CN108719067A - A kind of tissue culture and rapid propagation method of paris polyphylla - Google Patents
A kind of tissue culture and rapid propagation method of paris polyphylla Download PDFInfo
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- CN108719067A CN108719067A CN201810538915.6A CN201810538915A CN108719067A CN 108719067 A CN108719067 A CN 108719067A CN 201810538915 A CN201810538915 A CN 201810538915A CN 108719067 A CN108719067 A CN 108719067A
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- Prior art keywords
- paris polyphylla
- rapid propagation
- tissue
- tissue culture
- chiltern
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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 invention discloses a kind of tissue culture and rapid propagation methods of paris polyphylla,The tissue culture and rapid propagation method of the paris polyphylla includes paris polyphylla callus tissue culture,Paris polyphylla crowd shoots are cultivated,Paris polyphylla tissue-culturing rapid propagation seedling is cultivated and transplant step,Wherein,The chiltern callus tissue culture base,Chiltern crowd shoots culture medium and chiltern tissue-culturing rapid propagation seedling culture medium composition include river sand,Plant growth regulator,Nutrient solution,Ascorbic acid,Polyvinylpyrrolidone and fungicide,In terms of 1L,50 ~ 500 ml/L of MS nutrient solutions,0 ~ 15 g/L of sucrose,0.1 ~ 10.0 ml/L of plant growth regulator,0.1 ~ 5.0 g/L of ascorbic acid,0.1 ~ 5.0 g/L of polyvinylpyrrolidone,1 ~ 15 g/L of fungicide,0.4 ~ 0.6g/L of activated carbon,Remaining is river sand.The chiltern paris polyphylla tissue culture medium (TCM) of the present invention is using thin net river sand as solid dielectric, substitute agar, the cost of culture medium can be reduced, river sand cycle repeatedly utilizes, and need not accurately adjust the acid-base value of nutrient solution in plant tissue culture media, auxin and organic nutrition of plant liquid.
Description
Technical field
The invention belongs to paris polyphylla technical field of cultivation, and in particular to a kind of tissue culture and rapid propagation method of paris polyphylla.
Background technology
Paris polyphylla, alias Typhonium giganteum are herbaceos perennials.Root-like stock is sturdy, stem high 20-2500px, hairless, often
Band aubergine, base portion have 1~3 film quality leaf sheath to embrace stem.There is the effect of clearing heat and detoxicating, swelling and pain relieving, cool liver arresting convulsion, is used for carbuncle
The diseases such as swollen, abscess of throat, venomous snake bite, injuries from falls as well, convulsion.Paris polyphylla, alias Typhonium giganteum are that perennial herb is planted
Object.Root-like stock is sturdy, and the high 20-2500px of stem, hairless, often band aubergine, base portion have 1~3 film quality leaf sheath to embrace stem.Leaf 5~11
Piece, green is verticillate, long 7~425px, wide 2.2~150px, for the oblong of falling ovate or lanceolar, the sharp point or tapering of apex,
Base portion wedge shape to circle, full edge goes out arteries and veins, the long 0-50px of petiole often with a pair of apparent base.Flower basidixed is central in impeller, both sexes,
Bennet extends, perianth two-wheeled, and foreign steamer is by piece 4~6, green, oval or lanceolar, lubrication groove tapel and foreign steamer tapel with number,
Linear or Filamentous, the narrow cochlear of wide 2-5mm is often expanded as on yellow green, top.Stamen 2~4 is taken turns, 8~12 pieces, and anther length 5~
10mm, connective is more apparent, long 1~2mm.Ovary is subsphaeroidal, and green has rib or wing, Room 1.Style base purple, thickens, normal angle disk
Shape.Style purple, it is upright when spending, it is rolled up outside fruiting period.Fruit is subsphaeroidal, green, irregular to crack.Seed is most, and ovoid has scarlet
Exosper.4~July of florescence, fruit cracking in 10~November.
Paris polyphylla has a habit of " preferably shady fear is shone, hygrophilous avoid dry ", hygrophilous profit, the environment covered, topography is flat, irrigation side
Just, draining is good, well-grown in, the higher loose fertile sand loam of the content of organic matter more containing humus.Paris polyphylla is given birth to
In growth process, it is desirable that higher air humidity and depth of defilade.Suitable growth is in the area that height above sea level is 1600~3100m, annual
Temperature is 12~13 DEG C, 270 days frost-free periods or more.850~1200mm of annual rainfall, rainfall concentrated between June to September, air
For humidity 75% or more, soil Evening Tide can meet the needs of paris polyphylla growth and development is to soil moisture content.When planting paris polyphylla,
For the cool canopy degree of shading of construction between 60%-70%, scattering luminous energy effectively facilitates the growth of paris polyphylla.
Rhizoma Paridis there is no the product of extensive artificial growth still essentially from wild.It is quick with Chinese Medicine Industry
Development, increases considerably by manufacturing enterprise's dosage of raw material of Paris polyphylla, and long-term predatory excavation keeps wild Paris polyphylla fewer and fewer.
