CN103798138B - A kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium - Google Patents
A kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium Download PDFInfo
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- CN103798138B CN103798138B CN201410037331.2A CN201410037331A CN103798138B CN 103798138 B CN103798138 B CN 103798138B CN 201410037331 A CN201410037331 A CN 201410037331A CN 103798138 B CN103798138 B CN 103798138B
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Abstract
The present invention relates to micropropagation of plants technical field, it is desirable to provide a kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium.This kind of method includes step: material is taked, calli induction media configures, outer implant inoculates and callus induction, plantlet regeneration, the bottle outlet plantation of the successive transfer culture of plantlet, plantlet.The present invention highlights has the advantages that pollution rate is low, Callus induction rate is high, wound healing agglomerate big, wound healing embryo is good, regeneration capacity is strong, the rate of increase is high, it is outer implant especially for the present invention with the gynoecium in young tender petal, pollution rate can be reduced to 0, the Callus induction rate of up to 100% can be obtained, cultivate 130 days callus agglomerate diameters up to nearly 1cm, wound healing agglomerate regeneration rate 100%, embryo is high, and the most each gynoecium can obtain 10 40 strain plantlets.
Description
Technical field
The present invention is about micropropagation of plants technical field, is that outer implant sets up Bulbus Lilii embryo particularly to one with gynoecium
The method of wound healing regenerating system.
Background technology
Bulbus Lilii embryo callus regenerating system compares other regenerating systems such as shoot regeneration system, have the highest rate of increase and
Expanding propagation rate, preserves significance to elite germplasm.Additionally, Bulbus Lilii embryo callus regenerating system is also Bulbus Lilii something lost
Pass the decisive basis of transformation system, and then affect the genetic engineering improvement of Bulbus Lilii.
Setting up the many scales with field bulb of Bulbus Lilii wound healing regenerating system at present is outer implant, and its defect is that bulb is due to for a long time
Being in soil environment, it is many to carry pathogenic bacteria, makes tissue culture's sterilization require strict, and pollution rate is higher.Additionally, when needing
When setting up the in vitro system of laboratory of Lilium Germplasm kind matter, it is outer implant with bulb, after the florescence can only be used, takes kind of a ball,
Its prominent deficiency has two: first, the determination of Lilium Germplasm position and the identification of kind are largely dependent on eye-catching flower
Organ and feature thereof, after the florescence, Lilium Germplasm aerial parts is the most completely or partially withered, especially floral organ decay and flower
Come off by sheet and make its kind be difficult to recognize, easily cause obscuring of germ plasm resource；Secondly, take kind of a ball and be equal to wild money
Source gathers completely, Precious, Rare, Endangered kind is difficult to especially to the destruction replied.
Summary of the invention
Present invention is primarily targeted at and overcome deficiency of the prior art, it is provided that a kind of pollution rate is low, Callus induction rate is high,
Wound healing agglomerate is big, wound healing embryo is good, regeneration capacity is strong, the rate of increase is high, and destroys germ plasm resource and little set up Bulbus Lilii
The method of embryo callus subculture regenerating system.For solving above-mentioned technical problem, the solution of the present invention is:
Thering is provided a kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium, comprises the following steps:
Step one: material is taked:
At the Bulbus Lilii florescence, take (children is tender) alabastrum of Bulbus Lilii length 0.5～2.0cm, be placed in sealed bag at 4 degrees Celsius
In refrigerator after cold preservation 1 week, then carry out subsequent operation；
Step 2: calli induction media configures:
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.25～
The 2 of 1.5mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), calli induction media solution
PH value be 5.8；
After the calli induction media solution sterilization that will have configured, superclean bench carries out subpackage, will wound healing induction
Culture medium solution is poured in (glass or plastics) culture dish, treats that calli induction media solution is frozen into wound healing inducing culture
After base, the culture dish that at least one fills calli induction media with preservative film or tinfoil wraps, and is placed on aseptic
Environment is deposited under (such as group training room)；
Step 3: the inoculation of outer implant and callus induction:
Alabastrum after processing in step one, soaks 5～30min in the tap water with the addition of detergent, then at flowing water
Lower flushing 0.5～2h cleans up, and then carry out disinfection on superclean bench inoculation；
Sterilization method is as follows: use sodium hypochlorite (NaClO) solution of the 1%w/v that with the addition of 0.1%v/v tween, will
The alabastrum cleaned up carries out surface sterilization 8～15min, then uses aseptic water washing 3 times；
Inoculate after sterilization: with tweezers and dissecting knife, alabastrum is separated, gynoecium peeled off from alabastrum, excise stigma,
The gynoecium having excised stigma is inoculated in step 2 on the culture dish filling calli induction media made, wherein, female
The inoculation of stamen is as the criterion with contact media surface；By be vaccinated with the culture medium of outer implant 25 degrees Celsius, under dark condition
Cultivate 8～16 days, callus agglomerate occurs；
Step 4: plantlet regenerates:
Outer implant is cultivated 120 days on calli induction media, and the callus that part calli induction media is cultivated is opened
Beginning differentiation and regeneration goes out complete plantlet, and the callus that part calli induction media is cultivated still keeps dedifferentiation shape
State；Outer implant is transferred on fresh calli induction media, at complete darkness together with callus agglomerate, plantlet
Under the conditions of cultivate after 90～120 days, plantlet breaks up in a large number, differentiation rate 95～100%, in average each outer implant point
Dissolve 10～40 plantlets；Plantlet is separated from callus agglomerate by superclean bench, proceeds to continue
Culture base is cultivated；
Plantlet grow on subculture medium 4 weeks after (average diameter reaches more than 1cm), can not bottle outlet, continue into
Successive transfer culture in row step 5 is to maintain Bulbus Lilii in vitro bulb regenerating system, then carries out the bottle outlet plantation in step 6, often
4 weeks subcultures are once；Also the bottle outlet plantation in step 6 can directly be carried out；
Described subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter
Subculture medium is added with 4.43gMS powder, in subculture medium possibly together with 60g/L sucrose, 4～8g/L agar, 0～
0.2mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0～0.2mg/L α-naphthaleneacetic acid
(1-Naphthaleneacetic acid, NAA), the pH value of subculture medium is 5.8；(α-naphthaleneacetic acid is not required
, but certain density naphthalene acetic acid is beneficial to plantlet root growth, and 6-benzyl aminoadenine is optional, but one
The 6-benzyl aminoadenine determining concentration is beneficial to plantlet propagation)
Step 5: the successive transfer culture of plantlet:
Plantlet in step 4 grow on subculture medium 4 weeks after (average diameter reaches more than 1cm), not bottle outlet,
The method carrying out subculture is: plantlet grows on subculture medium, and base is taken root robust growth, root upper end turn green after,
Carry out the switching of plantlet；Forwarding method is: on superclean bench, is taken out by plantlet, by its root complete resection,
Blade also excises from base portion, only retains clove and a small amount of plateau, is transferred to by clove on fresh subculture medium,
Continue the cultivation of step 4；
Step 6: the bottle outlet plantation of plantlet:
Cold acclimation is carried out before bottle outlet, the plantlet will cultivated on the subculture medium in group culture container, Celsius in 1～5
The K cryogenic treatment of 30～50 days is carried out under degree, dark condition；After K cryogenic treatment, by whole for the plantlet in group culture container
Take out, rinse under flowing water, clean the culture medium of root, then plantlet is put in the string bag, carbendazim 1000 times
Soaking disinfection 15～30min in solution, then plant into nursery dedicated medium (such as Custom growing mix, Fafard)
In, plantlet kind enters the degree of depth in nursery dedicated medium in the range of 1～2cm, i.e. clove top is high away from soil table one ball
Left and right, carries out cultivation management, i.e. completes the foundation of Bulbus Lilii embryo callus subculture regenerating system.
Thering is provided a kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture genetic conversion system with gynoecium, will induce in step 3
The callus agglomerate obtained cuts or presss from both sides with tweezers, as the receptor of genetic conversion system, through Agrobacterium infection or
Biolistic mediated transformation, through screening and identify, and on regeneration culture medium differentiation take root, seedling exercising, transplanting obtain Bulbus Lilii
Transfer-gen plant；
Regeneration culture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter regeneration
Culture medium add 4.43gMS powder, in regeneration culture medium possibly together with 30～60g/L sucrose, 4～8g/L agar, 0～2.0
Mg/L α-naphthaleneacetic acid (1-Naphthaleneacetic acid, NAA), the pH value of regeneration culture medium is 5.8～6.0.
