CN103798138A - Method for establishing lily embryogenic callus regeneration system by using pistils as explants - Google Patents
Method for establishing lily embryogenic callus regeneration system by using pistils as explants Download PDFInfo
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- CN103798138A CN103798138A CN201410037331.2A CN201410037331A CN103798138A CN 103798138 A CN103798138 A CN 103798138A CN 201410037331 A CN201410037331 A CN 201410037331A CN 103798138 A CN103798138 A CN 103798138A
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Abstract
The invention relates to the technical field of quick reproduction of plants, and aims to provide a method for establishing a lily embryogenic callus regeneration system by using pistils as explants. The method comprises the following steps: material acquisition, callus-induced culture medium preparation, explant inoculation and callus induction, small plant regeneration, successive transfer culture of small plants, and transplanting of small plants. The method has the characteristics of low pollution rate, high callus inductivity, large callus conglomeration, favorable callus embryogenic property, high regeneration capacity and high reproducibility; especially, as pistils in the young bud are used as the explants, the pollution rate can be lowered to 0, the callus inductivity of up to 100% can be obtained, the callus conglomeration diameter can be approximate to 1cm after culture for 130 days, the callus conglomeration reproducibility is 100%, the embryogenic property is high, and each pistil can obtain 10-40 small plants on the whole.
Description
Technical field
The invention relates to micropropagation of plants technical field, particularly a kind of method of setting up lily embryo callus subculture regenerating system take gynoecium as explant.
Background technology
Lily embryo callus regenerating system is compared other regenerating systems as shoot regeneration system, has the highest rate of increase and expands numerous rate, to the significance of preserving of elite germplasm.In addition, lily embryo callus regenerating system is also the decisive basis of lily genetic conversion system, and then affects the gene engineering improvement of lily.
Setting up at present lily callus regenerating system is explant mainly with the scale of field bulb, and its defect is bulb due to for a long time in soil environment, carries germ many, makes tissue cultivate sterilization and requires strictly, and pollution rate is higher.In addition, in the time need to setting up the laboratory in vitro system of Lilium Germplasm germplasm, take bulb as explant, after can only adopting the florescence, take kind of a ball, its outstanding deficiency has two: first, determine and the identification of kind of Lilium Germplasm position are largely to rely on eye-catching floral organ and feature thereof, and after the florescence, Lilium Germplasm acrial part is all or part of withered, especially decaying of floral organ comes off and makes its kind almost illegible with tepal, easily causes obscuring of germ plasm resource; Secondly, take kind of a ball and be equal to wild resource is gathered completely, be difficult to especially the destruction of replying for Precious, Rare, Endangered kind.
Summary of the invention
Main purpose of the present invention is to overcome deficiency of the prior art, provide that a kind of pollution rate is low, callus of induce rate is high, callus agglomerate is large, callus embryo is good, regeneration capacity is strong, the rate of increase is high, and the method for setting up lily embryo callus subculture regenerating system little to the destruction of germ plasm resource.For solving the problems of the technologies described above, solution of the present invention is:
A kind of method of setting up lily embryo callus subculture regenerating system take gynoecium as explant is provided, comprises the following steps:
Step 1: material is taked:
At the lily florescence, get (children is tender) bud of the long 0.5～2.0cm of lily, be placed in sealed bag and hide after 1 week the refrigerator and cooled of 4 degrees Celsius, then carry out subsequent operation;
Step 2: calli induction media configuration:
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 2 of 0.25～1.5mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), the pH value of calli induction media solution is 5.8;
By after the calli induction media solution sterilization configuring, on superclean bench, carry out packing, pour into by calli induction media solution in (glass or plastics) culture dish, become after calli induction media until calli induction media solution solidifies, wrap with the culture dish that at least one is filled calli induction media by preservative film or tinfoil, be placed under aseptic clean environment (as group training chamber) and deposit;
