CN107711502A - A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction - Google Patents
A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction Download PDFInfo
- Publication number
- CN107711502A CN107711502A CN201711087128.6A CN201711087128A CN107711502A CN 107711502 A CN107711502 A CN 107711502A CN 201711087128 A CN201711087128 A CN 201711087128A CN 107711502 A CN107711502 A CN 107711502A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- bud
- adventitious bud
- culture
- adventitious
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction.Culture medium box set of the present invention includes being used for culture medium, the culture medium for clump bud induction and propagation, the culture medium for adventitious bud inducing, the culture medium for Elongation of adventitious bud induction, the culture medium for taking root for establishing sterile system, and volume ratio is 3：The matrix that 1 yellow mud and peat soil mixes.Present invention firstly discovers that stem with bud flattening induction adventitious bud development ways and pass through culture medium box set of the present invention carry out Vitex rotundifolia cultured in vitro, clump bud breeding multiplying power reaches as high as 21.9, the adventitious bud of average each flattening explant occur quantity be up to 84.9, Elongation of adventitious bud up to 3.0cm, rooting rate with averagely take root number respectively up to 70.5% with 86.4, transplant 30 days after survival rate reach as high as 86%, have a good application prospect.
Description
Technical field
The invention belongs to Vitro Plant quick breeding technology field, it is more particularly related to a kind of climing for single leaf
Jing Congya induces the culture medium box set and method of the Vitro Quick Reproduction with forming adventitious shoot regeneration approach in flattening stem section.
Background technology
Vitex rotundifolia (Vitex rotundifolia L.) is Verenaceae Vitex machaka.It is distributed widely in
On the waste continent on the ground such as the sandy beach of tropical and subtropical region, seashore and lakeside, its is adaptable, not strict to environmental requirement
And with characteristics such as alkaline-resisting, high temperature resistants, can be as coastal sandy land, the pioneer vegetation of wind gap district dune-fixating forestation.Vitex rotundifolia is also
With higher medical value, its fruit is one of source of China's conventional Chinese medicine " fructus viticis ", has dispelling wind and heat from the body, head clearing
The effect of.Modern pharmacological research shows that fructus viticis has the chemistry such as amphene, firpene, flavonoids, casticin and cyanidenon
Composition has significantly analgesia, anti-inflammatory, decompression, the antitumor and anti-ageing effect of waiting for a long time.And its stem bar can be also used for large bamboo or wicker basket
Establishment, spends extractable fragrance etc..
With the utilization of fructus viticis new application and new product in recent years, its market demand increasingly increases.Cause it
Resource increasingly depleted.《Key Protected medicinal material species register》In, it is in imminent danger that Vitex rotundifolia has been listed in III grade of country
Protect plant.At present, in terms of the research both at home and abroad for fructus viticis is concentrated mainly on chemical composition and pharmacological action, manually protecting
The research for protecting breeding is less.The propagation method of Vitex rotundifolia mainly has seminal propagation, cutting propagation, division propagation, mound layering
And non-test tube nutrition organs fast seedling growing etc..The report of Vitex rotundifolia tissue culture quick breeding is had no at present.
The content of the invention
It is an object of the invention to：Overcome the problems such as lacking the artificial fecundation method to Vitex rotundifolia in the prior art, carry
One kind has been supplied to inquire into various concentrations, the plant growth regulator and its group of species using Vitex rotundifolia stem segment with axillary bud as explant
Close to its inducing clumping bud, the inclined graduation induction of stem section and adventitious bud generation, Elongation of adventitious bud and the influence taken root, establish height
The Vitex rotundifolia tissue culture sprout quick propagating technology system of effect.
In order to realize foregoing invention purpose, be used to establish the culture medium of sterile system the invention provides a kind of, its be with
Culture medium based on MS culture mediums, it with the addition of 25~35g/L sucrose and 6.5~7.5g/L agar.
It is used for a kind of optimal technical scheme of culture medium for establishing sterile system as the present invention, the addition of sucrose is
30g/L, the addition of agar is 7g/L.
Present invention also offers a kind of culture medium for inducing and breeding for clump bud, it is for establishing sterile system
Culture medium based on culture medium, it with the addition of 0.5~2.5mg/L 6-benzyladenines.
Present invention also offers a kind of culture medium for adventitious bud inducing, and it is for establishing the culture of sterile system
Culture medium based on base, it with the addition of 2.5mg/L 6-benzyladenines.
Present invention also offers a kind of culture medium for Elongation of adventitious bud induction, it is for establishing sterile system
Culture medium based on culture medium, it with the addition of 1.5~2.5mg/L methyl α-naphthyl acetates.
Present invention also offers a kind of culture medium for taking root, and it is the culture medium based on 1/2MS culture mediums, addition
3.0mg/L indolebutyric acids, 2.0~3.0mg/L methyl α-naphthyl acetates, 25~35g/L sucrose and 6.5~7.5g/L agar.
BA, i.e. 6-benzyladenine, it is a kind of widely used basic element of cell division for making an addition to plant growth culture medium, tool
There is the decomposition for suppressing leaves of plants inner chlorophyll, nucleic acid, protein, protect green anti-old；By amino acid, auxin, inorganic salts etc. to place
A variety of efficiency such as position allocation and transportation are managed, are widely used in each stage of agricultural, fruit tree and garden crop from germination to harvest；In plant
Rapid propagation in vitro in can promote cell division and by breaking up adventitious bud on callus or organ, while help to make axillary bud by pushing up
Freed under the suppression of end advantage.
