CN104396742A - Five-step method for inducing lilium sulphureum bulbil callus to re-differentiate aseptic seedlings - Google Patents
Five-step method for inducing lilium sulphureum bulbil callus to re-differentiate aseptic seedlings Download PDFInfo
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Abstract
The invention relates to a five-step method for inducing lilium sulphureum bulbil callus to re-differentiate aseptic seedlings. According to the invention, lilium sulphureum bulbils are adopted as explants; through transition culturing, pollution-free explants are transferred to a callus culturing medium; a proliferation culturing medium is used for proliferating the callus; clustered buds are differentiated from the callus; and the clustered buds are transferred to a rooting and seedling-developing culturing medium. In each step, optimization is carried out upon different explant growth regulator types, concentrations and ratios. With the method, pollution-free explants can be obtained. Callus can be induced in 7 days, wherein an induction rate reaches 100%. The callus can be sub-cultured more than 10 times. Clustered buds can be induced in 15 days, wherein an induction rate reaches 98%. Robust aseptic seedlings with roots can be obtained in 14 days, wherein an induction rate reaches 100%. Compared to conventional propagation method of sowing, bulbil and scale cutting, and the like, the method provided by the invention has the advantages of short period, low cost, and high propagation coefficient.
Description
Technical field
The invention belongs to the tissue cultures of biological technical field, particularly relate to the production method differentiating aseptic seedling with five-step approach induction Lilium sulphureum Baker bulbil callus again.
Background technology
Lilium sulphureum Baker (Li lium sulphureum Baker) is wild species in Liliaceae lilium, originates in Guizhou Province, is perennial root herbaceous plant, is medicine-food two-purpose type, has higher economic worth.But owing to developing excessively, be in Critical Condition at present.Lilium sulphureum Baker is conventional with breedings such as bulb separation, scale cuttage, seeds, but reproduction coefficient is low, and long-term nutrition breeding easily causes virus infections thus has a strong impact on quality and yield, and tissue culture quick breeding is the effective way solved the problem.The existing report about different lily cultivar tissue cultures both at home and abroad, but less to the report of Lilium sulphureum Baker.
Li Dai (2012) etc. adopt wild Lilium sulphureum Baker bulb scale to be that material carries out cottage propagation research and shows, adopt Lilium sulphureum Baker middle level scale, moisturizing cuttage is carried out in the sealing of flowerpot mouth plastic film, and survival rate is to 86.66%, and value-added coefficient is 1.85; Zhang Wene (2008) etc., particle diameter is taked to be more than or equal to the bulbil of 1 cm with late August, the inducing effect being seeded in the indefinite bud on the 1/2MS medium of additional 0.1 mgLIBA after peelling off outer scale is best, type of culture medium and Subculture Time have remarkable impact to Multiplying culture, to transfer in 40 d, the cultivation effect of subculture on the medium of 1/2MS+0.1 mg/LKT+0.1 mg/LIBA is best, its callus induction rate reaches 97.3%, Bulblet induction rate is up to 100%, and indefinite bud robust growth.On 1/2MS+0.1 mg/LIBA medium, growth and the situation of taking root are well; Zhou Hongying (2007) etc. adopt humus soil to be matrix, and the cutting of " ten " font is sent out, and Lilium sulphureum Baker monolithic bulb reproduction coefficient can reach more than 4; Yang Xueqing (2007) etc. with the scale of Lilium sulphureum Baker, blade, petiole for explant, establish the fast asexual propagation system of Lilium sulphureum Baker, scale is best explant, MS+2. 0mg/L 6-BA+0. 3mg/L NAA is the best inducing culture of clove, inductivity is up to 95. 0 %, average clove growth coefficient reaches as high as 8. 6, and the best Bulblet induction medium of blade, petiole is MS+2.0mg/L6-BA+0.5mg/L NA A; Li Dai (2005) etc. are minimal medium with MS, research variable concentrations sucrose, 6-BA and NAA are on the impact of Lilium sulphureum Baker Shoot propagation and callus growth, and result shows: the optimal medium of Shoot propagation is MS+0.5mg/L6-BA+0.5mg/L NAA+4% sucrose.The optimum medium of callus proliferation is MS+0.5mg/L6-BA+1mg/L NAA+3% sucrose; Qiu Ninghong (2004) etc. are with the scale of Lilium sulphureum Baker for explant carries out Tube propagation, and the medium filtering out each cultivation stage suitable is respectively: 1) inducing clumping bud, MS+2.0 mg/L 6-BA+0.2 mg/L NAA+3% sucrose; 2) shoot proliferation, MS+ 1.5 mg/L 6-BA+0.1mg/L NAA+3% sucrose; 3) take root and clove growth, 1/2 MS+ 0.5 ~ 1.0 mg/L NAA+3% sucrose+0.1% activated carbon, 1/2 MS+ 0.5mg/L NAA+6% sucrose+0.1% activated carbon.
