CN105075860B - A kind of fleabane flower tissue culture method - Google Patents
A kind of fleabane flower tissue culture method Download PDFInfo
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Abstract
The present invention relates to a kind of fleabane flower tissue culture method, belong to field of plant tissue culture technique.This method is specifically included：The selection of explant, the sterilization of explant, Fiber differentiation, Multiplying culture, culture of rootage and the big step of hardening six.The more traditional fleabane flower seeling industry technology of the present invention, it is easy with production technology, breed that the time is short, breeding potential is high, not by season and territory restriction the advantages of, available for fleabane flower seedling factorial praluction.If calculated by one plant of fleabane flower plant, one plant of fleabane flower can gather 20 30 explants, and callus and the per generation of Multiplying culture 68 in 30 days generations, then the culture of rootage through 25 days and the hardening of 14 21 days can be obtained within 40 days through Fiber differentiation.Calculated with breeding for 6 generations, one plant of fleabane flower seedling is being lasted less than that in year, can obtain about 30 50 ten thousand commercial seedlings, with higher economic benefit.
Description
Technical field
The invention belongs to field of plant tissue culture technique, it is related to a kind of important source material medicinal plant method for tissue culture,
The medicinal plant method for tissue culture of more particularly to entitled fleabane flower.
Background technology
Fleabane flower（Erigeron breviscapus）, Erigeron breviscapus (Vant.) Hand.-Mazz., be composite family for many years
Raw herbaceous plant, is also erigeron breviscapus.The high mountain and Subalpine region for being distributed mainly on height above sea level 1200-3500m open spacious hillside, meadow or
Border.Herb is pungent, slight bitter, temperature, with dispelling wind and eliminating dampness, active pain relieving, effect of invigorating the spleen disperse accumulations, for paralysing, rheumatic arthritis,
Toothache, stomachache, infantile malnutrition, polio and meningitis sequela etc. are that China is used to treat angiocardiopathy Chinese patent drug
Important source material plant.Fleabane flower medicinal material market demand increases year by year, and the limited and excessive excavation of wild resource, fleabane flower
Resource is just reduced year by year.In recent years the artificial planting technique exploitation of fleabane flower has been carried out, it is necessary to be efficiently people's work post in some areas
Plant and stable, sufficient good seed is provided.The quick breeding of fleabane flower seedling is carried out using tissue culture technology, can be the rule of fleabane flower
Modelling artificial growth provides high-quality, stable abundant introduces a collection, again can for the synthesis mechanism of fleabane flower medicinal ingredient, functional gene,
The research such as molecular breeding lays the foundation, and can also protect the wild resource of wild fleabane flower.
The content of the invention
The present invention is directed to the deficiency of above-described fleabane flower seedling Traditional breeding processes, invented a kind of component it is simple,
It is easy to operate, with short production cycle, it can directly produce the tissue culture technique of fleabane flower plant in batches.
The present invention unless otherwise stated, percentage sign represent for mass percent, proportional representation is mass ratio.
The present invention is achieved by the following technical solutions：
A kind of fleabane flower tissue culture method, comprises the following steps：
Step（1）, the selection of explant：Explant is used as from the tender floral disc of fleabane flower or axillary bud；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, is cleaned clean with liquid soap and running water
Afterwards, add to 2-5%（v/v）8-12 min are soaked in the Tween-80 aqueous solution, then with running water shower 2h, wine is finally used successively
Essence, sodium hypochlorite and mercuric chloride sterilization；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+
Cultivated on 8.0-12.0 mg/L IAA+5.0-8.0 mg/L 6-BA inducing culture, culture environment is 23-25 DEG C of temperature,
Intensity of illumination 1500-2000 lux, odd-numbered day light application time obtains Cong Miao to carry out Fiber differentiation under conditions of 12 h；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong of the 2-3 strains with callus, so
It is seeded on MS+0.5-1.0 mg/L IAA+4.0-7.0 mg/L KT proliferated culture medium and cultivates afterwards, culture environment is temperature
23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is grown thickly to carry out Multiplying culture under conditions of 12 h
Seedling；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+0.8-1.2 mg/L IAA+
On the root media of 2.0 g/L activated carbons, at 23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is 12
Culture of rootage is carried out in the environment of h, rooted seedling is obtained；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the greenhouse that the rate of sheltering from heat or light is 80% together with tissue culture bottle
In-bottle seeding is carried out in greenhouse 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is moved
Plant and plastic covering film, transplanting medium uses fertile soil：Perlite=1：Air humidity is maintained at 70%- after 0.9-1.1, transplanting
80%, by 2-3 weeks cellar culture, you can be used as commercial seedling.
