CN106879474A - A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation - Google Patents
A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation Download PDFInfo
- Publication number
- CN106879474A CN106879474A CN201710263998.8A CN201710263998A CN106879474A CN 106879474 A CN106879474 A CN 106879474A CN 201710263998 A CN201710263998 A CN 201710263998A CN 106879474 A CN106879474 A CN 106879474A
- Authority
- CN
- China
- Prior art keywords
- protocorm
- culture
- vitro
- medium
- plantlet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
-
- 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
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
Abstract
The invention discloses a kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation, stem apex is stripped from the pseudobulb that cold orchid newly grows, it is placed on protocorm induction medium surface and is trained protocorm, protocorm is cut and squamous subculture repeatedly, so as to breed the protocorm of requirement, then cultivated using Plantlet in vitro culture medium, after protocorm enters the development growth stage, the light culture under the conditions of 0~5 DEG C；Protocorm in light culture is transferred on differential medium, seedling is produced by protocorm differentiation.The present invention utilizes cold orchid Shoot Tip Culture technology, induces protocorm, then obtains cold orchid seedling by protocorm differentiation.Cord blood protocorm, with reference to appropriate paclobutrazol is added, can greatly prolong the holding time, reduce Subculture times, effectively maintain the hereditary capacity of germ plasm resource.After preserving certain hour, culture under normal temperature and illumination condition is placed in, the rapid restoration ecosystem of energy differentiates seedling.
Description
Technical field
The present invention relates to a kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation.Belong to plant tissue
Culture technique field.
Background technology
Cold orchid（Scientific name：Cymbidium kanran Makino）It is orchid family Cymbidium ground plant, the narrow ovoid of pseudobulb,
Contain within phyllopodium.The slender fitness of plant type, leaf appearance gracefulness is pretty, and leaf band shape, thin keratin, dirty-green, forward edge often has carefully
Tooth.Spend often for pistac and with faint yellow lip, there is enhanced aroma.Its propagation method is generally division propagation, 3~4 years
Can just carry out once, reproduction speed is slower.Cold orchid seed is very tiny, in powdery, there is no an endosperm, the simple embryo of only one of which,
Outside enclose it is loose, transparent, be difficult permeable kind skin, embryo is contained within little nutriment, and the overwhelming majority is lipid material, from
Cold orchid seed is difficult to sprout under the conditions of so.Therefore, the inapplicable cold orchid Germ-plasma resources protection of traditional Seed storage technology.
Cold orchid majority cultivar is screened by wild species domestication or natural variation.For from resource, due to
A large amount of excavations to wild state orchid resource, natural resources have suffered considerable damage, and collectable wild resource is fewer and feweri, then
Extremely slow plus self-reproduction speed, the wild cold orchid resource of China is increasingly in short supply, and some rare kinds are endangered, because
This, sets up science, cold orchid Germ-plasma resources protection technology effectively, practical and compels in agility.By planned cultivation, breeding, hair
Open up, preserve peculiar species in imminent danger, be just avoided that the species extinction.
Tissue cultures are widely used as plant germplasm resource Plantlet in vitro technology, in germ plasm resource exchange process
In, swap that not only cost of transportation is low using test tube seedling, can also avoid the propagation of the pest and disease damage in exchange process, it is also possible to solve
Certainly crop field preserve floor space it is big, it is costly the problems such as.Frequently in vitro squamous subculture Techniques of preserving, in squamous subculture each time
During be likely to cause cross pollution, multiple squamous subculture can also trigger hereditary variation, meanwhile, with squamous subculture number
The increase of amount will expend substantial amounts of manpower and materials.
The content of the invention
The purpose of the present invention is to overcome above-mentioned the deficiencies in the prior art, there is provided a kind of cold orchid germ plasm resource Plantlet in vitro side
Method.
The method of restoration ecosystem after being preserved present invention also offers the in-vitro conservation method.
