CN108184678A - A kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent - Google Patents
A kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent Download PDFInfo
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- CN108184678A CN108184678A CN201810296053.0A CN201810296053A CN108184678A CN 108184678 A CN108184678 A CN 108184678A CN 201810296053 A CN201810296053 A CN 201810296053A CN 108184678 A CN108184678 A CN 108184678A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent, the growth promoting bacteria agent is made of Strepiomyces lavendulae, bacillus amyloliquefaciens, bacillus pumilus, Nafusaku, activated carbon, sucrose.The present invention promotes to establish the Regeneration in Vitro rapid propagation system of Cultivars of Chinese Herbaceous Peony paenoiae alba, and one of important channel to solve the shortage of Chinese herbaceous peony seedling all has very important significance to the shifting of Chinese herbaceous peony south and the exploitation of Chinese herbaceous peony and utilization and extention, has broad application prospects.
Description
Technical field
The present invention relates to the propagation methods of Chinese herbaceous peony tissue cultures, belong to the technical field of herbaceous ornamental sapling multiplication,
More particularly to a kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent.
Background technology
Chinese herbaceous peony（Scientific name：Paeonia lactiflora Pall.）, alias takes leave of grass, spends middle chancellor, belongs to Dilleniales, Chinese herbaceous peony
Medicine section Paeonia Chinese herbaceous peony group perennial herb.For root tuber by bearing below rootstock, meat is sturdy, in spindle or long cylindricality, slightly
0.6~3.5cm.Chinese herbaceous peony petal is in obovate, and floral disc is tazza shape, spends the general top for being born in stem or near in 5~June of florescence
At the axil of top, the grey color of original seed, 5~13 pieces of petal.Garden-variety pattern enriches, and has white, powder, red, purple, yellow, green, black and multiple
Color etc., 10~30 centimetres of Hua Jing, petal is up to up to a hundred pieces.Fruit is in spindle, and seed is rounded, oblong or point are round.Chinese herbaceous peony
Medicine light is shone, drought-enduring.For Chinese herbaceous peony plant in 1 year, the phasic development variation generated with the variation of the weather rhythm and pace of moving things is main
Show as the alternating variation of growth period and rest period.Wherein closed the most with the photostage in the period of vernalization of rest period and growth period
It is strong.The period of vernalization of Chinese herbaceous peony, it is desirable that under 0 DEG C of low temperature, by that could complete for 40 days or so and then mixed bud can sprout life
Long Paeonias long-day plant, bud will be developed under the long-day and be bloomed, after mixed bud is sprouted, if light application time it is insufficient or
It usually only comes into leaves and does not bloom or bloom exception under the conditions of short-day.
Chinese herbaceous peony is distributed in the areas such as China northeast, North China, Shaanxi and SOUTH OF GANSU, and in China, economic height is flourishing, lives
The very big the south of the lower reaches of the Yangtze River of horizontal relatively high, spiritual cultural demand is not always the center of Chinese herbaceous peony cultivation and landscape application, so far
The kind for being capable of expanded reference is very rare, and there is serious hot evil, damage or crop failure caused by waterlogging, the bad, pest and disease damage of blooming is serious and underground
The problems such as root tuber is degenerated, also short there are the florescence, the problem of growing way dies down year by year.
Chinese herbaceous peony only has the skilful medicine garden in Fish Wonder at Huagang Crook park, western green dike of small stream wetland park etc. in the concentration in Hangzhou plantation point at present, is made
Kind, gimmick, place and form Characteristic landscape quantity far away from tree peony, the reason of causing the present situation also mainly by
Cause cold accumulation insufficient so as to influence to sprout with blooming in Jiangnan winter temperature is higher, spring temperature is higher to lead to scape apprentice
It is long, hang one's head with the florescence it is too short and and summer it is extremely hot it is moist cause burn serious day, hot evil and the sick insect pest of evil etc..
' paenoiae alba ' blooms relatively early, and it is many that the flowers are in blossom, and pollen amount is big, and ' paenoiae alba ' breaks the minimum of bud dormancy under natural low temperature
Chilling requirement is 540CH and 665CU, can ensure that faster germination rate, more stem number and single plant are averagely bloomed number；Bud dormancy
The best chilling requirement released is 795CH and 906CU, can be from the not low-priced of bud is faster ingeniously broken, and guarantee is flourishing, it is numerous to bloom
It contains.Processing of the chilling requirement for 672CH unified with nature and manual temperature in the case where artificial hypothermia is handled, breaks ' paenoiae alba ' bud and stops
Free time ensures that smooth growth and the chilling requirement bloomed are 672-795CH, is the preferable kind of growth adaptability, is worth in Hangzhou
The south of the lower reaches of the Yangtze River is waited to consolidate and are promoted the use of or be preferably used as the parent for cultivating outstanding Jiangnan product section in woods.
