CN109156360A - A kind of method for tissue culture of Paris polyphylla - Google Patents
A kind of method for tissue culture of Paris polyphylla Download PDFInfo
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- CN109156360A CN109156360A CN201811263620.9A CN201811263620A CN109156360A CN 109156360 A CN109156360 A CN 109156360A CN 201811263620 A CN201811263620 A CN 201811263620A CN 109156360 A CN109156360 A CN 109156360A
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- paris polyphylla
- seedling
- culture medium
- illumination
- 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
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention belongs to medicine bioengineering or drug planting technology fields, and in particular to a kind of method for tissue culture of Paris polyphylla；This method is based on different culture medium, after bud is inoculated into culture medium I, obtains sprouting, sprouting is inoculated into culture medium II, seedling is obtained, by seedling inoculation into strengthening seedling and rooting culture medium, obtains that Paris polyphylla seedling can be cultivated；Method for tissue culture of the invention uses fresh Radix Notoginseng juice, Poria cocos juice and ground moss, for in culture medium, research shows that, it is short that Paris polyphylla method for tissue culture of the invention obtains the Paris polyphylla growth of seedling period, not only contain effective component Dioscin in further Paris polyphylla seedling, also contains other effective components.
Description
Technical field
The invention belongs to medicine bioengineering or drug planting technology fields, and in particular to a kind of method for tissue culture of Paris polyphylla.
Background technique
Rhizoma Paridis is liliaceous plant Yunnan Rhizoma Paridis (paris polyphylla) Parispolyphylla Smith
Var.yunnanensis (Franch) Hand.-Mazz. or paris polyphylla (magnificent Paris polyphylla) Paris polyphylla
The dry rhizome of Smithvar.chinensis (Franch) Hara.Steroid saponin is the main active substances of Paris polyphylla, is had anti-
The pharmacological actions such as tumour, anti-inflammatory, hemostasis, anti-oxidant, promotion uterine contraction and protection vascular endothelial cell, clinically take orally and are used for
Hemostasis, analgesic, antitumor, external applied anti-infectious, anti-inflammatory and antalgic etc..Since Paris polyphylla has multiple pharmacological effect and good drug efficacy, pharmacy
Industry is to the demand cumulative year after year of Paris polyphylla, so that supply falls short of demand.So artificial cultivation Paris polyphylla is imperative.
However Paris polyphylla vegetable seeds natural propagation rate is low, embryo development is incomplete, and endosperm is hard and plumular axis after-ripening etc. has
The obviously characteristic of " dual suspend mode ", after planting it is generally necessary to undergo two winters that could sprout, seedling growth time is long, is allusion quotation
" the biennial seed " of type.The ontogenetic process of Paris polyphylla plant is very slow, is sprouted by seed to blossoming and bearing fruit, at least needs 4-
5 years, wherein seedling stage (brephic) can continue 4 years or more.Therefore, and low reproduction rate long using seminal propagation elapsed cycles,
It can not be mass produced to meet the needs of people are to Paris polyphylla medicinal material.Plant tissue culture technique, which is one, can make Paris polyphylla fast
One of the effective way of speed breeding.
Therefore, new Paris polyphylla tissue culture technique is studied, is of great significance.
Summary of the invention
For these reasons, applicant passes through multiple creative research, obtains a kind of Paris polyphylla tissue culture technique, the skill
Art is based on different culture medium, after bud is inoculated into culture medium I, obtains sprouting, sprouting is inoculated into culture medium II, is obtained
Seedling by seedling inoculation into strengthening seedling and rooting culture medium obtains that Paris polyphylla seedling can be cultivated；Method for tissue culture of the invention is using new
Fresh Radix Notoginseng juice, Poria cocos juice and ground moss is used in culture medium, studies have shown that Paris polyphylla tissue cultures of the invention
The method acquisition Paris polyphylla growth of seedling period is short, and effective component Dioscin is not only contained in further Paris polyphylla seedling, also contains it
His effective component.