The said method that the present invention provides may be used in Bulbus Lilii callus regeneration system and genetic conversion system, specifically
For: to said method obtain callus carry out successive transfer culture after cut into fritter, be inoculated into corresponding subculture medium
Upper cultivation；Breaking up on regeneration culture medium with this callus and take root, seedling exercising, transplanting can obtain the tissue training of Bulbus Lilii
Support plant；Maybe using this callus as the receptor of genetic conversion system, regeneration culture medium breaks up and takes root, refining
Seedling, transplanting can obtain the transfer-gen plant of Bulbus Lilii.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention is outer implant with Bulbus Lilii gynoecium, has the advantages that pollution rate is low, material is easy to get, and its prominent advantage is
System high efficiency, outer implant children are tender, less virus.For the Germ-plasma resources protection of Lilium Germplasm, take at the Bulbus Lilii florescence
Obtain appropriate gynoecium to be used for setting up laboratory wound healing regenerating system rather than after the florescence, taking kind of a ball, be easier to gynoecium feature
Confirm the kind of Lilium Germplasm, and considerably reduce the destruction to Wild ornamental resources.The present invention is also prominent has pollution
The feature that rate is low, Callus induction rate is high, wound healing agglomerate big, wound healing embryo is good, regeneration capacity is strong, the rate of increase is high, especially
Being outer implant for the present invention with the gynoecium in young tender petal, pollution rate can be reduced to 0, can obtain up to 100% more
Hindering inductivity, cultivate 130 days callus agglomerate diameters up to nearly 1cm, wound healing agglomerate regeneration rate 100%, embryo is high,
The most each gynoecium can obtain 10-40 strain plantlet.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in further detail:
A kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium, comprises the following steps:
Step one: material is taked:
At the Bulbus Lilii florescence, take the tender alabastrum of children of Bulbus Lilii length 0.5～2.0cm, be placed in sealed bag the refrigerator at 4 degrees Celsius
After middle cold preservation 1 week, then carry out subsequent operation.
Step 2: calli induction media configures:
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.25～
The 2 of 1.5mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D)；Calli induction media solution
PH value be 5.8.
After the calli induction media solution sterilization that will have configured, superclean bench carries out subpackage, will wound healing induction
Culture medium solution is poured in the culture dish of glass or plastics, treats that calli induction media solution is frozen into calli induction media
After, the culture dish that at least one fills calli induction media with preservative film or tinfoil wraps, be placed on group training room or its
Deposit under his aseptic environment.
Step 3: the inoculation of outer implant and callus induction:
Alabastrum after processing in step one, soaks 5～30min in the tap water with the addition of detergent, then at flowing water
Lower flushing 0.5～2h cleans up, and then carry out disinfection on superclean bench inoculation；
Sterilization method is as follows: use sodium hypochlorite (NaClO) solution of the 1%w/v that with the addition of 0.1%v/v tween, will
The alabastrum cleaned up carries out surface sterilization 8～15min, then uses aseptic water washing 3 times；
Inoculate after sterilization: with tweezers and dissecting knife, alabastrum is separated, gynoecium peeled off from alabastrum, excise stigma,
The gynoecium having excised stigma is inoculated in step 2 on the culture dish filling calli induction media made, wherein, female
The inoculation of stamen is as the criterion with contact media surface.By be vaccinated with the culture medium of outer implant 25 degrees Celsius, under dark condition
Cultivate 8～16 days, callus agglomerate occurs.
Step 4: plantlet regenerates:
After outer implant grows 120 days on calli induction media, the callus that part calli induction media is cultivated
Starting differentiation and regeneration and go out complete plantlet, the callus that part calli induction media is cultivated still keeps dedifferentiation shape
State.Outer implant is transferred on fresh calli induction media, at complete darkness together with callus agglomerate, plantlet
Under the conditions of cultivate after 90～120 days, plantlet breaks up in a large number, differentiation rate 95-100%, and average each outer implant is broken up
Go out 10-40 plantlet.Plantlet is separated from callus agglomerate by superclean bench, proceeds to subculture training
Support base；
After plantlet grows 4 weeks on subculture medium, average diameter reaches more than 1cm, can not bottle outlet, proceed
Successive transfer culture in step 5 is to maintain Bulbus Lilii in vitro bulb regenerating system, then carries out the bottle outlet plantation in step 6, and every 4
Zhou Jidai is once；Also the bottle outlet plantation in step 6 can directly be carried out.