Although paenoiae alba demand is huge, how fast and efficiently to be carried out expanding it is numerous, be pendulum be badly in need of in face of grower
The technical issues of solution.
The current most common propagation method of Chinese herbaceous peony is division propagation and seed propagation, although the former can keep the property of original kind
Shape, but need plant strain growth that could divide for 3~5 years once, and the period is longer, breeding coefficient is relatively low, and the maternal plant after plant division watches
Value is decreased obviously；The latter can neither keep the character of original kind, and need just bloom by 4~5 juvenile phase, therefore, two
Person is not suitable for being commercially produced on a large scale.Plant Tissue Breeding is a kind of maximally efficient side of micropropagation of plants
Method, compared with Traditional breeding processes, tissue cultures can keep original kind and merit, expand its breeding coefficient, and not
It is influenced, can be operated with the anniversary by season, the efficiency of breeding has been significantly increased.CN102428872 discloses one kind and passes through
The method that Seed embryo culture obtains complete Chinese herbaceous peony plant, but since embryo can not keep maternal character completely, cannot use
It is numerous in the expansion of improved seeds.Beautiful et al. the papers delivered of Wu Hong：Herbaceous peony ' big rich and honour ' inducing clumping bud and rooting technique,
' Chinese herbaceous peony different cultivars Primary culture the compares and research of ' cinnabar is sentenced ' inducing clumping bud ' is reported using underground resting bud as explant
The method that body induces Multiple Buds, this method have the following disadvantages for the expansion of improved seeds is numerous：1. the ground that a plant is included
Lower resting bud limited amount is not high using its reproductive efficiency as explant；2. every plant of tissue-cultured seedling can only generate 3~5 Multiple Buds,
Breeding coefficient is not high；3. whole process is subjected to the process of a underground bud Primary culture, a large amount of human and material resources are expended,
Improve cost.The inductive technology of paper ' Chinese herbaceous peony adventitious bud that Hu Yingquan et al. is delivered ' (Hu Yingquan, Feng Haihua, when treasured insult Chinese herbaceous peonies
The inductive technology of adventitious bud, Shanxi Forestry science and technology, 2003, supplementary issue:23-24,33.) tissue cultures are carried out to Chinese herbaceous peony tender stem, in M
On S+BA 3.5mg/L+ sucrose 30g/L+ agar 6g/L culture mediums can a step into bud, shorten into bud time, but the paper data
More ambiguous, the time of statistics does not also hand over, and the inductivity highest of final bud only has 74%.The paper that Sun Xiao plums et al. are delivered
' research of Chinese herbaceous peony callus induction and adventitious buds differentiation ' (Sun Xiaomei, Cheng Wan, Liu Ping, Zhou Wenqiang, wangdan, Yang Hong light Chinese herbaceous peonies
The research Agricultural University Of Shenyang journal of callus induction and adventitious buds differentiation, 2014-02,45 (1):24-27.) to Chinese herbaceous peony
Leaf, stem, blade are cultivated, and using base portion stem as optimal adventitious buds differentiation explant, but differentiation rate only has 35%, and need by
Callus induction period.In conclusion in Chinese herbaceous peony tissue culture procedures, the prior art successfully differentiates adventitious bud, but he
Mostly with embryo, underground resting bud, stem section etc. for explant, not only differentiation rate is low, but also subculture number is more, and experimentation is numerous
Trivial, the period is longer, can not obtain ideal effect.
To sum up, the Regeneration in Vitro rapid propagation system of Cultivars of Chinese Herbaceous Peony paenoiae alba is established, to solve the important way of Chinese herbaceous peony seedling shortage
One of diameter all has very important meaning to the shifting of Chinese herbaceous peony south and the exploitation of Chinese herbaceous peony and utilization and extention.
Invention content
The present invention solves the technical problem of disclose a kind of to promote paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent
Method.