NAA, i.e. methyl α-naphthyl acetate, it is a kind of broad spectrum type plant growth regulator, is used to induce in Vitro Plant incubation
The division of cell and the differentiation of root, at the same can influence stem and section elongation, tropism, apical dominance, leaf abscission phenomena such as.
TDZ, i.e. Thidiazuron, it is a kind of efficient basic element of cell division, can be used for promoting callus group in Vitro Plant is numerous soon
The generation of growth, lateral bud and adventitious bud is knitted, promotes the formation of embryoid.
2,4-D, i.e. 2,4- dichlorphenoxyacetic acid, it is a kind of artificial synthesized auxin analog.Under low consistency conditions,
It highly effective can promote callus mitogen, but the generation of energy strong inhibition organ.Its simultaneously or systemic herbicide.
KT, i.e. kinetin, it is the artificial synthesized basic element of cell division, cell differentiation, division, growth can be promoted, promotes thin
Born of the same parents divide, and break up adventitious bud on callus or organ.
ZT, i.e. zeatin, it is the natural phytohormone extracted from the seed of corn pustulation period, but now can be with
It is artificial synthesized, callus can be promoted to germinate.
IAA, i.e. heteroauxin, it is plant endogenous auxin, is used to promote cell division during Vitro Plant is numerous soon
With cell growth, induced synthesis adventitious root etc..
IBA, i.e. indolebutyric acid, it is plant endogenous auxin, is used to promote cell division during Vitro Plant is numerous soon
With cell growth, induced synthesis adventitious root, fruit setting can be increased in agricultural production, prevent shedding, change female, male flower ratio etc..
In order to realize foregoing invention purpose, present invention also offers a kind of culture medium box set, it includes above-mentioned being used to build
Found the culture medium of sterile system, induced for clump bud with the culture medium of propagation, the culture medium for adventitious bud inducing, for indefinite
Culture medium, the culture medium for taking root of bud elongation induction, and volume ratio is 3：The base that 1 yellow mud and peat soil mixes
Matter.
The above-mentioned culture medium box set of the present invention can be used for promoting in Vitex rotundifolia Vitro Quick Reproduction.
In order to realize foregoing invention purpose, present invention also offers a kind of side for promoting Vitex rotundifolia Vitro Quick Reproduction
Method, it comprises the following steps：
(1) choose Vitex rotundifolia semi-lignified seedling stem segment with axillary bud be explant (preferably take length be 1.5~3cm simultaneously
Stem section with 2 lateral buds)；
(2) aseptically, the described training for being used to establish sterile system is inoculated in after explant is sterilized with mercuric chloride
Support on base, obtain aseptic seedling；
(3) aseptic seedling that step (2) obtains is cut into the stem with bud of 1, band section (2 lateral buds), is inoculated in described
The culture medium with breeding is induced for clump bud, obtains clump bud；
(4) clump bud that step (3) obtains is cut into the stem section of 1, band section, is inoculated in and described is used for adventitious bud inducing
Culture medium on, induce stem section flattening, obtain adventitious bud；
(5) adventitious bud that step (4) obtains is inoculated in the described culture medium for being used for Elongation of adventitious bud induction, obtained
Extend for the adventitious bud of normality bud；
(6) elongation for obtaining step (5) is inoculated in the described culture medium for taking root for the adventitious bud of normality bud,
Culture of rootage is carried out, tissue-cultured seedling is obtained, its root culture medium is cleaned up, it is 3 to transplant in volume ratio：1 yellow soil and mud
In the matrix that charcoal soil mixes, the Vitro Quick Reproduction of Vitex rotundifolia is completed.
As the present invention promote Vitex rotundifolia Vitro Quick Reproduction method a kind of optimal technical scheme, step (2)~
(6) condition of culture in is：(25 ± 1) DEG C, light application time 12h/12h (L/D), 1500~2000lx of intensity of illumination.
Promote a kind of optimal technical scheme of the method for Vitex rotundifolia Vitro Quick Reproduction as the present invention, in step (2)
The process of the sterilization includes：10~15s, rinsed with sterile water 1 time, with 0.1% mercuric chloride soaking disinfection are sterilized in 75% alcohol
8min, rinsed with sterile water 7 times, then blot surface moisture with aseptic filter paper.
