CN108308033A - Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique - Google Patents
Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique Download PDFInfo
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- CN108308033A CN108308033A CN201810370587.3A CN201810370587A CN108308033A CN 108308033 A CN108308033 A CN 108308033A CN 201810370587 A CN201810370587 A CN 201810370587A CN 108308033 A CN108308033 A CN 108308033A
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- explant
- atractylis lancea
- callus
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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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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique, Atractylis lancea callus tissue culture base include MS minimal mediums and add respectively in MS minimal mediums 2,4 dichlorphenoxyacetic acids and methyl α-naphthyl acetate, wherein, addition 2 in every liter of MS minimal medium, 4 1.8~2.2mg of dichlorphenoxyacetic acid, 0.9~1.1mg of methyl α-naphthyl acetate.The Atractylis lancea callus tissue culture base of the present invention can improve explant callus induction rate so that explant forms a large amount of yellow greens and the close callus of quality, is conducive to the Analytical Chemical Experiment in later stage, improves economic benefit.
Description
Technical field
The present invention relates to a kind of Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique.
Background technology
Atractylis lancea is composite family [WTBX herbaceos perennial, also known as RHIZOMA ATRACTYLODIS IANCEAE, is distributed mainly on Jiangsu, Hubei and river
The provinces such as south, Region in Maoshan, Jiangsu Province are the producing regions of Atractylis lancea genunie medicinal materials.Atractylis lancea is used as medicine with rhizome, have it is drying damp and strengthening spleen, dispel
Wind dehumidifying and other effects.It is clinically used for the rheumatism that dampness taste, loss of appetite, the bored evil of vomitting of institute, abdominal pain diarrhea, damp evil are laid particular stress on,
Primary medicinal component is：Atisine chloride Atractydin, β-cineol.
The modes of reproduction of Atractylis lancea is mainly seminal propagation and root division, and seed is not easy to maintain, and germination rate is low, divides root numerous
It grows and there are problems that variety deterioration and heavy workload.In recent years, as the research to Atractylis lancea is gradually goed deep into, Atractylis lancea market needs
The amount of asking increasingly increases, and traditional modes of reproduction limits the Atractylis lancea quickly work such as breeding and breeding of new variety, rising in recent years
Plant tissue culture technique can be very good to solve Atractylis lancea variety deterioration, the shortcomings of breeding cycle is long, plant knits culture skill
Art is the totipotency according to cell, using in vitro tissue, organ or cell, raw plastid etc., by sterile working, appropriate
Culture medium and and suitable external condition under carry out the process that culture obtains regenerated intact plant, from each device in incubation
Generate callus on official, callus forms intact plant using being differentiated to form Multiple Buds again, and callus lures
It is a step extremely crucial during entire tissue culture to lead.
Invention content
The technical problem to be solved by the present invention is to overcome the deficiencies of existing technologies, a kind of Atractylis lancea callus training is provided
Base is supported, it can improve explant callus induction rate so that explant forms a large amount of yellow greens and the close callus of quality
Tissue, is conducive to the Analytical Chemical Experiment in later stage, improves economic benefit.
In order to solve the above-mentioned technical problem, the technical scheme is that：A kind of Atractylis lancea callus tissue culture base, it is wrapped
Minimal medium containing MS and 2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate in MS minimal mediums are added respectively, wherein every liter
2,4- of addition 1.8~2.2mg of dichlorphenoxyacetic acid, 0.9~1.1mg of methyl α-naphthyl acetate in MS minimal mediums.
Further, MS minimal mediums add comprising MS culture mediums and respectively sucrose and agar in MS culture mediums,
In, 25~35g of sucrose is added in every liter of MS culture medium, the PH of agar 4.26~6.26g, MS minimal medium is 5.6~6.
The present invention also provides a kind of Atractylis lancea callus induction techniques, it applies Atractylis lancea callus tissue culture base.
Further, include in processing step：
(1) explant samples：Take the tender spire piece of Atractylis lancea as explant；
(2) explant sterilizes；
(3) explant is handled：The explant that explant is cut into small pieces；
(4) explant culture：The explant of the fritter cut in step (3) is seeded in Atractylis lancea callus tissue culture base
On, it is placed in incubator and carries out callus induction, wherein condition of culture is light culture.
Further, also include between step (1) and step (2)：Explant is rinsed well with tap water, with tap water
The process banister brush of flushing removes the outer layer bud skin of bud, then clean with pure water rinsing.
Further, in step (2), first explant is placed in the alcoholic solution that mass concentration is 75% and impregnates 10s, it is rear to turn
Enter in sterile water and rinse 3~4 times, then the mercuric chloride solution 4~5min of soaking disinfection for being 0.1% with mass concentration, it finally again will be outer
Implant is transferred in sterile water and rinses 3~5 times, that is, completes the disinfection to explant.
