CN110063259A - A kind of method of panax japonicus callus fast-propagation - Google Patents
A kind of method of panax japonicus callus fast-propagation Download PDFInfo
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- CN110063259A CN110063259A CN201910451468.5A CN201910451468A CN110063259A CN 110063259 A CN110063259 A CN 110063259A CN 201910451468 A CN201910451468 A CN 201910451468A CN 110063259 A CN110063259 A CN 110063259A
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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 present invention provides a kind of method of panax japonicus callus fast-propagation, the present invention passes through best hormone ratio needed for optimization panax japonicus callus growth, filter out a kind of panax japonicus callus tissue culture base, contain archusia 2,4-D and basic element of cell division KT in the culture medium, when the concentration of the two is respectively 3mg/L, 0.10mg/L, panax japonicus callus cell increment is maximum, the speed of growth is fast, and healthy appearance, without browning, 20 days callus biomass of a cycle are 8.395g.Panax japonicus callus fast-propagation is carried out using culture medium provided by the invention, panax japonicus callus can quickly be bred, improve panax japonicus biomass, to improve the survival rate of panax japonicus artificial cultivation, promote the accumulation of panax japonicus active constituent, the quality for improving artificial cultivation panax japonicus, the problem of making up market caused by wild panax japonicus resource reserve declines supply falls short of demand.
Description
Technical field
The present invention relates to a kind of methods of panax japonicus callus fast-propagation, belong to field of plant tissue culture technique.
Background technique
Panax japonicus (Panax japonicus C.A.Meyer) is the root and rhizome of araliaceae ginseng plant panax japonicus,
Also known as panax japonicus, arhat Radix Notoginseng, wild Radix Notoginseng, rhizoma panasis japonici etc., good reputations such as " kings of herbal medicine " are enjoyed, there is activating microcirculation and removing stasis medicinal and nourishing
The effect of strong is rare " seven classes " medicinal material (north of volume 54 Chinese Plants will editorial board Chinese Plants will of Precious, Rare, Endangered
Capital: Science Press, 1978:185.).The main effective component of panax japonicus is saponin(e, has anti-inflammatory and antalgic, immunological regulation, resists
The multiple biological activities such as myocardial ischemia, hypoglycemic, anti-aging oxidation, antifatigue, anti-inflammatory, anticancer and improvement learning and memory function
(protection mechanism study Hangzhou of the Li Yougui panax japonicus saponin(e to Ethanol hepatic injury: Zhejiang University, 2011:108.Dun
YY, Liu M, Chen J, et al.Regulatory effects of saponins from Panax japonicus on
epithelial tight junctions in aging rats.J Ginseng Res,20107,42(1):50.)。
The predatoriness of wild panax japonicus was excavated in recent years, causes the resource reserve of wild panax japonicus to decline, causes to supply
It should not ask.Meanwhile artificial cultivation is one of the major measure for meeting the panax japonicus market demand, but panax japonicus growing and cultivation and harvesting
Process is lack of standardization, and plantation panax japonicus lack of standardization causes active constituent accumulation to be greatly affected (Chen Jiali, Tan Mengxia, Zou
It is vertical to think, wait multicomponent reactive ingredient while measurement and grey relational grade analysis CHINA JOURNAL OF CHINESE MATERIA MEDICA in difference processing panax japonicus
.2018；11).
Plant tissue culture technique is the technology to grow up early 20th century, is referred to aseptically, by vitro plant
Sundries official, tissue, cell, protoplast etc. carry out in vitro culture in suitable condition, and induction generates callus, adventitious bud
Deng, later formed intact plant process.Tissue culture technique can use various culture conditions controls carefully because it has
The advantages that growth of born of the same parents, differentiation, plant physiology, pathology, pharmacy, science of heredity, breeding and bioid are effectively pushed
The cross development of subjects such as learn, and a variety of industries such as be widely used in agricultural, forestry, gardening, industry, pharmaceutical sector, generation is huge
Economic benefit and social benefit, be summed up, there is the following aspects: 1, the rapid propagation in vitro of nursery stock；2, detoxic seedling is cultivated；3,
Culture medium transgenic breeding etc. applied to plant breeding such as haploid breeding, protoplast fusion, embryo and endosperm；4, production time
Raw metabolite such as traditional Chinese medicine ingredients etc.；5, artificial seed and preserving seed；6, gene functional research etc..
By callus tissue culture technology, panax japonicus seedling not only can be quickly bred, but also can directly extract secondary metabolism
Product is one of the approach to solve the above problems.
Summary of the invention
The object of the present invention is to provide a kind of quick propagation methods of panax japonicus callus and one kind can be improved expansion
The panax japonicus callus tissue culture base of numerous efficiency.