In order to achieve the above object, it originally adopts the following technical scheme that：
（1）The acquisition of explant：Tender paenoiae alba blade is chosen at the beginning of late March to 4 months, is rinsed well with clear water, Ran Houyong
The rinsing of Amway detergent is primary, and 15-20min is vibrated on shaking table；Flowing water rinse 3min after, on aseptic operating platform with 75% ethyl alcohol
10-20s is sterilized, pours into 0.1% mercuric chloride immediately, is 2cm by the blade cuts of sanitized finally with sterile water washing 2 times
Long segment.
（2）It is prepared by callus：Take step（1）It is middle to handle obtained explant segment, be inoculated in inducing culture into
The induction of row callus, cultivation temperature are 24-28 DEG C, first light culture 7-10d, then carry out illumination cultivation 25d；The light
It is according to condition of culture：Light application time is 14h/d, intensity of illumination 1500lx；The inducing culture is MS+ sucrose 30g/L+KT
1mg/L+IBA0.02mg/L, pH value 5.4-6.0；
（3）Adventitious buds differentiation：By step（2）It is middle to induce obtained callus, it is cut on explant, accesses differential medium
Adventitious buds differentiation is carried out, cultivation temperature is 24-28 DEG C；Light application time 14h/d, intensity of illumination 2500lx；The differential medium
For 1/2MS+NAA0.6mg/L+CPPU 0.3mg/L, pH value 5.4-6.0；
（4）Squamous subculture：After obtained adventitious bud is induced to cut, band 0.3cm or so stem section and 2-3 piece blades, access per bud
Squamous subculture 25d is carried out in subculture multiplication medium, cultivation temperature is 24-28 DEG C, light application time 14h/d, and intensity of illumination is
2500lx；The subculture multiplication medium is 1/2MS+NAA0.5mg/L, pH value 5.4-6.0；
（5）The induction of root：By step（4）Culture of rootage is carried out in the test tube seedling access root media that middle shoot proliferation obtains,
Cultivation temperature is 24-28 DEG C, light application time 14h/d, intensity of illumination 2000lx；The root media is 1/2MS+
2.0mg/L IBA, pH value 5.4-6.0；It treats that adventitious root covers with bottom of bottle, when plant integrally grows steady, is ready for bottle outlet of transplanting seedlings.
(6) bottle cap of culture bottle is opened, root is immersed in preparation of taking root, 15min by hardening 2-3 days.
The preparation of taking root promotees root-growing agent for microorganism, and composition includes：
Strepiomyces lavendulae, bacillus amyloliquefaciens, bacillus pumilus, Nafusaku, activated carbon, sucrose；
The Strepiomyces lavendulae (Streptomyces lavendulae) is CGMCC NO.2130；
The bacillus amyloliquefaciens are（Bacillus amyloliquefaciens）ATCC23843；
The bacillus pumilus is（Bacillus pumilus）ATCC7061；
The preparation of taking root specifically comprises：
Strepiomyces lavendulae zymotic fluid：Solve starch gemma bar zymotic fluid：Bacillus pumilus zymotic fluid：Nafusaku：Activated carbon；
Sucrose weight ratio is 4:2:5:1:1:2
Preparation method is：Strepiomyces lavendulae, bacillus amyloliquefaciens, bacillus pumilus are lived in a conventional manner first
Change, then cultivate into bacterium solution viable count and reach 107A/gram acquisition zymotic fluid, by above-mentioned zymotic fluid according to mass ratio 4:2:5
Mixing, according to weight proportion addition Nafusaku, activated carbon, sucrose to obtain the final product.
（7）It is transplanted into cutting medium after hardening, the cutting medium is preferably：According to 1:1 volume ratio is by pearl
Rock and peat mixing spray the MS dilutions of 50 times of dilution, daily water spray 2 times, ensure humidity, avoid direct sunlight.
The present invention overcomes pollution rate during Chinese herbaceous peony tissue-culturing rapid propagation is high, melting brown rate is high, take root difficulty, transplanting survival rate are low
The problems such as.
With disinfecting, present inventor passes through a large amount of creative works and factorial experiment for the selection of 1 explant,
Major tuneup operating parameter of the prior art, it is found that according to conventional disinfection way, there are problems that certain pollution rate,
And explant position and acquisition time in the operating process of the application are used, not only achieve ideal sterilization effect, additionally it is possible to
Ensure the explant number needed for Fiber differentiation.