To achieve the above object, the present invention uses following technical proposals：
A kind of cold orchid germ plasm resource in-vitro conservation method, stem apex is stripped from the pseudobulb that cold orchid newly grows, and is placed on VW trainings
Support Kiev and protocorm is trained with the protocorm induction medium surface of 0.5~1mg/L 6-benzyladenines, protocorm is entered
Row cuts and squamous subculture repeatedly, and it is fast that the subculture medium for being used is aided with 0.2~0.4mg/L 6- benzyl glands for VW culture mediums
Purine, so as to breed the protocorm of requirement, is then aided with 2.5mg/L paclobutrazols using VW culture mediums（PP333）It is in vitro
Storaged media is cultivated, after protocorm enters the development growth stage, the light culture under the conditions of 0~5 DEG C.
Preferably, the stem apex to strip method as follows：Selection length is the pseudobulb of 5~7cm, and table is cleaned with running water
Dusty complexion soil, peels off outermost 1~2 blade, is cleaned 3~4 seconds with the alcohol of volumetric concentration 75%, is put into mass concentration 0.1%
Mercuric chloride solution in sterilize 20 minutes, aseptic water washing 3~4 times dries or blots surface moisture with filter paper, shells under the microscope
Take the size about stem apex of 0.5mm.
Preferably, the surface of protocorm induction medium in stem apex being placed in into test tube or blake bottle, it is light to press stem apex to contact
Surface to protocorm induction medium is advisable, it is to avoid embedment, and then causes death by suffocation, seals and carry out mark in time.
Preferably, the cultural method of protocorm is：By stem apex in 25 DEG C, illumination 2000lux, the bar of daily illumination 10 hours
After being cultivated 15 days under part, appearance is substantially expanded, and occurs several white projections after 30 days, after 45 days, the lug volume increase
Form protocorm.
Preferably, the specific method of cutting and squamous subculture is repeatedly：The protocorm that 4~6mg will be grown to cuts into 4
Block, is subsequently placed in subculture medium and continues amplification cultivation, sets up clone, repeatedly cutting and squamous subculture, so that short
Time breeds the protocorm of requirement.
Preferably, the development growth stage is selection development protocorm healthy and strong, of uniform size, is placed in Plantlet in vitro training
Support in base, at 25 DEG C, illumination 2000lux, daily illumination is cultivated 7 days under conditions of 10 hours.
Preferably, the light culture stage should routine observation growing state, reject has the blake bottle of pollution at any time.
Preferably, the Plantlet in vitro culture medium that the light culture stage is used, thickness is about 4cm, also, in vitro for placing
The test tube or blake bottle of Storaged media should be closed good, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
It is further preferred that test tube or blake bottle are sealed with sealed membrane, also, in outer layer 3 layers of plastics of winding of sealed membrane
Antistaling film.
The method of restoration ecosystem, differentiation culture is transferred to by the protocorm in light culture after above-mentioned in-vitro conservation method preservation
On base, seedling is produced by protocorm differentiation；The differential medium is the bananas juice that KC culture mediums are aided with mass concentration 10%, training
25 DEG C of temperature, intensity of illumination 2000lux are supported, daily light application time is 10 hours.
Preferably, after protocorm is inoculated with 30 days on differential medium, occur being sent out by phyllopodium in protocorm tip portion
The spire of incubation, subsequent spire is mushroomed out, and during the 2nd~3 leaf to appear, protocorm extends downwards, and has the 1st article of root to give birth to
Go out；After 50 days, when seedling has 2~3 roots, 5~6 leaves grow into 10cm or so, complete plant are developed into, by test tube
Transplantation of seedlings grows into normal plant under field conditions (factors) in seedling medium.
Beneficial effects of the present invention：
The present invention is, using cold orchid Shoot Tip Culture technology, to induce protocorm, then obtains cold orchid seedling by protocorm differentiation.