What the present invention was achieved through the following technical solutions.
A kind of Paris polyphylla method for tissue culture, the cultural method are as follows:
(1) Paris polyphylla plant terminal bud is taken, deionized water is rinsed, and sterilizing, water for injection rinses；
(2) bud is inoculated into culture medium I, at 25-30 DEG C, intensity of illumination 2000-2500Lx, illumination 12h/d, culture
70-90 days, obtain the sprouting that pumping shoot is in clump bud shape；
(3) sprouting is inoculated into culture medium II, culture 50-60 days, 20-30 DEG C of cultivation temperature, luminous intensity 2500-
4000Lx, 10-14 hours/day of illumination, obtains seedling；
(4) seedling being transferred in strengthening seedling and rooting culture medium, illumination is reinforced to 2500-4000Lx, illumination 10-14 hours/
It, cultivates 70-90 days, obtains that Paris polyphylla seedling can be cultivated.
Wherein culture medium I are as follows:
1/2MS, 6-BA 5-10mg/L, NAA 5-10mg/L, ground moss 100-200g/L, Poria cocos juice 1-3g/L,
On the agar medium of sucrose 10-20g/L；PH is 4-6.
Wherein culture medium II are as follows:
1/2MS, 6-BA5-10mg/L, NAA5-10mg/L, Radix Notoginseng juice 1-2g/L, ground moss 100-200g/L are living
Property charcoal 5-10g/L, sucrose 40-50g/L, agar 20-30g/L, pH4-6.
Wherein strengthening seedling and rooting culture medium are as follows:
1/2MS, NAA5-10mg/L, ground bog moss 150-200g/L, Poria cocos juice 4-6g/L, sucrose 40-50g/L,
Active carbon 5-10g/L, agar 20-30g/L.
" Radix Notoginseng juice " of the present invention are as follows: fresh Radix Notoginseng after deionized water is cleaned, squeezed using juice extractor by sterilizing
Radix Notoginseng juice.
" Poria cocos juice " of the present invention are as follows: fresh Poria cocos after deionized water is cleaned, squeezed using juice extractor by sterilizing
Poria cocos juice.
" ground moss " of the present invention are as follows: take fresh moss, be washed with deionized water, ground using grinder
It obtains.
Method for tissue culture of the present invention has the Paris polyphylla seedling obtained in a relatively short period of time for plantation, entire to train
The method of supporting is completed in 240 days, effective to shorten the production cycle.
Specific embodiment
Below the technical scheme of the invention is illustrated by a specific example, but the scope of the present invention is not limited thereto.
Content described in this specification embodiment is only enumerating to the way of realization of inventive concept, protection of the invention
Range should not be construed as being limited to the specific forms stated in the embodiments, and protection scope of the present invention is also and in art technology
Personnel conceive according to the present invention it is conceivable that equivalent technologies mean.Although embodiment of the invention below is retouched
It states, but the invention is not limited to above-mentioned specific embodiments and applications field, following specific embodiments is only to show
It is meaning property, guiding, rather than it is restrictive.Those skilled in the art under the enlightenment of this specification and are not taking off
In the case where the range protected from the claims in the present invention, a variety of forms can also be made, these belong to the present invention
The column of protection.
The following tests of the present invention are on the basis of multiple creative test, with the claimed technical solution of the present invention
Based on, the conclusive test of the research staff of summary.
Prepare embodiment
Embodiment 1
A kind of Paris polyphylla method for tissue culture, the cultural method are as follows:
(1) Paris polyphylla plant terminal bud is taken, deionized water is rinsed, and sterilizing, water for injection rinses；
(2) bud is inoculated into culture medium I, at 25 DEG C, intensity of illumination 2000Lx, illumination 12h/d, cultivates 90 days, obtain
It is in the sprouting of clump bud shape to pumping shoot；
(3) sprouting is inoculated into culture medium II, is cultivated 60 days, 20 DEG C of cultivation temperature, luminous intensity 2500Lx, illumination 14 is small
When/day, obtain seedling；
(4) seedling is transferred in strengthening seedling and rooting culture medium, illumination is reinforced to 2500Lx, 14 hour/day of illumination, culture 90
It, obtains that Paris polyphylla seedling can be cultivated.