It is further preferred that step（2）The middle specific method that is cleaned with liquid soap and running water is：Explant is used
Liquid soap is rinsed after 10-20 min, and rinse 3-4 times with running water remains to without liquid soap.
It is further preferred that step（2）The specific method of middle sterilization is：First with 75%（v/v）Alcohol disinfecting 10-12
S, then 10-20 min are sterilized with saturation aqueous sodium hypochlorite solution, it is rear to use aseptic water washing 1-2 times, then use aseptic water washing 1-2
It is secondary, then with 0.05%（w/v）Mercuric chloride solution sterilizes 5-10 min, finally with aseptic water washing 4-5 times.
It is further preferred that step（2）Middle sterilization is carried out on superclean bench.
In above-mentioned technical proposal, step（3）The time of described Fiber differentiation is 25-35 days.
In above-mentioned technical proposal, step（4）The subculture cycle of described Multiplying culture is 25-35 days.
In above-mentioned technical proposal, step（5）The incubation time of described culture of rootage is 25-30 days.
It is further preferred that described fleabane flower tissue culture method, comprises the following steps：
Step（1）, the selection of explant：Fleabane flower is colonized in greenhouse, and routinely management method, which is poured, applies through fully fermentation
The biological organic solution become thoroughly decomposed, chooses healthy and strong, no disease and pests harm plant, clip its young tender floral disc or axillary bud are used as explant；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, 10-20 min are rinsed with liquid soap,
3-4 times is rinsed with running water to remain to without liquid soap, then adds explant to 2-5%（v/v）Soaked in the Tween-80 aqueous solution
8-12 min are steeped, then are sterilized with after running water shower 2h, being transferred on superclean bench；During sterilization, first with 75%（v/v）Alcohol
10-12 s are sterilized, it is rear to use aseptic water washing 1-2 times, then 10-20 min are sterilized with saturation aqueous sodium hypochlorite solution, then with nothing
Bacterium water is rinsed 1-2 times, then with 0.05%（w/v）Mercuric chloride solution sterilizes 5-10 min, finally with aseptic water washing 4-5 times；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+
Cultivated on 8.0-12.0 mg/L IAA+5.0-8.0 mg/L 6-BA inducing culture, culture environment is 23-25 DEG C of temperature,
Intensity of illumination 1500-2000 lux, odd-numbered day light application time is carries out Fiber differentiation under conditions of 12 h, and latter step is lured within 25-35 days
Export the Cong Miao with callus；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong of the 2-3 strains with callus, so
It is seeded on MS+0.5-1.0 mg/L IAA+4.0-7.0 mg/L KT proliferated culture medium and cultivates afterwards, culture environment is temperature
23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time be 12 h under conditions of carry out Multiplying culture, 25-35 days
For a subculture cycle, proliferation rate is up to 1:5-1:7, obtain tufted seedling；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+0.8-1.2 mg/L IAA+
On the root media of 2.0 g/L activated carbons, at 23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is 12
Culture of rootage is carried out in the environment of h, the rooted seedling of stalwartness can be obtained after 25-30 days；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the greenhouse that the rate of sheltering from heat or light is 80% together with tissue culture bottle
In-bottle seeding is carried out in greenhouse 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is moved
Plant in through pipeline high-temperature steam sterilization 1.8-2.2 h with 1：Fertile soil and the perlite mixing of 0.9-1.1 quality proportioning
In matrix, and build air humidity after Small plastic shed film, transplanting and be maintained at 70%-80%, by 2-3 weeks cellar culture, you can as
Commercial seedling.