It is the intermediate for breaking up seedling as the protocorm in Subculture, by Cord blood protocorm, in Plantlet in vitro training
Appropriate paclobutrazol is added in foster base, the holding time can be greatly prolonged, reduce Subculture times, effectively maintain germ plasm resource
Hereditary capacity.After preserving certain hour, cultivated under culture is placed in into normal temperature and illumination condition, can rapidly recover raw
It is long, differentiate seedling.
The seed for generally using in the prior art is sprouted and quick breeding technology, although can obtain a large amount of plant, but breeding
Progeny variation coefficient is larger, and genetic stability and kind uniformity have problem, do not meet the preservation condition of germ plasm resource.This hair
Used as explant, stem apex is that most fast separate living tissue is grown in plant to bright use Shoot Tip Culture, gene stabilization, power of regeneration
By force, virus and other pathogeny tissues are substantially free of（Such as bacterium, fungi）, by can be obtained after tissue cultures inheritance stability,
Kind it is consistent and healthy and strong it is virus-free, without germ plant, reached the purpose degermed and quickly breed, possess germplasm money
The necessary condition of source Plantlet in vitro.
General germ plasm resource Plantlet is mostly preserved using test tube seedling, but test tube seedling is compared thermotonus
Sensitivity, storage temperature is unfavorable for that lower temperature is preserved substantially all more than 12 DEG C.In addition to cold orchid plant, other plant induction,
The intermediate for breaking up test tube seedling is mostly callus, by callus induction into embryoid or adventitious bud, final differentiation production
Go out test tube seedling, and callus induction rate is general than relatively low, it is relatively low in the ratio for continuing to differentiate test tube seedling after renewal cultivation,
Relative to the differentiation rate of cold orchid protocorm more than 90%, preserving callus needs cost higher.
The present invention carries out conservation in vitro using protocorm, and protocorm is substantially reduced to the sensitiveness of temperature, Ke Yiyou
Effect reduces storage temperature, and temperature is lower, and the speed of growth is slower, and the holding time is more long, and protocorm is in 0~5 DEG C of model of temperature
Enclosing interior can slowly grow；Preserve facility also relatively easy, light culture Techniques of preserving is used in low temperature refrigerator, eliminate illumination
Facility, temperature control is also relatively easy, reduces luminous energy loss, reduces power consumption, effectively reduces preservation cost；Protected in vitro
Deposit and be aided with paclobutrazol in culture medium, paclobutrazol is plant growth retardent, by suppressing the synthesis of gibberellin, reduce cell point
Split and extend, reduce the speed of growth, reduce the consumption to cultivating composition, extend the holding time of isolated culture, during preservation
Between can reach more than 40 months.
Specific embodiment
With reference to embodiment, the present invention will be further elaborated, it should explanation, the description below merely to
The present invention is explained, its content is not defined.
Embodiment 1：
The method of restoration ecosystem, comprises the following steps that after a kind of cold orchid germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that cold orchid newly grows, being placed on VW culture mediums, to be aided with 0.5mg/L 6- benzyl glands fast
The protocorm induction medium surface of purine is trained protocorm.
Wherein, stem apex to strip method as follows：Selection length is the pseudobulb of 5cm, and surface dirt is cleaned with running water, is shelled
Outermost 1 blade is removed, is cleaned 3 seconds with the alcohol of volumetric concentration 75%, be put into sterilizing in the mercuric chloride solution of mass concentration 0.1%
20 minutes, aseptic water washing 3 times was dried or blots surface moisture with filter paper, and the size about stem of 0.5mm is stripped under the microscope
Point.
The surface of protocorm induction medium during stem apex is placed in into test tube or blake bottle, it is light to press stem apex to touch protocorm
The surface of inducing culture is advisable, it is to avoid embedment, and then causes death by suffocation, seals and carry out mark in time.