It is to obtain more raw materials that Paris polyphylla, which needs the time of 8-10, pharmacy corporation from plantation to harvesting utilization, increases substantially Paris polyphylla purchase
Price, purchasing price is up to 1000 yuan/kg or more.Purchasing price rise medicinal herb grower excavates enthusiasm, and excavation speed is considerably beyond certainly
The right speed of growth, it has been in rare state to lead to Paris polyphylla resource.According to data, it is distributed in the domestic wild Paris polyphylla 80% in China
It is exploited, only remaining 20% resource.China consumes 1000 tons of Paris polyphylla or more, and all wild Paris polyphylla, existing money every year
It source may be exhausted using excavation in 5 years or so.Paris polyphylla resource it is rare be likely to become restrict Yunnan correlation pharmaceutical industries can
The bottleneck of sustainable development.Therefore, the needs of market far can not be met by only relying on wild resource, and artificial growth, which becomes, solves weight
The inevitable choice of building scarcity of resources.And with Chinese Medicinal Materials Markets it is continuous expansion and Paris polyphylla itself possessed by good drug effect,
The demand of Paris polyphylla will necessarily increase substantially, and price can also raise up.Traditional method for planting of paris polyphylla far can not at present
Meet the market demand, therefore a kind of method that can solve above-mentioned technical problem of exploitation is very important.
Invention content
The purpose of the present invention is to provide a kind of tissue culture and rapid propagation methods of paris polyphylla.
The object of the present invention is achieved like this, and the tissue culture and rapid propagation method of the paris polyphylla includes paris polyphylla callus
Culture, paris polyphylla crowd shoots are cultivated, paris polyphylla tissue-culturing rapid propagation seedling is cultivated and transplant step, are specifically included：
A, paris polyphylla callus tissue culture：With sterile inoculating tool after explant is disinfected paris polyphylla seed or
Root-like stock is inoculated into chiltern callus tissue culture base, is cultivated under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination
Obtain within 10 ~ 60 days lurid paris polyphylla callus；
B, paris polyphylla crowd shoots are cultivated：Paris polyphylla callus or cell mass are inoculated into chiltern clump with sterile inoculating tool
It sprouts in seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla
Crowd shoots；
C, paris polyphylla tissue-culturing rapid propagation seedling is cultivated：Paris polyphylla crowd shoots are inoculated into chiltern tissue-culturing rapid propagation with sterile inoculating tool
In seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla tissue culture
Fast propagating seedling；
D, it transplants：When paris polyphylla tissue-culturing rapid propagation seedling is grown to 10 ~ 25cm, hardening is taken out after 10 ~ 30 days, by paris polyphylla tissue-culturing rapid propagation
Seedling moves into plantation in the cool canopy rich in organic matter seedbed, moisturizing of watering, and carries out conventional heat and moisture preserving ventilation and pest management；
Wherein, the chiltern callus tissue culture base, chiltern crowd shoots culture medium and chiltern tissue-culturing rapid propagation seedling culture medium group
Cheng Jun includes river sand, plant growth regulator, nutrient solution, ascorbic acid, polyvinylpyrrolidone and fungicide, in terms of 1L, MS
50 ~ 500 ml/L of nutrient solution, 0 ~ 15 g/L of sucrose, 0.1 ~ 10.0 ml/L of plant growth regulator, ascorbic acid 0.1 ~
5.0 g/L, 0.1 ~ 5.0 g/L of polyvinylpyrrolidone, fungicide 1 ~ 15 g/L, 0.4 ~ 0.6g/L of activated carbon, remaining is river
Sand.
Paris polyphylla tissue culture and rapid propagation method of the present invention carries out the cultivation of paris polyphylla, culture using sandy paris polyphylla tissue culture medium (TCM)
Base is to utilize thin net, dry river sand and Plant Tissue Breeding nutrient solution, plant growth regulator, ascorbic acid, polyethylene pyrrole
Pyrrolidone and fungicide composition, are fabricated to novel solid chiltern culture medium.In the present invention, thin net, dry river sand generation is utilized
Solid medium medium is made for agar, can not only reduce the cost of the coagulators such as agar, river sand can also recycle multiple profit
With, and replace agar to make in solid dielectric with thin net river sand, be added each plant nutrient liquor, plant growth regulator, carbohydrate,
After organic nutrition of plant liquid, the required various nutrients of Plant Tissue Breeding material normal growth can be supplied, need not also be used
Acid solution or lye carry out the accurate acid-base value for adjusting culture medium, really up to the mark after solid medium sterilization can also be avoided to cool down
Or the too soft limitation for being unfavorable for Plant Tissue Breeding, and enhance using the nutty structure of river sand the gas permeability of matrix, it reaches
To fixation, moisturizing, ventilative purpose in Plant Tissue Breeding, meet moisture in the tissue culture procedures of plant, nutrient, hormone and
The functions such as ventilative are more advantageous to the Plant Tissue Breeding of plant, plant tissue culture is quickly bred and modern soilless culture.