Described subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter
Subculture medium is added with 4.43gMS powder, in subculture medium possibly together with 60g/L sucrose, 4～8g/L agar, 0～
0.2mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0～0.2mg/L α-naphthaleneacetic acid
(1-Naphthaleneacetic acid, NAA), the pH value of subculture medium is 5.8.Wherein, α-naphthaleneacetic acid is not
Required, but certain density naphthalene acetic acid is beneficial to plantlet root growth, and 6-benzyl aminoadenine is optional,
But certain density 6-benzyl aminoadenine is beneficial to plantlet propagation.
Step 5: the successive transfer culture of plantlet:
After plantlet in step 4 grows 4 weeks on subculture medium, average diameter reaches more than 1cm, not bottle outlet,
The method carrying out subculture is: plantlet grows on subculture medium, and base is taken root robust growth, root upper end turn green after,
Carry out the switching of plantlet；Forwarding method is: on superclean bench, is taken out by plantlet, by its root complete resection,
Blade also excises from base portion, only retains clove and a small amount of plateau, is transferred to by clove on fresh subculture medium,
Continue the cultivation of step 4.
Step 6: the bottle outlet plantation of plantlet:
Cold acclimation is carried out before bottle outlet, the plantlet will cultivated on the subculture medium in group culture container, Celsius in 1～5
The K cryogenic treatment of 30～50 days is carried out under degree, dark condition；After K cryogenic treatment, by whole for the plantlet in group culture container
Take out, rinse under flowing water, clean the culture medium of root, then plantlet is put in the string bag, carbendazim 1000 times
Soaking disinfection 15～30min in solution, then plant into nursery dedicated medium (such as Custom growing mix, Fafard)
In, plantlet kind enters the degree of depth in nursery dedicated medium in the range of 1～2cm, i.e. clove top is high away from soil table one ball
Left and right, carries out cultivation management, i.e. completes the foundation of Bulbus Lilii embryo callus subculture regenerating system.
The following examples can make the professional and technical personnel of this specialty that the present invention be more fully understood, but never in any form
Limit the present invention.
Embodiment 1 is that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with east lilium Sorbonne gynoecium
(1) bud stage takes the Bulbus Lilii tender petal of children of 1.7cm length, and the bennet of belt length 1cm, in sealed bag, cold preservation is in 4
In degree Celsius refrigerator, cold preservation is taken out after one week and is processed.
(2) petal is soaked in the tap water with the addition of detergent 30min, under flowing water, rinse 75min, clean dry
After Jing, superclean bench used the 1%w/v liquor natrii hypochloritis that with the addition of 0.1%v/v tween, carries out surface sterilization
8min, then uses aseptic water washing 3 times.Gynoecium is peeled off from alabastrum, excises stigma, be inoculated in step 2 making
The culture dish filling calli induction media on, wherein, the inoculation of gynoecium with contact media surface be as the criterion；Will inoculation
The culture medium of outer implant 25 degrees Celsius, cultivate 8 days under dark condition, callus agglomerate occurs, pollution rate is 0.
Callus induction rate reaches 96%.
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 1.5mg/L
2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D)；The pH value of calli induction media solution
It is 5.8.
If diameter is maximum up to 9.81mm after wound healing agglomerate is cultivated 130 days on calli induction media.Wherein, wound healing
Agglomerate size is with vernier caliper measurement, owing to wound healing agglomerate is subsphaeroidal or length and width are equal, therefore using its diameter as wound healing agglomerate
Size criterion, and along with the differentiation of plantlet.
(3), after outer implant grows 120 days on calli induction media, start to differentiate plantlet.By outer implant even
On superclean bench, the fresh calli induction media in cylindricality bottle it is transferred to together with callus agglomerate, plantlet
On, to cultivate 90 days under dark condition, continue to differentiate a large amount of plantlet, averagely every piece outer implant can differentiate 25
Plantlet, differentiation rate is 100%.Plantlet is separated in outer implant by superclean bench, is transferred to fresh
Subculture medium on, under illumination is the dark illumination condition of 1800lux, 12h illumination/12h, carry out successive transfer culture.
Subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter subculture
Culture medium adds 4.43gMS powder, possibly together with sucrose, the 8g/L agar of 60g/L in subculture medium, subculture medium
PH value is 5.8.
(4) plantlet in step (3) grows 4 weeks on subculture medium, and bulb average diameter reaches more than 1cm,
Plantlet directly carries out bottle outlet plantation.Plantlet on subculture medium is placed under 5 degrees Celsius of dark conditions and carries out low temperature
Process 40 days；By the whole taking-up of plantlet in group culture container, rinse under flowing water, clean the culture medium of root, then
Plantlet is put in the string bag, soaking disinfection 25min in 1000 times of solution of carbendazim, then plant into special Jie of nursery
In matter (Custom growing mix, Fafard), plantlet kind enters the degree of depth in seedling medium at 1cm, cultivates
Management.Plantlet robust growth, survival rate 100%.This system propagation efficiency reaches 1:25, the most every piece outer planting
Body finally produces 25 plantlets.
Embodiment 2 is that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with east lilium Sorbonne gynoecium
(1) bud stage takes the Bulbus Lilii tender petal of children of 2cm length, the bennet of band 1.5cm length, be enclosed in pocket cold preservation in
4 degrees Celsius of refrigerator cold-storages are after one week, then carry out subsequent operation.
(2) petal is soaked in the tap water with the addition of detergent 18min, under flowing water, rinse 30min, clean dry
After Jing, with the addition of 0.1%(v/v on superclean bench) 1% (w/v) liquor natrii hypochloritis of tween carries out surface and disappears
Poison 12min, then uses aseptic water washing 3 times.Gynoecium is peeled off from alabastrum, excises stigma, be inoculated in step 2
On the culture dish filling calli induction media made, wherein, the inoculation of gynoecium is as the criterion with contact media surface；Will
Be vaccinated with the culture medium of outer implant 25 degrees Celsius, cultivate 13 days under dark condition, callus agglomerate occurs, pollutes
Rate is 0.Callus induction rate reaches 88.7%.
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.5mg/L
2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D)；The pH value of calli induction media solution
It is 5.8.
If diameter is maximum up to 6.85mm after wound healing agglomerate is cultivated 130 days on calli induction media.Wherein, wound healing
Agglomerate size is with vernier caliper measurement, owing to wound healing agglomerate is subsphaeroidal or length and width are equal, therefore using its diameter as wound healing agglomerate
Size criterion, and along with the differentiation of plantlet.
(3), after outer implant grows 120 days on calli induction media, start to differentiate plantlet.By outer implant even
On superclean bench, the fresh calli induction media in cylindricality bottle it is transferred to together with callus agglomerate, plantlet
On, after cultivating 120 days under dark condition, plantlet breaks up in a large number, differentiation rate 97%, and average each outer implant is divided
Dissolve 40 plantlets；Plantlet is separated in outer implant, is that 1800lux, 12h illumination/12h is black in illumination
Successive transfer culture is carried out under dark illumination condition.
Subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter subculture
Culture medium adds 4.43gMS powder, possibly together with the sucrose of 60g/L, 6g/L agar, 0.1mg/L6-in subculture medium
Benzyl aminoadenine (6-benzyladenine, 6-BA), 0.1mg/L α-naphthaleneacetic acid (1-Naphthaleneacetic acid,
NAA), the pH value of subculture medium is 5.8.
(5) plantlet in step (3) grows 4 weeks on subculture medium, and bulb average diameter, at 1.4cm, reaches
To more than 1cm, plantlet is directly carried out bottle outlet plantation.Plantlet on subculture medium is placed in 4 degrees Celsius of dark
Under the conditions of carry out K cryogenic treatment 50 days；By the whole taking-up of plantlet in group culture container, rinse under flowing water, clean root
The culture medium in portion, then plantlet is put in the string bag, soaking disinfection 30min in 1000 times of solution of carbendazim, then
Planting in nursery dedicated medium (Custom growing mix, Fafard), the degree of depth that plantlet kind enters in seedling medium exists
2cm, carries out cultivation management.Plantlet robust growth.Generally, this system propagation efficiency reaches 1:18, the most averagely
Every piece of outer implant finally produces 18 plantlets.