The cultural method of protocorm is：By stem apex at 25 DEG C, illumination 2000lux, daily illumination is trained under conditions of 10 hours
After supporting 15 days, appearance is substantially expanded, and occurs several white projections after 30 days, and after 45 days, the lug volume increases to form original
Bulb.Table 1 shows the influence that different 6-benzyladenine concentration are formed to protocorm.
The influence that the 6-benzyladenine content of table 1. is formed to protocorm
（2）Protocorm is cut and squamous subculture repeatedly, the subculture medium for being used is aided with 0.2mg/L for VW culture mediums
6-benzyladenine, so as to breed the protocorm of requirement.
Specific method is：The protocorm that 4mg will be grown to cuts into 4 pieces, and continuation is expanded in being subsequently placed in subculture medium
Culture, sets up clone, repeatedly cutting and squamous subculture, so that the short time breeds the protocorm of requirement.
（3）The Plantlet in vitro culture medium for being aided with 2.5mg/L paclobutrazols using VW culture mediums is cultivated, and treats that protocorm enters
After the development growth stage, the light culture under the conditions of 0 DEG C.
Wherein, the development growth stage is selection development protocorm healthy and strong, of uniform size, is placed in Plantlet in vitro culture medium
In, at 25 DEG C, illumination 2000lux, daily illumination is cultivated 7 days under conditions of 10 hours.
The light culture stage should routine observation growing state, reject has the blake bottle of pollution at any time.
The Plantlet in vitro culture medium that the light culture stage is used, thickness is about 4cm, also, for placing Plantlet in vitro training
The test tube or blake bottle for supporting base should be closed good（Test tube or blake bottle are sealed with sealed membrane, also, outer layer in sealed membrane is twined
Around 3 layers of plastics antistaling film）, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
（4）Restoration ecosystem after preservation：Protocorm in light culture is transferred on differential medium, is produced by protocorm differentiation
Raw seedling；The differential medium is the bananas juice that KC culture mediums are aided with mass concentration 10%, 25 DEG C of cultivation temperature, intensity of illumination
2000lux, daily light application time is 10 hours.
Wherein, after protocorm is inoculated with 30 days on differential medium, occur being developed by phyllopodium in protocorm tip portion
Into spire, subsequent spire mushrooms out, and during the 2nd leaf to appear, protocorm extends downwards, and has the 1st article of root to bear；50
After it, when seedling has 2 roots, 5 leaves grow into 10cm or so, complete plant are developed into, by test tube transplantation of seedlings in nursery
In matrix, normal plant is grown under field conditions (factors).
Each culture medium that embodiment 1 is related to is as follows：Protocorm induction medium（VW culture mediums are aided with 0.5mg/L 6- benzyls
Adenine）, subculture medium（VW culture mediums are aided with 0.2mg/L 6-benzyladenines）, Plantlet in vitro culture medium（VW culture mediums
It is aided with 2.5mg/L paclobutrazols）, differential medium（KC culture mediums are aided with the bananas juice of mass concentration 10%）.
Embodiment 2：
The method of restoration ecosystem, comprises the following steps that after a kind of cold orchid germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that cold orchid newly grows, VW culture mediums is placed on and is aided with 1mg/L 6-benzyladenines
Protocorm induction medium surface be trained protocorm.
Wherein, stem apex to strip method as follows：Selection length is the pseudobulb of 7cm, and surface dirt is cleaned with running water, is shelled
Outermost 2 blades are removed, is cleaned 4 seconds with the alcohol of volumetric concentration 75%, be put into sterilizing in the mercuric chloride solution of mass concentration 0.1%
20 minutes, aseptic water washing 4 times was dried or blots surface moisture with filter paper, and the size about stem of 0.5mm is stripped under the microscope
Point.
The surface of protocorm induction medium during stem apex is placed in into test tube or blake bottle, it is light to press stem apex to touch protocorm
The surface of inducing culture is advisable, it is to avoid embedment, and then causes death by suffocation, seals and carry out mark in time.