Present invention is generally directed in traditional Plant Tissue Breeding, need that suitable fine jade is added when preparing solid medium
Fat is added after carbohydrate, organic nutrition of plant liquid, and solid medium itself is easy to go mouldy, and when preparing solid medium, needs
To the acid-base value of the culture solutions such as agar, macro-element nutrients liquid, microelement nutritious liquid, liquid glucose, organic additive matter and hormone
It is accurately adjusted with acid solution or lye, if the acid-base value regulation and control of solid culture base fluid are inaccurate, is easy to cause solid medium
It is really up to the mark or excessively soft after sterilization cooling, it is unfavorable for Plant Tissue Breeding or leads to the tissue cultures problems such as unsuccessfully.
The present invention is to substitute agar using thin net river sand as solid dielectric, can not only reduce cost, but also utilize
River sand adsorbs the nutrient solution, auxin and organic nutrition of plant liquid of Plant Tissue Breeding, meanwhile, preparing solid training
The acid-base value of nutrient solution, auxin and organic nutrition of plant liquid need not be accurately adjusted very much when supporting base, culture medium is just
To provide support solid dielectric, nutrient solution and the organic principle of Plant Tissue Breeding, supply Plant Tissue Breeding material is just
Be frequently grown, and at the same time enhance the gas permeability of matrix using the nutty structure of river sand, reach in Plant Tissue Breeding it is fixed,
Moisturizing, ventilative purpose meet moisture in the tissue culture procedures of plant, nutrient, hormone and the functions such as ventilative.
The tissue culture and rapid propagation method of paris polyphylla of the present invention, concrete operations are as follows：
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Callus induction formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA of MS nutrient solutions+6-BA, 0.1 ml/L+poly- second
1.0 g/L of alkene pyrrolones+1.0 g/L of carbendazim+0.5 g/L of activated carbon+ascorbic acid 0.5g/L, after mixing well
It is added in thin net river sand and is uniformly mixed, be sub-packed in culture bottle, every bottle of 50cm3, it covers tightly bottle cap and is put in sterilizing in autoclave,
Sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to spare in superclean bench；With sterile inoculation
Tool after explant is disinfected paris polyphylla seed or root-like stock be inoculated in superclean bench sterilizing after sand
In matter culture medium, every bottle is inoculated with 2-5（Block）, tighten bottle cap and move to culture in culturing room（Cultivation temperature is controlled at 20 ± 2 DEG C,
Light application time daily 8-12,1000-3000 Lux of intensity of illumination, aftermentioned condition of culture are identical）, by 10-60 days, you can
Long lurid paris polyphylla callus.
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river
Sand, by inducing clumping bud formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA 0.2-0.5 of MS nutrient solutions+6-BA
Ml/L+KT 0.2-1.0 ml/L+1.0 g/L of polyvinyl pyrrolidone+1.0 g/L of carbendazim+activated carbon, 0.5 g/L
+ ascorbic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle of 50cm3,
Cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to
It is spare in superclean bench.Sterile callus or cell mass are inoculated in superclean bench with sterile inoculating tool and gone out
In sandy culture medium after bacterium, every bottle of inoculation 2-5 block tightens bottle cap and moves to culture in culturing room, by 10-60 days, you can long
Go out that leaf color is dark green, paris polyphylla crowd shoots of robust growth；
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Indefinite root system induction formula in paris polyphylla tissue-culturing rapid propagation, by 0.2 ml/L+NAA 1.0- of plant 1/2MS nutrient solutions+6-BA
5.0 1.0 g/L of ml/L+IBA 0.2-2.0 ml/L+ polyvinyl pyrrolidones+1.0 g/L of carbendazim+activated carbon 0.5
G/L+ascorbic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle
50cm3, cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minutes, is taken after sterilizing
Go out to move to spare in superclean bench.Sterile paris polyphylla crowd shoots are inoculated in superclean bench with sterile inoculating tool
In sandy culture medium after sterilizing, every bottle is inoculated with 2-5 clumps, tightens bottle cap and moves to culture in culturing room, by 10-60 days, i.e.,
It can grow that root system is complete, leaf color is dark green, the complete tissue-culturing rapid propagation seedling of the paris polyphylla root, stem and leaf of robust growth；When the indoor Yunnan of culture
When Paris polyphylla tissue-culturing rapid propagation seedling grows to 10-25cm, hardening is taken out after 10-30 days, it will be with sandy paris polyphylla tissue-culturing rapid propagation seedling
Plantation in the cool canopy rich in organic matter seedbed, watering moisturizing are moved into, and carries out relevant heat and moisture preserving ventilation and pest management.
The operations such as the preparation of culture medium, explant material processing and inductive technology are as follows in the above process：
1, the preparation of the sandy culture medium of paris polyphylla
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Paris polyphylla tissue-culturing rapid propagation formula, by plant growth regulator, MS nutrient solutions, ascorbic acid, polyvinylpyrrolidone, activated carbon and
Fungicide is added in thin net river sand and is uniformly mixed, and river sand water content 50-70% is sub-packed in culture bottle, every bottle of 30-200cm3,
Cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to
It is spare in superclean bench, it obtains containing the required nutrient solution of paris polyphylla tissue-culturing rapid propagation, plant growth regulator, Vitamin C
The sterile sandy culture medium of acid, polyvinylpyrrolidone, activated carbon and fungicide.
