CN107711502B - 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
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- CN107711502B CN107711502B CN201711087128.6A CN201711087128A CN107711502B CN 107711502 B CN107711502 B CN 107711502B CN 201711087128 A CN201711087128 A CN 201711087128A CN 107711502 B CN107711502 B CN 107711502B
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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
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Abstract
The invention discloses a kind of culture medium box set and its applications in Vitex rotundifolia Vitro Quick Reproduction.Culture medium box set of the present invention includes for establishing the culture medium of sterile system, inducing the matrix mixed with the culture medium being proliferated, the culture medium for adventitious bud inducing, culture medium, the culture medium for taking root and the volume ratio for Elongation of adventitious bud induction for the yellow mud and peat soil of 3:1 for clump bud.Present invention firstly discovers that the adventitious bud development ways of stem with bud flattening induction and the through the invention in vitro culture of culture medium box set progress Vitex rotundifolia, 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 fields, it is more particularly related to a kind of climing for single leaf
The culture medium box set and method of Jing Congya induction and the Vitro Quick Reproduction that adventitious shoot regeneration approach is formed in flattening stem section.
Background technique
Vitex rotundifolia (Vitex rotundifolia L.) is Verenaceae Vitex machaka.It is distributed widely in
It is adaptable on the waste continent on the ground such as the sandy beach of tropical and subtropical region, seashore and lakeside, it is not stringent to environmental requirement
And there are the characteristics such as alkaline-resisting, high temperature resistant, it can be used as the pioneer vegetation of coastal sandy land, wind gap district dune-fixating forestation.Vitex rotundifolia is also
With higher medical value, fruit is one of the source of China's conventional Chinese medicine " fructus viticis ", has dispelling wind and heat from the body, head clearing
The effect of.Modern pharmacological studies have shown that fructus viticis has amphene, firpene, flavonoids, casticin and luteolin etc. chemical
Ingredient has the effects that significantly analgesia, anti-inflammatory, decompression, antitumor and anti-aging.And its stem item can be also used for large bamboo or wicker basket
Establishment, spends extractable fragrance etc..
With the development and utilization of fructus viticis new application and new product in recent years, market demand increasingly increases.Lead to it
Resource is increasingly depleted.In " Key Protected medicinal material species register ", it is in imminent danger that Vitex rotundifolia has been cited as III grade of country
Protect plant.Currently, manually being protected in terms of being concentrated mainly on chemical component and pharmacological action for the research of fructus viticis both at home and abroad
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.
Summary 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, mentions
It has supplied one kind using Vitex rotundifolia stem segment with axillary bud as explant, has inquired into the plant growth regulator and its group of various concentration, type
It closes 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, establishes height
The Vitex rotundifolia tissue culture sprout quick propagating technology system of effect.
In order to achieve the above-mentioned object of the invention, the present invention provides a kind of for establishing the culture medium of sterile system, be with
MS culture medium is basic culture medium, is added to 25~35g/L sucrose and 6.5~7.5g/L agar.
A kind of optimal technical scheme of culture medium as the present invention for establishing sterile system, the additive amount of sucrose are
30g/L, the additive amount of agar are 7g/L.
The present invention also provides a kind of culture mediums induced for clump bud with proliferation, are for establishing sterile system
Culture medium is basic culture medium, is added to 0.5~2.5mg/L 6-benzyladenine.
It is with the culture for establishing sterile system the present invention also provides a kind of culture medium for adventitious bud inducing
Base is basic culture medium, is added to 2.5mg/L 6-benzyladenine.
The present invention also provides a kind of culture mediums for Elongation of adventitious bud induction, are for establishing sterile system
Culture medium is basic culture medium, is added to 1.5~2.5mg/L methyl α-naphthyl acetate.
It is with 1/2MS culture medium for basic culture medium, addition the present invention also provides a kind of culture medium for taking root
3.0mg/L indolebutyric acid, 2.0~3.0mg/L methyl α-naphthyl acetate, 25~35g/L sucrose and 6.5~7.5g/L agar.