The cultural method of protocorm is：By stem apex at 25 DEG C, illumination 2000lux, daily illumination is trained under conditions of 10 hours
After supporting 15 days, appearance is substantially expanded, and occurs several white projections after 30 days, and after 45 days, the lug volume increases to form original
Bulb.
（2）Protocorm is cut and squamous subculture repeatedly, the subculture medium for being used is aided with for VW culture mediums
0.4mg/L 6-benzyladenines, so as to breed the protocorm of requirement.
Specific method is：The protocorm that 6mg will be grown to cuts into 4 pieces, and continuation is expanded in being subsequently placed in subculture medium
Culture, sets up clone, repeatedly cutting and squamous subculture, so that the short time breeds the protocorm of requirement.
（3）The Plantlet in vitro culture medium for being aided with 2.5mg/L paclobutrazols using VW culture mediums is cultivated, and treats that protocorm enters
After the development growth stage, the light culture under the conditions of 5 DEG C.
Wherein, the development growth stage is selection development protocorm healthy and strong, of uniform size, is placed in Plantlet in vitro culture medium
In, at 25 DEG C, illumination 2000lux, daily illumination is cultivated 7 days under conditions of 10 hours.
The light culture stage should routine observation growing state, reject has the blake bottle of pollution at any time.
The Plantlet in vitro culture medium that the light culture stage is used, thickness is about 4cm, also, for placing Plantlet in vitro training
The test tube or blake bottle for supporting base should be closed good（Test tube or blake bottle are sealed with sealed membrane, also, outer layer in sealed membrane is twined
Around 3 layers of plastics antistaling film）, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
（4）Restoration ecosystem after preservation：Protocorm in light culture is transferred on differential medium, is produced by protocorm differentiation
Raw seedling；The differential medium is the bananas juice that KC culture mediums are aided with mass concentration 10%, 25 DEG C of cultivation temperature, intensity of illumination
2000lux, daily light application time is 10 hours.
Wherein, after protocorm is inoculated with 30 days on differential medium, occur being developed by phyllopodium in protocorm tip portion
Into spire, subsequent spire mushrooms out, and during the 3rd leaf to appear, protocorm extends downwards, and has the 1st article of root to bear；50
After it, when seedling has 3 roots, 6 leaves grow into 10cm or so, complete plant are developed into, by test tube transplantation of seedlings in nursery
In matrix, normal plant is grown under field conditions (factors).
Each culture medium that embodiment 2 is related to is as follows：Protocorm induction medium（VW culture mediums are aided with 1mg/L 6- benzyl glands
Purine）, subculture medium（VW culture mediums are aided with 0.4mg/L 6-benzyladenines）, Plantlet in vitro culture medium（VW cultivates Kiev
With 2.5mg/L paclobutrazols）, differential medium（KC culture mediums are aided with the bananas juice of mass concentration 10%）Ju Ti Pei Fang be shown in Table 2.
Embodiment 3：
The method of restoration ecosystem, comprises the following steps that after a kind of cold orchid germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that cold orchid newly grows, being placed on VW culture mediums, to be aided with 0.8mg/L 6- benzyl glands fast
The protocorm induction medium surface of purine is trained protocorm.
Wherein, stem apex to strip method as follows：Selection length is the pseudobulb of 6cm, and surface dirt is cleaned with running water, is shelled
Outermost 1 blade is removed, is cleaned 4 seconds with the alcohol of volumetric concentration 75%, be put into sterilizing in the mercuric chloride solution of mass concentration 0.1%
20 minutes, aseptic water washing 3 times was dried or blots surface moisture with filter paper, and the size about stem of 0.5mm is stripped under the microscope
Point.
The surface of protocorm induction medium during stem apex is placed in into test tube or blake bottle, it is light to press stem apex to touch protocorm
The surface of inducing culture is advisable, it is to avoid embedment, and then causes death by suffocation, seals and carry out mark in time.