2, Yunnan weighs the processing of seed explant material in sandy tissue-culturing rapid propagation
The paris polyphylla seed just sprouted is taken out with tweezers, 20min is rinsed under tap water, 30% washing powder solution impregnates 1h, originally
It is put into after water rinsed clean in bottle and suitable distilled water concussion, washing 4-5 times is added, removing is attached to the surface of the seed sundries, uses
It is rinsed 3-5 times added with the distilled water of 2-3 drop Tween 80s, moves into ultra violet lamp 20min in superclean bench；Again with 75% ethyl alcohol
10-30s are impregnated, sterile water washing 3-5 times impregnates 5-10min with 0.1% mercury chloride, and sterile water washing 5 times uses aseptic filter paper
The moisture for blotting the surface of the seed is seeded to the sandy culture culture in glassware of paris polyphylla with aseptic inoculation tool.
3, Yunnan weighs the processing of root-like stock explant material in sandy tissue-culturing rapid propagation
With grubbing up, shovel takes out the paris polyphylla rhizome with young shoot or bud eye, strikes off appearance impurity with pocket knife or scissors, is put in saturation
After impregnating 20min in detergent liquid, 30min is rinsed under tap water, then slightly shaken, washed 4-5 times with distilled water, remove attached
It in root-like stock surface soil and sundries, is rinsed 3-5 times, moved into superclean bench with the distilled water added with 2-3 drop Tween 80s
Ultra violet lamp 30min；10-30s are impregnated with 75% ethyl alcohol again, sterile water washing 3-5 times impregnates 10- with 0.1% mercury chloride
30min, sterile water washing 5 times, the moisture on root-like stock surface is blotted with aseptic filter paper, and paris polyphylla is seeded to aseptic inoculation tool
Sandy culture culture in glassware.
4, callus induction technology in the sandy tissue-culturing rapid propagation of Yunnan weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Callus induction formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA of MS nutrient solutions+6-BA, 0.1 ml/L+poly- second
1.0 g/L of alkene pyrrolones+1.0 g/L of carbendazim+0.5 g/L of activated carbon+ascorbic acid 0.5g/L, after mixing well
It is added in thin net river sand and is uniformly mixed, be sub-packed in culture bottle, every bottle of 50cm3, it covers tightly bottle cap and is put in sterilizing in autoclave,
Sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to spare in superclean bench；With sterile inoculation
Tool after explant is disinfected paris polyphylla seed or root-like stock be inoculated in superclean bench sterilizing after sand
In matter culture medium, every bottle is inoculated with 2-5（Block）, tighten bottle cap and move to culture in culturing room（Cultivation temperature is controlled at 20 ± 2 DEG C,
Light application time daily 8-12,1000-3000 Lux of intensity of illumination, aftermentioned condition of culture are identical）, by 10-60 days, you can
Grow the lurid paris polyphylla callus of rough surface, graininess, quality or cell mass.
5, inducing clumping bud technology in the sandy tissue-culturing rapid propagation of Yunnan weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Inducing clumping bud formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA 0.2-0.5 ml/L of MS nutrient solutions+6-BA
+ KT 0.2-1.0 ml/L+1.0 g/L of polyvinyl pyrrolidone+1.0 g/L of carbendazim+activated carbon, 0.5 g/L+anti-
Bad hematic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle of 50cm3, cover tightly
Bottle cap is put in sterilizing in autoclave, and sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to ultra-clean
It is spare in workbench.After sterile callus or cell mass are inoculated in sterilizing in superclean bench with sterile inoculating tool
Sandy culture medium in, every bottle of inoculation 2-5 block tightens bottle cap and moves to culturing room in culture, by 10-60 days, you can grow leaf
Color is dark green, robust growth paris polyphylla crowd shoots；
6, root induction technology in the sandy tissue-culturing rapid propagation of Yunnan weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Indefinite root system induction formula in paris polyphylla tissue-culturing rapid propagation, by 0.2 ml/L+NAA 1.0- of plant 1/2MS nutrient solutions+6-BA
5.0 1.0 g/L of ml/L+IBA 0.2-2.0 ml/L+ polyvinyl pyrrolidones+1.0 g/L of carbendazim+activated carbon 0.5
G/L+ascorbic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle
50cm3, cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minutes, is taken after sterilizing
Go out to move to spare in superclean bench.Sterile paris polyphylla crowd shoots are inoculated in superclean bench with sterile inoculating tool
In sandy culture medium after sterilizing, every bottle is inoculated with 2-5 clumps, tightens bottle cap and moves to culture in culturing room, by 10-60 days, i.e.,
It can grow that root system is complete, leaf color is dark green, the complete tissue-culturing rapid propagation seedling of the paris polyphylla root, stem and leaf of robust growth；When the indoor Yunnan of culture
When Paris polyphylla tissue-culturing rapid propagation seedling grows to 10-25cm, hardening is taken out after 10-30 days, it will be with sandy paris polyphylla tissue-culturing rapid propagation seedling
Plantation in the cool canopy rich in organic matter seedbed, watering moisturizing and related heat and moisture preserving and pest management are moved into, by 3-5's
Cultivation management, the sandy tissue-culturing rapid propagation seedling of paris polyphylla blossom and bear fruit, and cycle carries out the sand of paris polyphylla after collecting seed or taking root-like stock
Matter tissue-culturing rapid propagation.