BA, i.e. 6-benzyladenine are 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 inhibiting leaves of plants inner chlorophyll, nucleic acid, protein, protects green anti-old；By amino acid, auxin, inorganic salts etc. to place
A variety of efficiency such as position allocation and transportation are managed, each stage of agricultural, fruit tree and garden crop from germination to harvest is widely used in；In plant
Rapid propagation in vitro in can promote cell division and by breaking up adventitious bud on callus or organ, while helping to make axillary bud by pushing up
It is freed under the inhibition of end advantage.
NAA, i.e. methyl α-naphthyl acetate are a kind of broad spectrum type plant growth regulator, for inducing in Vitro Plant incubation
The division of cell and the differentiation of root, while phenomena such as the elongation of stem and section, tropism, apical dominance, leaf abscission can be influenced.
TDZ, i.e. Thidiazuron are a kind of efficient basic elements of cell division, can be used for promoting callus group in Vitro Plant is numerous fastly
The generation for knitting growth, lateral bud and adventitious bud promotes the formation of embryoid.
2,4-D, i.e. 2,4- dichlorphenoxyacetic acid are a kind of artificial synthesized auxin analogs.Under low consistency conditions,
The highly effective promotion callus mitogen of energy, but the generation of energy strong inhibition organ.Its simultaneously or systemic herbicide.
KT, i.e. kinetin are the artificial synthesized basic elements of cell division, can promote cell differentiation, division, growth, are promoted thin
Born of the same parents divide, and break up adventitious bud on callus or organ.
ZT, i.e. zeatin are the natural phytohormones 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 are plant endogenous auxin, fast numerous in the process for promoting cell division in Vitro Plant
It is grown with cell, induced synthesis adventitious root etc..
IBA, i.e. indolebutyric acid are plant endogenous auxin, fast numerous in the process for promoting cell division in Vitro Plant
It is grown with cell, induced synthesis adventitious root, fruit setting can be increased in agricultural production, prevent shedding, change female, male flower ratio etc..
In order to achieve the above-mentioned object of the invention, the present invention also provides a kind of culture medium box sets comprising above-mentioned is used to build
The culture medium of vertical sterile system, for clump bud induce with the culture medium being proliferated, for adventitious bud inducing culture medium, be used for it is indefinite
The yellow mud that culture medium, the culture medium for taking root and the volume ratio of bud elongation induction are 3:1 and the base that 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 achieve the above-mentioned object of the invention, the present invention also provides a kind of sides for promoting Vitex rotundifolia Vitro Quick Reproduction
Method comprising following steps:
(1) choose Vitex rotundifolia semi-lignified seedling stem segment with axillary bud be explant (preferably take length for 1.5~3cm simultaneously
Stem section with 2 lateral buds)；
(2) aseptically, the training for being used to establish sterile system is inoculated in after explant being sterilized with mercuric chloride
It supports on base, obtains aseptic seedling；
(3) aseptic seedling that step (2) obtain is cut into the stem with bud of 1, band section (2 lateral buds), be inoculated in described in
The culture medium with proliferation is induced for clump bud, obtains clump bud；
(4) clump bud that step (3) obtain is cut into the stem section of 1, band section, is inoculated in described for adventitious bud inducing
Culture medium on, induce stem section flattening, obtain adventitious bud；
(5) adventitious bud that step (4) obtain is inoculated in the culture medium for Elongation of adventitious bud induction, is obtained
Elongation is the adventitious bud of normality bud；
(6) adventitious bud that the elongation that step (5) obtain is normality bud is inoculated in the culture medium for taking root,
Culture of rootage is carried out, tissue-cultured seedling is obtained, culture medium cleans up by its root, transplants in yellow soil and mud that volume ratio is 3:1
In the matrix that charcoal soil mixes, the Vitro Quick Reproduction of Vitex rotundifolia is completed.
