CN110432150A - A kind of method of the acquisition and fast breeding of lamiophlomis rotata aseptic seedling - Google Patents
A kind of method of the acquisition and fast breeding of lamiophlomis rotata aseptic seedling Download PDFInfo
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- CN110432150A CN110432150A CN201910842860.2A CN201910842860A CN110432150A CN 110432150 A CN110432150 A CN 110432150A CN 201910842860 A CN201910842860 A CN 201910842860A CN 110432150 A CN110432150 A CN 110432150A
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- lamiophlomis rotata
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The preparation method of present invention offer lamiophlomis rotata aseptic seedling, steps are as follows: (1) first using tap water flowing water to rinse in lamiophlomis rotata seed, then it disinfects on the super-clean bench, first sterilized with 75% ethanol solution, aseptic water washing, it is impregnated with 30% hydrogenperoxide steam generator, then is impregnated with 10% hydrogenperoxide steam generator, germination culture medium is inoculated into after aseptic water washing and carries out dark culture；(2) after seed is sprouted, it is transferred to illumination cultivation；Aseptic seedling cotyledon and hypocotyl are cut into small pieces/section on superclean bench after cotyledon stretching, extension, lie against the induction that inducing clumping bud culture medium carries out Multiple Buds；Also it can be used the seedling for growing 4-5 piece true leaf or the test tube seedling in squamous subculture, its blade of clip to be cut into small pieces, be inoculated in the induction that inducing clumping bud culture medium carries out Multiple Buds.The present invention also provides its enrichment procedures.The present invention improves lamiophlomis rotata tissue cultures proliferation rate, and the acquisition time of aseptic seedling foreshortens to 3-4 months.
Description
Technical field
The invention belongs to tissue cultures, secondary metabolite evaluation and exploration technology fields, and in particular to one kind passes through tissue
Culture induces lamiophlomis rotata Multiple Buds and rapid propagation method.
Background technique
Currently, lamiophlomis rotata medicinal material is mainly derived from wild resource, with the extensive use of lamiophlomis rotata clinically, medicine is needed
The amount of asking increases, the predatory ground excessively harvesting to wild resource, so that the wild resource of lamiophlomis rotata already close to exhaustion, is arranged
Enter Tibetan medicine material in imminent danger.On the other hand, great destruction also is caused to local ecological environment to the excessive excavation of wild resource,
The desertification on meadow is caused in Alpine Grasslands excavation, accelerates grassland degeneration.Lamiophlomis rotata is mainly by Propagation of Rhizomes, the germination percentage of seed
It is lower, due to that cannot regenerate after excavation, cause largely reducing for wild resource.Forbid excavating under ground portion at present, but on the ground
Part is restored to medical value after excavating and is generally 4 years, and high altitude localities takes 5 years or more, but in fact, many main producing regions are basic
The excavation with regard to carrying out a new round in 4 years is not achieved, this is the common issue that many plateau medicinal plants face.The artificial of lamiophlomis rotata is tamed and dociled
Changing cultivation, also there are many limiting factors, due to the special annidation of lamiophlomis rotata, are only capable of planting in high altitudes and cold area, this
Undoubtedly increase cultivation difficulty.The altitude environment season of growth is short, and cultivation medicinal material reaches medicinal standard and needed for 6 years, and the period is long
Large-scale production is limited, artificial cultivation not can solve mesh regardless of all showing as slow growth in laboratory or crop field
The demand of preceding pharmaceuticals industry.
The border that wild resource faces extinction to the destruction of ecological environment and lamiophlomis rotata germ plasm resource is excessively excavated in order to prevent
Ground, it is very necessary to open up new approach, solves the problems, such as this just using biotechnological method and becomes lamiophlomis rotata medicinal material and is most reliable
Source.The tissue cultures for carrying out lamiophlomis rotata are not only that the large-scale the factorial production of lamiophlomis rotata provides theoretical foundation, will yet
For the development of ethnic drug, the protection of lamiophlomis rotata wild resource and Alpine Grasslands ecological environmental protection are played an important role.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides the preparation methods of lamiophlomis rotata aseptic seedling, and lead to
The mode for crossing induction Multiple Buds is quickly bred, and can get a large amount of aseptic seedlings in short term.