Promote a kind of optimal technical scheme of the method for Vitex rotundifolia Vitro Quick Reproduction, methods described tool as the present invention
Body is：The stem section of Vitex rotundifolia health branch is taken, cuts off unnecessary blade, takes long 1.5~3cm and with the stem section of 2 lateral buds
For explant, after surface sterilization is handled, the culture medium for establishing sterile system is seeded in, obtains sterile sprout；Take
The stem section of 1 section (2 lateral buds) of band of sterile sprout is inoculated in the culture medium for inducing and breeding for clump bud respectively, with clump bud
Inductivity, bud proliferation times, the average index such as plant height and growth conditions determine suitable clump bud induction and proliferated culture medium；
Take and the clump bud to grow fine with being formed on the culture medium of propagation induced for clump bud, the stipes with axillary bud is cut with blade,
It is inoculated in and is used for adventitious bud induction culture base, passes through adventitious bud incidence (stem section flattening inductivity), average each explant
Adventitious bud number and growth conditions etc. are reference on body, it is determined that suitable stem section flattening induction is matched somebody with somebody with adventitious bud
Side；Take and the flattening stem section of adventitious bud is being formed on Elongation of adventitious bud inducing culture, aseptically, cut with blade
Take adventitious bud number identical flattening stem section, be inoculated in for Elongation of adventitious bud inducing culture, with Elongation of adventitious bud rate,
The index such as adventitious bud average length and upgrowth situation, it is determined that the formula of suitable Elongation of adventitious bud inducing culture.Take
The indefinite sprout of elongation to be grown fine on Elongation of adventitious bud inducing culture, it is inoculated in for being carried out on the culture medium taken root
Rooting induction, with rooting rate, averagely take root quantity, average root long and plant growth condition etc. be index, it is determined that it is suitable
Rooting induction formula.To be taken root good stand, after the culture medium for washing away the attachment of its root, be transplanted in being mixed with yellow mud and peat soil
(volume ratio 3：1) in matrix, culture can obtain the tissue-cultured seedling to grow fine in 30 days.It is climing that the present invention establishes single leaf first
The rapid propagation system that the Cut stem clump bud induction of chaste tree occurs with adventitious bud in flattening stem section, by the foundation of sterile system, clump
Bud induce with propagation, stem section flattening induction and adventitious bud generations, Elongation of adventitious bud, the processing such as take root, acquisition is completely after transplanting
A considerable number of Vitex rotundifolia tissue-cultured seedling.
Relative to prior art, the invention has the advantages that：
(1) present invention be used for establish in the culture medium of sterile system, using any plant growth regulator is not added, only with
MS is that minimal medium induces explant axillary bud sprouting, establishes the sterile system of Vitex rotundifolia.
(2) present invention is used for clump bud induction with the culture medium of propagation, cutting for establishing in the culture medium of sterile system
The stem section of one section of the sterile sprout band formed is explant, with MS minimal mediums, the BA of addition different quality concentration, with clump
Bud induction rate, bud proliferation times and plant height are index, have screened suitable clump bud induction formula, and be found that single leaf is climing first
Chaste tree stem with bud can occur flattening and form the phenomenon of adventitious bud.With 30 days for cultivation cycle, clump bud inductivity, bud propagation times
Number and plant height reach as high as 81.11%, 21.91 and 5.25cm respectively, and the clump bud blade formed is light green, and internode is compact, long
Gesture is good.
(3) because being induced for clump bud with being found that Vitex rotundifolia stem with bud flattening and not in the culture medium bred
The generation of normal bud, then, as starting point, in order to inquire into Vitex rotundifolia stem with bud flattening induction and adventitious bud generation
Matrix, be used for for the induction of Vitex rotundifolia stem with bud inclined graduation with what adventitious bud occurred in adventitious bud induction culture base, with
It is explant to be induced in clump bud with the in vitro stem with bud for the sprout that grown thickly in the culture medium of propagation, using MS as minimal medium, is added
High concentration (>=2.5mg/L) BA is added, to form adventitious bud number as finger on adventitious bud incidence, average each explant
Mark, has screened suitable adventitious bud induction culture base.With 30 days for cultivation cycle, stem section flattening induction and adventitious bud occur
Rate, the adventitious bud number highest of average each explant obtain short and small close adventitious bud respectively up to 60% and 84.9.
(4) present invention is used in Elongation of adventitious bud inducing culture, cuts for occurring not in adventitious bud induction culture base
The flattening stem section of normal bud is explant, and using MS as minimal medium, addition 1.5~2.5mg/L NAA or BA combine with NAA,
Using the average length of bud elongation and adventitious bud as index, the Optimum formulae of Vitex rotundifolia Elongation of adventitious bud induction has been screened.With
It is within 30 days cultivation cycle, the average length highest of Elongation of adventitious bud rate and adventitious bud is short and small respectively up to 58.9% and 3.0cm
Close sprout can form normality bud, grow fine.
(5) by for the elongation of gained in Elongation of adventitious bud inducing culture and the sprout of robust growth in containing IBA or
Culture of rootage is carried out in NAA and combinations thereof 1/2MS culture mediums, finally carries out rooting culture.Rooting rate is with averagely taking root several points
Not up to 70.5% and 86.4.Survival rate reaches as high as 86% after transplanting 30 days.
(6) present invention firstly discovers that the adventitious bud of Vitex rotundifolia stem section flattening induction occurs, and its clump bud is established
The tissue culture quick breeding system of traces adventitious bud approach, is generated with gene identity, can keep the group of maternal plant merit
Seedling is trained, and the breeding multiplying power for solving cutting propagation and seminal propagation is low, is limited by the time cycle and environmental restrictions etc. are asked
Topic.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, the inventive method and beneficial effect are described in detail.
