CN105145365B - A kind of tissue culture propagation method of China Caulis cyatheae spinulosae green spheroids approach - Google Patents
A kind of tissue culture propagation method of China Caulis cyatheae spinulosae green spheroids approach Download PDFInfo
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Abstract
The present invention discloses the tissue culture propagation method of a kind of China Caulis cyatheae spinulosae green spheroids approach.The method includes the induction of the acquisition of aseptic explant, green spheroids, its induction is cultivated through green spheroids inducing culture after being cultivated by two induction pre-culture again, obtain green spheroids, afterwards, through two proliferated culture medium alternate cultures, then obtain tissue cultured seedling through differentiation culture, root culture.The present invention successfully induces tree-shaped woody pteridophyta green spheroids, reproductive efficiency is high, its green spheroids inductivity reaches 82～88%, proliferation times reaches 7.5～9.3 times, and the differentiation rate of green spheroids reaches 100%, and rooting rate reaches 100%, breeding cycle is short, for the expansion of Precious, Rare, Endangered fern China Alsophila spinulosa population quantity, and the alleviation to degree in imminent danger is significant, is simultaneously achieved the in vitro conservation of Endangered species germ plasm resource.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to China's Caulis cyatheae spinulosae green spheroids induction and propagation
Method.
Technical background
China Caulis cyatheae spinulosae (Alsophila costularis) is under the jurisdiction of Cyatheaceae Caulis cyatheae spinulosae and belongs to (Caulis cyatheae spinulosae belongs to wood).The aerial stem of plant is straight
Vertical, tree-shaped, may be up to 10m, be woody pteridophyta few in number.
The Triassic period in that Cyatheaceae plant coming across Mesozoic Jurassic Period morning or evening the earliest, it it is the coeval thing of dinosaur
Kind.Later due to geology transition and climate change, a large amount of kinds are become extinct, and finally exist only in Perenniporia martius
" refuge " that some weather is particularly suitable, is therefore referred to as botanic " Relict Plant ", " living fossil ".
All kinds of China Caulis cyatheae spinulosae and Cyatheaceae are all classified as two grades of Top-rated protected wild plants " special-protection-by-the-State of country
Wild plant register (first) ".
Under natural conditions, China Caulis cyatheae spinulosae uses spore to breed, but spore germination and Development of Gametophytes are to environment
The requirement of condition is more strict, and the natural propagation ability causing China Caulis cyatheae spinulosae is extremely low, and environmental disruption is with artificial in addition
Digging, the wild stocks quantity of China Caulis cyatheae spinulosae drastically reduces, the most endangered.
At present, the tissue culture of China Caulis cyatheae spinulosae is limited only to sporogenesis approach, and its method specifically includes that spore
, there is breeding cycle length, breeding effect in the steps such as cultivation, original foliage cultivation, original foliage propagation, sporinite cultivation
The defects such as rate is low, therefore, find a kind of breeding cycle short and breed the method that efficiency is high, for treasuring Herba pteridii latiusculi in imminent danger
The expansion of class China Alsophila spinulosa population quantity, and the alleviation of degree in imminent danger is significant.
Pteridophyta green spheroids refer to by pteridophyta outer implant induction obtain by numerous green particles groups
The spheroid become, its essence is separate living tissue aggregation, is inoculated on division culture medium and can obtain a large amount of fern
Plant seedlings, once induction obtains green spheroids, and its breeding coefficient will be significantly higher than that sporogenesis approach, and
Breeding cycle shortens.
Owing to outer implant selects and suitable phytohormone screening aspect, at present, the most successfully induce
The kind of green spheroids is relatively limited, and in green spheroids breeding, only selects suitable plant
Hormone prescription and training method, could realize fast breeding while suppression green spheroids differentiation.Therefore,
At present, there is no the large-scale tree-shaped woody pteridophyta such as China Caulis cyatheae spinulosae to be studied by green spheroids approach tissue culture propagation
Relevant report.
Summary of the invention
The technical problem to be solved in the present invention is to overcome China's Caulis cyatheae spinulosae natural propagation power low, by sporogenesis approach
The tissue culture propagation cycle is longer, and the technical problem such as breeding coefficient is relatively low.