The present invention provides a kind of preparation method of lamiophlomis rotata aseptic seedling, comprising the following steps:
(1) explant culture: first using tap water flowing water to rinse in lamiophlomis rotata seed, then disinfect on the super-clean bench,
First with 75% ethanol solution sterilizing 90-120s, aseptic water washing 2-3 times, 15-20min is impregnated with 30% hydrogenperoxide steam generator, then
30-60min is impregnated with 10% hydrogenperoxide steam generator, aseptic water washing 2-3 is inoculated into germination culture medium after and carries out dark culture；
(2) inducing clumping bud: after seed is sprouted, it is transferred to illumination cultivation；By aseptic seedling cotyledon and lower embryo after cotyledon stretching, extension
Axis is cut into 1.8-2.2mm fritter/section on superclean bench, lies against inducing clumping bud culture medium, carries out the induction of Multiple Buds；
Also the seedling for growing 4-5 piece true leaf or the test tube seedling in squamous subculture, its blade of clip can be used to be cut into the fritter of 2-5mm, connect
Kind carries out the induction of Multiple Buds in inducing clumping bud culture medium.
Preferably, first using tap water flowing water to rinse in lamiophlomis rotata seed, then on superclean bench in step (1)
Disinfection treatment aseptic water washing 3 times, impregnates 20min with 30% hydrogenperoxide steam generator first with 75% ethanol solution sterilizing 2min,
30min is impregnated with 10% hydrogenperoxide steam generator again, germination culture medium is inoculated into after aseptic water washing 3 times and carries out germination culture.
Preferably, the culture medium of the germination culture is 1/2MSB culture medium in step (1), condition of culture is dark training
It supports, 25 ± 2 DEG C of cultivation temperature.
Preferably, in step (2), the illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h are dark
10h, 25 ± 2 DEG C of cultivation temperature.
Preferably, the inducing clumping bud culture medium is to add 300-800mg in MSB culture medium in step (2)
L-1CH, 2.2g Gelrite, pH value 5.8, and additional 1.0-2.0mgL-1KT+0.1-0.5mg·L-1IBA；
Preferably, the inducing clumping bud culture medium is to add 500mgL in MSB culture medium in step (2)- 1CH, 2.2g Gelrite, pH value 5.8, and additional 1.0mgL-1KT+0.1mg·L-1IBA。
The present invention provides a kind of quick proliferation method of lamiophlomis rotata aseptic seedling, using the method for any one of claim 1-5
Obtain lamiophlomis rotata aseptic seedling, then under the conditions of illumination cultivation by lamiophlomis rotata aseptic seedling transfer into subculture multiplication medium carry out after
For Multiplying culture；The subculture multiplication medium formula are as follows: add 300-800mgL in MSB culture medium-1CH, 2.2g
Gelrite, and additional 0.2-1.0mgL-1KT+0.05-0.2mg·L-1IBA, will be in subculture multiplication medium after subculture 2 times
Hormone concentration, which halves, to be continued to cultivate.
Preferably, the subculture multiplication medium formula are as follows: add 500mgL in MSB culture medium-1CH, and add
0.2mg·L-1KT+0.05mg·L-1IBA。
Preferably, the illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, cultivation temperature
25±2℃。
The present invention provides a kind of rooting method of lamiophlomis rotata aseptic seedling, only one obtained using the method for claim 6 or 7
Taste aseptic seedling is selected and grows vigorous, healthy and strong bud, cuts from base portion, be inoculated into root media under the conditions of illumination cultivation and lure
It leads and takes root, root media is 1/2MSB culture medium, and adds 0.05-0.2mgL-1NAA+0.5-1.0mg·L-1IBA。
Preferably, the illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, cultivation temperature
25±2℃。
The present invention successfully overcomes lamiophlomis rotata Seed sterilization and is not thorough the problem low with germination rate, improves lamiophlomis rotata tissue
Cultivate proliferation rate.The present invention develops acquisition and the rapid propagation system for the aseptic seedling for being adapted to lamiophlomis rotata, so that aseptic seedling
The acquisition time foreshortens to 3-4 months, and by inducing clumping bud, shoot proliferation multiple reaches 10-25 times.