Embodiment 3 is that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with east lilium Sorbonne gynoecium
(1) bud stage takes the Bulbus Lilii tender petal of children of 1cm length, the bennet of band 1.3cm length, be enclosed in pocket cold preservation in
4 degrees Celsius of refrigerators, cold preservation is taken out outer implant after one week and is processed.
(2) petal is soaked in the tap water with the addition of detergent 5min, under flowing water, rinse 120min, clean dry
After Jing, with the addition of 0.1%(v/v on superclean bench) 1%(w/v of tween) liquor natrii hypochloritis carries out surface
Sterilization 15min, then uses aseptic water washing 3 times.Gynoecium is peeled off from alabastrum, excises stigma, be inoculated in step 2
On the culture dish filling calli induction media of middle making, wherein, the inoculation of gynoecium is as the criterion with contact media surface；
By be vaccinated with the culture medium of outer implant 25 degrees Celsius, cultivate 16 days under dark condition, callus agglomerate occurs, dirty
Dye rate is 0.Callus induction rate reaches 81.7%.
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.25mg/L
2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D)；The pH value of calli induction media solution
It is 5.8.
If after wound healing agglomerate cultivates 130 days on calli induction media, diameter is maximum up to 7.00mm.Wherein, more
Hinder agglomerate size with vernier caliper measurement, owing to wound healing agglomerate is subsphaeroidal or length and width are equal, therefore using its diameter as wound healing group
Block size criterion, and along with the differentiation of plantlet.
(3), after outer implant grows 120 days on calli induction media, start to differentiate plantlet.By outer implant even
On superclean bench, the fresh calli induction media in cylindricality bottle it is transferred to together with callus agglomerate, plantlet
On, after cultivating 100 days under dark condition, plantlet breaks up in a large number, differentiation rate 95%, and average each outer implant is divided
Dissolve 10 plantlets；Plantlet is separated in outer implant by superclean bench, illumination be 1800lux,
Successive transfer culture is carried out under the illumination condition that 12h illumination/12h is dark.
Subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter subculture
Culture medium adds 4.43gMS powder, and subculture medium is basic with MS culture medium (Murashige and Skoog, 1962)
Culture medium, i.e. every liter subculture medium adds 4.43gMS powder, possibly together with sucrose, the 4g/L of 60g/L in subculture medium
Agar, 0.2mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.2mg/L α-naphthaleneacetic acid
(1-Naphthaleneacetic acid, NAA), the pH value of subculture medium is 5.8.
(4) plantlet in step (3) grows 4 weeks on subculture medium, and bulb average diameter, at 1.3cm, reaches
To more than 1cm, plantlet is directly carried out bottle outlet plantation.Plantlet on subculture medium is placed in 1 degree Celsius of dark
Under the conditions of carry out K cryogenic treatment 30 days；By the whole taking-up of plantlet in group culture container, rinse under flowing water, clean root
The culture medium in portion, then plantlet is put in the string bag, soaking disinfection 15min in 1000 times of solution of carbendazim, then
Planting in nursery dedicated medium (Custom growing mix, Fafard), the degree of depth that plantlet kind enters in seedling medium exists
1.5cm, carries out cultivation management.Plantlet robust growth, survival rate 100%.This system propagation efficiency reaches 1:10,
The most every piece outer implant finally produces 10 plantlets.
Embodiment 4 is that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with east lilium Sorbonne gynoecium
(1) bud stage takes the Bulbus Lilii tender petal of children of 0.5cm length, the bennet of band 1.3cm length, is enclosed in cold preservation in pocket
In 4 degrees Celsius of refrigerators, cold preservation is taken out outer implant after one week and is processed.
(2) petal is soaked in the tap water with the addition of detergent 5min, under flowing water, rinse 120min, clean dry
After Jing, with the addition of 0.1%(v/v on superclean bench) 1%(w/v of tween) liquor natrii hypochloritis carries out surface
Sterilization 15min, then uses aseptic water washing 3 times.Gynoecium is peeled off from alabastrum, excises stigma, be inoculated in step 2
On the culture dish filling calli induction media of middle making, wherein, the inoculation of gynoecium is as the criterion with contact media surface；
By be vaccinated with the culture medium of outer implant 25 degrees Celsius, cultivate 16 days under dark condition, callus agglomerate occurs, dirty
Dye rate is 0.Callus induction rate reaches 81.7%.