Wherein culture medium I are as follows:
1/2MS, 6-BA 5mg/L, NAA 5mg/L, ground moss 100g/L, Poria cocos juice 1g/L, sucrose 10g/L's
On agar medium；PH is 4.
Wherein culture medium II are as follows:
1/2MS, 6-BA5mg/L, NAA5mg/L, Radix Notoginseng juice 1g/L, ground moss 100g/L, active carbon 5g/L, sugarcane
Sugared 40g/L, agar 20g/L, pH4.
Wherein strengthening seedling and rooting culture medium are as follows:
1/2MS, NAA5mg/L, ground bog moss 150g/L, Poria cocos juice 4g/L, sucrose 40g/L, active carbon 5g/L,
Agar 20g/L.
Embodiment 2
A kind of Paris polyphylla method for tissue culture, the cultural method are as follows:
(1) Paris polyphylla plant terminal bud is taken, deionized water is rinsed, and sterilizing, water for injection rinses；
(2) bud is inoculated into culture medium I, at 30 DEG C, intensity of illumination 2500Lx, illumination 12h/d, cultivates 70 days, obtain
It is in the sprouting of clump bud shape to pumping shoot；
(3) sprouting is inoculated into culture medium II, is cultivated 50 days, 30 DEG C of cultivation temperature, luminous intensity 4000Lx, illumination 14 is small
When/day, obtain seedling；
(4) seedling is transferred in strengthening seedling and rooting culture medium, illumination is reinforced to 4000Lx, 14 hour/day of illumination, culture 70
It, obtains that Paris polyphylla seedling can be cultivated.
Wherein culture medium I are as follows:
1/2MS, 6-BA 10mg/L, NAA 10mg/L, ground moss 200g/L, Poria cocos juice 3g/L, sucrose 20g/L
Agar medium on；PH is 6.
Wherein culture medium II are as follows:
1/2MS, 6-BA10mg/L, NAA 10mg/L, Radix Notoginseng juice 2g/L, ground moss 200g/L, active carbon 10g/
L, sucrose 50g/L, agar 230g/L, pH6.
Wherein strengthening seedling and rooting culture medium are as follows:
1/2MS, NAA10mg/L, ground bog moss 200g/L, Poria cocos juice 6g/L, sucrose 50g/L, active carbon 10g/
L, agar 30g/L.
Embodiment 3
A kind of Paris polyphylla method for tissue culture, the cultural method are as follows:
(1) Paris polyphylla plant terminal bud is taken, deionized water is rinsed, and sterilizing, water for injection rinses；
(2) bud is inoculated into culture medium I, at 27 DEG C, intensity of illumination 2200Lx, illumination 12h/d, cultivates 80 days, obtain
It is in the sprouting of clump bud shape to pumping shoot；
(3) sprouting is inoculated into culture medium II, is cultivated 55 days, 25 DEG C of cultivation temperature, luminous intensity 3500Lx, illumination 12 is small
When/day, obtain seedling；
(4) seedling is transferred in strengthening seedling and rooting culture medium, illumination is reinforced to 3500Lx, 12 hour/day of illumination, culture 80
It, obtains that Paris polyphylla seedling can be cultivated.
Wherein culture medium I are as follows:
1/2MS, 6-BA 8mg/L, NAA 8mg/L, ground moss 150g/L, Poria cocos juice 2g/L, sucrose 15g/L's
On agar medium；PH is 5.