The cultural method of protocorm is：By stem apex at 25 DEG C, illumination 2000lux, daily illumination is trained under conditions of 10 hours
After supporting 15 days, appearance is substantially expanded, and occurs several white projections after 30 days, and after 45 days, the lug volume increases to form original
Bulb.
（2）Protocorm is cut and squamous subculture repeatedly, the subculture medium for being used is aided with for VW culture mediums
0.3mg/L 6-benzyladenines, so as to breed the protocorm of requirement.
Specific method is：The protocorm that 5mg will be grown to cuts into 4 pieces, and continuation is expanded in being subsequently placed in subculture medium
Culture, sets up clone, repeatedly cutting and squamous subculture, so that the short time breeds the protocorm of requirement.
（3）The Plantlet in vitro culture medium for being aided with 2.5mg/L paclobutrazols using VW culture mediums is cultivated, and treats that protocorm enters
After the development growth stage, the light culture under the conditions of 2 DEG C.
Wherein, the development growth stage is selection development protocorm healthy and strong, of uniform size, is placed in Plantlet in vitro culture medium
In, at 25 DEG C, illumination 2000lux, daily illumination is cultivated 7 days under conditions of 10 hours.
The light culture stage should routine observation growing state, reject has the blake bottle of pollution at any time.
The Plantlet in vitro culture medium that the light culture stage is used, thickness is about 4cm, also, for placing Plantlet in vitro training
The test tube or blake bottle for supporting base should be closed good（Test tube or blake bottle are sealed with sealed membrane, also, outer layer in sealed membrane is twined
Around 3 layers of plastics antistaling film）, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
（4）Restoration ecosystem after preservation：Protocorm in light culture is transferred on differential medium, is produced by protocorm differentiation
Raw seedling；The differential medium is the bananas juice that KC culture mediums are aided with mass concentration 10%, 25 DEG C of cultivation temperature, intensity of illumination
2000lux, daily light application time is 10 hours.
Wherein, after protocorm is inoculated with 30 days on differential medium, occur being developed by phyllopodium in protocorm tip portion
Into spire, subsequent spire mushrooms out, and during the 3rd leaf to appear, protocorm extends downwards, and has the 1st article of root to bear；50
After it, when seedling has 2 roots, 5 leaves grow into 10cm or so, complete plant are developed into, by test tube transplantation of seedlings in nursery
In matrix, normal plant is grown under field conditions (factors).
Each culture medium that embodiment 3 is related to is as follows：Protocorm induction medium（VW culture mediums are aided with 0.8mg/L 6- benzyls
Adenine）, subculture medium（VW culture mediums are aided with 0.3mg/L 6-benzyladenines）, Plantlet in vitro culture medium（VW culture mediums
It is aided with 2.5mg/L paclobutrazols）, differential medium（KC culture mediums are aided with the bananas juice of mass concentration 10%）.
The protocorm induction medium that embodiment 1~3 is related to（It is fast that VW culture mediums are aided with 0.5~1mg/L 6- benzyl glands
Purine）, subculture medium（VW culture mediums are aided with 0.2~0.4mg/L 6-benzyladenines）, Plantlet in vitro culture medium（VW culture mediums
It is aided with 2.5mg/L paclobutrazols）, differential medium（KC culture mediums are aided with the bananas juice of mass concentration 10%）Ju Ti Pei Fang be shown in Table
2。
The Ju Ti Pei Fang of the various culture mediums of table 2.
Although above-mentioned be described to specific embodiment of the invention, not limiting the scope of the invention,
On the basis of technical scheme, the various modifications that those skilled in the art make by need not paying creative work
Or deformation is still within protection scope of the present invention.