Its step of 2 method for tissue culture all has similitude, and result but makes a marked difference, tissue known in this field
Cultural method is generally divided into two stages of light culture and optical culture, and light culture is usually evoked callus, and optical culture induction divides
Change.Light culture requires to be protected from light, and is the healing cell aging for preventing from dividing rapidly in fact, present inventor by testing repeatedly
Go out, Cultivars of Chinese Herbaceous Peony paenoiae alba first passes through the light culture of a period of time, and explant is made to divide rapidly, then photoinduction culture, sprouts
Hair rate highest, callus budding is rapider, shortens the tissue culture time.
Effect is not satisfactory always for the tissue culture work of 3 this field Chinese herbaceous peonies, such as CN102124946, two steps is used to sterilize
Method, the survival rate only 25% of acquirement, and Chinese herbaceous peony can be taken the material for doing explant to include flower, embryo, leaf etc., the application is with can
The paenoiae alba blade of southern climates is fully adapted to as explant, physiological habits, the warp such as fully studies its callus, adventitious bud, take root
Cross the step of largely groping to obtain the application Systems Theory with experiment.
4, it is known in the art that for plant tissue culture area research how using plant tissue differentiation regeneration plant, improve plant
Reproductive number, be that study important technology emphasis be also technological difficulties to scientist, and hormone kind and hormone concentration use model
It encloses, type of culture medium is even more the most important thing, and present inventor passes through a large amount of creative works, and best paenoiae alba is arrived in screening
The ingredient and hormone combinations of inducing culture, proliferated culture medium and root media, not only induction, cultivation effect are apparent, and go out
The bud time greatly shortens, and only 7+25d just can grow callus, then induces 25 adventitious bud that can go out, and proliferation, rooting efficiency are notable.
Specific embodiment
Embodiment one
1 Screening of Media embodiment
During the vitro Regeneration System of callus approach is established, the success of callus differentiation namely inducing culture are again raw bodies
The committed step of system.Whether callus differentiation rate directly affects regeneration tissue culture system feasible.Callus differential medium by training substantially
Supporting degree that base and hormone kind and concentration collocation break up callus and differentiation rate has very big correlation.The present invention attempts
Different minimal mediums and hormon concentration proportioning, the influence to paenoiae alba blade induction degree are shown in Table 1.It can be with from table 1
Find out, MS+ sucrose 30g/L+KT 1mg/L+IBA0.02mg/L, paenoiae alba phenylacetic acid highest, for best callus point
Change culture medium.
Table 1
| Basal medium type | Hormone combinations mg/L | Callus divergaence time | Callus differentiation rate % |
| MS | Sucrose 30g/L+KT 1mg/L+IBA0.02mg/ | 32 | 87.5 |
| MS | Sucrose 30g/L | 50 | 62.13 |
| MS | TDZ1 mg/L+ coconut juice 5%+ sucrose 30% | 55 | 83.27 |
| B5 | Sucrose 30g/L+KT 1mg/L+IBA0.02mg/ | 45 | 43.21 |
| B5 | TDZ1 mg/L+ coconut juice 5%+ sucrose 30% | 60 | 48.44 |
2 take root growth promoting bacteria agent screening
The present invention has attempted different influences of the preparation to paenoiae alba rooting of vitro seedling of taking root and has been shown in Table 2：
Table 2
The present invention takes root preparation for the nutritional preparation specifically for Cultivars of Chinese Herbaceous Peony paenoiae alba, by a large amount of investigation and tests,
Strepiomyces lavendulae, solution starch gemma bar, bacillus pumilus form a good microecosystem, and matrix is brought into after immersion
In be built with conducive to plant growth, improve plant rhizosphere nutrient environment, metabolism can also strengthen the decomposition of soil organic matter, each bacterium
Reasonable compatibility between kind, symbiosis are coordinated, and mutually antagonism, Nafusaku do not promote the formation of adventitious root and the formation of root, promote to insert skewer
Take root, promote branches and leaves it is luxuriant, improve yield, the effect of improving quality, improve crop it is resistant to lodging wait anti-adversity abilities.Sucrose is continuous
Nutrition is supplied, the robust seedling impetus is maintained, greatly improves the adaptability of plant, improves entire survival rate, activated carbon aeration is good, inhales
Attached ability is strong, conducive to microbial activities, enhances biological property, rich in organic matter, humic acid and other nutritional ingredients.The prior art
Patent CN105123517 although inducing adventitious bud using callus, however, it is limited only to laboratory tissue culture, does not have yet
Continue its acclimatization and transplants to outside scenery, although root-inducing powder ABT can also obtain 88.7% rooting efficiency, price compared with
For costliness, it is unfavorable for large-scale production.