Through finding existing lily relate art literature retrieval, number of patent application: 201110071332.5, application publication number: CN102210266A, denomination of invention: a kind of medium cultivated for lilium pumilum tissues, these medium are respectively and are made up of just for inducing culture MS+1.0mg/L6-BA+0.1mg/LNAA, the subculture multiplication medium be made up of MS+0.2mg/L6-BA+0.01mg/LNAA, the root media be made up of 1/2MS+0.5mg/LIBA, the balling medium be made up of 1/2MS+0.5mg/LIBA+ 2% sugar.Medium described in this patent medium used from this technology is different, and described in this patent, the differentiation-inducing rate of subculture multiplication medium is 60%, and this technology uses NAA, TDZ, KT, GA of variable concentrations
3carry out the medium of matched combined formation, its inductivity is up to 98%, root media root induction rate described in this patent is 88.89%, and this technology uses NAA, IBA, the bananas juice combination of variable concentrations, its rooting rate reaches 100%, and this technology adopts is MS minimal medium, and described in this patent, be 1/2MS minimal medium.At present, more research is had for aspect tissue cultures such as oriental hybrid lily, new PE curriculum, Huzhou lily, Lilium tenuifolium, lanzhou lily, rattlebush, and different lily is because of its difference such as genotype, growing environment, technical conditions difference needed for it is larger, particularly in the process of carrying out tissue-culturing quick-propagation, great difference is all had to the kind, concentration etc. of the minimal medium type of use and the external source plant growth regulator of interpolation, and also less to the system research of Lilium sulphureum Baker group culturation rapid propagating technology at present.Through finding Lilium sulphureum Baker group training Literature Consult, medium used and the medium described in this technology have obviously different, the employing five-step approach described in this technology is not more had to carry out the relevant report of Lilium sulphureum Baker tissue culture, transition described in this technology is cultivated, be showed no in the document of relevant lily group training and mention, and the use of transition cultural method, callus induction can be reduced greatly, callus proliferation is cultivated, inducing clumping bud, the pollution rate in the stages such as Rooting and hardening-off culture and the stability of material, and in Rooting and hardening-off culture base, add the NAA of variable concentrations, IBA, after bananas juice, its rooting rate reaches 100%, root system is sturdy, Gen Maofada, grow vigorous, greatly improve the survival rate of transplanting.Method of the present invention, can meet that Lilium sulphureum Baker is extensive, the demand of standardized planting, has certain originality and novelty.
summary of the invention
The object of the invention is to set up to differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, build and do not rely on simple seminal propagation or bulbil breeding to carry out the cropping pattern of cultivating, the mode of bulbil being carried out callus induction differentiation and seedling emergence again carries out extensive nursery, the proliferative speed of bulbil and the reproduction coefficient of bulbil can greatly be improved, the stability of plant can be ensured again to greatest extent, not easily as seminal propagation, produce degradation phenomena, a large amount of proterties can be obtained in a short time consistent simultaneously, the Lilium sulphureum Baker seedling of detoxification, for on a large scale, standardized planting Lilium sulphureum Baker provides seedling and ensures.
The present invention realizes by following technology
1) explant obtains
The bulbil tweezers grown by Lilium sulphureum Baker blade base pick puts into preprepared beaker, and the collection of bulbil explant is suitable for and is carried out at the 8:00 ~ 10:00 of fine day or the 10:00 ~ 12:00 at cloudy day.
