CN105145364B - A kind of tissue culture propagation method of Cibotium barometz (L.) J. Sm. green spheroids approach - Google Patents
A kind of tissue culture propagation method of Cibotium barometz (L.) J. Sm. green spheroids approach Download PDFInfo
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Abstract
The present invention discloses the tissue culture propagation method of a kind of Cibotium barometz (L.) J. Sm. 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 Precious, Rare, Endangered fern Cibotium barometz (L.) J. Sm. green spheroids, reproductive efficiency is high, its green spheroids inductivity reaches 90～94%, proliferation times reaches 9.5～10.2 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 Cibotium barometz (L.) J. Sm. 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 the induction of Cibotium barometz (L.) J. Sm. green spheroids and enrichment procedure.
Technical background
Cibotium barometz (L.) J. Sm. (Cibotium barometz), is under the jurisdiction of Dicksoniaceae Cibotium, and root stock is flat sleeping, thick, end
Upwarping, part of basseting is close, and by golden yellow villus, the Cibotium barometz (L.) J. Sm. that shape is seemingly thrown oneself on the ground, form is unique, has ornamental value, golden fine and soft
Hair can be used as medicine, and is incorporated into the Pharmacopoeia of the People's Republic of China." national key protected wild plants register (first) " is by concha Anodonta seu Cristaria
All kinds of Pteridiaceae are all classified as national II Top-rated protected wild plants, and CONTINENTAL AREA OF CHINA Dicksoniaceae only 1 belongs to a kind, it may be assumed that gold
The Cibotium barometz (L.) J. Sm. that hair Canis familiaris L. belongs to.
Under natural conditions, Cibotium barometz (L.) J. Sm. uses spore to breed, but spore germination and Development of Gametophytes are to environmental condition
Require more strict, cause its natural propagation ability extremely low, in addition environmental disruption and artificial digging, the wild stocks number of Cibotium barometz (L.) J. Sm.
Amount drastically reduces, endangered.
At present, the tissue culture of Cibotium barometz (L.) J. Sm. is limited to sporogenesis approach more, its method specifically include that spore germination cultivate,
, there is the defects such as breeding cycle length, sporinite inductivity be low in the steps such as original foliage cultivation, sporinite cultivation, root culture, because of
This, find a kind of breeding cycle and short breed the method that efficiency is high, for Precious, Rare, Endangered fern Cibotium barometz (L.) J. Sm. population quantity simultaneously
Expand, and the alleviation of degree in imminent danger is significant.
Pteridophyta green spheroids referred to by pteridophyta outer implant induction being made up of numerous green particles of obtaining
Spheroid, its essence is separate living tissue aggregation, is inoculated on division culture medium and can obtain a large amount of pteridophyta seedling, once
Induction obtains green spheroids, and its breeding coefficient will be significantly higher than that sporogenesis approach, and the breeding cycle shortens.
Owing to outer implant selects and suitable phytohormone screening aspect, at present, the most successfully induce green
The kind of spheroid is relatively limited, and in green spheroids breeding, only selects suitable phytohormone formula and training
The mode of supporting, could realize fast breeding while suppression green spheroids differentiation.Therefore, at present, there is no Precious, Rare, Endangered fern
Cibotium barometz (L.) J. Sm. is by the relevant report of green spheroids approach tissue culture propagation research.