A kind of optimal technical scheme of method as present invention promotion Vitex rotundifolia Vitro Quick Reproduction, step (2)~
(6) condition of culture in is equal are as follows: (25 ± 1) DEG C, light application time 12h/12h (L/D), 1500~2000lx of intensity of illumination.
A kind of optimal technical scheme for promoting the method for Vitex rotundifolia Vitro Quick Reproduction as the present invention, in step (2)
The process of the disinfection includes: that 10~15s is sterilized in 75% alcohol, and rinsed with sterile water 1 time, with 0.1% mercuric chloride soaking disinfection
8min, rinsed with sterile water 7 times, then surface moisture is blotted with aseptic filter paper.
Promote a kind of optimal technical scheme of the method for Vitex rotundifolia Vitro Quick Reproduction, the method tool as the present invention
Body are as follows: the stem section for taking Vitex rotundifolia health branch cuts off extra blade, takes long 1.5~3cm and has the stem section of 2 lateral buds
It is seeded in the culture medium for establishing sterile system after surface sterilization is handled for explant, obtains sterile sprout；It takes
The stem section of 1, the band section (2 lateral buds) of sterile sprout is inoculated in the culture medium for inducing and being proliferated for clump bud respectively, with clump bud
Inductivity, bud proliferation times, the average indexs such as plant height and growth conditions have determined suitable clump bud induction and proliferated culture medium；
The clump bud to grow fine formed on the culture medium for inducing and being proliferated for clump bud is taken, cuts the stipes with axillary bud with blade,
It is inoculated in 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 with adventitious bud
Side；It takes and is aseptically cut with blade in the flattening stem section for forming adventitious bud in Elongation of adventitious bud induced medium
Take the identical flattening stem section of adventitious bud number, be inoculated in for Elongation of adventitious bud induced medium, with Elongation of adventitious bud rate,
The indexs such as adventitious bud average length and upgrowth situation, it is determined that the formula of suitable Elongation of adventitious bud induced medium.It takes
The indefinite sprout of the elongation grown fine in Elongation of adventitious bud induced medium is inoculated on the culture medium for taking root and carries out
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 take root good stand, and after the culture medium for washing away the attachment of its root, transplanting is in being mixed with yellow mud and peat soil
In the matrix of (volume ratio 3:1), culture can be obtained the tissue-cultured seedling to grow fine in 30 days.It is climing that the present invention establishes single leaf for the first time
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 induction and proliferation, 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.
Compared with the existing technology, the present invention has the advantage that
(1) present invention is for establishing 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 induced for clump bud with the culture medium of proliferation, is cut in the culture medium for establishing sterile system
The stem section of one, the sterile sprout band section formed is explant, with MS minimal medium, the BA of different quality concentration is added, with clump
Bud induction rate, bud proliferation times and plant height are index, have screened suitable clump bud induction formula, and have found that single leaf is climing for the first time
Chaste tree stem with bud there is a phenomenon where flattening and can form adventitious bud.With 30 days for cultivation cycle, clump bud inductivity, bud proliferation times
Several and plant height reaches as high as 81.11%, 21.91 and 5.25cm respectively, is formed by that clump bud blade is light green, and internode is compact, long
Gesture is good.
(3) because being induced for clump bud and having found Vitex rotundifolia stem with bud flattening and not in the culture medium that is proliferated
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, for the induction of Vitex rotundifolia stem with bud inclined graduation and adventitious bud occur in adventitious bud induction culture base, with
It induces in clump bud and adds for explant using MS as minimal medium with the in vitro stem with bud for the sprout that grows thickly in the culture medium of proliferation
The BA of high concentration (>=2.5mg/L) is added, has been to refer to form adventitious bud number 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 for cutting for occurring not in adventitious bud induction culture base in Elongation of adventitious bud induced medium
The flattening stem section of normal bud is explant, and using MS as minimal medium, addition 1.5~2.5mg/L NAA or BA are combined 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
30 days are cultivation cycle, and 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) will be used to extend obtained in Elongation of adventitious bud induced medium and the sprout of robust growth in containing IBA or
Culture of rootage is carried out in the 1/2MS culture medium of NAA and combinations thereof, finally carries out rooting culture.It rooting rate and averagely takes 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, produces 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.
