CN107079813A - A kind of method of restoration ecosystem after sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation - Google Patents
A kind of method of restoration ecosystem after sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation Download PDFInfo
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- CN107079813A CN107079813A CN201710264016.7A CN201710264016A CN107079813A CN 107079813 A CN107079813 A CN 107079813A CN 201710264016 A CN201710264016 A CN 201710264016A CN 107079813 A CN107079813 A CN 107079813A
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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
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- A—HUMAN NECESSITIES
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- A01H4/008—Methods for regeneration to complete plants
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- 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
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Abstract
The invention discloses a kind of method of restoration ecosystem after sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation, stem apex is stripped from the pseudobulb that sword-leaved cymbidium newly grows, it is placed on protocorm induction medium surface and is trained protocorm, protocorm is subjected to cutting 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 sword-leaved cymbidium Shoot Tip Culture technology, induces protocorm, then obtains sword-leaved cymbidium 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.Preserve after certain time, be placed under normal temperature and illumination condition and cultivate, the rapid restoration ecosystem of energy differentiates seedling.
Description
Technical field
The present invention relates to a kind of method of restoration ecosystem after sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation.Belong to plant tissue
Culture technique field.
Background technology
Sword-leaved cymbidium（Scientific name：Cymbidium ensifolium (L. ) Sw.）For ground plant, also known as male blue, fine horse river it is blue,
Sword a species of orchid etc., also referred to as autumn are blue, and pseudobulb ovoid is contained within phyllopodium, and main flower-shape often has perfume (or spice) into generally in season gold autumn, spending
Gas, color and luster is changed greatly, usually chartreuse and have purple plague purpura.Its propagation method is generally division propagation, can just enter within 3~4 years
Once, reproduction speed is slower for row.Sword-leaved cymbidium seed is very tiny, in powdery, without endosperm, the simple embryo of only one of which, outer bread
It is loose, transparent, be difficult permeable kind skin, seldom nutriment is contained in embryo, the overwhelming majority is lipid material, natural conditions
Lower sword-leaved cymbidium seed is difficult to sprout.Therefore, traditional Seed storage technology does not apply to sword-leaved cymbidium Germ-plasma resources protection.
Sword-leaved cymbidium majority cultivar is by wild species domestication or natural variation screening.For from resource, due to
A large amount of excavations to the blue resource of wild state, natural resources are by considerable damage, and collectable wild resource is fewer and fewer, then
Extremely slow plus self-reproduction speed, the wild sword-leaved cymbidium resource of China is increasingly in short supply, and some rare kinds are endangered, because
This, sets up science, sword-leaved cymbidium Germ-plasma resources protection technology effectively, practical and compels in agility.Pass through 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, can also 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 that there is provided a kind of sword-leaved cymbidium germ plasm resource Plantlet in vitro side to overcome above-mentioned the deficiencies in the prior art
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 sword-leaved cymbidium germ plasm resource in-vitro conservation method, stem apex is stripped from the pseudobulb that sword-leaved cymbidium 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 is cut and squamous subculture repeatedly, and it is fast that the subculture medium 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.
It is preferred that, the stem apex to strip method as follows：The pseudobulb for selecting length to be 5~7cm, 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 stem apex that size is about 0.5mm.
It is preferred that, stem apex is placed in the surface of protocorm induction medium in 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 in time and carries out mark.
It is preferred that, 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.
It is preferred that, the specific method of cutting and squamous subculture is repeatedly：The protocorm for being grown to 4~6mg is cut 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.
It is preferred that, 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.
It is preferred that, the light culture stage should routine observation growing state, reject has the blake bottle of pollution at any time.
It is preferred that, Plantlet in vitro culture medium used in the light culture stage, 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, 3 layers of plastics are wound in the outer layer of sealed membrane
Antistaling film.
The method of restoration ecosystem after above-mentioned in-vitro conservation method is preserved, differentiation culture is transferred to by the protocorm in light culture
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.
It is preferred that, protocorm is inoculated with after 30 days on differential medium, occurs 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 life
Go out；After 50 days, when seedling has 2~3 roots, 5~6 leaves grow into 10cm or so, develop into complete plant, 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 to utilize sword-leaved cymbidium Shoot Tip Culture technology, induces protocorm, then obtains sword-leaved cymbidium 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
Support in base and add appropriate paclobutrazol, the holding time can be greatly prolonged, reduce Subculture times, effectively maintain germ plasm resource
Hereditary capacity.Preserve after certain time, culture is placed under normal temperature and illumination condition and cultivated, can recover raw rapidly
It is long, differentiate seedling.
The seed generally used 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
Bright use Shoot Tip Culture is as explant, and stem apex is that most fast separate living tissue is grown in plant, and gene are stable, power of regeneration
By force, virus and other pathogeny tissues are substantially free of（Such as bacterium, fungi）, 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 He quickly bred, possessed 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 lower temperature preservation substantially all more than 12 DEG C.In addition to sword-leaved cymbidium 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 sword-leaved cymbidium protocorm more than 90%, preserving callus needs higher cost.
The present invention carries out conservation in vitro using protocorm, and protocorm is substantially reduced to the sensitiveness of temperature, Ke Yiyou
Effect reduction storage temperature, temperature is lower, and the speed of growth is slower, and the holding time is longer, 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, illumination is eliminated
Facility, temperature control is also relatively easy, reduces luminous energy loss, reduces power consumption, effectively reduce 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 point of cell
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.