Step 3: explant inoculation and callus induction:
By bud after treatment in step 1, in the running water that has added detergent, soak 5～30min, then under flowing water, rinse 0.5～2h and clean up, inoculation then carries out disinfection on superclean bench;
Sterilization method is as follows: use clorox (NaClO) solution of the 1%w/v that has added 0.1%v/v tween, the bud cleaning up is carried out to surface sterilization 8～15min, then use aseptic water washing 3 times;
After sterilization, inoculate: bud is separated with tweezers and scalpel, gynoecium is peeled off from bud, and excision column cap, is inoculated in the gynoecium that has excised column cap on the culture dish that fills calli induction media of making in step 2, wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 8～16 days under 25 degrees Celsius, dark condition, occurred callus agglomerate;
Step 4: plantlet regeneration:
Explant is cultivated 120 days on calli induction media, and the callus of cultivating on part calli induction media starts differentiation and regeneration and goes out complete plantlet, and the callus of cultivating on part calli induction media still keeps dedifferentiation state; Explant is transferred on fresh calli induction media together with callus agglomerate, plantlet, under complete darkness condition, cultivate after 90～120 days, plantlet breaks up in a large number, and differentiation rate 95～100% differentiates 10～40 plantlets on average each explant; On superclean bench, plantlet is separated from callus agglomerate, proceed to subculture medium and cultivate;
Plantlet is grown after 4 weeks (average diameter reach 1cm more than) on subculture medium, not bottle outlet, and the subculture of proceeding in step 5 cultivates to maintain the in vitro bulb regenerating system of lily, then carries out the Planting out of test-tube in step 6, and every 4 weeks subcultures are once; Also can directly carry out the Planting out of test-tube in step 6;
Described subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be in every liter of subculture medium, to be added with 4.43gMS powder, in subculture medium, also contain sucrose, 4～8g/L agar, the 0～0.2mg/L6-benzyl aminoadenine (6-benzyladenine of 60g/L, 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 optional, but certain density methyl α-naphthyl acetate will be conducive to plantlet root growth, and 6-benzyl aminoadenine is optional, but certain density 6-benzyl aminoadenine will be conducive to plantlet propagation)
Step 5: the subculture of plantlet is cultivated:
Plantlet in step 4 grow on subculture medium after 4 weeks (average diameter reach 1cm more than), not bottle outlet, the method for carrying out subculture is: plantlet is grown on subculture medium, the base robust growth of taking root, root upper end turn green after, carry out the switching of plantlet; Forwarding method is: on superclean bench, plantlet is taken out, by its root complete resection, blade, also from base portion excision, only retains clove and a small amount of plateau, and clove is transferred on fresh subculture medium, continues the cultivation of step 4;
Step 6: the Planting out of test-tube of plantlet:
Before bottle outlet, carry out cold acclimation, the plantlet of cultivating on the subculture medium by group in culture container carries out the low temperature treatment of 30～50 days under 1～5 degree Celsius, dark condition; After low temperature treatment, by the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 15～30min in 1000 times of solution of carbendazim, then plant into the dedicated medium of growing seedlings (as Custom growing mix, Fafard) in, the degree of depth that plantlet kind enters to grow seedlings in dedicated medium is within the scope of 1～2cm, be clove top apart from native table one ball height left and right, carry out cultivation management, complete the foundation of lily embryo callus subculture regenerating system.
A kind of method of setting up lily embryo callus subculture genetic conversion system take gynoecium as explant is provided, the callus agglomerate that in step 3, induction obtains is cut or press from both sides with tweezers, as the acceptor of genetic conversion system, through Agrobacterium transfection or particle gun mediated transformation, through screening and identification, and the transfer-gen plant that differentiation is taken root, hardening, transplanting obtain lily on regeneration culture medium;
Regeneration culture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of regeneration culture medium adds 4.43gMS powder, in regeneration culture medium, also contain sucrose, 4～8g/L agar, 0～2.0mg/L α-naphthaleneacetic acid (the 1-Naphthaleneacetic acid of 30～60g/L, NAA), the pH value of regeneration culture medium is 5.8～6.0.