2) explant sterilization
First ramentum is peeled with tweezers by adopting the bulbil of returning, again ramentum is placed in clean beaker, instillation 2-3 pours on preprepared gauze after dripping common liquid detergent or appropriate washing powder cleaning 2 ~ 3 times, is bundled in by the gauze wrapping ramentum on tap with tap water 1 ~ 3h; By the explant material after flushing on superclean bench, first use 75% alcohol-pickled 30s, period constantly stirs with tweezers, to enable alcohol submergence explant, then use aseptic water washing 3 times, then pour in the mercuric chloride of 0.1% explant after flushing into the 8 ~ 10min that sterilizes, aseptic water washing 6 times, each 2min, finally pours into explant in preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material;
3) transition is cultivated
Explant after sterilizing is inoculated in transitional culture medium, when 7 ~ 15d is cultivated in transition;
4) callus induction
Explant free of contamination in transitional culture medium is transferred in callus inducing medium, during inoculation, ramentum is disposed across on medium, and press down scale gently with tweezers and make it fully contact with medium; Culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, cultivate visible light green Lax callus after 7d to grow from ramentum base portion, when callus grows to about 3.5cm wide fritter, proceeded to callus proliferation medium;
5) callus proliferation is cultivated
The callus blade induced is cut into the wide fritter of about 0.5cm, then fritter is transferred in proliferated culture medium cultivates, be transferred in inducing clumping bud medium when callus grows to about 2.5cm;
6) inducing clumping bud and propagation
Loose, peak green, the callus that grows fine are transferred on inducing clumping bud medium, callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, after cultivating 10d, visible a large amount of Calli Differentiation is cells,primordial, then cultivates about 5d i.e. visible Multiple Buds and grow in a large number, continues to cultivate 10d, well differentiated can be obtained, the Multiple Buds that growing way is vigorous;
7) Rooting and hardening-off culture
The Multiple Buds of high 3 ~ 5cm is intercepted with blade, be inoculated on Rooting and hardening-off culture base, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, can obtain healthy and strong Lilium sulphureum Baker aseptic seedling after cultivating 14d.
The present invention is in inoculation operating process, because bulbil is made up of scale from level to level, grow again in the wild, be easy to carry bacterium secretly in the middle of scale, therefore before carrying out explant sterilization, bulbil first need be peeled ramentum with tweezers, the pollution adopting whole bulbil sterilizing thoroughly not cause can be avoided like this; When carrying out induction of callus inoculation, ramentum need be disposed across on medium, and press down scale gently with tweezers and make it fully contact with medium; When carrying out inducing clumping bud, callus is transferred on inducing clumping bud medium, needs gently to press callus to make it fully to contact with medium with tweezers.Avoid that inoculation material out-of-shape causes contact insufficient with medium and growth that is that cause is bad even dead.
Technical scheme of the present invention, sows relative to utilizing prior seed or bulbil is directly sowed, and has following benefit: reproduction speed is fast, only needs the time of about 3 months from explant to seedling; Reproduction coefficient is high, and Rhizoma Trillii Tschonoskii bud can constantly carry out callus proliferation to be broken up again, and reproduction coefficient is the hundreds of times of single bulbil; Not by region, external environment and the impact of time, seedling breeding can be carried out according to cultivated area and cultivation demand; Seedling good stability, the situation of degeneration occurs less; Seedling is by group training, and be all detoxic seedling, the phene of seedling is basically identical, is beneficial to high yield, few damage by disease and insect, and the lepisphere stem size of production is basically identical, does not need to carry out classification process; Small investment; only need a small amount of primary bulbil explant just can obtain a large amount of aseptic seedling; high financial profit; can large-scale planting, solve and use seed or slow and coefficient this problem low of bulbil breeding merely, the plantlet in vitro that the method is produced not only is conducive on a large scale, standardized planting; also be conducive to the recovery of wild Lilium sulphureum Baker population simultaneously; excavating of containment wild resource, improves the ecological environment, can produce good economy, society and ecological benefits.
Embodiment
Embodiment:
The bulbil tweezers grown by Lilium sulphureum Baker blade base pick to be put into preprepared beaker and takes back, first on testing stand, first ramentum is peeled with tweezers, again ramentum is placed in clean beaker, pour on preprepared gauze after instilling 2 ~ 3 common liquid detergents or appropriate washing powder cleaning 2 ~ 3 times, the gauze wrapping ramentum is bundled on tap with tap water 1 ~ 3h, again by rinse after explant material on superclean bench, first use 75% alcohol-pickled 30s, period constantly stirs with tweezers, to enable alcohol submergence explant, then use aseptic water washing 3 times, then pour in the mercuric chloride of 0.1% explant after flushing into the 8-10min that sterilizes, aseptic water washing 6 times, each 2min, finally pours into explant in preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material, explant after sterilizing is inoculated in transitional culture medium, until cultivate 7 ~ 15d in transitional culture medium time, the free of contamination explant of picking is transferred in callus inducing medium, during inoculation, ramentum is disposed across on medium, and press down scale gently with tweezers and make it fully contact with medium, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, after cultivating 7d, visible light green Lax callus grows from ramentum base portion, when callus grows to about 3.5cm wide fritter, callus is taken out, the wide fritter of about 0.5cm is cut into blade, again fritter is transferred in proliferated culture medium, when callus grows to about 2.5cm, to loosen, peak green, the callus grown fine is transferred on inducing clumping bud medium, callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, after cultivating 10d, visible a large amount of Calli Differentiation is cells,primordial, cultivate about 5d i.e. visible Multiple Buds again to grow in a large number, continue to cultivate 10d, well differentiated can be obtained, the Multiple Buds that growing way is vigorous, the Multiple Buds of high 3 ~ 5cm is finally intercepted with blade, be inoculated on Rooting and hardening-off culture base, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, can obtain healthy and strong Lilium sulphureum Baker aseptic seedling after cultivating 14d.