Specifically, the method for a kind of panax japonicus callus fast-propagation provided by the invention, using panax japonicus callus
Tissue culture medium (TCM) cultivates panax japonicus explant, and the panax japonicus callus tissue culture base contains archusia 2,4-D and thin
Born of the same parents' mitogen KT.
The panax japonicus callus tissue culture base includes but is not limited to MS culture medium, N6 culture medium or B5 medium etc.,
In one embodiment of the present of invention, the panax japonicus callus minimal medium used is MS culture medium.In the embodiment of the present invention
The MS basal medium is term generally in the art, is commercially available, can also voluntarily prepare, and formula is common knowledge.
In the method for panax japonicus callus fast-propagation of the invention, the panax japonicus explant is panax japonicus root, stem
Or leaf.
Explant disinfectant program: taking explant, and tap water sufficiently rinses, and clear water blots the water on surface with blotting paper after cleaning
Point；Then 60s is impregnated with 75% alcohol, 0.1%HgCl2 impregnates 15min, is thoroughly cleaned with aqua sterilisa flushing 4~5 times remaining
Mercury ion；The moisture for blotting surface with blotting paper again, is cut into the slice of 2mm, is connected to the above-mentioned panax japonicus callus of the present invention
In culture medium.
Preferably, the panax japonicus callus tissue culture base contains archusia 2, and the concentration of 4-D is 2-4mg/L.
Preferably, the concentration that the panax japonicus callus tissue culture base contains basic element of cell division KT is 0.05-0.3mg/L.
It is highly preferred that the panax japonicus callus tissue culture base contains archusia 2, the concentration of 4-D is 2-4mg/L,
Concentration containing basic element of cell division KT is 0.05-0.3mg/L.
Most preferably, the panax japonicus callus tissue culture base contains archusia 2, and the concentration of 4-D is 3mg/L, contains
The concentration for having basic element of cell division KT is 0.1mg/L.
In a kind of method of panax japonicus callus fast-propagation provided by the invention, panax japonicus callus tissue culture base
PH value is 5-6, preferably 5.8, cultivation temperature is 25 DEG C, dark culture.
The present invention provides a kind of panax japonicus callus tissue culture bases, in panax japonicus callus minimal medium
Add archusia 2,4-D and basic element of cell division KT.
The panax japonicus callus minimal medium includes but is not limited to MS culture medium, N6 culture medium or B5 medium.
Panax japonicus callus tissue culture base provided by the invention is in panax japonicus callus minimal medium MS culture medium
The KT that the 2,4-D and concentration that middle addition concentration is 2-4mg/L are 0.05-0.3mg/L.
Preferably, panax japonicus callus tissue culture base provided by the invention is that addition concentration is 3mg/L's in MS culture medium
The KT that 2,4-D and concentration are 0.1mg/L.
Further, the present invention provides panax japonicus callus tissue culture bases in fast-propagation panax japonicus callus
Using.
The present invention provides above-mentioned panax japonicus callus tissue culture bases to improve answering in panax japonicus callus biomass
With.
The present invention aseptically, using tender explant, by exploring hormon to panax japonicus callus
Effect, adjust hormone dosage ratio, optimize panax japonicus callus growth culture medium optimum formula, establish a kind of panax japonicus
The method of callus fast-propagation not only makes seedling healthy appearance without brown, and the speed of growth is fast, and growing way is good, and biomass is high,
20 days callus biomass of a cycle are 8.395g.It is fast that panax japonicus callus is carried out using culture medium provided by the invention
Speed expansion is numerous, can quickly breed panax japonicus callus, panax japonicus biomass is improved, to improve depositing for panax japonicus artificial cultivation
Motility rate promotes the accumulation of panax japonicus active constituent, improves the quality of artificial cultivation panax japonicus, make up under wild panax japonicus resource reserve
The problem of supply falls short of demand in market caused by dropping.
Detailed description of the invention
Fig. 1 is the impact effect figure that grows to panax japonicus of culture medium of different formulations in embodiment 1, described in A. embodiment 1
A formula panax japonicus callus grow picture, B. B described in embodiment 1 formula panax japonicus callus grows picture, 1 institute of C1. embodiment
C (1) the formula panax japonicus callus growth picture stated, C2. C described in embodiment 1 (2) are formulated panax japonicus callus and grow picture.
Fig. 2 is KT concentration when being 0.5mg/L, and 2,4-D various concentrations are to (20 days) panax japonicus callus in a cycle
The influence of biomass.