The method of the acquisition and fast breeding of lamiophlomis rotata aseptic seedling provided by the invention, from the disinfection of wild seed to seed
It sprouts, using cotyledon, hypocotyl slice (section), blade induces Multiple Buds, induces it, the condition of culture in breeding carries out
Optimization, finally establishes Aseptic seedling culture system.The preparation method of lamiophlomis rotata aseptic seedling of the invention can obtain in 3-4 months
A large amount of aseptic seedlings, inducing clumping bud rate can reach 80%, and further reach 50%-70% using rooting method rooting rate.Kind
The disinfection of son is the first step of tissue cultures, and 30% hydrogenperoxide steam generator immersion 20min is conducive to the softening of kind of skin and surface disappears
Poison changes 10% hydrogenperoxide steam generator into, can play the role of thoroughly eliminating for the intradermal portion's endophyte of kind after softening.It selects
Hydrogenperoxide steam generator is conducive to environmental protection as disinfectant, without the use of mercuric chloride solution.Mercuric chloride is mercuric chloride solution, is extensive
The plant explant disinfectant used will cause pollution, the mistake that the present invention passes through environmental protection since mercuric chloride solution waste liquid enters environment
Hydrogen peroxide solution substitutes mercuric chloride, and preferably completes the disinfection of seed.
In inducing clumping bud, addition CH is conducive to the formation of Multiple Buds, compared to the processing of no addition CH, Multiple Buds
Quantity can be improved 5% or so.The formation of secondary metabolite in medicinal plant incubation, so that browning in incubation
It is generally existing, the culture medium of agar is added, the culture medium of inoculation lamiophlomis rotata leafcutting 20d or so blade section contact starts brown
To change, is spread immediately to surrounding, inducing clumping bud rate is low, and only 40% or so, and slow growth；And it adds in the medium
Gelrite replaces traditional agar as coagulator, can significantly reduce the browning of culture materials base portion, is conducive to blade to battalion
The absorption formed point, inducing clumping bud rate is high, can reach 80%, and grow vigorous.Due to lamiophlomis rotata wild seed germination rate
It is low, and endophyte is complex, during the cultivation process, takes the blade of test tube seedling, and the induction of Multiple Buds is carried out after stripping and slicing, can be increased
Proliferation rate, and effectively shorten cultivation cycle.Hormone concentration is reduced in Subculture and adjustment hormone kind is advantageous
In the healthy growth of Multiple Buds.In the rooting induction stage, rooting rate is can be improved in the mode for having used two kinds of auxin to cooperate, and is tried
After pipe seedling rooting, nutrient absorption is abundant, hence it is evident that shows as blade area increase, thickness increases, and plant strain growth is vigorous, after being conducive to
Other researchs are transplanted or carried out to phase.
In conclusion disinfectant selects during the present invention has fully considered seed disinfection, in inducing clumping bud and culture
The influence of auxin and mitogen, by the screening of optimal medium, the addition of CH, Gelrite replaces traditional agar, and
The adjustment of exogenous hormone concentration and type in incubation, it is final to obtain lamiophlomis rotata aseptic seedling, and establish the training of fast breeding
The method of supporting.This method time-consuming is short, and inductivity is high, and Aseptic Seedling Growth is healthy and strong.This method is suitable for lamiophlomis rotata wild seed, to only one
The development and utilization of taste and molecular biology research have positive effect.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the sprouting of lamiophlomis rotata seed.
Fig. 2 is lamiophlomis rotata leafcutting induced synthesis Multiple Buds.
Fig. 3 is lamiophlomis rotata Multiple Buds.
Fig. 4 is lamiophlomis rotata aseptic seedling Multiplying culture.
Fig. 5 is lamiophlomis rotata intact plant.
Fig. 6 is lamiophlomis rotata in vitro cuttings in culturing rack culture.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
In the present invention:
6-BA -6-benzyl aminopurine, KT--N6- isopentennyladenine；NAA-methyl α-naphthyl acetate；IAA-heteroauxin,
IBA-indolebutyric acid, Gelrite-plant gel, CH-caseinhydrolysate；Above-mentioned is analytical reagents, public purchased from Sigma
Department.
The preparation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
(1) material to be tested: the wild lamiophlomis rotata seed of Gansu Maqu Alpine Grasslands acquisition.
(2) explant culture: by the lamiophlomis rotata seed of field acquisition, 2h first is rinsed with tap water flowing water, then ultra-clean
It is disinfected on workbench, it is aseptic water washing 2-3 times, molten with 30% hydrogen peroxide first with 75% ethanol solution sterilizing 90-120s
Liquid impregnates 15-20min, changes 10% hydrogenperoxide steam generator into and impregnates 30-60min, aseptic water washing 2-3 is inoculated into germination training after
Support base, germination culture medium be 1/2MSB culture medium, dark culture, 25 ± 2 DEG C of cultivation temperature.