Generally required after transplanting of the present invention through 2-3 weeks cellar culture and gradually decrease water spray frequency, reduce rate of sheltering from heat or light
And plastic sheeting is progressively opened, rate of sheltering from heat or light can be finally reduced to 40%.
Compared with prior art, its advantage is the present invention：
（1）The acquisition time of explant of the present invention be March to October, can acquisition time it is long；In gatherer process, do not injure into
The subterraneous root of medicine, it is acquired after can continued growth, the plant that also can not gathered with other to the harvest season while harvesting and not shadow
Ring yield and quality；
（2）The group culturation rapid propagating technology that the present invention is used, component is simple, easy to operate, not by region and season limit；
（3）Fleabane flower tissue culture method of the present invention, reproduction speed is fast, breeding potential is high.If by one plant of fleabane flower
Plant calculates that one plant of fleabane flower can gather 20-30 explant, and callus and per generation 30 can be obtained within 40 days through Fiber differentiation
It Multiplying culture 6-8 generations, then the culture of rootage through 25 days and the hardening of 14-21 days.Calculated with breeding for 6 generations, one plant of fleabane flower
Seedling, is lasting less than in year, can obtaining the commercial seedlings of about 30-50 ten thousand.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, are that can be obtained by buying
Conventional products.
Embodiment 1
A kind of fleabane flower tissue culture method, comprises the following steps：
Step（1）, the selection of explant：Fleabane flower is colonized in greenhouse, and routinely management method, which is poured, applies through fully fermentation
The biological organic solution become thoroughly decomposed, chooses healthy and strong, no disease and pests harm plant, and its young tender floral disc 100 of clip is used as explant；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, 10 min are rinsed with liquid soap, with certainly
Water rinses 3 times to without liquid soap residual, then adds explant to 2%（v/v）8 min are soaked in the Tween-80 aqueous solution,
Use again after running water shower 1.9h, be transferred on superclean bench and sterilize；During sterilization, first with 75%（v/v）The s of alcohol disinfecting 10,
Afterwards with aseptic water washing 2 times, then with saturation aqueous sodium hypochlorite solution sterilization 10min, then with aseptic water washing 1 time, then use
0.05%（w/v）Mercuric chloride solution sterilizes 5 min, finally with aseptic water washing 4 times；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+8.0
On mg/L IAA+5.0mg/L 6-BA inducing culture cultivate, culture environment be 23 DEG C of temperature, the lux of intensity of illumination 1500,
Odd-numbered day light application time is carries out Fiber differentiation under conditions of 12 h, after 35 days, obtains 100 pieces of the Cong Miao with bud callus；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong of the 2-3 strains with callus, so
It is seeded on MS+0.5 mg/L IAA+4.0 mg/L KT proliferated culture medium and cultivates afterwards, culture environment is 23 DEG C of temperature,
The lux of intensity of illumination 1500, odd-numbered day light application time is carries out Multiplying culture under conditions of 12 h, culture simultaneously continues 5 generations of transferring in 30 days
Afterwards, the tufted seedling 17500 of callus is obtained（100*5*7*5=17500）Clump；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+0.8 mg/L IAA+2.0
On the root media of g/L activated carbons, at 23 DEG C, intensity of illumination 1500lux, odd-numbered day light application time is to enter in the environment of 12 h
Row culture of rootage, obtains high about 5-8 cm, has about 120,000 plants of the fleabane flower rooted seedling of 3-4 bar roots after 25 days；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the greenhouse that the rate of sheltering from heat or light is 80% together with tissue culture bottle
In-bottle seeding is carried out in greenhouse 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is pressed
5cm*5cm planting density transplant in through the h of pipeline high-temperature steam sterilization 1.8 with 1：The fertile soil of 0.9 quality proportioning and
In perlite mixed-matrix, and build air humidity after Small plastic shed film, transplanting and be maintained at 70%-80%, conventional trained by 2-3 weeks
Support, you can as commercial seedling, transplant afterwards in crop field, transplanting survival rate is 92.6%.