Fig. 3 be 2,4-D concentration be 3mg/L when, influence of the various concentration KT to (20 days) biomass in a cycle.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
In the embodiment of the present invention, 2,4-D (CAS:D-7299) are bought from sigma company, and precision weighs 10mg, first plus NaOH
After solution (1mol/L) dissolution, adds 10mL water constant volume, be made into 1mg/mL solution, and be stored in -20 DEG C of refrigerators.KT (CAS:
K3378) from sigma company, precision weighs 10mg for purchase, first plus after HCl solution (1mol/L) dissolution, adds 10mL water constant volume, matches
At 1mg/mL solution, and it is stored in -20 DEG C of refrigerators.NAA (CAS:N-0640) is bought from sigma company, and precision weighs 10mg, first
Solubilization adds 10mL water constant volume, is made into 1mg/mL solution in a small amount of ethyl alcohol, and is stored in -20 DEG C of refrigerators.
Embodiment 1
1, test method
The present embodiment uses the root, stem and leaf of panax japonicus for explant, filters out the optimal callus tissue culture of panax japonicus
Based formulas, a kind of method for establishing panax japonicus callus fast-propagation.
Explant disinfectant program: taking explant, and tap water sufficiently rinses, and clear water blots the water on surface with blotting paper after cleaning
Point；Then 60s is impregnated with 75% alcohol, 0.1%HgCl2 impregnates 15min, is thoroughly cleaned with aqua sterilisa flushing 4~5 times remaining
Mercury ion；The moisture for blotting surface with blotting paper again, is cut into the slice of 2mm, is connected in the MS culture dish of different proportion hormone.Training
Supporting ware pH is 5.8, and incubator temperature is 25 DEG C, dark culture.
The panax japonicus callus growth amount for counting hormon ratio, determines best hormone ratio.The present embodiment uses 3
Kind formula is compared analysis.Wherein formula A and B is that (Luo Zhengwei, Zhang Lai, Lv Cuiping wait ring for the formula reported in article
Ginseng in vitro culture and plant regeneration Chinese medicine, 2011；34 (12): 1818-1823):
It is formulated A (MS+2,4-D1.5mg/L+KT0.2mg/L+NAA1.0mg/L)
It is formulated B (MS+2,4-D1.0mg/L+NAA1.5mg/L)
C (MS+2,4-D X mg/L+KT Y mg/L) is formulated as the present invention formula system of re-optimization:
Being formulated C (1) is when KT concentration is X=0.1mg/L, and 2,4-D (Y) concentration are respectively 1,2,3,4mg/L；It is formulated C
(2) for when 2,4-D concentration is Y=3mg/L, KT (X) concentration is respectively 0.05,0.1,0.25,0.5mg/L.
2, result
The formula of 2.1 hormon ratios compares
The result shows that hormon ratio formula weaves more with the influence of growth different (see Fig. 1) panax japonicus group.A matches
The callus just induced goes out more slowly, and the more time was up to 1-2 months out, and callus browning is more serious, grows slow (Fig. 1
A).The callus of B formula induction is formulated out faster compared with A, and more the time is 30 days or so out, callus it is more fluffy (Fig. 1's
B).The callus of C formula (KT=0.1mg/L, 2,4-D=3mg/L when) induction goes out faster, grows vigorous, more the time is only out
Graininess, short texture and without browning situation (C1 and C2 of Fig. 1) is presented in 20 days or so callus.
2.2 KT concentration are 0.1mg/L and various concentration 2, and 4-D combines the influence to panax japonicus callus biomass
In the present embodiment, when KT concentration is 0.1mg/L, 2,4-D various concentrations are to (20 days) biomass in a cycle
Influence different (see Fig. 2).
When 2,4-D concentration is 1.00mg/L, biomass is minimum, is 2.621g；
When 2,4-D concentration are 2.00mg/L, biomass increases, and is 4.107g；
When 2,4-D concentration are 3.00mg/L, biomass is maximum, is 4.781g；
When 2,4-D concentration are 4.00mg/L, biomass reduces, and is 3.194g.
The result shows that 2,4-D be 3.00mg/L+0.1mg/L, panax japonicus callus with the optium concentration of KT hormone prescription
Increase very fast.
Influence of KT and 2,4-D the various concentration ratio to cell yield is as shown in table 1, and 2,4-D concentration are 1.00mg/L
When, biomass repeats three times are as follows: 2.627,2.435,2.801g；When 2,4-D concentration are 2.00mg/L, biomass repeats three times
Are as follows: 4.200,4.031,4.090g；When 2,4-D concentration are 3.00mg/L, biomass repeats three times are as follows: and 4.932,4.664,
4.747g；When 2,4-D concentration are 3.00mg/L, biomass repeats three times are as follows: 3.541,2.841,3.201g.
Influence of the 1 KT and 2,4-D various concentration ratio of table to cell yield
2.3 2,4-D concentration are that 3mg/L combines the influence to panax japonicus callus biomass with various concentration KT
On above-mentioned 2.2 result basis, when using 2,4-D concentration being 3mg/L, various concentration KT is analyzed in a cycle
The influence of interior (20 days) to biomass (see Fig. 3).