Further, in step (3), the explant is cut into 0.5cm × 0.5cm sizes.
Further for the probability for improving callus induction, in step (3), in the explant that explant is cut into fritter
Petiole and main lobe arteries and veins are avoided during body.
After using above-mentioned technical proposal, the present invention is to be added to 2, the 4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate of appropriate weight
MS minimal mediums as Atractylis lancea callus tissue culture base, improve explant callus induction rate so that the explant bodily form
At a large amount of yellow greens and the close callus of quality, is conducive to the Analytical Chemical Experiment in later stage, improves economic benefit；The present invention's
Atractylis lancea callus induction technique is using the young leaflet tablet of Atractylis lancea as explant, convenient material drawing, and processing step through testing repeatedly
Card optimization, it is efficient and rational.
Specific implementation mode
In order that the present invention can be more clearly and readily understood, below according to specific embodiment, to the present invention make into
One step is described in detail.
Embodiment one
A kind of Atractylis lancea callus tissue culture base, it includes MS minimal mediums and adds respectively in MS minimal mediums
2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate, wherein 2,4- dichlorphenoxyacetic acid 1.8mg are added in every liter of MS minimal medium,
Methyl α-naphthyl acetate 0.9mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 25g is added in MS culture mediums, the PH of agar 4.26g, MS minimal medium is 5.6.
A kind of Atractylis lancea callus induction technique, it applies Atractylis lancea callus tissue culture base.
Include in processing step：
(1) explant samples：Take the tender spire piece of Atractylis lancea as explant；
(2) explant sterilizes；
(3) explant is handled：The explant that explant is cut into small pieces；
(4) explant culture：The explant of the fritter cut in step (3) is seeded in Atractylis lancea callus tissue culture base
On, it is placed in incubator and carries out callus induction, wherein condition of culture is light culture.
Also include between step (1) and step (2)：Explant is rinsed well with tap water, what is rinsed with tap water
Process banister brush removes the outer layer bud skin of bud, then clean with pure water rinsing.
In step (2), first by explant be placed in mass concentration be 75% alcoholic solution in impregnate 10~20s, after be transferred to
It rinses 3 times in sterile water, then the mercuric chloride solution soaking disinfection 4min for being 0.1% with mass concentration, is finally again transferred to explant
It is rinsed 3 times in sterile water, that is, completes the disinfection to explant.
In step (3), the explant is cut into 0.5cm × 0.5cm sizes.It in the present embodiment, will be outer after disinfection
Implant is positioned on aseptic inoculation disk, is fixed with sterile dissecting needle, then explant is cut into small pieces with sterile scalpel outer
Implant, but not limited to this.
In order to improve the probability of callus induction, in step (3), in the mistake for the explant that explant is cut into fritter
Petiole and main lobe arteries and veins are avoided in journey.
Embodiment two
A kind of Atractylis lancea callus tissue culture base, it includes MS minimal mediums and adds respectively in MS minimal mediums
2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate, wherein 2,4- dichlorphenoxyacetic acid 2.0mg are added in every liter of MS minimal medium,
Methyl α-naphthyl acetate 1.0mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 30g is added in MS culture mediums, the PH of agar 5.26g, MS minimal medium is 5.8.
A kind of Atractylis lancea callus induction technique, it applies Atractylis lancea callus tissue culture base.
Include in processing step：
(1) explant samples：Take the tender spire piece of Atractylis lancea as explant；
(2) explant sterilizes；
(3) explant is handled：The explant that explant is cut into small pieces；
(4) explant culture：The explant of the fritter cut in step (3) is seeded in Atractylis lancea callus tissue culture base
On, it is placed in incubator and carries out callus induction, wherein condition of culture is light culture.
Also include between step (1) and step (2)：Explant is rinsed well with tap water, what is rinsed with tap water
Process banister brush removes the outer layer bud skin of bud, then clean with pure water rinsing.
In step (2), first by explant be placed in mass concentration be 75% alcoholic solution in impregnate 15s, after be transferred to it is sterile
It rinses 3 times, then the mercuric chloride solution soaking disinfection 5min for being 0.1% with mass concentration, is finally again transferred to explant sterile in water
It is rinsed 4 times in water, that is, completes the disinfection to explant.
In step (3), the explant is cut into 0.5cm × 0.5cm sizes.It in the present embodiment, will be outer after disinfection
Implant is positioned on aseptic inoculation disk, is fixed with sterile dissecting needle, then explant is cut into small pieces with sterile scalpel outer
Implant, but not limited to this.
In order to improve the probability of callus induction, in step (3), in the mistake for the explant that explant is cut into fritter
Petiole and main lobe arteries and veins are avoided in journey.