Claims (10)
1. a kind of cold orchid germ plasm resource in-vitro conservation method, it is characterised in that strip stem apex from the pseudobulb that cold orchid newly grows,
It is placed on VW culture mediums and is aided with the protocorm induction medium surface of 0.5~1mg/L 6-benzyladenines and is trained protocorm
Stem, protocorm is cut and squamous subculture repeatedly, and the subculture medium for being used is aided with 0.2~0.4mg/ for VW culture mediums
L 6-benzyladenines, so as to breed the protocorm of requirement, are then aided with 2.5mg/L paclobutrazols using VW culture mediums
Plantlet in vitro culture medium is cultivated, after protocorm enters the development growth stage, the light culture under the conditions of 0~5 DEG C.
2. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that the stripping of the stem apex
Take method as follows：Selection length is the pseudobulb of 5~7cm, and surface dirt is cleaned with running water, peels off outermost 1~2 leaf
Piece, is cleaned 3~4 seconds with the alcohol of volumetric concentration 75%, is put into the mercuric chloride solution of mass concentration 0.1% and is sterilized 20 minutes, aseptic
Water is rinsed 3~4 times, is dried or is blotted surface moisture with filter paper, and the size about stem apex of 0.5mm is stripped under the microscope.
3. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that stem apex is placed in examination
The surface of protocorm induction medium in pipe or blake bottle, light pressure stem apex is to touch the surface of protocorm induction medium
Preferably, it is to avoid imbed, and then cause death by suffocation, seal and carry out mark in time.
4. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that the culture of protocorm
Method is：By stem apex at 25 DEG C, illumination 2000lux after daily illumination is cultivated 15 days under conditions of 10 hours, occurs substantially swollen
Greatly, occur several white projections after 30 days, after 45 days, the lug volume increases to form protocorm.
5. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that repeatedly cutting and after
Foster specific method of being commissioned to train is：The protocorm that 4~6mg will be grown to cuts into 4 pieces, and continuation is expanded in being subsequently placed in subculture medium
Increase culture, set up clone, repeatedly cutting and squamous subculture, so that the short time breeds the protocorm of requirement.
6. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that the development growth
Stage is selection development protocorm healthy and strong, of uniform size, is placed in Plantlet in vitro culture medium, at 25 DEG C, illumination 2000lux,
Daily illumination is cultivated 7 days under conditions of 10 hours.
7. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that the light culture stage should
When routine observation growing state, the blake bottle for having pollution is rejected at any time.
8. a kind of cold orchid germ plasm resource in-vitro conservation method according to claim 1, it is characterised in that the light culture stage institute
The Plantlet in vitro culture medium for using, thickness is about 4cm, also, should for placing the test tube or blake bottle of Plantlet in vitro culture medium
When closed good, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
9. the method for restoration ecosystem after in-vitro conservation method described in claim 1~8 is preserved, it is characterised in that by light culture
Protocorm be transferred on differential medium, seedling is produced by protocorm differentiation；The differential medium is aided with for KC culture mediums
The bananas juice of mass concentration 10%, 25 DEG C of cultivation temperature, intensity of illumination 2000lux, daily light application time is 10 hours.