For solving above-mentioned technical problem, the present invention provides the tissue culture propagation of a kind of China Caulis cyatheae spinulosae green spheroids approach
Method, the method comprises the following steps:
(1) acquisition of China's Caulis cyatheae spinulosae aseptic explant
By aseptic for China Caulis cyatheae spinulosae spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80
Culture medium on, condition of culture is: 20～25 DEG C, the light application time of every day be 12 hours, intensity of illumination is
2000lx, obtains aseptic seedling, at superclean bench, with tweezers, aseptic seedling is separated stem apex under anatomical lens,
The aseptic explant that stem apex is induced as China's Caulis cyatheae spinulosae green spheroids；
(2) induction of China's Caulis cyatheae spinulosae green spheroids
1. preculture is induced for the first time: the stem apex that step (1) obtains is inoculated in the pre-training of green spheroids induction
Supporting base I, cultivate 5 days～7 days, condition of culture is identical with step (1), and the induction of described green spheroids is pre-
Culture medium I is: 1/4MS～1/2MS+6-BA 1.0～2.0mg/L+NAA 0.2～0.4mg/L++ sucrose
30.0g/L+ agar 7.0g/L, pH are 5.80；
2. second time induction preculture: the stem apex after 1. for the first time step (2) is induced preculture is inoculated in green
Color Globular body induction pre-culture II, incubation time is 5 days～7 days, and condition of culture is identical with step (1),
Described green spheroids induction pre-culture II is: 1/4MS～1/2MS+2-ip 0.1～0.3mg/L+NAA
0.2～0.4mg/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
Color spheroid inducing culture, condition of culture is identical with step (1), and incubation time is 14 days～20 days,
Obtaining green spheroids, described green spheroids inducing culture is: 1/4MS～1/2MS+2-ip 0.5～
0.8mg/L+NAA0.2～0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(3) propagation of China's Caulis cyatheae spinulosae green spheroids
Each proliferating cycle sequentially includes the following steps:
1. first step enrichment culture: the green spheroids that step (2) 3. obtains is cut into 1～3mm, connects
Planting in proliferated culture medium A, cultivate 21 days～22 days, condition of culture is identical with step (1), described propagation
Culture medium A is: 1/4MS～1/2MS+TDZ 0.8～1.0mg/L+NAA 0.1～0.2mg/L+ sucrose
30.0g/L+ agar 7.0g/L, pH are 5.80；
2. second step enrichment culture: the green spheroids after being bred by (3) 1. first step enrichment culture is inoculated in
Proliferated culture medium B, cultivates 21 days～22 days, and condition of culture is identical with step (1), described enrichment culture
Base B is: 1/4MS～1/2MS+2-ip 0.1～0.2mg/L+6-BA 1.0～1.2mg/L+NAA0.05～
0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(4) differentiation of China's Caulis cyatheae spinulosae green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into diameter 1～3mm
Spheroid, is inoculated in division culture medium, cultivates 28 days～29 days, obtains breaking up seedling, condition of culture and step
Suddenly (1) is identical, and described division culture medium is: 1/6MS～1/4MS+ activated carbon 1～2g/L+ caseinhydrolysate
200～300mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(5) root culture
Differentiation seedling inoculation step (4) obtained is China in root media, differentiation seedling after taking root
Caulis cyatheae spinulosae tissue cultured seedling, condition of culture is identical with step (1), and described root media is: 1/2MS+ activated carbon 1～
2g/L+ caseinhydrolysate 200～300mg/L+ sucrose 30.0g/L+ agar concentration 7.0g/L, pH is 5.80.
The innovative point of the present invention:
1, the green spheroids of large-scale tree-shaped woody pteridophyta China Caulis cyatheae spinulosae has successfully been induced, green spherical
The inductivity of body reaches 82～88%.
The present invention passes through great many of experiments, has filtered out suitable large-scale tree-shaped woody pteridophyta China Caulis cyatheae spinulosae
Two green spheroids induction pre-culture and a green spheroids inducing culture.And devise twice induction
Preculture and an inducing culture step, successfully induced Chinese Caulis cyatheae spinulosae green spheroids, and green spherical
The inductivity of body reaches 82～88%.
