CN107667859A - A kind of rhizome of cyrtomium tissue culture and rapid propagation method - Google Patents
A kind of rhizome of cyrtomium tissue culture and rapid propagation method Download PDFInfo
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- CN107667859A CN107667859A CN201710971681.XA CN201710971681A CN107667859A CN 107667859 A CN107667859 A CN 107667859A CN 201710971681 A CN201710971681 A CN 201710971681A CN 107667859 A CN107667859 A CN 107667859A
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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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- 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)
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Abstract
The invention discloses a kind of rhizome of cyrtomium tissue culture and rapid propagation method, Cyrtomium perennial herb are high 30 ~ 80 centimetres.Root-like stock is upright, together with when the dense sepia of handle base portion, the big phosphorus piece of ovum shape lanceolar.Winglike compound leaf fasciation, blade fall lanceolar, herbaceous stem.Sporangiorus is distributed on accessory pinna more than middle part, is born in following in the middle part of scun.Indusium justifies kidney shape.Spring, autumn excavation, prune fibrous root and petiole, dry or using fresh herb.All parts of the country are grown on, are born in the dark and damp place of sylvan life.Rhizome of cyrtomium bitter, it is cool in nature, it is slightly poisonous.With clearing heat and detoxicating, hemostasis, disinsection efficiency.The present invention by callus induction, propagation, break up, take root, the process such as acclimatization and transplantses successfully obtains the in vitro plant again of rhizome of cyrtomium, establish rhizome of cyrtomium tissue culture rapid propagation technique system, to save the sowing quantity in cultivation production, improve the yield of its medicinal material and further carry out Study on Genetic Transformation for rhizome of cyrtomium and lay the foundation.Technical method is simple, and operation is easy, quick.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, is related to a kind of rhizome of cyrtomium tissue culture
Quick-breeding method.
Background technology
Rhizome of cyrtomium also known as：Cyrtomium fortunei, fortune holly fern rhizome, Guan Zhong.Belong to perennial herb, it is high 30 ~ 80 centimetres.Root-like stock is upright, together with when
The dense sepia of handle base portion, the big phosphorus piece of ovum shape lanceolar.Winglike compound leaf fasciation, blade fall lanceolar, herbaceous stem, 10 ~ 20 pairs of leaflet,
Shallow incise is arranged at nearly full edge or top；Lateral vein pinniform bifurcated.Sporangiorus is distributed on accessory pinna more than middle part, is born in the middle part of scun
Below.Indusium justifies kidney shape.Spring, autumn excavation, prune fibrous root and petiole, dry or using fresh herb.All parts of the country are grown on to produce.It is born in
The dark and damp place of sylvan life.Rhizome of cyrtomium bitter, it is cool in nature, it is slightly poisonous.With clearing heat and detoxicating, hemostasis, disinsection efficiency.Dryopteris crassirhizoma Nakai resource can not
The needs of scale and standardized production are adapted to, turn into the maximum bottleneck for hindering rhizome of cyrtomium industry development.Both at home and abroad plant resources,
Chemical composition analysis, Pharmacognosy Studies, pharmacological action and clinical practice, callus tissue culture and break up again etc. has done substantial amounts of
Work, these researchs have established certain basis for the clinical practice using rhizome of cyrtomium as base source preparation.But still deposited in production
In many theory and practice urgent problems.In order to seek the development of industry, rhizome of cyrtomium industry is led to move towards high-tech, high rule
Lattice, high level science and technology road, rhizome of cyrtomium tissue cultures have become the focus researched and developed at present.Therefore, it is highly desirable
Rhizome of cyrtomium vitro Regeneration System is established, technical support is provided for the industrialized development of rhizome of cyrtomium.
The content of the invention
It is an object of the invention to provide one kind is gone out using rhizome of cyrtomium seed as explant, by callus induction, propagation, divide
Change, take root, the process such as acclimatization and transplantses have successfully been obtained the in vitro plant again of rhizome of cyrtomium, establish rhizome of cyrtomium tissue culture rapid propagation technique body
System, technical method is simple, and operation is easy, and seedling is pure sturdy, and growth is fast, and yield is high.