Fig. 1 is that Vitex rotundifolia aseptic seedling establishes culture effect figure；
Fig. 2 is the design sketch of clump bud induction；
Fig. 3 is the design sketch of stem section flattening and adventitious bud inducing；
Fig. 4 is the design sketch of Elongation of adventitious bud；
Fig. 5 is the plant to take root；
Fig. 6 is the plant of transplant survival.
Embodiment
In order that the purpose of the present invention, technical scheme and advantageous effects become apparent from, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
Bright, being not intended to limit the present invention, the parameter of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no substance to result
Influence.
In following examples, term " in vitro " refers to a part for organism is extracted and swum for various research purposes
From in the state of in vitro.
Term " explant " refers to the tissue segment in squamous subculture, moving into the culture of new culture medium.In the present invention
In, " explant " specifically refers to the stem segment with axillary bud of Vitex rotundifolia.
1/2 culture medium refers to the amount of a great number of elements in MS culture mediums is reduced to the culture medium prepared by original 1/2.
The composition of MS culture mediums is as shown in table 1 below：
The composition of table 1MS culture mediums
Unless otherwise specified, training involved in the method for Vitex rotundifolia Vitro Quick Reproduction is promoted in the embodiment of the present invention
Support base and matrix prepared by the following method respectively (need to contrast variety classes, concentration growth regulator when, then replace phase
Answer component)：
(1) 1L MS culture mediums are prepared：The accurate various compounds weighed described in table 1, add appropriate distilled water to dissolve, and use
Glass bar stirs dissolution, and pH to 6.0, last constant volume to 1L are adjusted with NaOH.
(2) it is formulated for establishing the culture medium of sterile system：Also add on the basis of the MS culture mediums prepared by step (1)
Add 25~35g/L sucrose and 6.5~7.5g/L agar；
(3) clump bud induction and the culture medium of propagation are formulated for：On the basis of the MS culture mediums prepared by step (2) also
Added with 0.5~2.5mg/L BA；
(4) it is formulated for the culture medium of adventitious bud inducing：Also added on the basis of the MS culture mediums prepared by step (2)
There are 2.5mg/L BA；
(5) it is formulated for the culture medium of Elongation of adventitious bud induction：On the basis of the MS culture mediums prepared by step (2) also
Added with 1.5~2.5mg/L methyl α-naphthyl acetates；
(6) it is formulated for the culture medium taken root：Also it is added with the basis of 1/2 of the MS culture mediums prepared by step (1)
3.0mg/L indolebutyric acids, 2.0~3.0mg/L methyl α-naphthyl acetates, 25~35g/L sucrose and 6.5~7.5g/L agar；
(7) matrix 1 is prepared:According to yellow mud：Peat soil=3:The ratio of 1 (volume ratio) is mixed.
Unless otherwise specified, condition of culture as described below is：(25 ± 1) DEG C, light application time 12h/12h (L/D), light
According to 1500~2000lx of intensity.
The foundation of the sterile system of embodiment 1
Experiment sets only 1 group, in April, 2017, takes Vitex rotundifolia stem with bud to carry out Vitex rotundifolia sterile system and establishes examination
Test, comprise the following steps that：
The selection of explant：The stem section of Vitex rotundifolia health branch is taken, cuts off unnecessary blade, takes long 1.5~3cm and band
The stem section for having 2 lateral buds is explant
Surface sterilization：Explant is put into 10~15s of sterilization, rinsed with sterile water 1 in 75% alcohol on superclean bench
It is secondary, with 0.1% mercuric chloride soaking disinfection 8min, rinsed with sterile water 7 times, dry surface moisture.
Inoculation：After cutting off stem section both ends on aseptic filter paper, the MS blank without any plant growth regulator is inoculated in
Cultivated on culture medium.
The sterile sprout (Fig. 1) that blade is emerald green to unfold, and grows fine can be obtained after culture 30d.
Embodiment 2~6 tests variety classes respectively, the growth regulator of concentration is added in any of the above culture medium, right
Influenceed in Vitex rotundifolia Vitro Quick Reproduction caused by each period, and draw preferably growth regulator selection.
The influence that the variety classes of embodiment 2, the plant growth regulator of concentration are induced Vitex rotundifolia Cut stem clump bud
Experiment sets 1~25 group, and using MS as minimal medium, 0.1 is individually with the addition of in different test groups
~2.5mg/L IAA, 0.1~2.5mg/L NAA, 0.1~2.5mg/L KT, 0.1~2.5mg/L ZT, 0.1~2.5mg/L
BA and 0.1~2.5mg/L TDZ；
Comprise the following steps that：
The stem section of 1 section (2 lateral buds) of band is taken to be inoculated in experiment 1~25 respectively from the sterile sprout in embodiment 1
In culture medium, each bottle is inoculated with stem section 3, each 10 bottles of formula.
Its growing state and the average germination rate of statistics bud, clump bud inductivity, bud proliferation times are observed and recorded after culture 30d
And average plant height.
The experiment is repeated after being spaced 15d twice.
Average bud germination rate=(explant number during explant number/inoculation that bud is sprouted) × 100%；Clump bud inductivity=
Produce explant number during explant number/inoculation of clump bud；Bud sum during bud proliferation times=bud sum/inoculation；Average strain
The quantity of explant during high=total plant height/inoculation.