Said method provided by the invention can, in lily callus regeneration system and genetic conversion system, be specially: the callus that said method is obtained carries out cutting into fritter after subculture cultivation, is inoculated on corresponding subculture medium and cultivates; On regeneration culture medium, break up and take root with this callus, hardening, transplant the tissue that can obtain lily and cultivate plant; The maybe acceptor using this callus as genetic conversion system breaks up and takes root on regeneration culture medium, and hardening, transplanting can obtain the transfer-gen plant of lily.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention, take lily gynoecium as explant, has the advantages that pollution rate is low, material is easy to get, and its outstanding advantage is that system high efficiency, explant children are tender, less virus.Germ plasm resource for Lilium Germplasm is preserved, obtain appropriate gynoecium for setting up laboratory callus regenerating system at the lily florescence, rather than after the florescence, take kind of a ball, more easily confirm the kind of Lilium Germplasm with gynoecium feature, and greatly reduced the destruction to wild germplasm resource.The present invention also gives prominence to has the advantages that pollution rate is low, callus of induce rate is high, callus agglomerate large, callus embryo is good, regeneration capacity is strong, the rate of increase is high, especially for the present invention take the gynoecium in the tender petal of children as explant, pollution rate can be reduced to 0, can obtain the callus of induce rate up to 100%, cultivate 130 days callus agglomerate diameters and can reach nearly 1cm, callus agglomerate regeneration rate 100%, embryo is high, and each gynoecium can obtain 10-40 strain plantlet generally.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
A method of setting up lily embryo callus subculture regenerating system take gynoecium as explant, comprises the following steps:
Step 1: material is taked:
At the lily florescence, get the tender bud of children of the long 0.5～2.0cm of lily, be placed in sealed bag and hide after 1 week the refrigerator and cooled of 4 degrees Celsius, then carry out subsequent operation.
Step 2: calli induction media configuration:
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 0.25～1.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D); The pH value of calli induction media solution is 5.8.
By after the calli induction media solution sterilization configuring, on superclean bench, carry out packing, pour into by calli induction media solution in the culture dish of glass or plastics, become after calli induction media until calli induction media solution solidifies, wrap with the culture dish that at least one is filled calli induction media by preservative film or tinfoil, be placed under group training chamber or other aseptic clean environments and deposit.
Step 3: explant inoculation and callus induction:
By bud after treatment in step 1, in the running water that has added detergent, soak 5～30min, then under flowing water, rinse 0.5～2h and clean up, inoculation then carries out disinfection on superclean bench;
Sterilization method is as follows: use clorox (NaClO) solution of the 1%w/v that has added 0.1%v/v tween, the bud cleaning up is carried out to surface sterilization 8～15min, then use aseptic water washing 3 times;
After sterilization, inoculate: bud is separated with tweezers and scalpel, gynoecium is peeled off from bud, and excision column cap, is inoculated in the gynoecium that has excised column cap on the culture dish that fills calli induction media of making in step 2, wherein, the inoculation of gynoecium is as the criterion with contact media surface.The medium of having inoculated explant is cultivated 8～16 days under 25 degrees Celsius, dark condition, occurred callus agglomerate.
Step 4: plantlet regeneration:
Explant was grown after 120 days on calli induction media, and the callus of cultivating on part calli induction media starts differentiation and regeneration and goes out complete plantlet, and the callus of cultivating on part calli induction media still keeps dedifferentiation state.Explant is transferred on fresh calli induction media together with callus agglomerate, plantlet, under complete darkness condition, cultivates after 90～120 days, plantlet breaks up in a large number, and differentiation rate 95-100% differentiates 10-40 plantlet on average each explant.On superclean bench, plantlet is separated from callus agglomerate, proceed to subculture medium;
Plantlet was grown after 4 weeks on subculture medium, more than average diameter reaches 1cm, and not bottle outlet, the subculture of proceeding in step 5 cultivates to maintain the in vitro bulb regenerating system of lily, then carries out the Planting out of test-tube in step 6, and every 4 weeks subcultures are once; Also can directly carry out the Planting out of test-tube in step 6.
Described subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be in every liter of subculture medium, to be added with 4.43gMS powder, in subculture medium, also contain sucrose, 4～8g/L agar, the 0～0.2mg/L6-benzyl aminoadenine (6-benzyladenine of 60g/L, 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 optional, but certain density methyl α-naphthyl acetate will be conducive to plantlet root growth, and 6-benzyl aminoadenine is optional, but certain density 6-benzyl aminoadenine will be conducive to plantlet propagation.
Step 5: the subculture of plantlet is cultivated:
Plantlet in step 4 was grown after 4 weeks on subculture medium, more than average diameter reaches 1cm, not bottle outlet, the method for carrying out subculture is: plantlet is grown on subculture medium, the base robust growth of taking root, root upper end turn green after, carry out the switching of plantlet; Forwarding method is: on superclean bench, plantlet is taken out, by its root complete resection, blade, also from base portion excision, only retains clove and a small amount of plateau, and clove is transferred on fresh subculture medium, continues the cultivation of step 4.