A kind of rhizome of cyrtomium tissue culture and rapid propagation method of the present invention, it is characterised in that comprise the following steps：
（1）The acquisition of aseptic seedling：Full rhizome of cyrtomium seed is chosen, kind of a skin is peelled off with surgical scissors in superclean bench, with 75%
Alcohol disinfecting 60s, then 16min is sterilized with 0.3% mercuric chloride solution, finally with rinsed with sterile water 6 times, surface is blotted with aseptic filter paper
It is inoculated into after moisture in seed germination medium, daily illumination 26 hours, intensity of illumination 2200lx, cultivation temperature are after inoculation
28 DEG C, relative air humidity counts germination rate after being cultivated 21 days under conditions of being 75%, and described germination medium is：1/2MS+
0.1mg/LNAA+32g/L sucrose+6.2g/L agar, pH 5.8；
（2）Callus induces：When the cotyledon of aseptic seedling just deploys, the stem apex of aseptic seedling is taken to be cut into as explant with scalpel
0.9cm or so length, it is inoculated in the culture medium for callus induction and cultivates, daily illumination 15 hours, intensity of illumination after inoculation
For 2100lx, cultivation temperature is 28 DEG C, and relative air humidity counts inductivity after being cultivated 30 days under conditions of being 75%, described
Calli induction media is：MS+3.1mg/L6-BA+0.6mg/LNAA+1.1mg/L KT+31g/L sucrose+6.1g/L agar, pH
For 5.8；
（3）Multiplying culture：By step（2）Obtained callus is induced to cut into 0.6cm3The fritter of size is simultaneously transferred to propagation training
Foster base carries out expanding numerous, daily illumination 15 hours, intensity of illumination 3100lx after inoculation, and cultivation temperature is 28 DEG C, and air is relatively wet
Spend to count proliferative conditions after cultivating 31 days under conditions of 75%, described proliferated culture medium is：MS+2.2mg/L6-BA+
0.6mg/L NAA+31g/L sucrose+6.1g/L agar, pH 5.8；
（4）Differentiation culture：By step（3）Breed obtained callus and cut into 0.9cm3The fritter of size is simultaneously transferred to differentiation training
The generation of base evoking adventive bud, daily illumination 15 hours, intensity of illumination 3100lx after inoculation are supported, cultivation temperature is 28 DEG C, empty
Gas relative humidity counts differentiation situation after being cultivated 31 days under conditions of being 75%, and described differential medium is：MS+1.1mg/LKT
+ 2.1mg/LNAA+31g/L sucrose+6.1g/L agar, pH 5.8；
（5）Culture of rootage：By step（4）Differentiation obtains adventitious bud and cuts and be seeded in root media from base portion to be induced
Take root, daily illumination 15 hours, intensity of illumination 3100lx after inoculation, cultivation temperature is 24 DEG C, and relative air humidity is 75%
Under the conditions of cultivate 33 days after statistics take root situation, described root media is：1/2MS+1.5mg/LIBA+1.0mg/L NAA+
1.5% activated carbon+35g/L sucrose+6.1g/L agar, pH 5.8；
（6）Acclimatization and transplantses：After taking root one month, its bottleneck is opened, and pours into sterilized water, to ensure its moisture supply, after 11 days
Take out rooted seedling and clean institute's band culture medium on root, be transplanted to by peat soil:Perlite:Vermiculite=3:1:In the matrix of 1 composition, use
The film covering of air-vent is left, shady and cool ventilative interior is placed on, throws off film within 16 days, then outside scenery, is poured every other day
Secondary water, transplanting count survival rate after 31 days.
It is an advantage of the invention that：Using rhizome of cyrtomium seed as explant, by callus induction, propagation, break up, take root, refine
The processes such as transplantation of seedlings have successfully been obtained the in vitro plant again of rhizome of cyrtomium, establish rhizome of cyrtomium tissue culture rapid propagation technique system, to save
Sowing quantity in cultivation production, improve the yield of its medicinal material and further carry out Study on Genetic Transformation for rhizome of cyrtomium and lay the foundation.Skill
Art method is simple, and operation is easy.Growth is fast, and yield is high, has good economic benefit and social benefit.