The clump bud that Vitex rotundifolia is carried out using the above method is induced, resulting result such as table 2：
The variety classes of table 2, the plant growth regulator of concentration induce Vitex rotundifolia clump bud the influence with breeding
As shown in Table 2：Variety classes, the plant growth regulator and combinations thereof of concentration processing under, bud germination rate, clump
Bud induction rate, proliferation times and plant height have significant difference (P<0.5).In the culture medium containing auxin (IAA and NAA)
The plant major part blade of upper culture is emerald green to unfold, part yellow, and the growth of plant individual plant, propagation multiplying power is relatively low, in high concentration bar
(>=1.0mg/L) minority can take root under part；
Contrastingly, then without life in the culture medium cultivated on the culture medium containing the basic element of cell division (KT, ZT, BA, TDZ)
Root phenomenon, but there were significant differences for action effect of different types of basic element of cell division to bud propagation and plant height.
In the plant major part individual plant growth containing KT or ZT medium culture, the propagation times of clump bud inductivity and bud
Rate is relatively low, but average plant height is significantly higher than other treatment groups, and blade is emerald green to unfold, and growing way is preferable.
The plant growing way cultivated in the culture medium containing BA is best, and proliferation times are significantly higher than other treatment groups, by
There is the callus that substantial amounts of clump bud grows and there is green densification part to occur at the axillary bud of nearly base portion.Clump bud inductivity and bud increase
Multiple and being proportionate property of concentration are grown, highest is respectively up to 81.11% and 21.91.When concentration higher than 0.5mg/L is that then difference is not
Significantly.Under the conditions of high concentration (>=0.5mg/L) BA, the inclined graduation of part cane, and adventitious bud is directly produced from cane, with
The extension of incubation time, adventitious bud can grow up to normality bud；
Callus is formed in most of lateral bud containing TDZ medium cultures, germination rate is minimum；The lateral bud that can be sprouted
It can downgrade, fasciation and form the callus of a large amount of high degree of water in base portion, growing way is worst.Therefore optimal clump bud propagation formula
For MS+0.5~2.5mg/L BA.With higher bud germination rate, clump bud inductivity and proliferation times and grow fine.
The various concentrations of embodiment 3, the plant growth regulator of species are to the adventitious bud inducing shadow of Vitex rotundifolia Cut stem
Ring
Experiment set 1~17 test group, 1~17 test group using MS as minimal medium, in different test groups
Or individually add 0.5-5.0mg/L BA, 1.0mg/L TDZ, 1.0mg/L 2,4-D or 1.0mg/L BA and 0.1mg/L NAA groups
Close and 1.0mg/L BA, 0.1mg/L NAA combine with 0.1mg/L TDZ；
Comprise the following steps that：
The stipes with axillary bud is taken to be inoculated in respectively in the culture medium of test group 1~17 from the sprout that grows thickly of embodiment 2, often
Individual bottle is inoculated with 3 sections, and each test group is inoculated with 10 bottles.
Its growing state is observed and recorded after culture 30d and counts adventitious bud induction frequency, averagely each explant is indefinite
Bud number.
The experiment is repeated after being spaced 15d twice.
Adventitious bud induction frequency=(explant number during explant number/inoculation of adventitious bud occur) × 100%；It is average indefinite
There is the explant number of adventitious bud in bud number=adventitious bud sum/.
The adventitious bud that Vitex rotundifolia stem section flattening induction is carried out using the above method is occurred, resulting result such as table 3.
The influence of the various concentrations of table 3, the plant growth regulator and combinations thereof of species to Vitex rotundifolia adventitious bud inducing
As shown in Table 3：Stem section cultivates base portion otch on addition 1.0mg/L 2,4-D or 1.0mg/L TDZ culture medium
Most of place and axillary bud are respectively formed callus, the generation of unbiased graduation and the formation of adventitious bud；However, in addition BA or high
Flattening can occur for the KT or ZT of concentration (>=1.0mg/L), form adventitious bud (Fig. 3).It is indefinite in addition BA culture medium
Bud incidence and average adventitious bud number highest, adventitious bud incidence increase with average adventitious bud number with the rise of BA concentration,
Highest is respectively up to 60% and 84.87.As concentration >=2.5mg/L, then difference is not notable and high concentration (>=5.0mg/L) BA can
Make leaf curl, and with slight vitrifying；As 1.0mg/L BA and NAA or when being combined with NAA, TDZ, its adventitious bud incidence
With individually addition 1.0mg/L BA when no significant difference, but its adventitious bud number when BA individually combines with NAA considerably less than
Other two treatment groups.Understand, NAA has certain negative consequence to adventitious bud.Optimal adventitious bud inducing formula should
For：MS+2.5mg/L BA.
The various concentrations of embodiment 4, the plant growth regulator of species are to Elongation of adventitious bud in Vitex rotundifolia flattening stem section
Influence
Experiment set 1~13 group, 1~13 test group using MS as minimal medium, in different test groups or point
Not with the addition of individually 0~2.5mg/L KT, 0.5~2.5mg/L BA, 0.5~2.5mg/L NAA or 0.5~2.5mg/L BA with
0.1mg/L NAA are applied in combination；
Specific test procedure is as follows：
(1) take to form the flat stem section of adventitious bud from embodiment 3, be aseptically cut into size identical adventitious bud
Block, about 10/block of adventitious bud number, adventitious bud is highly roughly the same, about 0.2cm/.
