.80.The fresh weight of this 2mm green spheroids is 10mg, 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 21 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 21 days is 75mg, and proliferation times is about 7.5 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 Chinese spinulose tree fern 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, Chinese spinulose tree fern 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 Chinese spinulose tree fern plantlet in vitro reaches 2.87cm.Differentiation seedling all takes root and survives formation plantlet in vitro, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro.
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 88%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 2.87cm of plantlet in vitro.Diameter is the green spheroids fresh weight 10mg of 2mm, and green spheroids every 21 days mean freshs increase 75mg, and proliferation times is about 7.5 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 Breeding results of embodiment 2: the inductivity of green spheroids is 85.33%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 3.10cm of plantlet in vitro.Diameter is the green spheroids fresh weight 5mg of 1mm, and green spheroids every 21 days mean freshs increase 46.67mg, and proliferation times is about 9.3 times.With the green spheroids obtained for middle brood body, through two step Multiplying culture for 44 days, differentiation cultivation 29 days, culture of rootage form plantlet in vitro 28 days, repoductive time for 101 days.
The Breeding results of embodiment 3: the inductivity of green spheroids is 82%, differentiation rate 100%, rooting rate 100%, i.e. the formation rate 100% of plantlet in vitro, culture of rootage 28 days, the average height 2.70cm of plantlet in vitro.Diameter is the green spheroids fresh weight 10mg of 2mm, and green spheroids every 21 days mean freshs increase 83.67mg, and proliferation times is about 8.4 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.
Embodiment 4 contrasts
Embodiment 4 is the propagation method of existing sporogenesis approach, and step is as follows:
(1) Chinese spinulose tree fern spore sterilization
12mg China spinulose tree fern 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 Chinese spinulose tree fern for 3-5 time.
(2) Chinese spinulose tree fern spore germination is cultivated
The aseptic spore inoculating of Chinese spinulose tree fern step (1) obtained is in 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80, and inoculation quantity is 10 bottles, and 20 ~ 25 DEG C of cultivations, the light application time of every day is 12 hours, and intensity of illumination is 2000lx.
After cultivating 6 weeks, spore starts to sprout, and germination rate is about 34.33%, continues cultivation after 9 ~ 10 weeks, forms prothallium bunch.Spore count × 100% of the spore count/inoculation of germination rate=sprouting.
(3) Chinese spinulose tree fern prothallium Multiplying culture
The prothallium bunch obtained in step (2) is divided into fritter, and continue to be inoculated in 1/4MS+ sucrose 30.0g/L, agar 7.0g/L, pH are 5.80, and inoculation quantity is 50 pieces, the same step of condition of culture (1).After cultivating 4 weeks, prothallium bunch propagation 2 ~ 3 times.
Prothallium bunch after propagation is divided into fritter, and continue to be inoculated in 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80, inoculation quantity is 50 pieces, and on prothallium, drip appropriate sterile water, and to promote the formation of sporophyte, the same step of condition of culture (1).
Cultivate after 8 ~ 12 weeks, have sporophyte seedling to be formed gradually, sporophyte seedling planting percent is 30.67%.Prothallium number × 100% of the prothallium number/inoculation of sporophyte seedling planting percent=generation sporophyte seedling.
(4) cultivation of Chinese spinulose tree fern sporophyte seedling
By the sporophyte seedling inoculation of acquisition in step (3) in 1/2MS+ active carbon 2g/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
To cultivate after 4 weeks plantlet in vitro, the average height of Chinese spinulose tree fern plantlet in vitro is 2.57cm.
Comparative examples 4 shows: 42 days time is cultivated in spore germination, form 63 ~ 70 days prothallium time, 28 days Multiplying culture time, the cultivation time that sporophyte is formed is 56 days ~ 84 days, cultivate 28 days plantlet in vitro time, therefore, the tissue-culturing rapid propagation of sporogenesis approach, needs 217 days ~ 252 days from spore germination to cultivating into plantlet in vitro.Spore germination rate is 34.33%, and prothallium bunch propagation 2 ~ 3 times, sporophyte seedling planting percent is 24.00%.Existing spore approach breeding, each breeding all need be bred from spore germination, and therefore, each breeding cycle all needs 217 days ~ 252 days.
Above-described embodiment 1 to-embodiment 3 shows: the inductivity of green spheroids of the present invention is 82 ~ 88%, and the proliferation times of green spheroids reaches 7.5 ~ 9.3 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 98 days ~ 101 days.
Claims (1)
1. a tissue culture propagation method for Chinese spinulose tree fern green spheroids approach, its feature is comprising the following steps:
(1) acquisition of Chinese spinulose tree fern aseptic explant
By aseptic for Chinese spinulose tree fern 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 Chinese spinulose tree fern green spheroids;
(2) induction of Chinese spinulose tree fern 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 ~ 2.0mg/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+2-ip0.5 ~ 0.8mg/L+NAA0.2 ~ 0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, and pH is 5.80;
(3) propagation of Chinese spinulose tree fern 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 1 ~ 3mm, be inoculated in proliferated culture medium A, cultivate 21 days ~ 22 days, condition of culture is identical with step (1), described proliferated culture medium A is: 1/4MS ~ 1/2MS+TDZ0.8 ~ 1.0mg/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 21 days ~ 22 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 Chinese spinulose tree fern 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, Chinese spinulose tree fern 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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| CN108157184A (en) * | 2018-02-11 | 2018-06-15 | 珠海诺德生物科技有限公司 | A kind of Lesley's new pteris fern seedling tissue culture and rapid propagation method |
| CN112293253A (en) * | 2020-10-28 | 2021-02-02 | 中国科学院昆明植物研究所 | Isolated preservation culture medium and isolated preservation method for Adiantum furiosaeanum |
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| CN108077081A (en) * | 2018-02-11 | 2018-05-29 | 珠海诺德生物科技有限公司 | A kind of tissue culture and rapid propagation method of Australia jewel seedling |
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| CN112293253A (en) * | 2020-10-28 | 2021-02-02 | 中国科学院昆明植物研究所 | Isolated preservation culture medium and isolated preservation method for Adiantum furiosaeanum |
| CN115029294A (en) * | 2022-07-31 | 2022-09-09 | 中国长江三峡集团有限公司 | Cyathea spore aseptic propagation method |
| CN115623987A (en) * | 2022-12-01 | 2023-01-20 | 贵州师范大学 | Method for inducing green spheroid approach plant tissue culture by cyathea spores |
| CN115623987B (en) * | 2022-12-01 | 2023-07-21 | 贵州师范大学 | Cyathea spores induced green spheroid plant tissue culture method |
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