Detailed description of the invention
With reference to the accompanying drawings and detailed description, the method for the present invention and beneficial effect are described in detail.
Fig. 1 is that Vitex rotundifolia aseptic seedling establishes culture effect figure；
Fig. 2 is the effect picture of clump bud induction；
Fig. 3 is the effect picture of stem section flattening and adventitious bud inducing；
Fig. 4 is the effect picture of Elongation of adventitious bud；
Fig. 5 is the plant to take root；
Fig. 6 is the plant of transplant survival.
Specific embodiment
In order to be more clear the purpose of the present invention, technical solution and advantageous effects, with reference to embodiments, to this
Invention is further elaborated.It should be understood that embodiment described in this specification is just for the sake of this hair of explanation
It is bright, be not intended to limit the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substance had no to result
It influences.
In following embodiment, term " in vitro " refers to for various research purposes and a part of organism is extracted and swum
From in the state of in vitro.
Term " explant " refers in squamous subculture, moves into the tissue dissection of 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 that the amount a great number of elements in MS culture medium is reduced to culture medium prepared by original 1/2.
The ingredient of MS culture medium is as shown in table 1 below:
The ingredient of table 1MS culture medium
Unless otherwise specified, it is trained involved in the method for promoting Vitex rotundifolia Vitro Quick Reproduction in the embodiment of the present invention
Support base and matrix prepared by the following method respectively (need to compare variety classes, concentration growth regulator when, then replace phase
Answer component):
(1) it prepares 1L MS culture medium: accurately weighing various compounds described in table 1, appropriate distilled water is added to dissolve, use
Glass bar stirs dissolution, with NaOH tune pH to 6.0, last constant volume to 1L.
(2) it is formulated for establishing the culture medium of sterile system: also add on the basis of the MS culture medium prepared by step (1)
Add 25~35g/L sucrose and 6.5~7.5g/L agar；
(3) it is formulated for the culture medium of clump bud induction and proliferation: on the basis of the MS culture medium 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 being added on the basis of the MS culture medium prepared by step (2)
There is 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 medium prepared by step (2) also
Added with 1.5~2.5mg/L methyl α-naphthyl acetate；
(6) be formulated for the culture medium taken root: the MS culture medium prepared by step (1) 1/2 on the basis of be also added with
3.0mg/L indolebutyric acid, 2.0~3.0mg/L methyl α-naphthyl acetate, 25~35g/L sucrose and 6.5~7.5g/L agar；
(7) prepare matrix 1: according to yellow mud: the ratio of peat soil=3:1 (volume ratio) is mixed.
Unless otherwise specified, condition of culture as described below is equal are as follows: (25 ± 1) DEG C, light application time 12h/12h (L/D), light
According to 1500~2000lx of intensity.
The foundation of 1 sterile system of embodiment
Only 1 group of test setting, in April, 2017 take Vitex rotundifolia stem with bud to carry out Vitex rotundifolia sterile system and establish examination
It tests, the specific steps are as follows:
The selection of explant: taking the stem section of Vitex rotundifolia health branch, cuts off extra 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 disinfection, 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, it is inoculated in the MS blank without any plant growth regulator
It is cultivated on culture medium.
It can get that blade is emerald green unfolds after culture 30d, the sterile sprout (Fig. 1) to grow fine.
Embodiment 2~6 tests variety classes respectively, the growth regulator of concentration is added in the above various culture mediums, right
It is influenced brought by each period in Vitex rotundifolia Vitro Quick Reproduction, and obtains preferably growth regulator selection.
The plant growth regulator influence that Vitex rotundifolia Cut stem clump bud is induced of 2 variety classes of embodiment, concentration
1~25 group of test setting, using MS as minimal medium, is individually added to 0.1 in different test groups
IAA, 0.1~2.5mg/L NAA, 0.1~2.5mg/L KT, 0.1~2.5mg/L ZT, the 0.1~2.5mg/L of~2.5mg/L
BA and 0.1~2.5mg/L TDZ；
Specific step is as follows:
The stem section of 1, band section (2 lateral buds) is taken to be inoculated in test 1~25 respectively from the sterile sprout in embodiment 1
In culture medium, each bottle inoculation stem section 3, each 10 bottles of formula.