(2) aseptically micro cuttage carries out Elongation of adventitious bud induction in test group 1~13 respectively.
(3) each bottle inoculation stem section 6, each 10 bottles of processing.Its growing state is observed and recorded after culture 30d, with indefinite
Bud length >=0.5cm is elongation bud, statistics Elongation of adventitious bud rate and the length of average each adventitious bud.Repeat to be somebody's turn to do after being spaced 15d
Experiment is twice.Elongation=(adventitious bud sum during adventitious bud number/inoculation that appearance extends) × 100%；It is average each indefinite
Adventitious bud number during total length/inoculation of length=adventitious bud of bud.
Influence of the different plant growth regulator of table 4 and combinations thereof to Elongation of adventitious bud and bud average length
As shown in Table 4：Elongation of adventitious bud effect on addition NAA culture medium is best, and sprout is elongated emerald green (Fig. 4),
Bud elongation and the length of elongation are all remarkably higher than other treatment groups.With the increase of NAA concentration, bud elongation is grown with adventitious bud
Degree also increases, and highest is respectively up to 58.9% and 3.0cm, but then difference is not notable as concentration >=1.5mg/L；KT or BA inductions
Elongation of adventitious bud effect is not notable with blank control group difference.And the adventitious bud internode of elongation is induced on addition BA culture medium
It is short, while new adventitious bud can be formed, so as to influence the elongation of adventitious bud and average bud length, the adventitious bud of KT induction elongations
Although blade is unfolded, internode length, Elongation of adventitious bud rate and average length are relatively low.When BA and NAA arrange in pairs or groups, bud elongation with
Length be above individually addition BA when, it is seen then that NAA for bud elongation induction plays an important roll.Optimal adventitious bud inducing
It is formulated and is：MS+1.5~2.5mg/L NAA.
The influence that embodiment 5NAA and IBA is taken root to Vitex rotundifolia sprout
Experiment sets 1~16 test group, and for 1~16 test group using 1/2MS as minimal medium, test group 1 is blank pair
According to group, any plant growth regulator is not added；Added respectively in test group 2~16 0.5~5.0mg/L NAA or IBA and
IBA (1.0 with 3.0mg/L) and NAA (1.0,2.0,3.0mg/L) are combined, and are specifically shown in Table 5.
Specific test procedure is as follows：
(1) 3~4cm of plant height, the sprout for the robust growth that belt segment is 3, aseptically from its base are taken from embodiment 4
Portion is cut off with maternal plant.
(2) sprout cut off is inoculated in respectively in the culture medium of test group 1~16, every bottle is inoculated with 6, each experiment
10 bottles of group.
(3) its rooting rate is counted after 40d is cultivated, is averaged every plant and is taken root number and average root long and record the length of its seedling
Gesture.Experiment is repeated 3 times.Rooting rate=when strain number/inoculation (take root total strain number) × 100%.Average every plant of number=root of taking root is total
The plant sum count/taken root；The plant sum that average root long=root long sum/is taken root.
The influence that IBA and NAA of the various concentrations of table 5 and combinations thereof are taken root to Vitex rotundifolia sprout
As shown in Table 5：Sprout can not take root in the test group 1 without any plant growth regulator, in other processing
Middle culture 10~15 days, sprout starts to take root successively.Sprout individually addition NAA culture medium in rooting induction effect compared with
Difference, as NAA >=1.0mg/L, just can induce take root, rooting induction rate is minimum compared with other treatment groups, once but root induction,
Then radical is very intensive, and lateral root is flourishing, but average root long is shorter (the left plant of Fig. 5).
In the sprout rooting induction rate individually cultivated on 0.5~5.0mg/L of addition IBA culture medium with IBA concentration in just
Correlation, as IBA concentration increases, the increase of rooting induction rate, as concentration >=3.0mg/L, then difference is not notable.Averagely take root
Number and average root long difference between different IBA concentration is not notable, and showing as taking root, coefficient is relatively low, and lateral root is undeveloped, but
Average root long is longer (the right plant of Fig. 5).
It is more satisfactory that the sprout rooting efficiency cultivated on IBA and NAA culture medium is added at the same time, is superior to individually add
NAA's or IBA.When the timing of NAA concentration one, rooting rate raises with the increase of IBA concentration.It is average when the timing of IBA concentration one
Quantity of taking root raises with the increase of NAA concentration, and highest rooting rate is with taking root coefficient respectively up to 70.48% and 86.37.
Optimal rooting induction is combined as 1/2MS+3.0mg/L IBA+2.0~3.0mg/L NAA.It, which has been taken into account, individually with the addition of with dense
The advantages of NAA or IBA of degree, not only there is higher rooting induction rate and coefficient of taking root, while lateral root is flourishing, average root long occupies
In.
6 different plant growth regulator of embodiment induce influence of the source to tissue culture transplantation of seedlings
Experiment sets 1~3 group of test group.With yellow mud and peat soil with volume ratio 3 in test group 1~3：1 is mixed into base
Matter, the tissue-cultured seedling that then IBA, NAA and IBA are combined with NAA respectively and is induced are transplanted to Medium Culture after hardening.