Embodiment 2
A kind of fleabane flower tissue culture method, comprises the following steps：
Step（1）, the selection of explant：Fleabane flower is colonized in greenhouse, and routinely management method, which is poured, applies through fully fermentation
The biological organic solution become thoroughly decomposed, chooses healthy and strong, no disease and pests harm plant, its axillary bud of clip（Blade and petiole are cut off, petiole is only left
Base portion）100 are used as explant；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, 20 min are rinsed with liquid soap, with certainly
Water rinses 4 times to without liquid soap residual, then adds explant to 5%（v/v）12 min are soaked in the Tween-80 aqueous solution,
Use again after running water shower 2.1h, be transferred on superclean bench and sterilize；During sterilization, first with 75%（v/v）The s of alcohol disinfecting 12,
Afterwards with aseptic water washing 1 time, then with saturation aqueous sodium hypochlorite solution 20 min of sterilization, then with aseptic water washing 2 times, then use
0.05%（w/v）Mercuric chloride solution sterilizes 10 min, finally with aseptic water washing 5 times；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+
Cultivated on 12.0 mg/L IAA+8.0 mg/L 6-BA inducing culture, culture environment is 25 DEG C of temperature, intensity of illumination 2000
Lux, odd-numbered day light application time is carries out Fiber differentiation under conditions of 12 h, after 25 days, obtains the Cong Miao 100 with bud callus
Block；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong of the 2-3 strains with callus, so
It is seeded on MS+1.0 mg/L IAA+7.0 mg/L KT proliferated culture medium and cultivates afterwards, culture environment is 25 DEG C of temperature, illumination
The lux of intensity 2000, odd-numbered day light application time is carries out Multiplying culture under conditions of 12 h, culture simultaneously continues after 5 generations of switching for 25 days,
Obtain the tufted seedling 10000 with callus（100*4*5*5=10000）Clump；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+1.2 mg/L IAA+2.0
On the root media of g/L activated carbons, at 25 DEG C, the lux of intensity of illumination 2000, odd-numbered day light application time is to enter in the environment of 12 h
Row culture of rootage, obtains high about 5-8 cm, has about 100,000 plants of the fleabane flower rooted seedling of 3-4 bar roots after 30 days；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the greenhouse that the rate of sheltering from heat or light is 80% together with tissue culture bottle
In-bottle seeding is carried out in greenhouse 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is pressed
5cm*5cm planting density transplant in through the h of pipeline high-temperature steam sterilization 2.2 with 1：The fertile soil of 1.1 quality proportioning and
In perlite mixed-matrix, and build air humidity after Small plastic shed film, transplanting and be maintained at 70%-80%, conventional trained by 2-3 weeks
Support, you can as commercial seedling, then transplant in crop field, transplanting survival rate is 90.7%.