Description of the drawings
Fig. 1 is paris polyphylla tissue-culturing rapid propagation Multiple Buds germination and growth schematic diagram of the present invention；
Fig. 2 is paris polyphylla tissue Multiple Buds germination and growth schematic diagram of the present invention；
Fig. 3 is paris polyphylla tissue-culturing rapid propagation root induction schematic diagram of the present invention；
Fig. 4 is that paris polyphylla tissue-culturing rapid propagation seedling greenhouse gardening of the present invention grows schematic diagram.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that made by it is any transform or replace, all belong to the scope of protection of the present invention.
The tissue culture and rapid propagation method of paris polyphylla of the present invention includes paris polyphylla callus tissue culture, paris polyphylla crowd shoots
Cultivation, paris polyphylla tissue-culturing rapid propagation seedling is cultivated and transplant step, specifically includes：
A, paris polyphylla callus tissue culture：With sterile inoculating tool after explant is disinfected paris polyphylla seed or
Root-like stock is inoculated into chiltern callus tissue culture base, is cultivated under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination
Obtain within 10 ~ 60 days lurid paris polyphylla callus；
B, paris polyphylla crowd shoots are cultivated：Paris polyphylla callus or cell mass are inoculated into chiltern clump with sterile inoculating tool
It sprouts in seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla
Crowd shoots；
C, paris polyphylla tissue-culturing rapid propagation seedling is cultivated：Paris polyphylla crowd shoots are inoculated into chiltern tissue-culturing rapid propagation with sterile inoculating tool
In seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla tissue culture
Fast propagating seedling；
D, it transplants：When paris polyphylla tissue-culturing rapid propagation seedling is grown to 10 ~ 25cm, hardening is taken out after 10 ~ 30 days, by paris polyphylla tissue-culturing rapid propagation
Seedling moves into plantation in the cool canopy rich in organic matter seedbed, moisturizing of watering, and carries out conventional heat and moisture preserving ventilation and pest management；
Wherein, the chiltern callus tissue culture base, chiltern crowd shoots culture medium and chiltern tissue-culturing rapid propagation seedling culture medium group
Cheng Jun includes river sand, plant growth regulator, nutrient solution, ascorbic acid, polyvinylpyrrolidone and fungicide, in terms of 1L, MS
50 ~ 500 ml/L of nutrient solution, 0 ~ 15 g/L of sucrose, 0.1 ~ 10.0 ml/L of plant growth regulator, ascorbic acid 0.1 ~
5.0 g/L, 0.1 ~ 5.0 g/L of polyvinylpyrrolidone, fungicide 1 ~ 15 g/L, 0.4 ~ 0.6g/L of activated carbon, remaining is river
Sand.
The plant growth regulator is 6-BA, NAA, IBA, IAA, Zt and Kt.
6-BA is 0.0 ~ 10.0mg/L in the plant growth regulator, NAA be 0.0 ~ 10.0mg/L, IBA be 0.01 ~
10.0mg/L, IAA are 0.01 ~ 10.0mg/L, and Zt is 1 ~ 5mg/L, and Kt is 1 ~ 5mg/L.
The fungicide be Bravo, carbendazim, thiophanate methyl, fenaminosulf, pentachloronitrobenzene, triadimefon, dislike it is mould
One or more of spirit, benomyl and probenazole.
The fungicide is Bravo and carbendazim.
Bravo is 0.5 ~ 15g/L in the fungicide, and carbendazim is 0.5 ~ 15g/L.
The chiltern callus tissue culture base in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 1.0 ~
0.05 ~ 0.15ml/L of 3.0ml/L, NAA, 0.5 ~ 1.5g/L of polyvinyl pyrrolidone, 0.5 ~ 1.5g/L of carbendazim, activated carbon 0.25 ~
0.4 ~ 0.6g/L of 0.75g/L and ascorbic acid.
The chiltern crowd shoots culture medium in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 1.0 ~
3.0ml/L, NAA 0.2 ~ 0. 5ml/L, 0.2 ~ 1.0ml/L of KT, 0.5 ~ 1.5g/L of polyvinyl pyrrolidone, carbendazim 0.5 ~
0.4 ~ 0.6g/L of 1.5g/L, 0.3 ~ 0.7g/L of activated carbon and ascorbic acid.
The chiltern tissue-culturing rapid propagation seedling culture medium in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 0.1 ~
0.3ml/L, NAA 1.0 ~ 5.0ml/L, IBA 0.2 ~ 2.0ml/L, 0.5 ~ 1.5g/L of polyvinyl pyrrolidone, carbendazim 0.5 ~
0.4 ~ 0.6g/L of 1.5g/L, 0.3 ~ 0.7g/L of activated carbon and ascorbic acid.