Step 6: the Planting out of test-tube of plantlet:
Before bottle outlet, carry out cold acclimation, the plantlet of cultivating on the subculture medium by group in culture container carries out the low temperature treatment of 30～50 days under 1～5 degree Celsius, dark condition; After low temperature treatment, by the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 15～30min in 1000 times of solution of carbendazim, then plant into the dedicated medium of growing seedlings (as Custom growing mix, Fafard) in, the degree of depth that plantlet kind enters to grow seedlings in dedicated medium is within the scope of 1～2cm, be clove top apart from native table one ball height left and right, carry out cultivation management, complete the foundation of lily embryo callus subculture regenerating system.
The following examples can make this professional professional and technical personnel's comprehend the present invention, but do not limit the present invention in any way.
Embodiment 1 sets up lily embryo callus subculture regenerating system take east lilium Sorbonne gynoecium as explant
(1) bud stage is got the long tender petal of lily children of 1.7cm, and the bennet of belt length 1cm refrigerates in 4 degrees Celsius of refrigerators in sealed bag, refrigerates one week rear taking-up and processes.
(2) petal is soaked to 30min in the running water that has added detergent, under flowing water, rinse 75min, after cleaning up, on superclean bench, use the 1%w/v liquor natrii hypochloritis who has added 0.1%v/v tween, carry out surface sterilization 8min, then use aseptic water washing 3 times.Gynoecium is peeled off from bud, and excision column cap, is inoculated on the culture dish that fills calli induction media of making in step 2, and wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 8 days under 25 degrees Celsius, dark condition, occurred callus agglomerate, pollution rate is 0.Callus induction rate reaches 96%.
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 1.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D); The pH value of calli induction media solution is 5.8.
If cultivating diameter maximum after 130 days on calli induction media, callus agglomerate can reach 9.81mm.Wherein, callus agglomerate size is with vernier caliper measurement, because callus agglomerate is subsphaeroidal or length and width equate, therefore using its diameter as callus agglomerate size criterion, and be accompanied by the differentiation of plantlet.
(3) explant was grown after 120 days on calli induction media, started to differentiate plantlet.Explant is transferred on the fresh calli induction media in cylindricality bottle together with callus agglomerate, plantlet on superclean bench, under dark condition, cultivate 90 days, continue to differentiate a large amount of plantlets, average every explant can differentiate 25 plantlets, and differentiation rate is 100%.On superclean bench, plantlet being separated from explant, be transferred on fresh subculture medium, is under the illumination condition of 1800lux, 12h illumination/12h dark in illumination, carries out subculture cultivation.
Subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, also contain sucrose, the 8g/L agar of 60g/L in subculture medium, the pH value of subculture medium is 5.8.
(4) plantlet in step (3) is grown 4 weeks on subculture medium, and more than bulb average diameter reaches 1cm, plantlet directly carries out Planting out of test-tube.Plantlet on subculture medium is placed under 5 degrees Celsius of dark conditions and carries out low temperature treatment 40 days; By the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 25min in 1000 times of solution of carbendazim, then plants in the dedicated medium of growing seedlings (Custom growing mix, Fafard), plantlet kind enters the degree of depth in seedling medium at 1cm, carries out cultivation management.Plantlet robust growth, survival rate 100%.This system proliferate efficiency reaches 1:25, i.e. 25 plantlets of the final generation of average every explant.
Embodiment 2 sets up lily embryo callus subculture regenerating system take east lilium Sorbonne gynoecium as explant
(1) bud stage is got the long tender petal of lily children of 2cm, with the bennet of 1.5cm length, is enclosed in pocket and refrigerates in 4 degrees Celsius of refrigerator cold-storages after one week, then carry out subsequent operation.
(2) petal is soaked to 18min in the running water that has added detergent, under flowing water, rinse 30min, after cleaning up, on superclean bench with having added 0.1%(v/v) 1% (w/v) liquor natrii hypochloritis of tween carries out surface sterilization 12min, then uses aseptic water washing 3 times.Gynoecium is peeled off from bud, and excision column cap, is inoculated on the culture dish that fills calli induction media of making in step 2, and wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 13 days under 25 degrees Celsius, dark condition, occurred callus agglomerate, pollution rate is 0.Callus induction rate reaches 88.7%.
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 0.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D); The pH value of calli induction media solution is 5.8.