Using above-mentioned sterilizing methods, lamiophlomis rotata Seed sterilization is thorough, and sterilization rate is between 70%~80%；Using above-mentioned hair
Bud method, lamiophlomis rotata percentage of seedgermination is high, between 20%~30%.
(3) induction of Multiple Buds: after 35~45 days, seed is sprouted, and is transferred to illumination cultivation, by aseptic seedling after cotyledon stretching, extension
Cotyledon and hypocotyl are cut into 1.8-2.2mm fritter/section on superclean bench, lie against inducing clumping bud culture medium；Also it can be used
The seedling of 4-5 piece true leaf or the test tube seedling of later period squamous subculture are grown, its blade of clip is cut into the fritter of 2-5mm, is inoculated in
Inducing clumping bud culture medium carries out the induction of Multiple Buds.After 20-30 days, Multiple Buds are successfully induced, have obtained the second generation only one
Taste aseptic seedling.
Wherein, illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
(Multiplying culture is identical with illumination cultivation condition in culture of rootage).
Inducing clumping bud culture medium is to add 300-800mgL in MSB culture medium-1CH, 2.2g Gelrite, pH value
5.8, and additional 1.0-2.0mgL-1KT+0.1-0.5mg·L-1IBA。
Using above-mentioned abductive approach, inducing clumping bud rate is 80%-85%.
The quick proliferation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
Multiple Buds are transferred and carry out shoot proliferation culture into subculture multiplication medium, make Multiple Buds continued growth, and can be from
Multiple Buds basal growth goes out new Multiple Buds.Subculture multiplication medium is to add 300-800mgL in MSB culture medium-1CH,
2.2g Gelrite, and additional 0.2-1.0mgL-1KT+0.05-0.2mg·L-1IBA reduces hormone concentration and is conducive to grow thickly
The healthy growth of bud；Hormone concentration in subculture multiplication medium is halved after subculture 2 times and continues to cultivate.Using above-mentioned Multiplying culture
Method, Multiplying culture time are 35-50 days, and proliferation times are 10-25 times, thus the lamiophlomis rotata aseptic seedling after being proliferated.Instead
The induction of Multiple Buds and the fast breeding step of lamiophlomis rotata aseptic seedling are carried out again, obtain large batch of lamiophlomis rotata aseptic seedling.
When needing to transplant, then the lamiophlomis rotata aseptic seedling of Multiplying culture is subjected to culture of rootage, culture of rootage can use
Following steps:
It selects and grows vigorous, healthy and strong bud, cut from base portion, be inoculated into root media root induction, culture 30~45
It, adventitious root, root long 3-5cm are born in lower end, and rooting rate 50%-70% obtains complete plant.Lamiophlomis rotata root media
For 1/2MSB culture medium, and additional 0.05-0.2mgL-1NAA+0.5-1.0mg·L-1IBA.Two kinds of auxin are used in combination,
Facilitate the raising of rooting rate.
Fig. 1-6 is lamiophlomis rotata Aseptic Seedling Growth process.Wherein, Fig. 1 is the sprouting of lamiophlomis rotata seed；Fig. 2 is lamiophlomis rotata blade
Stripping and slicing induced synthesis Multiple Buds；Fig. 3 is lamiophlomis rotata Multiple Buds；Fig. 4 is lamiophlomis rotata aseptic seedling Multiplying culture；Fig. 5 is that lamiophlomis rotata is complete
Whole plant；Fig. 6 is lamiophlomis rotata in vitro cuttings in culturing rack culture.
MSB culture medium used in the present invention is the organic element of the inorganic elements and B5 medium using MS culture medium
A kind of culture medium of combination.Its main component and dosage are as shown in table 1.
1/2MSB culture medium is the MSB culture medium that all the components content halves.
1 MSB culture medium prescription of table
Reagent is analytical reagents in table 1, is purchased from Sinopharm Chemical Reagent Co., Ltd..