Specific experiment step is as follows：
(1) take respectively and individually add the tissue-cultured seedling that IBA, NAA and IBA combine progress rooting induction with NAA.
(2) after tissue-cultured seedling grows to 3~4cm height, the culture bottle cap where it is opened, 3~5d of hardening, makes seedling preliminary
Adapt to extraneous environment.
(3) solid medium that its root system adheres to is washed away with running water, is then transplanted to mixing respectively and plants in containing yellow mud
With peat soil (volume ratio=3：1) in Medium Culture, every bag is transplanted one plant, each 100 plants of processing transplanting.
(4) root water is poured, the daily morning and evening keeps ground moistening using the method for spraying.After 30d count seedling into
Motility rate and its upgrowth situation.
The influence that 6 different plant growth regulator of table induction source is transplanted to training tissue culture seedling
The good group of taking root that will be induced respectively on the culture medium that individually addition NAA or IBA and NAA combines with IBA
Training seedling rinses out the agar of root system attachment in running water, finds the undeveloped tissue culture of lateral root induced in containing IBA culture mediums
The main root of seedling is easy to come off during agar is removed, or even the main root having all has come off, and is not used to transplant.And
Although the tissue-cultured seedling root containing the lateral root prosperity cultivated in NAA culture mediums also has the situation that root comes off, due to its root system
It is intensive, so the part root that comes off nor affects on transplanting.As shown in Table 6, observe and count after transplanting 30 days, find individually addition
The transplanting survival rate that NAA or IBA and NAA combines with IBA is respectively：82%th, 66.67% and 86%, tissue culture seedling leaf is emerald green
Green, cane is sturdy, grows fine (Fig. 6).
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than to the scope of the present invention
Limitation, although being explained in detail with reference to preferred embodiment to the present invention, it will be understood by those within the art that, can
To be modified to technical scheme or equivalent substitution, without departing from the spirit and scope of technical solution of the present invention.
Claims (9)
1. a kind of culture medium for being used to establish sterile system, it is characterised in that it is the culture medium based on MS culture mediums, addition
25~35g/L sucrose and 6.5~7.5g/L agar.
2. a kind of culture medium induced for clump bud with breeding, it is characterised in that it is to be used to establish nothing described in claim 1
Culture medium based on the culture medium of thalline system, it with the addition of 0.5~2.5mg/L 6-benzyladenines.
3. a kind of culture medium for adventitious bud inducing, it is characterised in that it is to be used to establish without thalline described in claim 1
Culture medium based on the culture medium of system, it with the addition of 2.5mg/L 6-benzyladenines.
4. a kind of culture medium for Elongation of adventitious bud induction, it is characterised in that it is to be used to establish nothing described in claim 1
Culture medium based on the culture medium of thalline system, it with the addition of 1.5~2.5mg/L methyl α-naphthyl acetates.
5. a kind of culture medium for taking root, it is characterised in that it is the culture medium based on 1/2MS culture mediums, be with the addition of
3.0mg/L indolebutyric acids, 2.0~3.0mg/L methyl α-naphthyl acetates, 25~35g/L sucrose and 6.5~7.5g/L agar.
A kind of 6. culture medium box set, it is characterised in that including described in claim 1 be used for establish sterile system culture medium,
The culture medium for being used for clump bud induction and propagation described in claim 2, the culture for adventitious bud inducing described in claim 3
The culture medium for being used for Elongation of adventitious bud induction described in base, claim 4, described in claim 5 for the culture medium taken root,
And volume ratio is 3：The matrix that 1 yellow mud and peat soil mixes.
7. application of the culture medium box set described in claim 6 in Vitex rotundifolia Vitro Quick Reproduction is promoted.
A kind of 8. method for promoting Vitex rotundifolia Vitro Quick Reproduction, it is characterised in that comprise the following steps：
(1) it is explant to choose Vitex rotundifolia semi-lignified seedling stem segment with axillary bud；
(2) aseptically, be inoculated in after explant is sterilized with mercuric chloride described in claim 1 be used for establish sterile system
Culture medium on, obtain aseptic seedling；
(3) aseptic seedling that step (2) obtains is cut into the stem with bud of 1 section of band, is inoculated in described in claim 2 and is used for
Clump bud is induced with the culture medium of propagation, obtaining clump bud；
(4) clump bud that step (3) obtains is cut into the stem section of 1 section of band, is inoculated in described in claim 3 and is used for adventitious bud
On the culture medium of induction, stem section flattening is induced, obtains adventitious bud；
(5) adventitious bud that step (4) obtains is inoculated in the culture medium for being used for Elongation of adventitious bud induction described in claim 4
On, obtain adventitious bud of the elongation for normality bud；
(6) elongation for obtaining step (5) for the adventitious bud of normality bud be inoculated in described in claim 5 for the culture taken root
On base, culture of rootage is carried out, tissue-cultured seedling is obtained, its root culture medium is cleaned up, it is 3 to transplant in volume ratio：1 yellow soil
In the matrix mixed with peat soil, the Vitro Quick Reproduction of Vitex rotundifolia is completed.