10. after preservation according to claim 9 restoration ecosystem method, it is characterised in that protocorm is in differential medium
After upper inoculation 30 days, in the spire that being developed into by phyllopodium occurs in protocorm tip portion, subsequent spire is mushroomed out, to appear
During the 2nd~3 leaf, protocorm extends downwards, and has the 1st article of root to bear；After 50 days, when seedling has 2~3 roots, 5~6
Piece leaf, grows into 10cm or so, develops into complete plant, by test tube transplantation of seedlings in seedling medium, gives birth under field conditions (factors)
Grow up to normal plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710263998.8A CN106879474A (en) | 2017-04-21 | 2017-04-21 | A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710263998.8A CN106879474A (en) | 2017-04-21 | 2017-04-21 | A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106879474A true CN106879474A (en) | 2017-06-23 |
Family
ID=59183421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710263998.8A Pending CN106879474A (en) | 2017-04-21 | 2017-04-21 | A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106879474A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050041818A (en) * | 2003-10-31 | 2005-05-04 | 제주도(농업기술원) | Selection of cymbidium kanran 'su-kwan' new variety |
| CN103651136A (en) * | 2013-12-06 | 2014-03-26 | 徐州生物工程职业技术学院 | Rhynchostylis tissue culture method |
| CN103718959A (en) * | 2013-12-18 | 2014-04-16 | 柳州市天姿园艺有限公司 | Quick propagation method of cymbidium kanran tissue culture seedling |
-
2017
- 2017-04-21 CN CN201710263998.8A patent/CN106879474A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050041818A (en) * | 2003-10-31 | 2005-05-04 | 제주도(농업기술원) | Selection of cymbidium kanran 'su-kwan' new variety |
| CN103651136A (en) * | 2013-12-06 | 2014-03-26 | 徐州生物工程职业技术学院 | Rhynchostylis tissue culture method |
| CN103718959A (en) * | 2013-12-18 | 2014-04-16 | 柳州市天姿园艺有限公司 | Quick propagation method of cymbidium kanran tissue culture seedling |
Non-Patent Citations (3)
| Title |
|---|
| KIRAN GOGOI ET.AL.,: "Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid, by asymbiotic seed germination", 《3 BIOTECH》 * |
| 刘佩佩: "大花蕙兰‘幻影’组培再生体系建立及离体保存技术研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 谷祝平等: "大花蕙兰茎尖组织培养及其形态建成的研究", 《实验生物学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108552056B (en) | Method for rapidly cultivating Baishan ancestor fir seedlings through embryo rescue technology | |
| CN106171985B (en) | A kind of liquid shallow quick-breeding method of ginger | |
| CN102696487A (en) | Method for building leaf in vitro regeneration system of begonia rex | |
| CN104855292B (en) | A kind of method of Cinnamomum kanahirai hay stem segment tissue culture fast breeding | |
| CN104145818B (en) | A kind of store method of flameray gerbera germ plasm resource | |
| CN102511388B (en) | Method for breeding anti-reversible tissue culture seedlings of anoectochilus roxburghii | |
| CN103141387A (en) | Method for cultivating haworthia maughanii tissue | |
| CN106258960B (en) | A kind of orchid seed sprouting quick-breeding method | |
| CN104145814B (en) | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) | |
| CN110073981A (en) | A method of it is saved using embryo and obtains poplar hybrid seedling | |
| CN106613993B (en) | A kind of cultural method of the tissue cultures regrowth of trifoliate orange | |
| CN103125398B (en) | A kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency | |
| CN105941147B (en) | Paper birch tissue culture of sprout mating system | |
| CN108142284A (en) | A kind of tissue culture and rapid propagation method of five leaflets maple | |
| CN106577280A (en) | Rapid propagation aseptic seedling by means of tender stem segments and blades of merrillanthus hainanensis | |
| CN105379621A (en) | Efficient in-vitro plant regeneration method of adult high-quality single-plant Xiaoqiao oriental cherry of cerasus lannesiana var. speciosa | |
| CN105684910B (en) | The tissue culture and rapid propagation method of dendrobium hancockii Rolfe | |
| CN106538385A (en) | A kind of extracorporeal culturing method of katsura tree | |
| CN106879474A (en) | A kind of method of restoration ecosystem after cold orchid germ plasm resource Plantlet in vitro and preservation | |
| CN104351051B (en) | A kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling | |
| CN103430851A (en) | Method for improving domestication survival rate of distant-hybridization tissue cultured seedlings of cucumbers | |
| CN103202228A (en) | One-step seedling and efficient in-vitro propagation method with gynura bicolor leaves | |
| CN106879475A (en) | A kind of method of restoration ecosystem after oncidiumLuridum germ plasm resource Plantlet in vitro and preservation | |
| CN107410021A (en) | A kind of method of restoration ecosystem after Chunlan germ plasm resource Plantlet in vitro and preservation | |
| CN107041305A (en) | A kind of method of restoration ecosystem after orchid germ plasm resource Plantlet in vitro and preservation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170623 |