Case is embodied, the present invention will be further described below：
The preparation of the sandy culture medium of 1 paris polyphylla of embodiment
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Paris polyphylla tissue-culturing rapid propagation formula, by plant growth regulator, MS nutrient solutions, ascorbic acid, polyvinylpyrrolidone, activated carbon and
Fungicide is added in thin net river sand and is uniformly mixed, and river sand water content 50-70% is sub-packed in culture bottle, every bottle of 30-200cm3,
Cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to
It is spare in superclean bench, it obtains containing the required nutrient solution of paris polyphylla tissue-culturing rapid propagation, plant growth regulator, Vitamin C
The sterile sandy culture medium of acid, polyvinylpyrrolidone, activated carbon and fungicide.
The processing of seed explant material in the sandy tissue-culturing rapid propagation of 2 Yunnan of embodiment weight
The paris polyphylla seed just sprouted is taken out with tweezers, 20-60 min are rinsed under tap water, is impregnated with washing powder saturated solution
1h is put into bottle after tap water rinse is clean and suitable distilled water concussion, washing 4-5 times is added, and removing is attached to the surface of the seed
Sundries is rinsed 3-5 times with the distilled water added with 2-3 drop Tween 80s, is moved into superclean bench with ultra violet lamp 20min；Again
10-30s are impregnated with 75% ethyl alcohol, sterile water washing 3-5 times impregnates 5-20min, sterile water washing 5-8 with 0.1% mercury chloride
It is secondary, the moisture of the surface of the seed is blotted with aseptic filter paper, is seeded to containing 50 ~ 500 ml/L of MS nutrient solutions with aseptic inoculation tool
0.1 ~ 3.0ml/L+ of+6-BA NAA, 0.2 ~ 0. 0.5 ~ 1.5g/ of 5ml/L+ KT 0.2 ~ 1.0ml/L+ polyvinyl pyrrolidones
The sandy culture bottle of paris polyphylla of 0.1 ~ 2.0g/L of L+ carbendazim 0.5 ~ 1.5g/L+ activated carbon 0.3 ~ 2.0g/L+ ascorbic acid
Interior culture, 20-25 DEG C of cultivation temperature, air relative temperature 80% are cultivated in the culturing room of 1000-3000Lux of illumination, are passed through
Seed can grow young leaves and root system within 20-60 days, obtain the paris polyphylla plant of normal growth.
Root-like stock explant material is handled in the sandy tissue-culturing rapid propagation of 3 Yunnan of embodiment weight
With grubbing up, shovel takes out the paris polyphylla rhizome with young shoot or bud eye from field or seedbed, and Yunnan weight is struck off with pocket knife or scissors
Building root-like stock appearance impurity is put in after impregnating 20-60 min in saturation detergent liquid, 30 min is rinsed under tap water, then use
Distilled water slightly shakes, washs 4-5 times, and removing is attached to root-like stock surface soil and sundries, with added with 2-3 drop Tween 80s
Distilled water rinses 3-5 times, moves into superclean bench with ultra violet lamp 30min；Again 10-30s, nothing are impregnated with 75% ethyl alcohol
Bacterium water washing 3-5 times impregnates surface sterilization with 0.1% mercury chloride and handles 10-30min, and sterile water washing 5-8 times uses aseptic filter paper
The moisture for blotting root-like stock surface is seeded to aseptic inoculation tool containing 50 ~ 500 ml/L+ 6-BA 0.1 of MS nutrient solutions
0.2 ~ 2.0 ml/L+ 0.2 ~ 2.0ml/L+ of KT polyvinyl pyrrolidones of ~ 3.0ml/L+ NAA, 0.5 ~ 3 more bacterium of .0 g/L+
Training in the sandy culture bottle of paris polyphylla of 0.1 ~ 2.0g/L of spirit 0.5 ~ 1.5g/L+ activated carbon 0.3 ~ 2.0g/L+ ascorbic acid
It supports, 20-25 DEG C of cultivation temperature, air relative temperature 80%, is cultivated in the culturing room of 1000-3000Lux of illumination, by 20-60
Its culture, paris polyphylla rhizome can grow young leaves and root system, obtain the paris polyphylla plant of normal growth.
Callus induction technology in the sandy tissue-culturing rapid propagation of 4 Yunnan of embodiment weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Callus induction formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA of MS nutrient solutions+6-BA, 0.1 ml/L+poly- second
1.0 g/L of alkene pyrrolones+1.0 g/L of carbendazim+0.5 g/L of activated carbon+ascorbic acid 0.5g/L, after mixing well
It is added in thin net river sand and is uniformly mixed, be sub-packed in culture bottle, every bottle of 50cm3, it covers tightly bottle cap and is put in sterilizing in autoclave,
Sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to spare in superclean bench；With sterile inoculation
Tool after explant is disinfected paris polyphylla seed or root-like stock be inoculated in superclean bench sterilizing after sand
In matter culture medium, seed can be longitudinal sectional or crosscutting, and root-like stock is cut into the stripping and slicing of the grain of rice or big beans size, and every bottle is inoculated with 2-5
（Block）, tighten bottle cap and move in culturing room and cultivate, cultivation temperature control is at 20 ± 2 DEG C, light application time daily 8-12, light
According to 1000-3000 Lux of intensity, by 30-60 days, the seed or root-like stock of paris polyphylla can grow rough surface,
Granular, the lurid paris polyphylla callus of quality or cell mass.