Wherein culture medium II are as follows:
1/2MS, 6-BA8mg/L, NAA8mg/L, Radix Notoginseng juice 1.5g/L, ground moss 150g/L, active carbon 7g/L,
Sucrose 45g/L, agar 25g/L, pH5.
Wherein strengthening seedling and rooting culture medium are as follows:
1/2MS, NAA8mg/L, ground bog moss 175g/L, Poria cocos juice 5g/L, sucrose 45g/L, active carbon 7g/L,
Agar 25g/L.
Test 1
The detection of Dioscin
Test method:
The preparation of reference substance solution: it takes dry Dioscin reference substance appropriate, accurately weighs 1mg, methanol is added to be configured to
The reference substance solution of 1mg/mL.
Sample preparation: taking different embodiments that can cultivate Paris polyphylla seedling 1g, and 85% ethyl alcohol 20mL refluxing extraction 40min is added, mentions
Taking liquid is sample solution.
Detection method: according to four 0502 thin-layered chromatography tests of the Pharmacopoeia of the People's Republic of China, 10 μ of reference substance is drawn
L, 10 μ L of sample solution are put respectively on same silica gel g thin-layer plate, with chloroform-methanol-water (volume ratio 15:5:1)
Lower layer's solution is solvent, takes out, dries, and it is clear that 10% ethanol solution of sulfuric acid of spray is heated to spot development at 105 DEG C, ultraviolet
It is inspected under lamp 365nm.
Test result is as follows: being shown in Table 1.
1 thin-layer chromatography mobility ratio of table compared with
| Group | Rf | Spot number |
| Reference substance | 0.357 | 1 |
| Embodiment 1 | 0.357 | 5 |
| Embodiment 2 | 0.357 | 5 |
| Embodiment 3 | 0.357 | 5 |
Conclusion (of pressure testing): above-mentioned TLC test shows method for tissue culture of the present invention, the Paris polyphylla seedling of acquisition
In contain effective component Dioscin, and contain other compositions simultaneously, the method for absolutely proving tissue cultures of the present invention has weight
Want scientific meaning.
Claims (4)
1. a kind of Paris polyphylla method for tissue culture, it is characterised in that: the cultural method are as follows:
(1) Paris polyphylla plant terminal bud is taken, deionized water is rinsed, and sterilizing, water for injection rinses；
(2) bud is inoculated into culture medium I, at 25-30 DEG C, intensity of illumination 2000-2500Lx, illumination 12h/d, cultivates 70-
90 days, obtain the sprouting that pumping shoot is in clump bud shape；
(3) sprouting is inoculated into culture medium II, culture 50-60 days, 20-30 DEG C of cultivation temperature, luminous intensity 2500-4000Lx,
10-14 hours/day of illumination, obtains seedling；
(4) seedling is transferred in strengthening seedling and rooting culture medium, illumination is reinforced to 2500-4000Lx, 10-14 hours/day of illumination, training
It supports 70-90 days, obtains that Paris polyphylla seedling can be cultivated.
2. a kind of Paris polyphylla method for tissue culture according to claim 1, wherein culture medium I are as follows:
1/2MS, 6-BA5-10mg/L, NAA5-10mg/L, ground moss 100-200g/L, Poria cocos juice 1-3g/L, sucrose
On the agar medium of 10-20g/L；PH is 4-6.
3. a kind of Paris polyphylla method for tissue culture according to claim 1, wherein culture medium II are as follows:
1/2MS, 6-BA5-10mg/L, NAA5-10mg/L, Radix Notoginseng juice 1-2g/L, ground moss 100-200g/L, active carbon
5-10g/L, sucrose 40-50g/L, agar 20-30g/L, pH4-6.
4. a kind of Paris polyphylla method for tissue culture according to claim 1, wherein strengthening seedling and rooting culture medium are as follows:
1/2MS, NAA5-10mg/L, ground bog moss 150-200g/L, Poria cocos juice 4-6g/L, sucrose 40-50g/L, activity
Charcoal 5-10g/L, agar 20-30g/L.
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