Summary of the invention
The technical problem to be solved in the present invention is to overcome Cibotium barometz (L.) J. Sm. natural propagation power low, numerous by the training of sporogenesis approach group
Cycle of growing 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 method of a kind of Cibotium barometz (L.) J. Sm. green spheroids approach,
The method comprises the following steps:
(1) acquisition of Cibotium barometz (L.) J. Sm. aseptic explant
By aseptic for Cibotium barometz (L.) J. Sm. spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is the culture medium of 5.80
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,
Superclean bench, separates stem apex with tweezers by aseptic seedling under anatomical lens, is induced as Cibotium barometz (L.) J. Sm. green spheroids by stem apex
Aseptic explant；
(2) induction of Cibotium barometz (L.) J. Sm. 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,
Cultivating 5 days～7 days, condition of culture is identical with step (1), and described green spheroids induction pre-culture I is: 1/4MS～1/2MS
+ 6-BA 1.0～1.5mg/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 spherical
Body induction pre-culture II, incubation time is 5 days～7 days, and condition of culture is identical with step (1), and described green spheroids is induced
Pre-culture II is: 1/4MS～1/2MS+2-ip 0.1～0.3mg/L+NAA0.2～0.4mg/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 spherical
Body inducing culture, condition of culture is identical with step (1), and incubation time is 14 days～20 days, obtains green spheroids, described green
Color spheroid inducing culture is: 1/4MS～1/2MS+TDZ 0.6～0.8mg/L+NAA0.2～0.4mg/L+ sucrose 30.0g/
L+ agar 7.0g/L, pH are 5.80；
(3) propagation of Cibotium barometz (L.) J. Sm. green spheroids
Each proliferating cycle is carried out in the following order:
1. first step enrichment culture: the green spheroids that step (2) 3. obtains is cut into 1～3mm, is inoculated in propagation training
Supporting base A, cultivate 18 days～21 days, condition of culture is identical with step (1), and described proliferated culture medium A is: 1/4MS～1/2MS+TDZ
0.8～1.2mg/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 propagation training
Supporting base B, cultivate 18 days～21 days, condition of culture is identical with step (1), and 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
It is 5.80；
(4) differentiation of Cibotium barometz (L.) J. Sm. green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into the spheroid of diameter 1～3mm,
Being inoculated in division culture medium, cultivate 28 days～29 days, obtain breaking up seedling, condition of culture is identical with step (1), described differentiation training
Foster base is: 1/6MS～1/4MS+ activated carbon 1～2g/L+ caseinhydrolysate 200～300mg/L+ sucrose 30.0g/L+ agar
7.0g/L, pH are 5.80；
(5) root culture
Differentiation seedling inoculation step (4) obtained is in root media, and differentiation seedling is the training of Cibotium barometz (L.) J. Sm. group after taking root
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 Precious, Rare, Endangered pteridophyta Cibotium barometz (L.) J. Sm., and the inductivity of green spheroids have successfully been induced
Reach 90～94%.
The present invention passes through great many of experiments, has filtered out two green spheroids of suitable Precious, Rare, Endangered pteridophyta Cibotium barometz (L.) J. Sm.
Induction pre-culture and a green spheroids inducing culture.And devise twice induction preculture and an inducing culture step
Suddenly, Cibotium barometz (L.) J. Sm. green spheroids has successfully been induced.
2, on enrichment culture, filter out two proliferated culture mediums of A and B, and design each proliferating cycle with successively with A and
Two proliferated culture mediums of B, both restrained effectively the differentiating phenomenon that breeding occurs, and had achieved again proliferation times and reach 9.5
～10.2 times of especially significant technique effects, on the premise of not breaking up, realize high efficiently multiplying, maintain higher
Multiplication rate.The enrichment culture in several cycle need to be carried out, can determine according to the seedling quantity needed on producing.The present inventor is grinding
Find during studying carefully, only with proliferated culture medium A, can Inhibited differentiation, but proliferation times is relatively low, and only with proliferated culture medium B,
Proliferation times is higher but differentiating phenomenon occurs, and hands over proliferated culture medium A and proliferated culture medium B successively as described in the inventive method
For the enrichment culture carried out, both inhibited the differentiating phenomenon that breeding occurs, the technique effect of high efficiently multiplying can have been achieved again.
And beneficially Cibotium barometz (L.) J. Sm. green spheroids is as the propagation of middle brood body and preservation.
By the innovation of the present invention, compared with prior art, the present invention has the beneficial effect that:
1, Precious, Rare, Endangered pteridophyta Cibotium barometz (L.) J. Sm. green spheroids, and the inductivity etc. of green spheroids are successfully induced
Reproductive efficiency is the highest, two grades of focused protections of China is treasured to the expansion of endangered fern Cibotium barometz (L.) J. Sm. group's quantity, and in imminent danger
The alleviation of degree is significant.
The inventive method makes green spheroids inductivity, green spheroids multiplication rate, green spheroids differentiation rate, single
The efficiency that green spherical differentiation Seedling number and differentiation Seedling are trained tissue cultured seedling is the highest, and 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 the highest；And the spore of existing sporogenesis approach
Body inductivity is only 28.30%, and compared with the breeding of existing spore approach, the reproductive efficiency of the present invention creates unforeseeable
Technique effect.
2, the breeding cycle is short
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 seedlinies, it is not necessary to repeating and start to cultivate from spore, therefore, the breeding cycle is short.Material is bred for centre with green spheroids
Material calculates, and the breeding cycle is 92 days～98 days, and the existing spore approach breeding cycle needs about 224 days～284 days (its miospores
Sprout and form original foliage 60-90 days, original foliage inductive formation sporinite 80-110 days, sporinite is bred 42 days, is taken 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 in vitro conservation of Endangered species Cibotium barometz (L.) J. Sm. germ plasm resource is achieved
Cibotium barometz (L.) J. Sm. green spheroids and Cibotium barometz (L.) J. Sm. tissue cultured seedling that the method provided by the present invention is obtained can be as kind of matter
Resource preserves, significant for realizing Precious, Rare, Endangered fern Cibotium barometz (L.) J. Sm. germ plasm resource in vitro conservation.