Sterilizing methods and culture medium prescription optimization experiment in the method for the present invention:
One, the sterilizing of lamiophlomis rotata seed
Field acquisition lamiophlomis rotata seed takes back laboratory and first cleans 2h with tap water flowing water, carries out on superclean bench
Sterilization treatment, 75% alcoholic solution sterilize 2min, aseptic water washing 3 times, are sterilized respectively with 3 kinds of methods, method 1: first with 30%
Hydrogenperoxide steam generator sterilize 20min, then with 10% hydrogen peroxide immersion 60min, aseptic water washing 3 times；Method 2:0.1% mercuric chloride
Solution sterilization 15min, aseptic water washing 5 times；Method 3:0.1% mercuric chloride solution sterilizes 15min, and aseptic water washing 5 times, then use
10% hydrogen peroxide impregnates 60min, aseptic water washing 3 times, as a result as follows:
Influence of the different sterilizing methods of table 2 to germination
Mercuric chloride and hydrogen peroxide composite sterilization pollution rate are minimum, but are easy to produce phytotoxicity, cause to damage to seed；Mercuric chloride pair
It is better than hydrogen peroxide in the sterilization effect of the surface of the seed, but sterilization time length will cause phytotoxicity, injure seed embryo, reduce germination,
Time, the short sterilization effect or even seed that the case where there are endophytes, cannot have accomplished intradermal for kind was killed, but mould
It still has；The hydrogen peroxide immersion of high-concentration and low-concentration can effectively reduce pollution, and low concentration hydrogen peroxide can be with the leaching of long period
Seed is steeped, kind of a skin can be softened, while permeability is preferable, endophyte can be killed without injuring seed, pollution rate is lower, germination percentage
It can reach 21.7%.Lamiophlomis rotata germination percentage is low, and there are also the reason is that maturity disunity, the germination of seed of wild seed are poor
Do not pre-process etc..
Two, the differentiation and Multiplying culture of lamiophlomis rotata
1, lamiophlomis rotata aseptic seedling cotyledon or blade are cut into the fritter of 1.8-2.2mm, are inoculated into inducing clumping bud culture medium,
Inducing clumping bud culture medium is MSB, adds CH 500mg, 2.2g Gelrite, pH value 5.8, wherein addition hormone combination is as follows
Table:
3 inducing clumping bud culture medium constituent (mgL of table-1)
Influence of the different culture medium to differentiation is counted, as a result as follows:
Influence of 4 different culture medium of table to differentiation
2, lamiophlomis rotata aseptic seedling cotyledon or blade are cut into the fritter of 1.8-2.2mm, are inoculated into inducing clumping bud culture medium,
Inducing clumping bud culture medium is MSB, adds CH 500mg, 5g agar, 1.0mgL-1KT+0.1mg·L-1IBA, pH value 5.8.
Inducing differentiation rate is 40%, and culture materials surrounding media is in yellowish-brown.
Embodiment 1
The preparation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
(1) material to be tested: the wild lamiophlomis rotata seed of Gansu Maqu Alpine Grasslands acquisition.
(2) explant culture: by the lamiophlomis rotata seed of field acquisition, 2h first is rinsed with tap water flowing water, in ultra-clean work
It disinfects on platform, first with 75% ethanol solution sterilizing 2min, aseptic water washing 3 times, is impregnated with 30% hydrogenperoxide steam generator
20min changes 10% hydrogenperoxide steam generator into and impregnates 30min, and germination culture medium, germination culture are inoculated into after aseptic water washing 3 times
Base be 1/2MSB culture medium, dark culture, 25 DEG C of cultivation temperature.Using above-mentioned sterilizing methods, lamiophlomis rotata Seed sterilization is thorough, sterilizing
Rate is 80%；Using above-mentioned germinating method, lamiophlomis rotata percentage of seedgermination is high, is 30%.
(3) induction of Multiple Buds: after 35 days, seed is sprouted, and is transferred to illumination cultivation, by the test tube seedling of later period squamous subculture,
Its blade of clip, is cut into the fritter of 4mm, lies against inducing clumping bud culture medium, carries out the induction of Multiple Buds.After 20 days, success
Multiple Buds are induced, lamiophlomis rotata aseptic seedling is obtained.
Wherein, illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 DEG C of cultivation temperature.(increase
It is identical with illumination cultivation condition in culture of rootage to grow culture).
Inducing clumping bud culture medium is to add 500mgL in MSB culture medium-1CH, 2.2g Gelrite, pH value 5.8,
And it adds.
Using above-mentioned abductive approach, inducing clumping bud rate is 85%.