2, on enrichment culture, filter out two proliferated culture mediums of A and B, and design each proliferating cycle
Successively with two proliferated culture mediums of A and B, both restrained effectively the differentiating phenomenon that breeding occurs, again
Achieve proliferation times and reach 7.5～9.3 times of especially significant technique effects, real on the premise of not breaking up
Existing high efficiently multiplying, maintains higher multiplication rate.The enrichment culture in several cycle need to be carried out, can basis
The seedling quantity needed in production determines.The present invention finds in research process, only with proliferated culture medium A,
Can Inhibited differentiation, but proliferation times is relatively low, and only with proliferated culture medium B, and proliferation times is higher but occurs point
Change phenomenon, and use proliferated culture medium A and proliferated culture medium B alternately successively as the inventive method described in
Enrichment culture, had both inhibited the differentiating phenomenon that breeding occurs, can achieve again the technique effect of high efficiently multiplying.
And be conducive to China's Caulis cyatheae spinulosae green spheroids as the propagation of middle brood body and preservation.
By the innovation of the present invention, compared with prior art, the present invention has a following benefit effect:
1, successfully induce tree-shaped woody pteridophyta green spheroids, and reproductive efficiency be the highest,
Two grades of focused protections of China are treasured to the expansion of endangered fern China Alsophila spinulosa population quantity, and journey in imminent danger
The alleviation of degree is significant.
The inventive method make green spheroids inductivity, green spheroids multiplication rate, green spheroids differentiation rate,
The efficiency that single green spherical differentiation Seedling number and differentiation Seedling are trained tissue cultured seedling is the highest, and its green is spherical
The inductivity of body is 82～88%, and the proliferation times of green spheroids reaches 7.5～9.3 times, dividing of green spheroids
Rate 100%, rooting rate 100%, the formation rate 100% of tissue cultured seedling, therefore, reproductive efficiency of the present invention is special
High.And the spore germination rate of existing sporogenesis approach is 34.33%, original foliage bunch breeds 2～3 times, spore
Body seedling planting percent is 24.00%.Compared with the breeding of existing spore approach, the reproductive efficiency of the present invention produces
Unforeseeable technique effect.
2, the breeding cycle is short
After the present invention induces green spheroids, i.e. efficiently can train using green spheroids as middle propagating materials
Bring out a large amount of tissue cultured seedling, it is not necessary to repeating and start to cultivate from spore, therefore, the breeding cycle is short.Spherical with green
Body is that middle propagating materials calculates, and the breeding cycle is 98 days～101 days, and the breeding of existing spore approach,
Breeding is both needed to start breeding from spore germination every time, and its breeding cycle needs 217 days～252 days, and the present invention is with often
The propagation method of rule sporogenesis approach is compared and is shortened 119～151 days, the breeding cycle shorten 54.8～
59.9%.
3, the in vitro conservation of Endangered species germ plasm resource is achieved
Chinese Caulis cyatheae spinulosae green spheroids and China's Caulis cyatheae spinulosae tissue cultured seedling that the method provided by the present invention is obtained can
Preserve as germ plasm resource, for realizing Precious, Rare, Endangered fern China Caulis cyatheae spinulosae germ plasm resource in vitro conservation tool
Significant.
Accompanying drawing explanation
Fig. 1 is China's Caulis cyatheae spinulosae green spheroids.Line segment in figure is scale, a length of 1mm of expression.
Fig. 2 is the Chinese Caulis cyatheae spinulosae green spheroids starting differentiation.Line segment in figure is scale, the length of expression
Degree is 1mm.
Specific implementation method
Below example facilitates a better understanding of the present invention, but does not limit the present invention.In following embodiment
Experimental technique, if no special instructions, is conventional method.Test material used in following embodiment is city
Sell.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
The preparation method of following embodiment culture medium be conventional method i.e. as described in culture medium prescription each component and
After the mixing of its content, pH is adjusted to be that the pH value that this culture medium requires i.e. is made with pH meter.
Embodiment 1 the inventive method
(1) acquisition of China's Caulis cyatheae spinulosae aseptic explant
By aseptic for China Caulis cyatheae spinulosae spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80
Culture medium on, condition of culture is: 20～25 DEG C, the light application time of every day be 12 hours, intensity of illumination is
2000lx, obtains the aseptic seedling that children is tender.In superclean bench, the aseptic seedling obtained is used tweezer under anatomical lens
Son separates stem apex, the aseptic explant induced as China's Caulis cyatheae spinulosae green spheroids by stem apex.