When KT concentration is 0.05mg/L, biomass is lower, is 5.656g；
When KT concentration is 0.1mg/L, biomass is maximum, is 8.395g；
When KT concentration is 0.25mg/L, biomass is reduced, and is 5.818g；
When KT concentration is 0.5mg/L, biomass is minimum, is 3.633g.
The result shows that 2,4-D be 3mg/L+0.10mg/L, panax japonicus callus growth with KT hormone concentration optium concentration
It is fastest.
Influence of 2, the 4-D and KT various concentration ratio to cell yield is as shown in table 2, when KT concentration is 0.05mg/L,
Biomass repeats three times are as follows: 6.156,5.180,5.632g；When KT concentration is 0.1mg/L, biomass repeats three times are as follows: and 8.040,
8.435 8.710g；When KT concentration is 0.25mg/L, biomass repeats three times are as follows: 5.837,6.550,5.068g；KT concentration is
When 0.5mg/L, biomass repeats three times are as follows: 3.125,4.341,3.433g.
Influence of the 2 2,4-D and KT various concentration ratio of table to cell yield
3, conclusion
The culture medium of panax japonicus callus is determined in the present invention are as follows: MS+2,4-D3mg/L+KT0.10mg/L.It utilizes
The culture medium can fast-propagation panax japonicus callus, for panax japonicus callus healthy appearance without browning, the speed of growth is fast, a week
20 days phases callus biomass is 8.395g.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of method of panax japonicus callus fast-propagation, which is characterized in that trained using panax japonicus callus tissue culture base
Panax japonicus explant is supported, the panax japonicus callus tissue culture base contains archusia 2,4-D and basic element of cell division KT.
2. the method as described in claim 1, which is characterized in that the panax japonicus explant is panax japonicus root, stem or leaf.
3. the method as described in claim 1, which is characterized in that the panax japonicus callus tissue culture base contains archusia
2,4-D concentration is 2-4mg/L.
4. the method as described in claim 1, which is characterized in that the panax japonicus callus tissue culture base contains the basic element of cell division
The concentration of KT is 0.05-0.3mg/L.
5. the method as described in claim 1-4 is any, which is characterized in that the panax japonicus callus tissue culture base contains cell
Auxin 2, the concentration of 4-D are 2-4mg/L, and the concentration containing basic element of cell division KT is 0.05-0.3mg/L.
6. method a method as claimed in any one of claims 1 to 5, which is characterized in that the panax japonicus callus tissue culture base contains cell
Auxin 2, the concentration of 4-D are 3mg/L, and the concentration containing basic element of cell division KT is 0.1mg/L.
7. a kind of panax japonicus callus tissue culture base, which is characterized in that it is to add in panax japonicus callus minimal medium
Add archusia 2,4-D and basic element of cell division KT.
8. panax japonicus callus tissue culture base as claimed in claim 7, which is characterized in that the panax japonicus callus is basic
Culture medium is MS culture medium；2,4-D concentration is 2-4mg/L, and the concentration of KT is 0.05-0.3mg/L.
9. panax japonicus callus tissue culture base as claimed in claim 7, which is characterized in that 2,4-D concentration is 3mg/L, KT
Concentration be 0.1mg/L.
10. any panax japonicus callus tissue culture base of claim 7-9 fast-propagation panax japonicus callus or
Improve the application in panax japonicus callus biomass.
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Citations (3)
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| JPH02234696A (en) * | 1989-03-08 | 1990-09-17 | Nitto Denko Corp | Production of anti-ulcer agent |
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| CN105755090A (en) * | 2016-03-18 | 2016-07-13 | 郭志刚 | Method for acquiring secondary metabolites including panax japonicus saponins and the like with large-scale culture and bioconversion technology for panax japonicus cells |
-
2019
- 2019-05-28 CN CN201910451468.5A patent/CN110063259A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02234696A (en) * | 1989-03-08 | 1990-09-17 | Nitto Denko Corp | Production of anti-ulcer agent |
| CN1883259A (en) * | 2006-07-10 | 2006-12-27 | 浙江省农业科学院 | Tissue-culturing quick propagation method of wild rhizoma panacis japonici |
| CN105755090A (en) * | 2016-03-18 | 2016-07-13 | 郭志刚 | Method for acquiring secondary metabolites including panax japonicus saponins and the like with large-scale culture and bioconversion technology for panax japonicus cells |
Non-Patent Citations (2)
| Title |
|---|
| NAOMI FUJIOKA等: "Production of oleanane saponins by callus tissue of panax japonicus", 《PLANTA MEDICA》 * |
| 王建华等: "人参发根的愈伤组织诱导", 《吉林农业大学学报》 * |
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Application publication date: 20190730 |