The quick proliferation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
Multiple Buds are transferred and carry out shoot proliferation culture into subculture multiplication medium, make Multiple Buds continued growth, and can be from
Multiple Buds basal growth goes out new Multiple Buds.Subculture multiplication medium is to add 500mgL in MSB culture medium-1CH, 2.2g
Gelrite, and additional 0.2mgL-1KT+0.05mg·L-1IBA, reduce that hormone concentration is conducive to Multiple Buds grows up to growth；
Hormone concentration in subculture multiplication medium is halved after subculture 2 times and continues to cultivate.Using above-mentioned Multiplying culture method, Multiplying culture
Time is 35 days, and proliferation times are 25 times, thus the lamiophlomis rotata aseptic seedling after being proliferated.Be repeated Multiple Buds induction and
The fast breeding step of lamiophlomis rotata aseptic seedling obtains large batch of lamiophlomis rotata aseptic seedling.
When needing to transplant, then the lamiophlomis rotata aseptic seedling of Multiplying culture is subjected to culture of rootage, culture of rootage can use
Following steps:
It selects and grows vigorous, healthy and strong bud, cut from base portion, be inoculated into root media root induction, cultivate 30 days, under
Adventitious root, root long 5cm are born in end, and rooting rate 70% obtains complete plant.Lamiophlomis rotata root media is 1/2MSB culture
Base, and additional 0.1mgL-1NAA+0.5mg·L-1IBA.Two kinds of auxin are used in combination, and facilitate the raising of rooting rate.
Embodiment 2
The preparation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
(1) material to be tested: the wild lamiophlomis rotata seed of Gansu Maqu Alpine Grasslands acquisition.
(2) explant culture: by the lamiophlomis rotata seed of field acquisition, 2h first is rinsed with tap water flowing water, in ultra-clean work
It disinfects on platform, first with 75% ethanol solution sterilizing 2min, aseptic water washing 2 times, is impregnated with 30% hydrogenperoxide steam generator
20min changes 10% hydrogenperoxide steam generator into and impregnates 30min, and germination culture medium, germination culture are inoculated into after aseptic water washing 2 times
Base be 1/2MSB culture medium, dark culture, 25 DEG C of cultivation temperature.Using above-mentioned sterilizing methods, lamiophlomis rotata Seed sterilization is thorough, sterilizing
Rate is 75%；Using above-mentioned germinating method, lamiophlomis rotata percentage of seedgermination is high, is 26%.
(3) induction of Multiple Buds: after 40 days, seed sprout, be transferred to illumination cultivation, with seedling grow up to intact plant or after
The test tube seedling of phase squamous subculture, its blade of clip, is cut into the fritter of 5mm, is inoculated in inducing clumping bud culture medium and carries out Multiple Buds
Induction.After 25 days, Multiple Buds are successfully induced, obtain lamiophlomis rotata aseptic seedling.
Wherein, illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
(Multiplying culture is identical with illumination cultivation condition in culture of rootage).
Inducing clumping bud culture medium is to add 300mgL in MSB culture medium-1CH, 2.2g Gelrite, pH value 5.8,
And additional 1.5mgL-1KT+0.3mg·L-1IBA。
Using above-mentioned abductive approach, inducing clumping bud rate is 82%.
The quick proliferation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
Multiple Buds are transferred and carry out shoot proliferation culture into subculture multiplication medium, make Multiple Buds continued growth, and can be from
Multiple Buds basal growth goes out new Multiple Buds.Subculture multiplication medium is to add 300mgL in MSB culture medium-1CH, 2.2g
Gelrite, and additional 0.5mgL-1KT+0.15mg·L-1IBA, reduce that hormone concentration is conducive to Multiple Buds grows up to growth；
Hormone concentration in subculture multiplication medium is halved after subculture 2 times and continues to cultivate.Using above-mentioned Multiplying culture method, Multiplying culture
Time is 42 days, and proliferation times are 20 times, thus the lamiophlomis rotata aseptic seedling after being proliferated.Be repeated Multiple Buds induction and
The fast breeding step of lamiophlomis rotata aseptic seedling obtains large batch of lamiophlomis rotata aseptic seedling.
When needing to transplant, then the lamiophlomis rotata aseptic seedling of Multiplying culture is subjected to culture of rootage, culture of rootage can use
Following steps:
It selects and grows vigorous, healthy and strong bud, cut from base portion, be inoculated into root media root induction, cultivate 40 days, under
Adventitious root, root long 4cm are born in end, and rooting rate 60% obtains complete plant.Lamiophlomis rotata root media is 1/2MSB culture
Base, and additional 0.05mgL-1NAA+0.8mg·L-1IBA.Two kinds of auxin are used in combination, and facilitate the raising of rooting rate.