Calli induction media is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. uses
In every liter of pure water dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution possibly together with
The sucrose of 30g/L, 5g/L agar, 0.25mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.25mg/L
2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D)；The pH value of calli induction media solution
It is 5.8.
If after wound healing agglomerate cultivates 130 days on calli induction media, diameter is maximum up to 7.00mm.Wherein, more
Hinder agglomerate size with vernier caliper measurement, owing to wound healing agglomerate is subsphaeroidal or length and width are equal, therefore using its diameter as wound healing group
Block size criterion, and along with the differentiation of plantlet.
(3), after outer implant grows 120 days on calli induction media, start to differentiate plantlet.By outer implant even
On superclean bench, the fresh calli induction media in cylindricality bottle it is transferred to together with callus agglomerate, plantlet
On, after cultivating 100 days under dark condition, plantlet breaks up in a large number, differentiation rate 95%, and average each outer implant is divided
Dissolve 10 plantlets；Plantlet is separated in outer implant by superclean bench, illumination be 1800lux,
Successive transfer culture is carried out under the illumination condition that 12h illumination/12h is dark.
Subculture medium is minimal medium with MS culture medium (Murashige and Skoog, 1962), i.e. every liter subculture
Culture medium adds 4.43gMS powder, and subculture medium is basic with MS culture medium (Murashige and Skoog, 1962)
Culture medium, i.e. every liter subculture medium adds 4.43gMS powder, possibly together with sucrose, the 4g/L of 60g/L in subculture medium
Agar, 0.2mg/L6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.2mg/L α-naphthaleneacetic acid
(1-Naphthaleneacetic acid, NAA), the pH value of subculture medium is 5.8.
(4) plantlet in step (3) grows 4 weeks on subculture medium, and bulb average diameter, at 1.3cm, reaches
To more than 1cm, part plantlet not bottle outlet, continuing to Bulbus Lilii in vitro bulb regenerating system, the method carrying out subculture is:
Plantlet grows on subculture medium, and base is taken root robust growth, root upper end turn green after, carry out the switching of plantlet；
Forwarding method is: on superclean bench, is taken out by plantlet, and by its root complete resection, blade also excises from base portion,
Only retain clove and a small amount of plateau, clove is transferred on the fresh subculture medium that configures by step (3)；
(5) clove in step (4) regenerates plantlet on subculture medium, carries out the plantlet regenerated
Bottle outlet is planted.Plantlet on subculture medium is placed under 1 degree Celsius of dark condition and carries out K cryogenic treatment 30 days；Will
The whole taking-up of plantlet in group culture container, rinses under flowing water, cleans the culture medium of root, then plantlet is put into net
In pocket, soaking disinfection 15min in 1000 times of solution of carbendazim, then plants into nursery dedicated medium (Custom growing
Mix, Fafard) in, plantlet kind enters the degree of depth in seedling medium at 1.5cm, carries out cultivation management.Plantlet grows
Stalwartness, survival rate 100%.This system propagation efficiency reaches 1:10, the most every piece outer implant finally produce 10 little
Plant.
It is only the specific embodiment of the present invention finally it should be noted that listed above.Obviously, the present invention does not limits
In above example, it is also possible to there is many variations.Those of ordinary skill in the art can be from present disclosure
The all deformation directly derived or associate, are all considered as protection scope of the present invention.