Embodiment
Following examples are the further explanations to the present invention, are not limitations of the present invention.
Embodiment 1
（1）The acquisition of aseptic seedling：Full rhizome of cyrtomium seed is chosen, kind of a skin is peelled off with surgical scissors in superclean bench, is used
75% alcohol disinfecting 31s, then 0.1% mercuric chloride solution sterilization 9min, finally with rinsed with sterile water 5 times, surface is blotted with aseptic filter paper
It is inoculated into after moisture in seed germination medium.Daily illumination 26 hours is placed in after inoculation, intensity of illumination 1200lx, is placed in training
It is 26 DEG C to support temperature, and germination rate reaches 110% after relative air humidity is cultivated 21 days under conditions of being 75%.Described sprouting culture
Base is：1/2MS+0.1mg/L NAA+19g/L sucrose+3.6g/L agar, pH 5.5.
（2）Callus induces：When the cotyledon of aseptic seedling just deploys, the stem apex of aseptic seedling is taken to use scalpel as explant
1.0cm or so length is cut into, is inoculated in the culture medium for callus induction and cultivates.Daily illumination 16 hours is placed in after inoculation,
Intensity of illumination is 1400lx, is placed in cultivation temperature as 25 DEG C, inductivity after relative air humidity is cultivated 31 days under conditions of being 75%
Reach 93%.Described calli induction media is：MS+1.9mg/L 6-BA+0.5mg/LNAA+0.8mg/L KT+26g/L sugarcanes
Sugar+4.6g/L agar, pH 5.6.
（3）Multiplying culture：By step（2）Obtained callus is induced to cut into 1.0cm3The fritter of size is simultaneously transferred to increasing
Grow culture medium expand it is numerous.Daily illumination is placed in after inoculation 11 hours, intensity of illumination 1900lx, is placed in cultivation temperature as 24
DEG C, proliferation times reach 4.6 times after relative air humidity is cultivated 31 days under conditions of being 75%.Described proliferated culture medium is：MS
+ 1.8mg/L 6-BA+0.5mg/L NAA+22g/L sucrose+4.0g/L agar, pH 5.5.
（4）Differentiation culture：By step（3）Breed obtained callus and cut into 0.4cm3The fritter of size is simultaneously transferred to point
Change the generation of culture medium evoking adventive bud.Daily illumination is placed in after inoculation 12 hours, intensity of illumination 2100lx, is placed in culture temperature
Spend for 25 DEG C, differentiation rate reaches 93% after relative air humidity is cultivated 31 days under conditions of being 75%, and average adventitious bud number is 8.
Described differential medium is：MS+1.0mg/LKT+1.9mg/L NAA+35g/L sucrose+5.0g/L agar, pH 5.6.
（5）Culture of rootage：By step（4）Differentiation obtains adventitious bud and cuts and be seeded in root media from base portion to carry out
Root induction.Daily illumination is placed in after inoculation 16 hours, intensity of illumination 2400lx, it is 25 DEG C to be placed in cultivation temperature, air phase
Rooting rate 94% after being cultivated 31 days under conditions of being 75% to humidity.Described root media is：1/2MS+1.0mg/L IBA+
0.6mg/L NAA+1.0% activated carbon+16g/L sucrose+3.9g/L agar, pH 5.6.
（6）Acclimatization and transplantses：After taking root one month, its bottleneck is opened, and pours into sterilized water, to ensure its moisture supply, 9 days
Rooted seedling is taken out afterwards and cleans institute's band culture medium on root, is transplanted to by peat soil:Perlite:Vermiculite=3:1:In the matrix of 1 composition.
Covered with the film for leaving air-vent, be placed on shady and cool ventilative interior, film thrown off in 15 days, be subsequently placed in outside scenery,
Pour a water every other day, survival rate is to more than 95% after transplanting 35 days.