9. promote the method for Vitex rotundifolia Vitro Quick Reproduction according to claim 8, it is characterised in that step (2)~(6)
In condition of culture be：(25 ± 1) DEG C, light application time 12h/12h (L/D), 1500~2000lx of intensity of illumination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711087128.6A CN107711502B (en) | 2017-11-07 | 2017-11-07 | A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711087128.6A CN107711502B (en) | 2017-11-07 | 2017-11-07 | A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107711502A true CN107711502A (en) | 2018-02-23 |
| CN107711502B CN107711502B (en) | 2019-07-26 |
Family
ID=61221776
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711087128.6A Expired - Fee Related CN107711502B (en) | 2017-11-07 | 2017-11-07 | A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107711502B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109122314A (en) * | 2018-08-07 | 2019-01-04 | 中国科学院华南植物园 | A kind of the culture medium box set and method of Scaevola aemula Vitro Quick Reproduction |
| CN112741001A (en) * | 2020-12-29 | 2021-05-04 | 嘉应学院 | Vitex negundo callus induction method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104996299A (en) * | 2015-07-10 | 2015-10-28 | 四川省农业科学院园艺研究所 | In-vitro regeneration method for cucumber ovary |
| CN105875410A (en) * | 2016-04-15 | 2016-08-24 | 上海市农业科学院 | Rapid propagation method for hybrid cymbidium seedlings of cymbidium sinense and cymbidium hookerianum |
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN106900552A (en) * | 2017-03-08 | 2017-06-30 | 仲恺农业工程学院 | A kind of the culture medium box set and method of promotion barna Vitro Quick Reproduction |
-
2017
- 2017-11-07 CN CN201711087128.6A patent/CN107711502B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104996299A (en) * | 2015-07-10 | 2015-10-28 | 四川省农业科学院园艺研究所 | In-vitro regeneration method for cucumber ovary |
| CN105875410A (en) * | 2016-04-15 | 2016-08-24 | 上海市农业科学院 | Rapid propagation method for hybrid cymbidium seedlings of cymbidium sinense and cymbidium hookerianum |
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN106900552A (en) * | 2017-03-08 | 2017-06-30 | 仲恺农业工程学院 | A kind of the culture medium box set and method of promotion barna Vitro Quick Reproduction |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109122314A (en) * | 2018-08-07 | 2019-01-04 | 中国科学院华南植物园 | A kind of the culture medium box set and method of Scaevola aemula Vitro Quick Reproduction |
| CN109122314B (en) * | 2018-08-07 | 2020-06-09 | 中国科学院华南植物园 | Culture medium kit and method for in-vitro rapid propagation of pittosporum tobira |
| CN112741001A (en) * | 2020-12-29 | 2021-05-04 | 嘉应学院 | Vitex negundo callus induction method |
| CN112741001B (en) * | 2020-12-29 | 2022-04-08 | 嘉应学院 | Vitex negundo callus induction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107711502B (en) | 2019-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102301952B (en) | Method for breeding chamomile | |
| CN102301951B (en) | Method for rapidly propagating roots of subprostrate sophora by tissue culture | |
| CN105340747A (en) | Asexual rapid propagation method for radix glycyrrhizae | |
| CN111758559B (en) | Sterile sowing and seedling raising method for distant hybrid seeds of phalaenopsis amabilis and rhynchophylla | |
| CN104012417B (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN104041273A (en) | Potting domestication method for traditional Chinese medicine stevia rebaudiana | |
| CN103988777B (en) | A kind of in-vitro culture method for tender stem segments of the wide yulan of leaflet dwarf form | |
| CN103444552A (en) | Method for inducing eggplant anther to regenerate haplobiont | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN107711502B (en) | A kind of culture medium box set and its application in Vitex rotundifolia Vitro Quick Reproduction | |
| CN101637130B (en) | Cephalotaxus hainanensis embryo culturing and seedling breeding method | |
| CN103477988A (en) | Culture in vitro and rapid propagation method for syzygium grijsii | |
| CN101015280B (en) | Tissue culture method for fast propagation of primula denticulata ssp.sino-denticulata | |
| CN103416302B (en) | Method for culturing regeneration plant of somatic embryo of osmanthus fragrans Lour | |
| CN112715367A (en) | Method for carrying out tissue culture subculture propagation on cornus walteri by utilizing lanthanum nitrate | |
| CN104094848A (en) | Induction of tung tree hypocotyls callus and method for efficiently regenerating plants | |
| CN101743908A (en) | Tissue culture, rapid propagation and cultivation method of grevillea banksii | |
| CN106489737A (en) | A kind of culture medium of Hybrid Tea tissue cultures and method | |
| CN103609444A (en) | Tissue culture method for hemerocallis sempervirens araki | |
| CN106804426A (en) | Promote the box set and method of Companumoea root vitro proliferation | |
| CN105766636A (en) | Tissue culture regeneration method of paeonia suffruticosa | |
| CN109156350A (en) | A kind of method of the numerous bud of wind resistance paulownia and root media and promotion wind resistance paulownia Vitro Quick Reproduction | |
| CN101595843A (en) | Kuh-seng tissue culture and method for quickly breeding | |
| CN100559935C (en) | The tissue culture and rapid propagation method that orange lamp stand is heralded spring | |
| CN109863998B (en) | Tissue culture method of tilia mongolica seedlings |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190726 Termination date: 20201107 |