Its growing state is observed and recorded after culture 30d and statistics bud is averaged germination rate, clump bud inductivity, bud proliferation times
And average plant height.
The test is repeated twice after being spaced 15d.
Average bud germination rate=(explant number when explant number/inoculation that bud is sprouted) × 100%；Clump bud inductivity=
Generate explant number when explant number/inoculation of clump bud；Bud sum when bud proliferation times=bud sum/inoculation；Average strain
The quantity of explant when high=total plant height/inoculation.
The clump bud induction of Vitex rotundifolia is carried out using the above method, obtained result such as table 2:
2 variety classes of table, concentration plant growth regulator to Vitex rotundifolia clump bud induce be proliferated influence
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 for 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 single plant, proliferation multiplying power is lower, in high concentration item
(>=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 function and effect of different types of basic element of cell division to bud proliferation and plant height.
It is grown in the plant major part single plant of the culture medium culture containing KT or ZT, the proliferation times of clump bud inductivity and bud
Rate is lower, but average plant height is significantly higher than other processing 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 processing groups, is leaning on
There is the callus that a large amount of clump bud is grown and part has green fine and close to occur at the axillary bud of nearly base portion.Clump bud inductivity and bud increase
Multiple and being positively correlated property of concentration are grown, highest is respectively up to 81.11% and 21.91.It is that then difference is not when concentration is higher than 0.5mg/L
Significantly.Under the conditions of the BA of high concentration (>=0.5mg/L), the inclined graduation of part stem, and adventitious bud is directly generated from stem, with
The extension of incubation time, adventitious bud can grow up to normality bud；
Callus is formed in most of lateral bud containing TDZ culture medium culture, 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 is proliferated formula
For MS+0.5~2.5mg/L BA.It bud germination rate, clump bud inductivity and proliferation times with higher and grows fine.
3 various concentration of embodiment, type plant growth regulator to the adventitious bud inducing shadow of Vitex rotundifolia Cut stem
It rings
Test setting 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 group
It closes and 1.0mg/L BA, 0.1mg/L NAA is combined with 0.1mg/L TDZ；
Specific step is as follows:
The stipes with axillary bud is taken to be inoculated in the culture medium of test group 1~17 respectively from the sprout that grows thickly of embodiment 2, often
A 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 test is repeated twice after being spaced 15d.
Adventitious bud induction frequency=(explant number when 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/.
Occurred using the adventitious bud that the above method carries out Vitex rotundifolia stem section flattening induction, obtained result such as table 3.
The influence to Vitex rotundifolia adventitious bud inducing of plant growth regulator and combinations thereof of 3 various concentration of table, type
As shown in Table 3: stem section cultivates base portion notch on the culture medium of addition 1.0mg/L 2,4-D or 1.0mg/L TDZ
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), be formed adventitious bud (Fig. 3).It is indefinite in the culture medium of addition BA
Bud incidence and average adventitious bud number highest, adventitious bud incidence increase with average adventitious bud number with the raising of BA concentration,
Highest is respectively up to 60% and 84.87.As concentration >=2.5mg/L, then difference is not significant and high concentration (>=5.0mg/L) BA can
Make leaf curl, and with slight vitrifying；When 1.0mg/L BA is combined with NAA or with NAA, TDZ, adventitious bud incidence
With no significant difference when independent addition 1.0mg/L BA, but its adventitious bud number when BA is individually combined with NAA considerably less than
Other two processing groups.It is found that NAA has certain negative consequence to adventitious bud.Optimal adventitious bud inducing formula is answered
Are as follows: MS+2.5mg/L BA.