Embodiment 2
（1）The acquisition of aseptic seedling：Full rhizome of cyrtomium seed is chosen, kind of a skin is peelled off with surgical scissors in superclean bench, with 75%
Alcohol disinfecting 50s, then 0.1% mercuric chloride solution sterilization 10min, are finally floated 67 times with sterilized water, surface moisture are blotted with aseptic filter paper
After be inoculated into seed germination medium.Daily illumination is placed in after inoculation 25 hours, intensity of illumination 1800lx, is placed in culture temperature
Spend for 28 DEG C, germination rate reaches 110% after relative air humidity is cultivated 21 days under conditions of being 75%.Described germination medium
For：1/2MS+0.08mg/L NAA+18g/L sucrose+3.8g/L agar, pH 5.6.
（2）Callus induces：When the cotyledon of aseptic seedling just deploys, the stem apex of aseptic seedling is taken to use scalpel as explant
0.6cm or so length is cut into, is inoculated in the culture medium for callus induction and cultivates.Daily illumination 12 hours is placed in after inoculation,
Intensity of illumination is 1800lx, is placed in cultivation temperature as 26 DEG C, inductivity after relative air humidity is cultivated 31 days under conditions of being 75%
Reach 94%.Described calli induction media is：MS+2.0mg/L 6-BA+0.6mg/LNAA+1.0mg/L KT+28g/L sugarcanes
Sugar+5.6g/L agar, pH 5.6.
（3）Multiplying culture：By step（2）Obtained callus is induced to cut into 1.0cm3The fritter of size is simultaneously transferred to increasing
Grow culture medium expand it is numerous.Daily illumination is placed in after inoculation 12 hours, intensity of illumination 2800lx, is placed in cultivation temperature as 25
DEG C, proliferation times reach 5.30 times after relative air humidity is cultivated 31 days under conditions of being 75%.Described proliferated culture medium is：
MS+2.1mg/L 6-BA+0.6mg/L NAA+26g/L sucrose+4.6g/L agar, pH 5.6.
（4）Differentiation culture：By step（3）Breed obtained callus and cut into 1.0cm3The fritter of size is simultaneously transferred to point
Change the generation of culture medium evoking adventive bud.Daily illumination is placed in after inoculation 12 hours, intensity of illumination 2800lx, is placed in culture temperature
Spend for 25 DEG C, differentiation rate reaches 94% after relative air humidity is cultivated 32 days under conditions of being 75%, and average adventitious bud number is 6.
Described differential medium is：MS+1.1mg/LKT+2.0mg/L NAA+30g/L sucrose+5.6g/L agar, pH 5.6.
（5）Culture of rootage：By step（4）Differentiation obtains adventitious bud and cuts and be seeded in root media from base portion to carry out
Root induction.Daily illumination is placed in after inoculation 15 hours, intensity of illumination 3300lx, it is 25 DEG C to be placed in cultivation temperature, air phase
Rooting rate 96% after being cultivated 30 days under conditions of being 75% to humidity.Described root media is：1/2MS+1.0mg/L IBA+
0.5mg/L NAA+1.0% activated carbon+21g/L sucrose+4.6g/L agar, pH 5.6.
（6）Acclimatization and transplantses：After taking root 28 days, its bottleneck is opened, and pours into sterilized water, to ensure its moisture supply, after 8 days
Take out rooted seedling and clean institute's band culture medium on root, be transplanted to matrix by peat soil:Perlite:Vermiculite=3:1:The matrix of 1 composition
In.Covered with the film for leaving air-vent, be placed on shady and cool ventilative interior, film thrown off in 16 days, be subsequently placed in outdoor training
Support, pour a water every other day, survival rate is to 96% after transplanting 30 days.