Accompanying drawing explanation
Fig. 1 is Cibotium barometz (L.) J. Sm. green spheroids.Line segment in figure is scale, a length of 1mm of expression.
Fig. 2 is the Cibotium barometz (L.) J. Sm. green spheroids starting differentiation.Line segment in figure is scale, a length of 1mm of expression.
Specific implementation method
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment is commercially available.Following example
In quantitative test, be respectively provided with three times repeat experiment, results averaged.
The preparation method of following embodiment culture medium is conventional method i.e. each component and containing as described in culture medium prescription
After amount mixing, 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 Cibotium barometz (L.) J. Sm. aseptic explant
By aseptic for Cibotium barometz (L.) J. Sm. spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is the culture medium of 5.80
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 children tender aseptic
Seedling.In superclean bench, the aseptic seedling obtained is separated stem apex with tweezers under anatomical lens, stem apex is green as Cibotium barometz (L.) J. Sm.
The aseptic explant of color Globular body induction.
The preparation method of the aseptic spore of Cibotium barometz (L.) J. Sm. is: is placed in 1.5ml centrifuge tube by 10mg spore, soaks 1-with sterilized water
2 hours, 6000r/min was centrifuged 2min, it is thus achieved that spore precipitate, and adding volume fraction is 5%NaClO solution sterilization 4min, nothing
Bacterium water cleans and i.e. obtains the aseptic spore of Cibotium barometz (L.) J. Sm. for 3-5 time.
(2) induction of Cibotium barometz (L.) J. Sm. 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
In, to cultivate 6 days, condition of culture is identical with step (1), and described green spheroids induction pre-culture I is: 1/4MS+6-BA
1.5mg/L+NAA 0.2mg/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 spherical
In body induction pre-culture II, incubation time is 6 days, and condition of culture is identical with step (1), the pre-training of described green spheroids induction
Supporting base II is: 1/4MS+2-ip 0.3mg/L+NAA 0.2mg/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 spherical
In body inducing culture, incubation time is 14 days, and condition of culture is identical with step (1), obtains green spheroids, and described green is spherical
Body inducing culture is: 1/4MS+TDZ 0.8mg/L+NAA 0.2mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 5.80.
After induction preculture and inducing culture, the stem apex of Cibotium barometz (L.) J. Sm. expands formation green spheroids, green spheroids
Inductivity is 94.00%.
Inductivity=(producing the aseptic explant number of the aseptic seedling number/inoculation of green spheroids) × 100%.
(3) propagation of Cibotium barometz (L.) J. Sm. 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 2mm, is inoculated in enrichment culture
On base A, cultivating 18 days, condition of culture is identical with step (1), and described proliferated culture medium A is: 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 8mg, 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 propagation training
Supporting on base B, cultivate 18 days, condition of culture is identical with step (1), and described proliferated culture medium B is: 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 is 5.80.
The green spheroids mean fresh increments of every 18 days is 76mg, average 9.5 times of proliferation times, and in breeding
Green spheroids differentiating phenomenon does not occurs.Each enrichment culture cycle is carried out by said sequence, need to carry out the propagation in several cycle
Cultivating, the tissue cultured seedling quantity according to needing on producing determines.
(4) differentiation of Cibotium barometz (L.) J. Sm. green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into the spheroid of diameter 2mm, inoculation
On division culture medium, cultivating 28 days, obtain breaking up seedling, condition of culture is identical with step (1)；Described division culture medium
For: 1/6MS+ activated carbon 1g/L+ caseinhydrolysate 200mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH is 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 is gold hair after taking root
Canis familiaris L. 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 Cibotium barometz (L.) J. Sm. tissue cultured seedling reaches 3.15cm.Differentiation seedling all takes root and survives the training of formation group
Seedling, the production rate of tissue cultured seedling reaches 100%.
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 measure is all with real
Execute example 1 identical, repeat 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 94%, differentiation rate 100%, rooting rate 100%, i.e.