4 various concentration of embodiment, type plant growth regulator to Elongation of adventitious bud in Vitex rotundifolia flattening stem section
It influences
Test setting 1~13 group, 1~13 test group using MS as minimal medium, in different test groups or point
Be not added to 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 is applied in combination；
Specific test procedure is as follows:
(1) it takes to form the flat stem section of adventitious bud from embodiment 3, is aseptically cut into the identical adventitious bud of size
Block, about 10/block of adventitious bud number, adventitious bud height is roughly the same, and 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, the length of statistics Elongation of adventitious bud rate and average each adventitious bud.It repeats to be somebody's turn to do after being spaced 15d
Test is twice.Elongation=(adventitious bud sum when adventitious bud number/inoculation that appearance extends) × 100%；It is average each indefinite
Adventitious bud number when length=adventitious bud total length/inoculation 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: the Elongation of adventitious bud effect on the culture medium of addition NAA is best, and sprout is elongated emerald green (Fig. 4),
Bud elongation and the length of elongation are all remarkably higher than other processing groups.With the increase of NAA concentration, bud elongation and adventitious bud are long
Degree also increases, and highest is respectively up to 58.9% and 3.0cm, but then difference is not significant as concentration >=1.5mg/L；KT or BA induction
Elongation of adventitious bud effect and blank control group difference is not significant.And the adventitious bud internode of elongation is induced on the culture medium of addition BA
It is short, while new adventitious bud can be formed, thus the adventitious bud that the elongation for influencing adventitious bud is extended with average bud length, KT induction
Although blade is unfolded, internode length, Elongation of adventitious bud rate and average length are lower.When BA and NAA arrange in pairs or groups, bud elongation with
Length is above when individually adding BA, it is seen then that NAA plays a significant role bud elongation induction.Optimal adventitious bud inducing
Formula are as follows: MS+1.5~2.5mg/L NAA.
The influence that embodiment 5NAA and IBA takes root to Vitex rotundifolia sprout
Test 1~16 test group of setting, 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
(1.0 combine IBA with 3.0mg/L) with NAA (1.0,2.0,3.0mg/L), 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 test
10 bottles of group.
(3) its rooting rate, average every plant of number of taking root are counted after cultivating 40d with average root long and record the length of its seedling
Gesture.Test 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 the IBA and NAA and combinations thereof of 5 various concentration of table take root to Vitex rotundifolia sprout
As shown in Table 5: sprout cannot 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 processing 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).
The sprout rooting induction rate and IBA concentration cultivated on the individually culture medium of addition 0.5~5.0mg/L IBA are in just
Correlation, as IBA concentration increases, rooting induction rate increases, and as concentration >=3.0mg/L, then difference is not significant.Averagely take root
Several and average root long difference between different IBA concentration is not significant, and showing as taking root, coefficient is lower, 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 the culture medium of IBA and NAA is added at the same time, is superior to individually add
NAA's or IBA.When one timing of NAA concentration, rooting rate is increased with the increase of IBA concentration.It is average when one timing of IBA concentration
Quantity of taking root is increased with the increase of NAA concentration, highest rooting rate and takes root coefficient respectively up to 70.48% and 86.37.
Optimal rooting induction group is combined into 1/2MS+3.0mg/L IBA+2.0~3.0mg/L NAA.It, which has been taken into account, is individually added to dense
The advantages of NAA or IBA of degree, not only rooting induction rate with higher and coefficient of taking root, while lateral root prosperity, average root long occupy
In.
Influence of the different plant growth regulator inductions of embodiment 6 source to tissue culture transplantation of seedlings
1~3 group of test group of test setting.Base is mixed into volume ratio 3:1 with yellow mud and peat soil in test group 1~3
Matter, the tissue-cultured seedling for then respectively combining IBA, NAA and IBA with NAA and inducing are transplanted to Medium Culture after hardening.
Steps are as follows for specific experiment:
(1) it takes respectively and individually adds the tissue-cultured seedling that IBA, NAA and IBA combine progress rooting induction with NAA.