Claims (2)
1. one kind is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium, it is characterised in that comprise the following steps:
Step one: material is taked:
At the Bulbus Lilii florescence, take the tender alabastrum of children of Bulbus Lilii length 0.5～2.0cm, after being placed in sealed bag in the refrigerator of 4 degrees Celsius cold preservation 1 week, then carry out subsequent operation；
Step 2: calli induction media configures:
Calli induction media is with MS culture medium (Murashige
And Skoog, 1962) it is minimal medium, i.e. use and every liter of pure water dissolves 4.43gMS powder as medium base liquid, in calli induction media solution possibly together with the sucrose of 30g/L, 5g/L agar, 0.25 mg/L 6-benzyl aminoadenine (6-benzyladenine, 6-BA), 0.25～1.5 mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), the pH value of calli induction media solution is 5.8；
After the calli induction media solution sterilization that will have configured, superclean bench carries out subpackage, will pour in culture dish by calli induction media solution, after calli induction media solution is frozen into calli induction media, the culture dish that at least one fills calli induction media with preservative film or tinfoil wraps, and is placed under aseptic environment and deposits；
Step 3: the inoculation of outer implant and callus induction:
Alabastrum after processing in step one, soaks 5～30min in the tap water with the addition of detergent, then flushing 0.5～2 h cleans up under flowing water, and then carry out disinfection on superclean bench inoculation；
Sterilization method is as follows: with sodium hypochlorite (NaClO) solution of 1% w/v that with the addition of 0.1% v/v tween, the alabastrum cleaned up carries out surface sterilization 8～15min, then uses aseptic water washing 3 times；
Inoculate after sterilization: with tweezers and dissecting knife, alabastrum is separated, gynoecium is peeled off from alabastrum, excises stigma, the gynoecium having excised stigma is inoculated in step 2 on the culture dish filling calli induction media made, wherein, the inoculation of gynoecium is as the criterion with contact media surface；By be vaccinated with the culture medium of outer implant 25 degrees Celsius, cultivate 8～16 days under dark condition, callus agglomerate occurs；
Step 4: plantlet regenerates:
Outer implant is cultivated 120 days on calli induction media, and the callus that part calli induction media is cultivated starts differentiation and regeneration and goes out complete plantlet, and the callus that part calli induction media is cultivated still keeps dedifferentiation state；Outer implant being transferred on fresh calli induction media together with callus agglomerate, plantlet, after cultivating 90～120 days under the conditions of complete darkness, plantlet breaks up in a large number, differentiation rate 95～100%, and average each outer implant differentiates 10～40 plantlets；Plantlet is separated from callus agglomerate by superclean bench, proceeds to subculture medium and cultivate；
After plantlet grows 4 weeks on subculture medium, not bottle outlet, proceed the successive transfer culture in step 5 to maintain Bulbus Lilii in vitro bulb regenerating system, then carry out the bottle outlet plantation in step 6, every 4 weeks subcultures are once；Or directly carry out the bottle outlet plantation in step 6；
Described subculture medium is with MS culture medium (Murashige
And Skoog, 1962) it is minimal medium, i.e. every liter subculture medium is added with 4.43gMS powder, possibly together with sucrose, 4～8g/L agar, 0～0.2 mg/L 6-benzyl aminoadenine (6-benzyladenine of 60g/L in subculture medium, 6-BA), 0～0.2 mg/L α-naphthaleneacetic acid (1-Naphthaleneacetic acid
NAA), the pH value of subculture medium is 5.8；
Step 5: the successive transfer culture of plantlet:
After plantlet in step 4 grows 4 weeks on subculture medium, not bottle outlet, the method carrying out subculture is: plantlet grows on subculture medium, and base is taken root robust growth, root upper end turn green after, carry out the switching of plantlet；Forwarding method is: on superclean bench, is taken out by plantlet, and by its root complete resection, blade also excises from base portion, only retains clove and a small amount of plateau, is transferred to by clove on fresh subculture medium, continues the cultivation of step 4；
Step 6: the bottle outlet plantation of plantlet:
Carry out cold acclimation before bottle outlet, the plantlet will cultivated on the subculture medium in group culture container, 1～5 degree Celsius, carry out the K cryogenic treatment of 30～50 days under dark condition；After K cryogenic treatment, by the whole taking-up of plantlet in group culture container, rinse under flowing water, clean the culture medium of root, then plantlet is put in the string bag, soaking disinfection 15～30min in 1000 times of solution of carbendazim, then planting in nursery dedicated medium, plantlet kind enters the degree of depth in nursery dedicated medium in the range of 1～2cm, and i.e. clove top is away from about soil table one ball height, carry out cultivation management, i.e. complete the foundation of Bulbus Lilii embryo callus subculture regenerating system；
And in the foundation of Bulbus Lilii embryo callus subculture regenerating system, pollution rate is 0, Callus induction rate reaches 100%, and wound healing agglomerate regeneration rate is 100%, and each gynoecium can obtain 10～40 strain plantlets.
The most according to claim 1 a kind of be the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with gynoecium, it is characterised in that described nursery dedicated medium use Custom growing mix, Fafard.
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