Embodiment 3
A kind of fleabane flower tissue culture method, comprises the following steps：
Step（1）, the selection of explant：Fleabane flower is colonized in greenhouse, and routinely management method, which is poured, applies through fully fermentation
The biological organic solution become thoroughly decomposed, chooses healthy and strong, no disease and pests harm plant, and its young tender floral disc 100 of clip is used as explant；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, 15 min are rinsed with liquid soap, with certainly
Water rinses 4 times to without liquid soap residual, then adds explant to 2-5%（v/v）10 are soaked in the Tween-80 aqueous solution
Min, then sterilized with after running water shower 2h, being transferred on superclean bench；During sterilization, first with 75%（v/v）Alcohol disinfecting 11
S, it is rear with aseptic water washing 2 times, then with saturation aqueous sodium hypochlorite solution 13 min of sterilization, then with aseptic water washing 1 time, then
With 0.05%（w/v）Mercuric chloride solution sterilizes 7 min, finally with aseptic water washing 5 times；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+
Cultivated on 10.0mg/L IAA+7.0 mg/L 6-BA inducing culture, culture environment is 24 DEG C of temperature, intensity of illumination 1800
Lux, odd-numbered day light application time is carries out Fiber differentiation under conditions of 12 h, after 30 days, obtains 100 pieces of band bud callus Cong Miao；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong of the 2-3 strains with callus, so
It is seeded on MS+0.8 mg/L IAA+5.0 mg/L KT proliferated culture medium and cultivates afterwards, culture environment is 24 DEG C of temperature, illumination
The lux of intensity 1700, odd-numbered day light application time is carries out Multiplying culture under conditions of 12 h, culture simultaneously continues after 5 generations of switching for 35 days,
Obtain the Cong Miao 17500 with callus（100*5*7*5=17500）Clump；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+1.1 mg/L IAA+2.0
On the root media of g/L activated carbons, at 24 DEG C, the lux of intensity of illumination 1900, odd-numbered day light application time is to enter in the environment of 12 h
Row culture of rootage, obtains high about 5-8 cm, has about 120,000 plants of the fleabane flower rooted seedling of 3-4 bar roots after 28 days；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the greenhouse that the rate of sheltering from heat or light is 80% together with tissue culture bottle
In-bottle seeding is carried out in greenhouse 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is pressed
5cm*5cm planting density transplant in through the h of pipeline high-temperature steam sterilization 2 with 1：The fertile soil of 1 quality proportioning and pearl
In rock mixed-matrix, and build air humidity after Small plastic shed film, transplanting and be maintained at 70%-80%, by 2-3 weeks cellar culture, i.e.,
Can then it be transplanted in crop field, transplanting survival rate is 91.7% as commercial seedling.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (4)
1. a kind of fleabane flower tissue culture method, it is characterised in that comprise the following steps：
Step（1）, the selection of explant：Explant is used as from the tender floral disc of fleabane flower or axillary bud；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, after being cleaned totally with liquid soap and running water,
Add to 2-5%（v/v）8-12 min are soaked in the Tween-80 aqueous solution, then with running water shower 1.9-2.1h, successively finally
Sterilized with alcohol, sodium hypochlorite and mercuric chloride；Wherein, sterilization is carried out on superclean bench；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+8.0-
Cultivated on 12.0 mg/L IAA+5.0-8.0 mg/L 6-BA inducing culture, culture environment is 23-25 DEG C of temperature, illumination
Intensity 1500-2000 lux, odd-numbered day light application time obtains Cong Miao to carry out Fiber differentiation under conditions of 12 h；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong, Ran Houjie of the 2-3 strains with callus
Plant and cultivated on MS+0.5-1.0 mg/L IAA+4.0-7.0 mg/L KT proliferated culture medium, culture environment is temperature 23-25
DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time obtains tufted seedling to carry out Multiplying culture under conditions of 12 h；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+0.8-1.2 mg/L IAA+2.0
On the root media of g/L activated carbon, at 23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is 12 h's
Culture of rootage is carried out under environment, incubation time is 25-30 days, obtains rooted seedling；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the warmhouse booth that the rate of sheltering from heat or light is 80% together with tissue culture bottle
Interior progress in-bottle seeding 2 days, after unscrew bottle cap finishing scouring seedling 1 day, seedling is then removed into culture medium and cleaned after culture medium, is transplanted simultaneously
Plastic covering film, transplanting medium uses fertile soil：Perlite=1：Air humidity is maintained at 70%-80% after 0.9-1.1, transplanting, after
Through 2-3 weeks cellar culture, you can be used as commercial seedling；
Wherein, step（2）The middle specific method that is cleaned with liquid soap and running water is：Explant is rinsed into 10-20 with liquid soap
After min, rinse 3-4 times with running water and remained to without liquid soap；
Step（2）The specific method of middle sterilization is：First with 75%（v/v）Alcohol disinfecting 10-12 s, use aseptic water washing 1-2 afterwards
It is secondary, then 10-20 min are sterilized with saturation aqueous sodium hypochlorite solution, then with aseptic water washing 1-2 times, then with 0.05%（w/v）
Mercuric chloride solution sterilizes 5-10 min, finally with aseptic water washing 4-5 times.