(2) after tissue-cultured seedling grows to 3~4cm high, the culture bottle cap where it is opened, 3~5d of hardening keeps seedling preliminary
Adapt to extraneous environment.
(3) then the solid medium that the attachment of its root system is washed away with tap water is transplanted to mixing respectively and plants in containing yellow mud
In the Medium Culture of peat soil (volume ratio=3:1), every bag is transplanted one plant, and each processing transplants 100 plants.
(4) it is watered with root water, the daily morning and evening keeps ground moistening using spraying method.After 30d count seedling at
Motility rate and its upgrowth situation.
The influence that training tissue culture seedling is transplanted in the different plant growth regulator inductions of table 6 source
It takes root what is induced on the culture medium that individually addition NAA or IBA and NAA is combined with IBA good group respectively
Training seedling rinses out the agar of root system attachment in tap water, finds the undeveloped tissue culture of lateral root induced in containing IBA culture medium
The main root of seedling is easy to fall off during removing agar, or even the main root having all has fallen off, and is not used to transplant.And
Although the tissue-cultured seedling root containing the lateral root prosperity cultivated in NAA culture medium also has the case where root falls off, due to its root system
Intensively, so the part root that falls off nor affects on transplanting.As shown in Table 6, it observes and counts after transplanting 30 days, find individually addition
The transplanting survival rate that NAA or IBA and NAA is combined with IBA is respectively as follows: 82%, 66.67% and 86%, and tissue culture seedling leaf is emerald green
Green, stem is sturdy, and grow 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 the invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that, can
With modification or equivalent replacement of the technical solution of the present invention are made, without departing from the spirit and scope of technical solution of the present invention.
Claims (1)
1. a kind of method for promoting Vitex rotundifolia Vitro Quick Reproduction, which comprises the steps of:
(1) choosing Vitex rotundifolia semi-lignified seedling stem segment with axillary bud is explant；
(2) aseptically, it is inoculated in the culture medium for being used to establish sterile system after explant being sterilized with mercuric chloride, obtains
Aseptic seedling；
(3) aseptic seedling that step (2) obtain is cut into the stem with bud of 1, band section, is inoculated in and is induced and be proliferated for clump bud
Culture medium on, obtain clump bud；
(4) clump bud that step (3) obtain is cut into the stem section of 1, band section, is inoculated in the culture medium for adventitious bud inducing,
Stem section flattening is induced, adventitious bud is obtained；
(5) adventitious bud that step (4) obtain is inoculated in the culture medium for Elongation of adventitious bud induction, obtaining elongation is normality
The adventitious bud of bud；
(6) adventitious bud that the elongation that step (5) obtain is normality bud is inoculated in the culture medium for taking root, carries out training of taking root
Support, obtain tissue-cultured seedling, by its root, culture medium is cleaned up, transplant in volume ratio be 3:1 yellow soil and peat soil mixing and
At matrix on, complete the Vitro Quick Reproduction of Vitex rotundifolia；
The culture medium for being used to establish sterile system is to be added to 25~35g/L with MS culture medium for basic culture medium
Sucrose and 6.5~7.5g/L agar；
Described induces the culture medium with proliferation for clump bud, is cultivated based on the culture medium for establishing sterile system
Base is added to 0.5~2.5mg/L6- benzyladenine；
The culture medium for adventitious bud inducing is with the culture medium for establishing sterile system for basic culture medium,
It is added to 2.5mg/L6- benzyladenine；
The culture medium for Elongation of adventitious bud induction, is cultivated based on the culture medium for establishing sterile system
Base is added to 1.5~2.5mg/L methyl α-naphthyl acetate；
The culture medium for taking root is to be added to 3.0mg/L indoles fourth with 1/2MS culture medium for basic culture medium
Acid, 2.0~3.0mg/L methyl α-naphthyl acetate, 25~35g/L sucrose and 6.5~7.5g/L agar；
Condition of culture in step (2)~(6) is equal are as follows: 25 ± 1 DEG C, light application time 12h/12h L/D, intensity of illumination 1500~
2000lx。
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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