Embodiment three
A kind of Atractylis lancea callus tissue culture base, it includes MS minimal mediums and adds respectively in MS minimal mediums
2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate, wherein 2,4- dichlorphenoxyacetic acid 2.2mg are added in every liter of MS minimal medium,
Methyl α-naphthyl acetate 1.1mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 35g is added in MS culture mediums, the PH of agar 6.26g, MS minimal medium is 6.
A kind of Atractylis lancea callus induction technique, it applies Atractylis lancea callus tissue culture base.
Include in processing step：
(1) explant samples：Take the tender spire piece of Atractylis lancea as explant；
(2) explant sterilizes；
(3) explant is handled：The explant that explant is cut into small pieces；
(4) explant culture：The explant of the fritter cut in step (3) is seeded in Atractylis lancea callus tissue culture base
On, it is placed in incubator and carries out callus induction, wherein condition of culture is light culture.
Also include between step (1) and step (2)：Explant is rinsed well with tap water, what is rinsed with tap water
Process banister brush removes the outer layer bud skin of bud, then clean with pure water rinsing.
In step (2), first by explant be placed in mass concentration be 75% alcoholic solution in impregnate 20s, after be transferred to it is sterile
It rinses 4 times, then the mercuric chloride solution soaking disinfection 5min for being 0.1% with mass concentration, is finally again transferred to explant sterile in water
It is rinsed 5 times in water, that is, completes the disinfection to explant.
In step (3), the explant is cut into 0.5cm × 0.5cm sizes.It in the present embodiment, will be outer after disinfection
Implant is positioned on aseptic inoculation disk, is fixed with sterile dissecting needle, then explant is cut into small pieces with sterile scalpel outer
Implant, but not limited to this.
In order to improve the probability of callus induction, in step (3), in the mistake for the explant that explant is cut into fritter
Petiole and main lobe arteries and veins are avoided in journey.
Such as table 1, when it is exogenous hormone that 2,4- dichlorphenoxyacetic acids, which are used alone, we are using culture under illumination and dark training
Two kinds of conditions are supported, start the callus induction rate, quality, color and the growing state that count explant after 14~15 days.
Wherein under the conditions of light culture, 2, the 4- dichlorphenoxyacetic acids that are added in every liter of MS minimal medium weigh less than
Under conditions of 2.0mg, only blade edge forms a small amount of callus；2, the 4- Dichlorophenoxy second added in every liter of MS minimal medium
Although the weight of acid in the case of 2.0mg higher than that can form complete callus, callus upgrowth situation is poor, and quality is relatively dredged
Pine is unfavorable for the induction differentiation in later stage；The weight of 2, the 4- dichlorphenoxyacetic acids added in every liter of MS minimal medium is
When 2.0mg, callus is green, and quality is close, is conducive to late-stage differentiation, but callus induction rate is still undesirable.And illumination cultivation
Under, no matter how the weight of 2,4- dichlorphenoxyacetic acids changes in every liter of MS minimal medium, most of explant is unable to shape
At well-grown callus, the callus that a small number of blades are formed is mostly yellowish-brown or even brown.Therefore it obtains, individually makes
When with 2,4- dichlorphenoxyacetic acids being exogenous hormone, the weight of 2,4- dichlorphenoxyacetic acids is in every liter of MS minimal medium
2.0mg, and use light culture is more conducive to the induction of Atractylis lancea callus.
Table 1：2, the 4- dichlorphenoxyacetic acids of addition different weight form callus to explant in every liter of MS minimal medium
Influence
Note：+ poor, ++ preferably, +++ it is vigorous
Such as table 2, since previous experiments prove, light culture is more suitable for the induction of Atractylis lancea callus, and subsequent experimental is directly given up
It is cultivated under illumination condition, selects light culture condition.Exclusive use methyl α-naphthyl acetate is exogenous hormone, starts to count explant after 14~15 days
Callus induction rate, quality, color and the growing state of body.
It was found that when the methyl α-naphthyl acetate added in every liter of MS minimal medium weighs less than 1.0mg, callus induction
Rate is relatively low；When the weight of the methyl α-naphthyl acetate added in every liter of MS minimal medium is higher than 1.0mg, callus forms a large amount of indefinite
Root is unfavorable for the Analytical Chemical Experiment in later stage；It, can be with when the weight of the methyl α-naphthyl acetate added in every liter of MS minimal medium is 1.0mg
A large amount of yellow greens and the close callus of quality are formed, but callus induction rate is still not up to desired value.
Table 2：The methyl α-naphthyl acetate of addition different weight forms explant the influence of callus in every liter of MS minimal medium
Note：+ poor, ++ preferably, +++ it is vigorous
Such as table 3, in order to more preferably reach experiment effect, we are by optimum weight in every liter of MS minimal medium in above-mentioned experiment
2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate be applied in combination, start after 14~15 days count explant callus induction rate,
Quality, color and growing state.