2. fleabane flower tissue culture method according to claim 1, it is characterised in that step（3）Described Fiber differentiation
Time be 25-35 days.
3. fleabane flower tissue culture method according to claim 1, it is characterised in that step（4）Described Multiplying culture
Subculture cycle be 25-35 days.
4. fleabane flower tissue culture method according to claim 1, it is characterised in that comprise the following steps：
Step（1）, the selection of explant：Fleabane flower is colonized in greenhouse, and routinely management method, which is poured, applies through abundant fermentation maturity
Biological organic solution, choose healthy and strong, no disease and pests harm plant, clip its young tender floral disc or axillary bud are used as explant；
Step（2）, the sterilization of explant：Will be through step（1）Selected explant, 10-20 min are rinsed with liquid soap, with certainly
Water rinses 3-4 times to without liquid soap residual, then adds explant to 2-5%（v/v）8- is soaked in the Tween-80 aqueous solution
12 min, then sterilized with after running water shower 2h, being transferred on superclean bench；During sterilization, first with 75%（v/v）Alcohol disinfecting
10-12 s, it is rear to use aseptic water washing 1-2 times, then 10-20 min are sterilized with saturation aqueous sodium hypochlorite solution, then use sterilized water
Rinse 1-2 times, then with 0.05%（w/v）Mercuric chloride solution sterilizes 5-10 min, finally with aseptic water washing 4-5 times；
Step（3）, Fiber differentiation：Will be through step（2）The explant disinfected is cut off after base portion wound, is seeded in MS+8.0-
Cultivated on 12.0 mg/L IAA+5.0-8.0 mg/L 6-BA inducing culture, culture environment is 23-25 DEG C of temperature, illumination
Intensity 1500-2000 lux, odd-numbered day light application time is carries out Fiber differentiation under conditions of 12 h, and latter one-step inducing goes out within 25-35 days
Cong Miao with callus；
Step（4）, Multiplying culture：By step（3）Obtained Cong Miao cuttings are the little Cong, Ran Houjie of the 2-3 strains with callus
Plant and cultivated on MS+0.5-1.0 mg/L IAA+4.0-7.0 mg/L KT proliferated culture medium, culture environment is temperature 23-25
DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is carries out Multiplying culture under conditions of 12 h, 25-35 days is once
Subculture cycle, obtains tufted seedling；
Step（5）, culture of rootage：It is individual plant by tufted seedling cutting, is then seeded in 1/2MS+0.8-1.2 mg/L IAA+2.0
On the root media of g/L activated carbon, at 23-25 DEG C, intensity of illumination 1500-2000 lux, odd-numbered day light application time is 12 h's
Culture of rootage is carried out under environment, the rooted seedling of stalwartness can be obtained after 25-30 days；
Step（6）, hardening：By step（5）Obtained rooted seedling is placed in the warmhouse booth that the rate of sheltering from heat or light is 80% together with tissue culture bottle
Interior progress in-bottle seeding 2 days, after unscrew bottle cap finishing scouring seedling 1 day, then seedling is removed culture medium and cleaned after culture medium, transplant in
Through pipeline high-temperature steam sterilization 1.8-2.2 h with 1：The fertile soil and perlite mixed-matrix of 0.9-1.1 quality proportioning
In, and build air humidity after Small plastic shed film, transplanting and be maintained at 70%-80%, by 2-3 weeks cellar culture, you can be used as commodity
Seedling.
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| CN201510476405.7A CN105075860B (en) | 2015-08-06 | 2015-08-06 | A kind of fleabane flower tissue culture method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510476405.7A CN105075860B (en) | 2015-08-06 | 2015-08-06 | A kind of fleabane flower tissue culture method |
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