Claims (1)
1. a kind of rhizome of cyrtomium tissue culture and rapid propagation method, it is characterised in that comprise the following steps：
（1）The acquisition of aseptic seedling：Full rhizome of cyrtomium seed is chosen, kind of a skin is peelled off with surgical scissors in superclean bench, with 75%
Alcohol disinfecting 60s, then 16min is sterilized with 0.3% mercuric chloride solution, finally with rinsed with sterile water 6 times, surface is blotted with aseptic filter paper
It is inoculated into after moisture in seed germination medium, daily illumination 26 hours, intensity of illumination 2200lx, cultivation temperature are after inoculation
28 DEG C, relative air humidity counts germination rate after being cultivated 21 days under conditions of being 75%, and described germination medium is：1/2MS+
0.1mg/LNAA+32g/L sucrose+6.2g/L agar, pH 5.8；
（2）Callus induces：When the cotyledon of aseptic seedling just deploys, the stem apex of aseptic seedling is taken to be cut into as explant with scalpel
0.9cm or so length, it is inoculated in the culture medium for callus induction and cultivates, daily illumination 15 hours, intensity of illumination after inoculation
For 2100lx, cultivation temperature is 28 DEG C, and relative air humidity counts inductivity after being cultivated 30 days under conditions of being 75%, described
Calli induction media is：MS+3.1mg/L6-BA+0.6mg/LNAA+1.1mg/L KT+31g/L sucrose+6.1g/L agar, pH
For 5.8；
（3）Multiplying culture：By step（2）Obtained callus is induced to cut into 0.6cm3The fritter of size is simultaneously transferred to propagation training
Foster base carries out expanding numerous, daily illumination 15 hours, intensity of illumination 3100lx after inoculation, and cultivation temperature is 28 DEG C, and air is relatively wet
Spend to count proliferative conditions after cultivating 31 days under conditions of 75%, described proliferated culture medium is：MS+2.2mg/L6-BA+
0.6mg/L NAA+31g/L sucrose+6.1g/L agar, pH 5.8；
（4）Differentiation culture：By step（3）Breed obtained callus and cut into 0.9cm3The fritter of size is simultaneously transferred to differentiation training
The generation of base evoking adventive bud, daily illumination 15 hours, intensity of illumination 3100lx after inoculation are supported, cultivation temperature is 28 DEG C, empty
Gas relative humidity counts differentiation situation after being cultivated 31 days under conditions of being 75%, and described differential medium is：MS+1.1mg/LKT
+ 2.1mg/LNAA+31g/L sucrose+6.1g/L agar, pH 5.8；
（5）Culture of rootage：By step（4）Differentiation obtains adventitious bud and cuts and be seeded in root media from base portion to be induced
Take root, daily illumination 15 hours, intensity of illumination 3100lx after inoculation, cultivation temperature is 24 DEG C, and relative air humidity is 75%
Under the conditions of cultivate 33 days after statistics take root situation, described root media is：1/2MS+1.5mg/LIBA+1.0mg/L NAA+
1.5% activated carbon+35g/L sucrose+6.1g/L agar, pH 5.8；
（6）Acclimatization and transplantses：After taking root one month, its bottleneck is opened, and pours into sterilized water, to ensure its moisture supply, after 11 days
Take out rooted seedling and clean institute's band culture medium on root, be transplanted to by peat soil:Perlite:Vermiculite=3:1:In the matrix of 1 composition, use
The film covering of air-vent is left, shady and cool ventilative interior is placed on, throws off film within 16 days, then outside scenery, is poured every other day
Secondary water, transplanting count survival rate after 31 days.
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| CN108496960A (en) * | 2018-05-17 | 2018-09-07 | 吉林农业科技学院 | A kind of dryopteris crassirhizoma Nakai cryopreservation method |
| CN109156355A (en) * | 2018-10-10 | 2019-01-08 | 东北农业大学 | A kind of method that IBA induces two strain callus from stem segment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108496960A (en) * | 2018-05-17 | 2018-09-07 | 吉林农业科技学院 | A kind of dryopteris crassirhizoma Nakai cryopreservation method |
| CN108496960B (en) * | 2018-05-17 | 2021-03-30 | 吉林农业科技学院 | Ultralow-temperature preservation method for rhizoma dryopteris crassirhizomae |
| CN109156355A (en) * | 2018-10-10 | 2019-01-08 | 东北农业大学 | A kind of method that IBA induces two strain callus from stem segment |
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