If cultivating diameter maximum after 130 days on calli induction media, callus agglomerate can reach 6.85mm.Wherein, callus agglomerate size is with vernier caliper measurement, because callus agglomerate is subsphaeroidal or length and width equate, therefore using its diameter as callus agglomerate size criterion, and be accompanied by the differentiation of plantlet.
(3) explant was grown after 120 days on calli induction media, started to differentiate plantlet.Explant is transferred on the fresh calli induction media in cylindricality bottle together with callus agglomerate, plantlet on superclean bench, under dark condition, cultivate after 120 days, plantlet breaks up in a large number, and differentiation rate 97% differentiates 40 plantlets on average each explant; Plantlet is separated from explant, is to carry out subculture cultivation under the illumination condition of 1800lux, 12h illumination/12h dark in illumination.
Subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, in subculture medium, also contain sucrose, 6g/L agar, the 0.1mg/L6-benzyl aminoadenine (6-benzyladenine of 60g/L, 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) is grown 4 weeks on subculture medium, and bulb average diameter, at 1.4cm, more than reaching 1cm, is directly carried out Planting out of test-tube to plantlet.Plantlet on subculture medium is placed under 4 degrees Celsius of dark conditions and carries out low temperature treatment 50 days; By the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 30min in 1000 times of solution of carbendazim, then plants in the dedicated medium of growing seedlings (Custom growing mix, Fafard), plantlet kind enters the degree of depth in seedling medium at 2cm, carries out cultivation management.Plantlet robust growth.Generally, this system proliferate efficiency reaches 1:18, i.e. 18 plantlets of the final generation of average every explant.
Embodiment 3 sets up lily embryo callus subculture regenerating system take east lilium Sorbonne gynoecium as explant
(1) bud stage is got the long tender petal of lily children of 1cm, with the bennet of 1.3cm length, is enclosed in pocket and refrigerates in 4 degrees Celsius of refrigerators, refrigerates one week rear explant that takes out and processes.
(2) petal is soaked to 5min in the running water that has added detergent, under flowing water, rinse 120min, after cleaning up, on superclean bench with having added 0.1%(v/v) 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 bud, and excision column cap, is inoculated on the culture dish that fills calli induction media of making in step 2, and wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 16 days under 25 degrees Celsius, dark condition, occurred callus agglomerate, pollution rate is 0.Callus induction rate reaches 81.7%.
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 0.25mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D); The pH value of calli induction media solution is 5.8.
If callus agglomerate was cultivated after 130 days on calli induction media, diameter maximum can reach 7.00mm.Wherein, callus agglomerate size is with vernier caliper measurement, because callus agglomerate is subsphaeroidal or length and width equate, therefore using its diameter as callus agglomerate size criterion, and be accompanied by the differentiation of plantlet.
(3) explant was grown after 120 days on calli induction media, started to differentiate plantlet.Explant is transferred on the fresh calli induction media in cylindricality bottle together with callus agglomerate, plantlet on superclean bench, under dark condition, cultivate after 100 days, plantlet breaks up in a large number, and differentiation rate 95% differentiates 10 plantlets on average each explant; On superclean bench, plantlet being separated from explant, is to carry out subculture cultivation under the illumination condition of 1800lux, 12h illumination/12h dark in illumination.
Subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, in subculture medium, also contain the sucrose of 60g/L, 4g/L 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) is grown 4 weeks on subculture medium, and bulb average diameter, at 1.3cm, more than reaching 1cm, is directly carried out Planting out of test-tube to plantlet.Plantlet on subculture medium is placed under 1 degree Celsius of dark condition and carries out low temperature treatment 30 days; By the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 15min in 1000 times of solution of carbendazim, then plants in the dedicated medium of growing seedlings (Custom growing mix, Fafard), plantlet kind enters the degree of depth in seedling medium at 1.5cm, carries out cultivation management.Plantlet robust growth, survival rate 100%.This system proliferate efficiency reaches 1:10, i.e. 10 plantlets of the final generation of average every explant.
Embodiment 4 sets up lily embryo callus subculture regenerating system take east lilium Sorbonne gynoecium as explant
(1) bud stage is got the long tender petal of lily children of 0.5cm, with the bennet of 1.3cm length, is enclosed in pocket and refrigerates in 4 degrees Celsius of refrigerators, refrigerates one week rear explant that takes out and processes.