Embodiment 3
The preparation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
(1) material to be tested: the wild lamiophlomis rotata seed of Gansu Maqu Alpine Grasslands acquisition.
(2) explant culture: by the lamiophlomis rotata seed of field acquisition, 2h first is rinsed with tap water flowing water, in ultra-clean work
It disinfects on platform, first with 75% ethanol solution sterilizing 2min, aseptic water washing 3 times, is impregnated with 30% hydrogenperoxide steam generator
20min changes 10% hydrogenperoxide steam generator into and impregnates 50min, and germination culture medium, germination culture are inoculated into after aseptic water washing 2 times
Base be 1/2MSB culture medium, dark culture, 23 DEG C of cultivation temperature.Using above-mentioned sterilizing methods, lamiophlomis rotata Seed sterilization is thorough, sterilizing
Rate is 70%；Using above-mentioned germinating method, lamiophlomis rotata percentage of seedgermination is high, is 20%.
(3) induction of Multiple Buds: after 45 days, seed is sprouted, and is transferred to illumination cultivation, and directly culture grows up to seedling, then uses
After cotyledon stretching, extension aseptic seedling cotyledon and hypocotyl are cut into 1.8mm fritter/section on superclean bench, lie against inducing clumping bud
Culture medium carries out the induction of Multiple Buds.After 30 days, Multiple Buds are successfully induced, obtain lamiophlomis rotata aseptic seedling.
Wherein, illumination cultivation condition are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
(Multiplying culture is identical with illumination cultivation condition in culture of rootage).
Inducing clumping bud culture medium is to add 800mgL in MSB culture medium-1CH, 2.2g Gelrite, pH value 5.8,
And additional 2.0mgL-1KT+0.5mg·L-1IBA。
Using above-mentioned abductive approach, inducing clumping bud rate is 80%.
The quick proliferation method of lamiophlomis rotata aseptic seedling of the invention are as follows:
Multiple Buds are transferred and carry out shoot proliferation culture into subculture multiplication medium, make Multiple Buds continued growth, and can be from
Multiple Buds basal growth goes out new Multiple Buds.Subculture multiplication medium is to add 800mgL in MSB culture medium-1CH, 2.2g
Gelrite, and additional 1.0mgL-1KT+0.2mg·L-1IBA, reduce that hormone concentration is conducive to Multiple Buds grows up to growth；After
Hormone concentration in subculture multiplication medium is halved after generation 2 times and continues to cultivate.Using above-mentioned Multiplying culture method, when Multiplying culture
Between be 50 days, proliferation times be 10 times, thus the lamiophlomis rotata aseptic seedling after being proliferated.Be repeated Multiple Buds induction and solely
The fast breeding step of aseptic seedling simply obtains large batch of lamiophlomis rotata aseptic seedling.
When needing to transplant, then the lamiophlomis rotata aseptic seedling of Multiplying culture is subjected to culture of rootage, culture of rootage can use
Following steps:
It selects and grows vigorous, healthy and strong bud, cut from base portion, be inoculated into root media root induction, cultivate 45 days, under
Adventitious root, root long 3cm are born in end, and rooting rate 50% obtains complete plant.Lamiophlomis rotata root media is 1/2MSB culture
Base, and additional 0.2mgL-1NAA+1.0mg·L-1IBA.Two kinds of auxin are used in combination, and facilitate the raising of rooting rate.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. a kind of preparation method of lamiophlomis rotata aseptic seedling, it is characterised in that: the following steps are included:
(1) it explant culture: first uses tap water flowing water to rinse in lamiophlomis rotata seed, then disinfects, first use on the super-clean bench
75% ethanol solution sterilizing 90-120s, aseptic water washing 2-3 times, impregnates 15-20min with 30% hydrogenperoxide steam generator, then use
10% hydrogenperoxide steam generator impregnates 30-60min, and aseptic water washing 2-3 is inoculated into germination culture medium after and carries out dark culture；
(2) inducing clumping bud: after seed is sprouted, it is transferred to illumination cultivation；Aseptic seedling cotyledon and hypocotyl are existed after cotyledon stretching, extension
It is cut into 1.8-2.2mm fritter/section on superclean bench, lies against inducing clumping bud culture medium, carries out the induction of Multiple Buds；It can also
With the test tube seedling in the seedling or squamous subculture for growing 4-5 piece true leaf, its blade of clip is cut into the fritter of 2-5mm, is inoculated in
Inducing clumping bud culture medium carries out the induction of Multiple Buds.