Embodiment two
（1）The acquisition of explant：Tender paenoiae alba blade is chosen at the beginning of late March to 4 months, is rinsed well with clear water, Ran Houyong
The rinsing of Amway detergent is primary, and 15-20min is vibrated on shaking table；Flowing water rinse 3min after, on aseptic operating platform with 75% ethyl alcohol
10-20s is sterilized, pours into 0.1% mercuric chloride immediately, is 2cm by the blade cuts of sanitized finally with sterile water washing 2 times
Long segment；
（2）It is prepared by callus：Take step（1）It is middle to handle obtained explant segment, it is inoculated in inducing culture and is cured
The induction of injured tissue accesses blade segment 40 altogether, and cultivation temperature is 24-28 DEG C, then light culture 7-10d first carries out light
According to culture 25d；The illumination cultivation condition is：Light application time is 14h/d, intensity of illumination 1500lx；The inducing culture
For MS+ sucrose 30g/L+KT 1mg/L+IBA0.02mg/L, pH value 5.4-6.0；Share 35 explant both ends strong point greens
Callus, the browning of remaining explant segment.
（3）Adventitious buds differentiation：By step（2）It is middle to induce obtained callus, it is cut on explant, access differentiation training
It supports base and carries out adventitious buds differentiation, cultivation temperature is 24-28 DEG C；Light application time 14h/d, intensity of illumination 2500lx；The differentiation training
It is 1/2MS+NAA0.6mg/L+CPPU 0.3mg/L, pH value 5.4-6.0 to support base；The callus fritter of access differential medium is total to
106 pieces, for callus differentiation rate up to 90.23%, every piece of callus can break up adventitious bud 3-4
（4）Squamous subculture：After obtained adventitious bud is induced to cut, band 0.3cm or so stem section and 2-3 piece blades, access per bud
Squamous subculture 25d is carried out in subculture multiplication medium, cultivation temperature is 24-28 DEG C, light application time 14h/d, and intensity of illumination is
2500lx；The subculture multiplication medium is 1/2MS+NAA0.5mg/L, pH value 5.4-6.0, and it is indefinite to be proliferated out per stem section
Bud 6-7
（5）The induction of root：By step（4）Culture of rootage is carried out in the test tube seedling access root media that middle shoot proliferation obtains,
Cultivation temperature is 24-28 DEG C, light application time 14h/d, intensity of illumination 2000lx；The root media is 1/2MS+
2.0mg/L IBA, pH value 5.4-6.0；It treats that adventitious root covers with bottom of bottle, when plant integrally grows steady, is ready for bottle outlet of transplanting seedlings.
(6) bottle cap of culture bottle is opened, root is immersed in preparation of taking root, 15min by hardening 2-3 days.
The preparation of taking root promotees root-growing agent for microorganism, and composition includes：
Strepiomyces lavendulae, solution starch gemma bar, bacillus pumilus, Nafusaku, activated carbon, sucrose；
The Strepiomyces lavendulae (Streptomyces lavendulae) is CGMCC NO.2130；
The bacillus amyloliquefaciens are（Bacillus amyloliquefaciens）ATCC23843；
The bacillus pumilus is（Bacillus pumilus）ATCC7061；
The preparation of taking root specifically comprises：
Strepiomyces lavendulae zymotic fluid：Solve starch gemma bar zymotic fluid：Bacillus pumilus zymotic fluid：Nafusaku：Activated carbon；
Sucrose weight ratio is 4:2:5:1:1:2
Preparation method is：Strepiomyces lavendulae, bacillus amyloliquefaciens, bacillus pumilus are lived in a conventional manner first
Change, then cultivate into bacterium solution viable count and reach 107A/gram acquisition zymotic fluid, by above-mentioned zymotic fluid according to mass ratio 4:2:
5:Mixing, according to weight proportion addition Nafusaku, activated carbon, sucrose to obtain the final product.
（7）It is transplanted into cutting medium after hardening, the cutting medium is preferably：According to 1:1 volume ratio is by pearl
Rock and peat mixing spray the MS dilutions of 50 times of dilution, daily water spray 2 times, ensure humidity, avoid direct sunlight, cultivate 15d
Afterwards, rooting rate reaches 97.8%, and survival rate is up to 94.3%.