(2) petal is soaked to 5min in the running water that has added detergent, under flowing water, rinse 120min, after cleaning up, on superclean bench with having added 0.1%(v/v) 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 bud, and excision column cap, is inoculated on the culture dish that fills calli induction media of making in step 2, and wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 16 days under 25 degrees Celsius, dark condition, occurred callus agglomerate, pollution rate is 0.Callus induction rate reaches 81.7%.
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 0.25mg/L 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D); The pH value of calli induction media solution is 5.8.
If callus agglomerate was cultivated after 130 days on calli induction media, diameter maximum can reach 7.00mm.Wherein, callus agglomerate size is with vernier caliper measurement, because callus agglomerate is subsphaeroidal or length and width equate, therefore using its diameter as callus agglomerate size criterion, and be accompanied by the differentiation of plantlet.
(3) explant was grown after 120 days on calli induction media, started to differentiate plantlet.Explant is transferred on the fresh calli induction media in cylindricality bottle together with callus agglomerate, plantlet on superclean bench, under dark condition, cultivate after 100 days, plantlet breaks up in a large number, and differentiation rate 95% differentiates 10 plantlets on average each explant; On superclean bench, plantlet being separated from explant, is to carry out subculture cultivation under the illumination condition of 1800lux, 12h illumination/12h dark in illumination.
Subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be that every liter of subculture medium adds 4.43gMS powder, in subculture medium, also contain the sucrose of 60g/L, 4g/L 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) is grown 4 weeks on subculture medium, bulb average diameter is at 1.3cm, more than reaching 1cm, not bottle outlet of part plantlet, continue to maintain the in vitro bulb regenerating system of lily, the method for carrying out subculture is: plantlet is grown on subculture medium, the base robust growth of taking root, root upper end turn green after, carry out the switching of plantlet; Forwarding method is: on superclean bench, plantlet is taken out, by its root complete resection, blade, also from base portion excision, only retains clove and a small amount of plateau, and clove is transferred to by the fresh subculture medium of step (3) configuration;
(5) plantlet of regenerating on subculture medium of the clove in step (4), carries out Planting out of test-tube to the plantlet bearing again.Plantlet on subculture medium is placed under 1 degree Celsius of dark condition and carries out low temperature treatment 30 days; By the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 15min in 1000 times of solution of carbendazim, then plants in the dedicated medium of growing seedlings (Custom growing mix, Fafard), plantlet kind enters the degree of depth in seedling medium at 1.5cm, carries out cultivation management.Plantlet robust growth, survival rate 100%.This system proliferate efficiency reaches 1:10, i.e. 10 plantlets of the final generation of average every explant.
Finally, it should be noted that above what enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above embodiment, can also have a lot of distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (1)
1. a method of setting up lily embryo callus subculture regenerating system take gynoecium as explant, is characterized in that, comprises the following steps:
Step 1: material is taked:
At the lily florescence, get the bud of the long 0.5～2.0cm of lily, be placed in sealed bag and hide after 1 week the refrigerator and cooled of 4 degrees Celsius, then carry out subsequent operation;
Step 2: calli induction media configuration:
Calli induction media is with MS medium (Murashige and Skoog, 1962) be minimal medium, adopt in every liter of pure water and dissolve 4.43gMS powder as calli induction media solution, in calli induction media solution, also contain sucrose, 5g/L agar, the 0.25mg/L6-benzyl aminoadenine (6-benzyladenine of 30g/L, 6-BA), 2 of 0.25～1.5mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), the pH value of calli induction media solution is 5.8;
By after the calli induction media solution sterilization configuring, on superclean bench, carry out packing, pour in culture dish by calli induction media solution, become after calli induction media until calli induction media solution solidifies, wrap with the culture dish that at least one is filled calli induction media by preservative film or tinfoil, be placed under aseptic clean environment and deposit;
Step 3: explant inoculation and callus induction:
By bud after treatment in step 1, in the running water that has added detergent, soak 5～30min, then under flowing water, rinse 0.5～2h and clean up, inoculation then carries out disinfection on superclean bench;