The result shows that both hormone combinations have gain effect, inducing effect best using to the induction of Atractylis lancea callus.
Table 3：Different exogenous hormones form explant the influence of callus
Note：+ poor, ++ preferably, +++ it is vigorous
Working principle of the present invention is as follows：
The present invention is to be added to 2, the 4- dichlorphenoxyacetic acids of appropriate weight and the MS minimal mediums of methyl α-naphthyl acetate as thatch
Rhizoma atractylodis callus tissue culture base improves explant callus induction rate so that explant forms a large amount of yellow greens and quality is tight
Close callus is conducive to the Analytical Chemical Experiment in later stage, improves economic benefit；The Atractylis lancea callus induction work of the present invention
Skill is using the young leaflet tablet of Atractylis lancea as explant, convenient material drawing, and processing step through verifying optimization repeatedly, efficient and rational.
Particular embodiments described above, pair present invention solves the technical problem that, technical solution and advantageous effect carry out
It is further described, it should be understood that the above is only a specific embodiment of the present invention, is not limited to this
Invention, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be included in this hair
Within bright protection domain.
Claims (8)
1. a kind of Atractylis lancea callus tissue culture base, it includes MS minimal mediums, it is characterised in that：Also include to add respectively
2,4- dichlorphenoxyacetic acids in MS minimal mediums and methyl α-naphthyl acetate, wherein 2,4- dichloros are added in every liter of MS minimal medium
1.8~2.2mg of phenoxy acetic acid, 0.9~1.1mg of methyl α-naphthyl acetate.
2. Atractylis lancea callus tissue culture base according to claim 1, it is characterised in that：MS minimal mediums are trained comprising MS
It supports base and adds sucrose and agar in MS culture mediums respectively, wherein add 25~35g of sucrose, fine jade in every liter of MS culture medium
The PH of fat 4.26~6.26g, MS minimal medium is 5.6~6.
3. a kind of Atractylis lancea callus induction technique, it is characterised in that：Its application such as claim 1~2 any one of them
Atractylis lancea callus tissue culture base.
4. Atractylis lancea callus induction technique according to claim 3, which is characterized in that include in processing step：
(1) explant samples：Take the tender spire piece of Atractylis lancea as explant；
(2) explant sterilizes；
(3) explant is handled：The explant that explant is cut into small pieces；
(4) explant culture：The explant of the fritter cut in step (3) is seeded on Atractylis lancea callus tissue culture base,
It is placed in incubator and carries out callus induction, wherein condition of culture is light culture.
5. to go the Atractylis lancea callus induction technique described in 4 according to right, which is characterized in that step (1) and step (2) it
Between also include：Explant is rinsed well with tap water, removes the outer layer bud of bud in the process banister brush rinsed with tap water
Skin is then clean with pure water rinsing.
6. Atractylis lancea callus induction technique according to claim 4, it is characterised in that：In step (2), first by explant
Body, which is placed in the alcoholic solution that mass concentration is 75%, impregnates 10~20s, after be transferred in sterile water and rinse 3~4 times, then use quality
Explant is finally transferred in sterile water and rinses 3~5 times, i.e., by a concentration of 0.1% mercuric chloride solution 4~5min of soaking disinfection again
Complete the disinfection to explant.
7. Atractylis lancea callus induction technique according to claim 4, it is characterised in that：In step (3), the explant
Body is cut into 0.5cm × 0.5cm sizes.
8. Atractylis lancea callus induction technique according to claim 4, it is characterised in that：In step (3), by explant
Body avoids petiole and main lobe arteries and veins during cutting into the explant of fritter.
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| CN114342810A (en) * | 2022-02-23 | 2022-04-15 | 内蒙古科学技术研究院 | Tissue culture method of rhizoma atractylodis with high disease resistance |
| CN114600774A (en) * | 2022-04-25 | 2022-06-10 | 河北民族师范学院 | Atractylodes chinensis tissue culture and rapid propagation method |
| CN116267620A (en) * | 2023-04-21 | 2023-06-23 | 德兴市中医研究院试验培训基地 | Method for constructing callus induction and adventitious bud regeneration system by utilizing atractylis lancea leaf stalks |
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| CN114342810A (en) * | 2022-02-23 | 2022-04-15 | 内蒙古科学技术研究院 | Tissue culture method of rhizoma atractylodis with high disease resistance |
| CN114600774A (en) * | 2022-04-25 | 2022-06-10 | 河北民族师范学院 | Atractylodes chinensis tissue culture and rapid propagation method |
| CN116267620A (en) * | 2023-04-21 | 2023-06-23 | 德兴市中医研究院试验培训基地 | Method for constructing callus induction and adventitious bud regeneration system by utilizing atractylis lancea leaf stalks |
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Application publication date: 20180724 |