2. the preparation method of lamiophlomis rotata aseptic seedling according to claim 1, it is characterised in that: in step (1), by lamiophlomis rotata
Seed first uses tap water flowing water to rinse, and then disinfects on superclean bench, first with 75% ethanol solution sterilizing 2min, nothing
Bacterium water rinses 3 times, impregnates 20min with 30% hydrogenperoxide steam generator, then impregnate 30min, sterile water with 10% hydrogenperoxide steam generator
It is inoculated into germination culture medium after rinsing 3 times and carries out germination culture.
3. the preparation method of lamiophlomis rotata aseptic seedling according to claim 1, it is characterised in that: in step (1), the germination
The culture medium of culture is 1/2MSB culture medium, and condition of culture is dark culture, 25 ± 2 DEG C of cultivation temperature.
4. the preparation method of lamiophlomis rotata aseptic seedling according to claim 1, it is characterised in that: in step (2), the illumination
Condition of culture are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
5. the preparation method of lamiophlomis rotata aseptic seedling according to claim 1-4, it is characterised in that: in step (2),
The inducing clumping bud culture medium is to add 300-800mgL in MSB culture medium-1CH, 2.2g Gelrite, pH value 5.8,
And additional 1.0-2.0mgL-1KT+0.1-0.5mg·L-1IBA；
Preferably, the inducing clumping bud culture medium is to add 500mgL in MSB culture medium in step (2)-1CH,
2.2g Gelrite, pH value 5.8, and additional 1.0mgL-1KT+0.1mg·L-1IBA。
6. a kind of quick proliferation method of lamiophlomis rotata aseptic seedling, it is characterised in that: obtained using the method for any one of claim 1-5
Lamiophlomis rotata aseptic seedling is obtained, then lamiophlomis rotata aseptic seedling is transferred under the conditions of illumination cultivation and carries out subculture into subculture multiplication medium
Multiplying culture；The subculture multiplication medium formula are as follows: add 300-800mgL in MSB culture medium-1CH, and additional 0.2-
1.0mg·L-1KT+0.05-0.2mg·L-1Hormone concentration in subculture multiplication medium is halved after subculture 2 times and continues to train by IBA
It supports.
7. the quick proliferation method of lamiophlomis rotata aseptic seedling according to claim 6, it is characterised in that: the shoot proliferation training
Support based formulas are as follows: add 500mgL in MSB culture medium-1CH, 2.2g Gelrite, and additional 0.2mgL-1KT+
0.05mg·L-1IBA。
8. the quick proliferation method of lamiophlomis rotata aseptic seedling according to claim 6, it is characterised in that: the illumination cultivation item
Part are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
9. a kind of rooting method of lamiophlomis rotata aseptic seedling, it is characterised in that: only one obtained using the method for claim 6 or 7
Taste aseptic seedling is selected and grows vigorous, healthy and strong bud, cuts from base portion, be inoculated into root media under the conditions of illumination cultivation and lure
It leads and takes root, root media is 1/2MSB culture medium, and adds 0.05-0.2mgL-1NAA+0.5-1.0mg·L-1IBA。
10. the rooting method of lamiophlomis rotata aseptic seedling according to claim 9, it is characterised in that: the illumination cultivation condition
Are as follows: 2000LuX illumination cultivation, illumination 14h, dark 10h, 25 ± 2 DEG C of cultivation temperature.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444551A (en) * | 2013-09-23 | 2013-12-18 | 成都大学 | Tissue culture test-tube plantlet obtaining method achieved through lamiophlomis rotata seed induced to sprout to be explant |
| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444551A (en) * | 2013-09-23 | 2013-12-18 | 成都大学 | Tissue culture test-tube plantlet obtaining method achieved through lamiophlomis rotata seed induced to sprout to be explant |
| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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|---|---|---|---|---|
| WO2022103341A1 (en) | 2020-11-11 | 2022-05-19 | Interkorn Semenarstvo In Obnovljivi Viri D.O.O. | Method for sterilization of crops |
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