The preparation method of China's aseptic spore of Caulis cyatheae spinulosae is: be placed in 1.5ml centrifuge tube by 10mg spore, by nothing
Bacterium water soaking 1～2 hours, 6000r/min is centrifuged 2min, it is thus achieved that spore precipitate, adds volume fraction and is
5%NaClO solution sterilization 4min, sterile water wash i.e. obtains the aseptic spore of Chinese Caulis cyatheae spinulosae for 3-5 time.
(2) induction of China's Caulis cyatheae spinulosae green spheroids
1. preculture is induced for the first time: the stem apex that step (1) obtains is inoculated in the pre-training of green spheroids induction
Supporting in base I, cultivate 7 days, condition of culture is identical with step (1), described green spheroids induction preculture
Base I is: 1/4MS+6-BA 1.5mg/L+NAA0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is
5.80；Inoculate several 50 stem apexs.
2. second time induction preculture: the stem apex after 1. for the first time step (2) is induced preculture is inoculated in green
In color Globular body induction pre-culture II, incubation time is 7 days, and condition of culture is identical with step (1), institute
Stating green spheroids induction pre-culture II is: 1/4MS+2-ip 0.3mg/L+NAA 0.2mg/L+ sucrose
30.0g/L+ agar 7.0g/L, pH are 5.80.
3. the induction of green spheroids: the stem apex after step (2) 2. second time induction preculture is inoculated in green
In color spheroid inducing culture, incubation time is 14 days, and condition of culture is identical with step (1), obtains green
Spheroid, described green spheroids inducing culture is: 1/4MS+2-ip 0.8mg/L+NAA 0.2mg/L+
Sucrose 30.0g/L+ agar 7.0g/L, pH are 5.80.
After induction preculture and inducing culture, the stem apex of China Caulis cyatheae spinulosae expands formation green spheroids, green ball
The inductivity of shape body is 88.00%.
Inductivity=(producing the aseptic explant number of the aseptic seedling number/inoculation of green spheroids) × 100%.
(3) propagation of China's Caulis cyatheae spinulosae green spheroids
Carry out enrichment culture each proliferating cycle according to the following steps:
1. first step enrichment culture: the green spheroids that step (2) 3. obtains is cut into 2mm, inoculation
On proliferated culture medium A, cultivating 21 days, condition of culture is identical with step (1), described proliferated culture medium A
For: 1/2MS+TDZ 0.8mg/L+NAA 0.1mg/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 enrichment culture: the green spheroids after being bred by (3) 1. first step enrichment culture is inoculated in
On proliferated culture medium B, cultivating 21 days, condition of culture is identical with step (1), described proliferated culture medium B
For: 1/2MS+2-ip 0.2mg/L+6-BA 1.0mg/L+NAA 0.1mg/L+ sucrose 30.0g/L+ agar
7.0g/L, pH are 5.80.
The green spheroids mean fresh increments of every 21 days is 75mg, and proliferation times is about 7.5 times, and increases
Green spheroids differentiating phenomenon does not occurs during growing.Each enrichment culture cycle is carried out by said sequence, need to enter
The enrichment culture in row several cycles, the tissue cultured seedling quantity according to needing on producing determines.
(4) differentiation of China's Caulis cyatheae spinulosae green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into the ball of diameter 2mm
Shape body, is inoculated on division culture medium, cultivates 28 days, obtains breaking up seedling, and condition of culture is and step (1)
Identical；Described division culture medium is: 1/6MS+ activated carbon 1g/L+ caseinhydrolysate 200mg/L+ sucrose
30.0g/L+ agar 7.0g/L, pH are 5.80.
Inoculating 50 green spheroids of number, average mark rate is 100%.
Differentiation rate=(quantity of the green spheroids of the green spheroids quantity/inoculation of differentiation occurs) × 100%.
(5) root culture
Being cultivated on root media by the differentiation seedling inoculation that step (4) obtains, differentiation seedling after taking root is
For China's Caulis cyatheae spinulosae tissue cultured seedling, condition of culture is identical with step (1), and described root media is: 1/2MS+
Activated carbon 1g/L+ caseinhydrolysate 200mg/L+ sucrose 30.0g/L+ agar concentration 7.0g/L, pH is 5.80.