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, is comprised the following steps that after a kind of sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that sword-leaved cymbidium 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：The pseudobulb for selecting length to be 5cm, 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%, is put into the mercuric chloride solution of mass concentration 0.1% and sterilizes
20 minutes, aseptic water washing 3 times dried or surface moisture is blotted with filter paper, the stem that size is about 0.5mm is stripped under the microscope
Point.
Stem apex is placed in the surface of protocorm induction medium in 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 in time and carries out mark.
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, 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 subjected to cutting and squamous subculture repeatedly, the subculture medium 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 for being grown to 4mg is cut into 4 pieces, is subsequently placed in subculture medium and continues to expand
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.
Plantlet in vitro culture medium used in the light culture stage, 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, are twined in the outer layer of sealed membrane
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, 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, protocorm is inoculated with after 30 days on differential medium, occurs 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, develop into complete plant, 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, is comprised the following steps that after a kind of sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that sword-leaved cymbidium 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：The pseudobulb for selecting length to be 7cm, 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%, is put into the mercuric chloride solution of mass concentration 0.1% and sterilizes
20 minutes, aseptic water washing 4 times dried or surface moisture is blotted with filter paper, the stem that size is about 0.5mm is stripped under the microscope
Point.
Stem apex is placed in the surface of protocorm induction medium in 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 in time and carries out mark.
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, after 45 days, the lug volume increases to form original
Bulb.
（2）Protocorm is subjected to cutting and squamous subculture repeatedly, the subculture medium 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 for being grown to 6mg is cut into 4 pieces, is subsequently placed in subculture medium and continues to expand
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.
Plantlet in vitro culture medium used in the light culture stage, 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, are twined in the outer layer of sealed membrane
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, 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, protocorm is inoculated with after 30 days on differential medium, occurs 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, develop into complete plant, 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%）Specific formula be shown in Table 2.
Embodiment 3：
The method of restoration ecosystem, is comprised the following steps that after a kind of sword-leaved cymbidium germ plasm resource Plantlet in vitro and preservation：
（1）Stem apex is stripped from the pseudobulb that sword-leaved cymbidium 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：The pseudobulb for selecting length to be 6cm, 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%, is put into the mercuric chloride solution of mass concentration 0.1% and sterilizes
20 minutes, aseptic water washing 3 times dried or surface moisture is blotted with filter paper, the stem that size is about 0.5mm is stripped under the microscope
Point.
Stem apex is placed in the surface of protocorm induction medium in 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 in time and carries out mark.
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, after 45 days, the lug volume increases to form original
Bulb.
（2）Protocorm is subjected to cutting and squamous subculture repeatedly, the subculture medium 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 for being grown to 5mg is cut into 4 pieces, is subsequently placed in subculture medium and continues to expand
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.
Plantlet in vitro culture medium used in the light culture stage, 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, are twined in the outer layer of sealed membrane
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, 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, protocorm is inoculated with after 30 days on differential medium, occurs 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, develop into complete plant, 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%）Specific formula be shown in Table
2。
The specific formula of the various culture mediums of table 2.
Although the above-mentioned embodiment to the present invention is described, not limiting the scope of the invention,
On the basis of technical scheme, those skilled in the art need not pay the various modifications that creative work can be made
Or deform still within protection scope of the present invention.
Claims (10)
1. a kind of sword-leaved cymbidium germ plasm resource in-vitro conservation method, it is characterised in that strip stem apex from the pseudobulb that sword-leaved cymbidium newly grows,
It is placed on VW culture mediums and is aided with the protocorm induction medium surfaces of 0.5~1mg/L 6-benzyladenines and is trained protocorm
Stem, cutting and squamous subculture repeatedly are carried out by protocorm, and the subculture medium 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 sword-leaved cymbidium 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：The pseudobulb for selecting length to be 5~7cm, cleans surface dirt 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 sterilizes 20 minutes, sterile
Water is rinsed 3~4 times, dries or surface moisture is blotted with filter paper, the stem apex that size is about 0.5mm is stripped under the microscope.
3. a kind of sword-leaved cymbidium 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 using touch the surface of protocorm induction medium as
Preferably, it is to avoid be embedded to, and then cause death by suffocation, seal in time and carry out mark.
4. a kind of sword-leaved cymbidium 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 sword-leaved cymbidium 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 for being grown to 4~6mg is cut into 4 pieces, is subsequently placed in subculture medium and continues to expand
Increase culture, set up clone, repeatedly cutting and squamous subculture, so that the short time breeds the protocorm of requirement.
6. a kind of sword-leaved cymbidium 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 sword-leaved cymbidium 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 sword-leaved cymbidium 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 used, thickness is about 4cm, also, should for the test tube or blake bottle for placing Plantlet in vitro culture medium
When closed good, to reduce the volatilization of moisture in Plantlet in vitro culture medium.
The method of restoration ecosystem after 9. 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 that KC culture mediums are aided with
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. the method for restoration ecosystem after preservation according to claim 9, it is characterised in that protocorm is in differential medium
After upper inoculation 30 days, the spire for occurring being developed into by phyllopodium 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.
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| KR20050041818A (en) * | 2003-10-31 | 2005-05-04 | 제주도(농업기술원) | Selection of cymbidium kanran 'su-kwan' new variety |
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