Sterilization method is as follows: use clorox (NaClO) solution of the 1%w/v that has added 0.1%v/v tween, the bud cleaning up is carried out to surface sterilization 8～15min, then use aseptic water washing 3 times;
After sterilization, inoculate: bud is separated with tweezers and scalpel, gynoecium is peeled off from bud, and excision column cap, is inoculated in the gynoecium that has excised column cap on the culture dish that fills calli induction media of making in step 2, wherein, the inoculation of gynoecium is as the criterion with contact media surface; The medium of having inoculated explant is cultivated 8～16 days under 25 degrees Celsius, dark condition, occurred callus agglomerate;
Step 4: plantlet regeneration:
Explant is cultivated 120 days on calli induction media, and the callus of cultivating on part calli induction media starts differentiation and regeneration and goes out complete plantlet, and the callus of cultivating on part calli induction media still keeps dedifferentiation state; Explant is transferred on fresh calli induction media together with callus agglomerate, plantlet, under complete darkness condition, cultivate after 90～120 days, plantlet breaks up in a large number, and differentiation rate 95～100% differentiates 10～40 plantlets on average each explant; On superclean bench, plantlet is separated from callus agglomerate, proceed to subculture medium and cultivate;
Plantlet was grown after 4 weeks on subculture medium, not bottle outlet, and the subculture of proceeding in step 5 cultivates to maintain the in vitro bulb regenerating system of lily, then carries out the Planting out of test-tube in step 6, and every 4 weeks subcultures are once; Also can directly carry out the Planting out of test-tube in step 6;
Described subculture medium is with MS medium (Murashige and Skoog, 1962) be minimal medium, be in every liter of subculture medium, to be added with 4.43gMS powder, in subculture medium, also contain sucrose, 4～8g/L agar, the 0～0.2mg/L6-benzyl aminoadenine (6-benzyladenine of 60g/L, 6-BA), 0～0.2mg/L α-naphthaleneacetic acid (1-Naphthaleneacetic acid, NAA), the pH value of subculture medium is 5.8;
Step 5: the subculture of plantlet is cultivated:
Plantlet in step 4 was grown after 4 weeks on subculture medium, not bottle outlet, the method for carrying out subculture is: plantlet is grown on subculture medium, the base robust growth of taking root, root upper end turn green after, carry out the switching of plantlet; Forwarding method is: on superclean bench, plantlet is taken out, by its root complete resection, blade, also from base portion excision, only retains clove and a small amount of plateau, and clove is transferred on fresh subculture medium, continues the cultivation of step 4;
Step 6: the Planting out of test-tube of plantlet:
Before bottle outlet, carry out cold acclimation, the plantlet of cultivating on the subculture medium by group in culture container carries out the low temperature treatment of 30～50 days under 1～5 degree Celsius, dark condition; After low temperature treatment, by the whole taking-up of plantlet in group culture container, under flowing water, rinse, clean the medium of root, again plantlet is put into the string bag, soaking disinfection 15～30min in 1000 times of solution of carbendazim, then plant into the dedicated medium of growing seedlings (as Custom growing mix, Fafard) in, the degree of depth that plantlet kind enters to grow seedlings in dedicated medium is within the scope of 1～2cm, be clove top apart from native table one ball height left and right, carry out cultivation management, complete the foundation of lily embryo callus subculture regenerating system.
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| CN106258963A (en) * | 2016-08-11 | 2017-01-04 | 浙江大学 | A kind of is the method that outer implant sets up aquamaine flower clove regenerating system with stamen |
| CN106258969A (en) * | 2016-08-11 | 2017-01-04 | 浙江大学 | A kind of is the method that outer implant sets up aquamaine flower clove regenerating system with petal |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104255711A (en) * | 2014-09-15 | 2015-01-07 | 上海交通大学 | Method for improving preservation effect of lily embryonic callus |
| CN104255711B (en) * | 2014-09-15 | 2016-03-09 | 上海交通大学 | A kind of method improving lily embryo callus preservation effect |
| CN106258963A (en) * | 2016-08-11 | 2017-01-04 | 浙江大学 | A kind of is the method that outer implant sets up aquamaine flower clove regenerating system with stamen |
| CN106258969A (en) * | 2016-08-11 | 2017-01-04 | 浙江大学 | A kind of is the method that outer implant sets up aquamaine flower clove regenerating system with petal |
| CN106258963B (en) * | 2016-08-11 | 2018-06-29 | 浙江大学 | A kind of method that aquamaine flower clove regenerating system is established using stamen as explant |
| CN106258969B (en) * | 2016-08-11 | 2018-06-29 | 浙江大学 | A kind of method that aquamaine flower clove regenerating system is established using petal as explant |
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