Cultivating 4 weeks, the average height of China's Caulis cyatheae spinulosae tissue cultured seedling reaches 2.87cm.Differentiation seedling all takes root and survives
Form tissue cultured seedling, the formation rate 100% of rooting rate 100%, i.e. tissue cultured seedling.
Embodiment 2 and embodiment 3 are the inventive method
Embodiment 2 is with embodiment 3 in addition in culture medium each listed by table 1, the concentration of each composition is different, and remaining is arranged
Shi Jun is same as in Example 1, repeats no more.
The each step of table 1 embodiment 2-embodiment 3 and the difference of 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 tissue cultured seedling, root culture 28 days, average height 2.87cm of tissue cultured seedling.
The green spheroids fresh weight 10mg of a diameter of 2mm, every 21 days mean freshs of green spheroids increase 75mg,
Proliferation times is about 7.5 times.With the green spheroids of acquisition for middle brood body, through two step enrichment cultures for 42
My god, differentiation culture 28 days, root culture form tissue cultured seedling 28 days, repoductive time for 98 days.
The Breeding results of embodiment 2: the inductivity of green spheroids is 85.33%, differentiation rate 100% is taken root
The formation rate 100% of rate 100%, i.e. tissue cultured seedling, root culture 28 days, average height 3.10cm of tissue cultured seedling.
The green spheroids fresh weight 5mg of a diameter of 1mm, every 21 days mean freshs of green spheroids increase
46.67mg, proliferation times is about 9.3 times.With the green spheroids of acquisition for middle brood body, increase through two steps
Grow cultivation confession 44 days, differentiation culture 29 days, root culture forms tissue cultured seedling 28 days, repoductive time supplies 101
My god.
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 tissue cultured seedling, root culture 28 days, average height 2.70cm of tissue cultured seedling.
The green spheroids fresh weight 10mg of a diameter of 2mm, every 21 days mean freshs of green spheroids increase
83.67mg, proliferation times is about 8.4 times.With the green spheroids of acquisition for middle brood body, increase through two steps
Grow cultivation confession 42 days, differentiation culture 28 days, root culture forms tissue cultured seedling 28 days, repoductive time supplies 98
My god.
The green spheroids of one 1～3mm is once broken up can obtain 20～25 strain tissue cultured seedlinies.
Embodiment 4 compares
Embodiment 4 is the propagation method of existing sporogenesis approach, and step is as follows:
(1) China's Caulis cyatheae spinulosae spore sterilization
12mg China Caulis cyatheae spinulosae spore is placed in 1.5ml centrifuge tube, soaks 1-2 hour with sterilized water,
6000r/min is centrifuged 2min, it is thus achieved that spore precipitate, and adding volume fraction is 5%NaClO solution sterilization 4min,
Sterile water wash i.e. obtains the aseptic spore of Chinese Caulis cyatheae spinulosae for 3-5 time.
(2) China's Caulis cyatheae spinulosae spore germination is cultivated
The aseptic spore inoculating of Chinese Caulis cyatheae spinulosae step (1) obtained is in 1/4MS+ sucrose 30.0g/L+ agar
7.0g/L, pH are 5.80, and inoculation quantity is 10 bottles, and 20～25 DEG C of cultivations, the light application time of every day is 12
Hour, intensity of illumination is 2000lx.
After cultivating 6 weeks, spore starts to sprout, and germination rate is about 34.33%, after continuing to cultivate 9～10 weeks,
Form original foliage bunch.Spore count × 100% of the spore count/inoculation of germination rate=sprouting.
(3) China's Caulis cyatheae spinulosae original foliage enrichment culture
The original foliage bunch obtained in step (2) is divided into fritter, continues 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,
Original foliage bunch breeds 2～3 times.
Original foliage bunch after propagation is divided into fritter, continues to be inoculated in 1/4MS+ sucrose 30.0g/L+ agar
7.0g/L, pH are 5.80, and inoculation quantity is 50 pieces, and drips appropriate sterilized water on original foliage, to promote
Enter the formation of sporinite, the same step of condition of culture (1).
After cultivating 8～12 weeks, gradually having sporinite seedling to be formed, sporinite seedling planting percent is 30.67%.