Inducing clumping bud technology in the sandy tissue-culturing rapid propagation of 5 Yunnan of embodiment weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Inducing clumping bud formula in paris polyphylla tissue-culturing rapid propagation, by 2.0 ml/L+NAA 0.2-0.5 ml/L of MS nutrient solutions+6-BA
+ KT 0.2-1.0 ml/L+1.0 g/L of polyvinyl pyrrolidone+1.0 g/L of carbendazim+activated carbon, 0.5 g/L+anti-
Bad hematic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle of 50cm3, cover tightly
Bottle cap is put in sterilizing in autoclave, and sterilising temp is 121 DEG C, sterilization time 15-60 minute, after sterilizing taking-up move to ultra-clean
It is spare in workbench.The sterile callus of paris polyphylla or cell mass obtained after induction is being surpassed with sterile inoculating tool
It being inoculated in net workbench in the sandy culture medium after sterilizing, every bottle of inoculation 2-5 block tightens bottle cap and moves to culture in culturing room,
By 10-60 days, you can grow that leaf color is dark green, paris polyphylla crowd shoots of robust growth.
Root induction culture technique in the sandy tissue-culturing rapid propagation of 6 Yunnan of embodiment weight
River sand is cleaned through clear water, soil and sundries is removed, dries or dry, the iron for crossing 10-50 mesh sieves to obtain thin net river sand, presses
Indefinite root system induction formula in paris polyphylla tissue-culturing rapid propagation, by 0.2 ml/L+NAA 1.0- of plant 1/2MS nutrient solutions+6-BA
5.0 1.0 g/L of ml/L+IBA 0.2-2.0 ml/L+ polyvinyl pyrrolidones+1.0 g/L of carbendazim+activated carbon 0.5
G/L+ascorbic acid 0.5g/L is added in thin net river sand after mixing well and is uniformly mixed, is sub-packed in culture bottle, every bottle
50cm3, cover tightly bottle cap and be put in sterilizing in autoclave, sterilising temp is 121 DEG C, sterilization time 15-60 minutes, is taken after sterilizing
Go out to move to spare in superclean bench.Sterile paris polyphylla crowd shoots are inoculated in superclean bench with sterile inoculating tool
In sandy culture medium after sterilizing, every bottle is inoculated with 2-5 clumps, tightens bottle cap and moves to culture in culturing room, by 10-60 days, i.e.,
It can grow that root system is complete, leaf color is dark green, the complete tissue-culturing rapid propagation seedling of the paris polyphylla root, stem and leaf of robust growth, to complete paris polyphylla
The quick propagating and cultivating of indefinite offspring.
The acclimatization and transplants technology of the sandy tissue-culturing rapid propagation seedling of 7 Yunnan of embodiment weight
When the indoor paris polyphylla tissue-culturing rapid propagation seedling of culture grows to 10-25cm, tissue culture bottle is removed in culturing room, seedling retains
In tissue culture bottle, places it in ventilation, avoids direct sunlight, hardening in clean indoor or cool canopy, make seedling gradually suitable
The natural environment of transplanting, allow its slowly reform of nature existence growing environment, bottle cap, then hardening are opened after 10-15 days
After 10-15 days, plantation in the cool canopy rich in organic matter seedbed, watering moisturizing will be moved into sandy paris polyphylla tissue-culturing rapid propagation seedling
With related heat and moisture preserving and pest management, by the cultivation management of 3-5, the sandy tissue-culturing rapid propagation seedling of paris polyphylla blossoms and bears fruit,
Cycle carries out the sandy tissue-culturing rapid propagation of paris polyphylla after collecting seed or taking root-like stock.