The formation rate 100% of tissue cultured seedling, root culture 28 days, average height 3.15cm of tissue cultured seedling.The green spheroids of a diameter of 2mm
Fresh weight 8mg, every 18 days mean freshs of green spheroids increase 76mg, average 9.5 times of proliferation times.With the green spheroids obtained
For middle brood body, through two step enrichment cultures for 36 days, differentiation culture 28 days, root culture form tissue cultured seedling 28 days, breeding time
Between 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 tissue cultured seedling, root culture 28 days, average height 3.08cm of tissue cultured seedling.A diameter of 1mm's is green
Color spheroid fresh weight 4.5mg, every 20 days mean freshs of green spheroids increase 45.9mg, average 10.2 times of proliferation times.To obtain
The green spheroids obtained is middle brood body, through two step enrichment cultures confessions 40 days, differentiation culture 29 days, the training of root culture formation group
Seedling 28 days, repoductive time were 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 tissue cultured seedling, root culture 28 days, average height 3.30cm of tissue cultured seedling.The green spheroids of a diameter of 2mm
Fresh weight 8mg, every 21 days mean freshs of green spheroids increase 77.6mg, average 9.7 times of proliferation times.Green with acquisition is spherical
Body is middle brood body, forms tissue cultured seedling 28 days, breeding through two step enrichment cultures confessions 42 days, differentiation culture 28 days, root culture
Time was for 98 days.
The green spheroids of one 1～3mm is once broken up can obtain 20～25 strain tissue cultured seedlinies.
Above-described embodiment 1 to embodiment 3 shows: the inductivity of green spheroids of the present invention is 90～94%, green spherical
The proliferation times of body reaches 9.5～10.2 times, the differentiation rate 100% of green spheroids, rooting rate 100%, the formation rate of tissue cultured seedling
100%, therefore, reproductive efficiency is the highest.After the present invention induces green spheroids, i.e. can be numerous using green spheroids as centre
Growing material, high effect culture goes 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.With green ball
Shape body is that middle propagating materials calculates, and the breeding cycle is 92 days～98 days.
Claims (1)
1. the tissue culture propagation method of a Cibotium barometz (L.) J. Sm. green spheroids approach, it is characterised in that comprise the following steps:
(1) acquisition of Cibotium barometz (L.) J. Sm. aseptic explant
By aseptic for Cibotium barometz (L.) J. Sm. spore inoculating at 1/4MS+ sucrose 30.0g/L+ agar 7.0g/L, pH is in the culture medium of 5.80, training
Foster condition is: 20～25 DEG C, the light application time of every day be 12 hours, intensity of illumination is 2000lx, obtains aseptic seedling, in ultra-clean work
Station, separates stem apex with tweezers by aseptic seedling under anatomical lens, using stem apex as Cibotium barometz (L.) J. Sm. green spheroids induce aseptic
Outer implant；
(2) induction of Cibotium barometz (L.) J. Sm. 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, cultivates 5
It～7 days, condition of culture is identical with step (1), and described green spheroids induction pre-culture I is: 1/4MS～1/2MS+6-BA
1.0～1.5mg/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 and lures
Leading pre-culture II, incubation time is 5 days～7 days, and condition of culture is identical with step (1), the pre-training of described green spheroids induction
Supporting base 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 are 5.80；
3. the induction of green spheroids: the stem apex after step (2) 2. second time induction preculture is inoculated in green spheroids and lures
Leading culture medium, condition of culture is identical with step (1), and incubation time is 14 days～20 days, obtains green spheroids, described green ball
Shape body inducing culture is: 1/4MS～1/2MS+TDZ 0.6～0.8mg/L+NAA 0.2～0.4mg/L+ sucrose 30.0g/L+
Agar 7.0g/L, pH are 5.80；
(3) propagation of Cibotium barometz (L.) J. Sm. green spheroids
Each proliferating cycle is carried out in the following order:
1. first step enrichment culture: the green spheroids that step (2) 3. obtains is cut into 1～3mm, is inoculated in proliferated culture medium
A, cultivates 18 days～21 days, and condition of culture is identical with step (1), and described proliferated culture medium A is: 1/4MS～1/2MS+TDZ 0.8
～1.2mg/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, cultivates 18 days～21 days, and condition of culture is identical with step (1), and 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 Cibotium barometz (L.) J. Sm. green spheroids
Green spheroids after being bred by step (3) 2. second step enrichment culture is divided into the spheroid of diameter 1～3mm, inoculation
In division culture medium, cultivating 28 days～29 days, obtain breaking up seedling, condition of culture is identical with step (1), described division culture medium
For: 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 Cibotium barometz (L.) J. Sm. tissue cultured seedling in root media, differentiation seedling after taking root, training
Support condition 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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