Original foliage number × 100% of the original foliage number/inoculation of sporinite seedling planting percent=generation sporinite seedling.
(4) cultivation of China Caulis cyatheae spinulosae sporinite seedling
By the sporinite seedling inoculation of acquisition in step (3) in 1/2MS+ activated carbon 2g/L+ sucrose 30.0g/L+
Agar 7.0g/L, pH are 5.80.
After cultivating 4 weeks tissue cultured seedling, China Caulis cyatheae spinulosae tissue cultured seedling average height be 2.57cm.
Comparative examples 4 shows: 42 days time is cultivated in spore germination, forms original foliage time 63～70 days,
28 days enrichment culture time, the cultivation time that sporinite is formed is 56 days～84 days, when cultivating tissue cultured seedling
Between 28 days, therefore, the tissue-culturing rapid propagation of sporogenesis approach, from spore germination to cultivate into tissue cultured seedling need 217
It～252 days.Spore germination rate is 34.33%, and original foliage bunch breeds 2～3 times, sporinite seedling planting percent
It is 24.00%.Existing spore approach is bred, and breeding is both needed to start to breed from spore germination every time, therefore,
Breeding cycle is both needed to 217 days～252 days every time.
Above-described embodiment 1 to-embodiment 3 shows: the inductivity of green spheroids of the present invention 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 tissue cultured seedling, therefore, reproductive efficiency is the highest.After the present invention induces green spheroids,
I.e. can be using green spheroids as middle propagating materials, high effect culture goes out a large amount of tissue cultured seedling, it is not necessary to repeat from spore
Son starts to cultivate, and therefore, the breeding cycle is short.Calculate with green spheroids for middle propagating materials, the breeding cycle
It it is 98 days～101 days.
Claims (1)
1. the tissue culture propagation method of a Chinese Caulis cyatheae spinulosae green spheroids approach, it is characterised in that: comprise the following steps:
(1) acquisition of China's Caulis cyatheae spinulosae aseptic explant
By aseptic for China Caulis cyatheae spinulosae spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is in the culture medium of 5.80, condition of culture is: 20～25 DEG C, the light application time of every day be 12 hours, intensity of illumination is 2000lx, obtain aseptic seedling, at superclean bench, aseptic seedling is separated stem apex with tweezers under anatomical lens, the aseptic explant induced by stem apex as China's Caulis cyatheae spinulosae green spheroids；
(2) induction of China's Caulis cyatheae spinulosae green spheroids
1. preculture is induced for the first time: the stem apex that step (1) obtains is inoculated in green spheroids induction pre-culture I, cultivate 5 days～7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture I is: 1/4MS～1/2MS+6-BA 1.0～2.0mg/L+NAA 0.2～0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
2. second time induction preculture: the stem apex after 1. for the first time step (2) is induced preculture is inoculated in green spheroids induction pre-culture II, incubation time is 5 days～7 days, condition of culture is identical with step (1), described green spheroids induction pre-culture II is: 1/4MS～1/2MS+2-ip 0.1～0.3mg/L+NAA 0.2～0.4mg/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, 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-ip 0.5～0.8mg/L+NAA 0.2～0.4mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(3) propagation of China's Caulis cyatheae spinulosae green spheroids
Each proliferating cycle sequentially includes the following steps:
1. first step enrichment culture: the green spheroids that step (2) 3. obtains is cut into 1～3mm, it is 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+TDZ 0.8～1.0mg/L+NAA 0.1～0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
2. second step enrichment culture: the green spheroids after being bred by (3) 1. first step enrichment culture 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-ip 0.1～0.2mg/L+6-BA 1.0～1.2mg/L+NAA 0.05～0.1mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(4) differentiation of China's Caulis cyatheae spinulosae green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into the spheroid of diameter 1～3mm, it is inoculated in division culture medium, cultivate 28 days～29 days, obtain breaking up seedling, condition of culture is identical with step (1), described division culture medium is: 1/6MS～1/4MS+ activated carbon 1～2g/L+ caseinhydrolysate 200～300mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80；
(5) root culture
Differentiation seedling inoculation step (4) obtained is in root media, differentiation seedling is China's Caulis cyatheae spinulosae tissue cultured seedling after taking root, condition of culture is identical with step (1), described root media is: 1/2MS+ activated 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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