Claims (9)
1. a kind of tissue culture and rapid propagation method of paris polyphylla, it is characterised in that the tissue culture and rapid propagation method of the paris polyphylla includes paris polyphylla
Callus tissue culture, paris polyphylla crowd shoots are cultivated, paris polyphylla tissue-culturing rapid propagation seedling is cultivated and transplant step, are specifically included：
A, paris polyphylla callus tissue culture：With sterile inoculating tool after explant is disinfected paris polyphylla seed or
Root-like stock is inoculated into chiltern callus tissue culture base, is cultivated under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination
Obtain within 10 ~ 60 days lurid paris polyphylla callus；
B, paris polyphylla crowd shoots are cultivated：Paris polyphylla callus or cell mass are inoculated into chiltern clump with sterile inoculating tool
It sprouts in seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla
Crowd shoots；
C, paris polyphylla tissue-culturing rapid propagation seedling is cultivated：Paris polyphylla crowd shoots are inoculated into chiltern tissue-culturing rapid propagation with sterile inoculating tool
In seedling culture medium, is cultivated 10 ~ 60 days under the conditions of 18 ~ 22 DEG C of temperature, 1000 ~ 3000Lux of intensity of illumination and obtain paris polyphylla tissue culture
Fast propagating seedling；
D, it transplants：When paris polyphylla tissue-culturing rapid propagation seedling is grown to 10 ~ 25cm, hardening is taken out after 10 ~ 30 days, by paris polyphylla tissue-culturing rapid propagation
Seedling moves into plantation in the cool canopy rich in organic matter seedbed, moisturizing of watering, and carries out conventional heat and moisture preserving ventilation and pest management；
Wherein, the chiltern callus tissue culture base, chiltern crowd shoots culture medium and chiltern tissue-culturing rapid propagation seedling culture medium group
Cheng Jun includes river sand, plant growth regulator, nutrient solution, ascorbic acid, polyvinylpyrrolidone and fungicide, in terms of 1L, MS
50 ~ 500 ml/L of nutrient solution, 0 ~ 15 g/L of sucrose, 0.1 ~ 10.0 ml/L of plant growth regulator, ascorbic acid 0.1 ~
5.0 g/L, 0.1 ~ 5.0 g/L of polyvinylpyrrolidone, fungicide 1 ~ 15 g/L, 0.4 ~ 0.6g/L of activated carbon, remaining is river
Sand.
2. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the plant growth regulator
For 6-BA, NAA, IBA, IAA, Zt and Kt.
3. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the plant growth regulator
Middle 6-BA is 0.0 ~ 10.0mg/L, and NAA is that 0.0 ~ 10.0mg/L, IBA are 0.01 ~ 10.0mg/L, and IAA is 0.01 ~ 10.0mg/L,
Zt is 1 ~ 5mg/L, and Kt is 1 ~ 5mg/L.
4. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the fungicide be Bravo,
One kind or several in carbendazim, thiophanate methyl, fenaminosulf, pentachloronitrobenzene, triadimefon, hymexazol, benomyl and probenazole
Kind.
5. the tissue culture and rapid propagation method of paris polyphylla according to claim 1 or 4, it is characterised in that the fungicide is hundred bacterium
Cleer and peaceful carbendazim.
6. the tissue culture and rapid propagation method of paris polyphylla according to claim 5, it is characterised in that Bravo in the fungicide
For 0.5 ~ 15g/L, carbendazim is 0.5 ~ 15g/L.
7. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the chiltern callus training
Base is supported in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 0.05 ~ 0.15ml/L of 1.0 ~ 3.0ml/L, NAA, polyethylene
0.4 ~ 0.6g/L of 0.5 ~ 1.5g/L of pyrrolones, 0.5 ~ 1.5g/L of carbendazim, 0.25 ~ 0.75g/L of activated carbon and ascorbic acid.
8. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the chiltern crowd shoots training
Base is supported in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 1.0 ~ 3.0ml/L, NAA 0.2 ~ 0. 5ml/L, KT 0.2 ~
1.0ml/L, 0.5 ~ 1.5g/L of polyvinyl pyrrolidone, 0.5 ~ 1.5g/L of carbendazim, 0.3 ~ 0.7g/L of activated carbon and ascorbic acid 0.4
~0.6g/L。
9. the tissue culture and rapid propagation method of paris polyphylla according to claim 1, it is characterised in that the chiltern tissue-culturing rapid propagation seedling
Culture medium is in terms of 1L, including MS nutrient solutions 50 ~ 500 ml/L, 6-BA 0.1 ~ 0.3ml/L, NAA 1.0 ~ 5.0ml/L, IBA 0.2
~ 2.0ml/L, 0.5 ~ 1.5g/L of polyvinyl pyrrolidone, 0.5 ~ 1.5g/L of carbendazim, 0.3 ~ 0.7g/L of activated carbon and ascorbic acid
0.4~0.6g/L。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111448963A (en) * | 2020-05-14 | 2020-07-28 | 盐津中港中药材专业合作社 | Quick-growing method for cutting root and keeping plant of Paris polyphylla |
| CN116267610A (en) * | 2023-03-14 | 2023-06-23 | 广西医科大学 | Aseptic treatment and in-vitro preservation method for Paris polyphylla explant |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548682A (en) * | 2013-10-31 | 2014-02-05 | 成都大学 | Rapid propagation method of Paris polyphylla plant |
-
2018
- 2018-05-30 CN CN201810538915.6A patent/CN108719067A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548682A (en) * | 2013-10-31 | 2014-02-05 | 成都大学 | Rapid propagation method of Paris polyphylla plant |
Non-Patent Citations (1)
| Title |
|---|
| 胡继金等: "对苎麻培养基改进的试验", 《湖南农学院学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111448963A (en) * | 2020-05-14 | 2020-07-28 | 盐津中港中药材专业合作社 | Quick-growing method for cutting root and keeping plant of Paris polyphylla |
| CN116267610A (en) * | 2023-03-14 | 2023-06-23 | 广西医科大学 | Aseptic treatment and in-vitro preservation method for Paris polyphylla explant |
| CN116267610B (en) * | 2023-03-14 | 2024-03-15 | 广西医科大学 | Aseptic treatment and in-vitro preservation method for Paris polyphylla explant |
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