Using paenoiae alba blade Regeneration in Vitro method for building up of the present invention, paenoiae alba seedling can be largely obtained, solves Chinese herbaceous peony
The problem of medicine is quickly bred by the screening to best explant, inducing hormone level, microbial inoculum of taking root, enables to a training
It supports in the period, a large amount of to obtain paenoiae alba seedlings, rooting rate reaches 97.8%, and survival rate also obtains 94.3%, proliferation rate and transplanting
The market vacancy has been filled up in the raising of survival rate significantly, is had broad application prospects.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent, the growth promoting bacteria agent is by Strepiomyces lavendulae, solution
Bacillus amyloliquefaciens, bacillus pumilus, Nafusaku, activated carbon, sucrose composition.
2. according to the method described in claim 1, it is characterized in that, the growth promoting bacteria agent specifically comprises：
Strepiomyces lavendulae zymotic fluid：Solve starch gemma bar zymotic fluid：Bacillus pumilus zymotic fluid：Nafusaku：Activated carbon；
The weight ratio of sucrose is 4:2:5:1:1:2.
3. according to the method described in claim 1-2, it is characterised in that：
The Strepiomyces lavendulae is (Streptomyces lavendulae) CGMCC NO.2130；
The bacillus amyloliquefaciens are（Bacillus amyloliquefaciens）ATCC23843；
The bacillus pumilus is（Bacillus pumilus）ATCC7061.
4. according to the method described in claim 3, it is characterized in that, the growth promoting bacteria agent preparation method is：First by lilac grey
Streptomycete, bacillus amyloliquefaciens, bacillus pumilus activate in a conventional manner, then cultivate to viable count and reach 107A/
Gram zymotic fluid, by above-mentioned zymotic fluid according to mass ratio 4:2:5 mixing, according to weight proportion addition Nafusaku, activated carbon,
Sucrose to obtain the final product.
5. according to the method described in claim 1, it is characterized in that：The method is as follows：
（1）The acquisition of explant：Tender Chinese herbaceous peony blade is chosen at the beginning of late March to 4 months, is rinsed well with clear water, then with washing
It is primary to wash agent rinsing, 15-20min is vibrated on shaking table；Flowing water rinse 3min after, on aseptic operating platform with 75% ethanol disinfection
10-20s pours into 0.1% mercuric chloride immediately, is 2cm's long by the blade cuts of sanitized finally with sterile water washing 2 times
Segment；
（2）It is prepared by callus：Take step（1）It is middle to handle obtained explant segment, it is inoculated in inducing culture and is cured
Then the induction of injured tissue, first light culture 7-10d carry out illumination cultivation 25d；The illumination cultivation condition is：Light application time
For 14h/d, intensity of illumination 1500lx；The inducing culture is MS+ sucrose 30g/L+KT 1mg/L+IBA0.02mg/L；
（3）Adventitious buds differentiation：By step（2）It is middle to induce obtained callus, it is cut on explant, accesses differential medium
Carry out adventitious buds differentiation, light application time 14h/d, intensity of illumination 2500lx；The differential medium is 1/2MS+NAA0.6mg/L+
CPPU 0.3mg/L；
（4）Squamous subculture：By induce obtain adventitious bud cutting after, access in the test tube containing subculture multiplication medium carry out after
Be commissioned to train foster 25d, and the subculture multiplication medium is 1/2MS+NAA0.5mg/L；
（5）The induction of root：By step（4）It is given birth in bottle of the test tube seedling access containing root media that middle shoot proliferation obtains
Root culture, the root media are 1/2MS+ IBA 2.0mg/L；Treat that adventitious root covers with bottom of bottle, plant integrally grows steadily and surely
When, it is ready for bottle outlet of transplanting seedlings；
(6) bottle cap of culture bottle is opened, root is immersed in preparation of taking root, 15min by hardening 2-3 days；
（7）Transplanting.
6. according to the method described in claim 5, it is characterized in that, inducing culture, differential medium, shoot proliferation culture
Base, root media pH value are 5.4-6.0.
7. according to the method described in claim 6, it is characterized in that, the Chinese herbaceous peony is paenoiae alba kind.
8. a kind of for promoting the growth promoting bacteria agent of paenoiae alba tissue culture expanding propagation, the growth promoting bacteria agent is by Strepiomyces lavendulae, solution starch
Gemma bar, bacillus pumilus, Nafusaku, activated carbon, sucrose composition.
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