Medium that scheme uses is:
Transitional culture medium:
MS (without inositol)+0.01 ~ 1.5mg/LNAA+4.5 ~ 6.5g/L agar.
Callus induction optimal medium:
MS+0.01mg/L ~ 1.5mg/LNAA+0.01 ~ 2.0mg/L2,4-D+0.01 ~ 1.5mg/L6-BA+25g/L sucrose+5 ~ 7g/L agar.
Callus proliferation optimal medium: MS+0.05 ~ 2.0mg/LNAA+0.01 ~ 2.0mg/L2,4-D+0.01mg/L ~ 1.2mg/LTDZ+0.01 ~ 1.5mg/LKT ,+25g/L sucrose+5 ~ 7g/L agar.
Inducing clumping bud optimal medium:
MS+0.05 ~ 2.0mg/L NAA+0.01mg/L ~ 2.0mg/LTDZ+0.01 ~ 2.5mg/LKT+0.01 mg/L ~ 3.0mg/L GA
3+ 0.01 ~ 3.0mg/LTIBA+30g/L sucrose+5 ~ 7g/L agar.
Strong plantlets and rootage optimal medium:
Sucrose+4.5 ~ 6.5g/L agar of the MS+0.01 ~ 3.0mg/LNAA+0.01mg/L ~ appropriate bananas juice of 2.5mg/LIBA++30g/L.
Claims (6)
1. differentiate aseptic seedling again, its feature: be following steps with five-step approach induction Lilium sulphureum Baker bulbil callus:
1) explant obtains: the bulbil tweezers grown by Lilium sulphureum Baker petiole base pick to be put into preprepared beaker and take back;
2) explant sterilization: first peel ramentum with tweezers by adopting the bulbil of returning, again ramentum is placed in clean beaker, instillation 2-3 drip common liquid detergent or appropriate washing powder cleaning 2-3 time after pour on preprepared gauze, the gauze wrapping ramentum is bundled on tap and uses tap water 1-3h; By the explant material after flushing on superclean bench, first use 75% alcohol-pickled 30s, period constantly stirs with tweezers, to enable alcohol submergence explant, then use aseptic water washing 3 times, then pour in the mercuric chloride of 0.1% explant after flushing into the 8-10min that sterilizes, aseptic water washing 6 times, each 2min, finally pours into explant in preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material;
3) transition is cultivated: be inoculated in transitional culture medium by the explant after sterilizing, and until cultivate 7 ~ 15d in transitional culture medium time, the free of contamination explant of picking carries out callus induction;
4) callus induction: explant free of contamination in transitional culture medium is transferred in callus inducing medium, during inoculation, ramentum is disposed across on medium, and press down scale gently with tweezers and make it fully contact with medium; Culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, cultivate visible light green Lax callus after 7d to grow from ramentum base portion, when callus grows to about 3.5cm wide fritter, proceeded to callus proliferation and subculture medium;
5) callus proliferation and squamous subculture: the callus blade induced is cut into the wide fritter of about 0.5cm, then fritter is transferred in subculture medium cultivates, be transferred in inducing clumping bud medium when callus grows to about 2.5cm;
6) inducing clumping bud: loose, peak green, the callus that grows fine are transferred on inducing clumping bud medium, callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, after cultivating 10d, visible a large amount of Calli Differentiation is cells,primordial, then cultivates about 5d i.e. visible Multiple Buds and grow in a large number, continues to cultivate 10d, well differentiated can be obtained, the Multiple Buds that growing way is vigorous;
7) Rooting and hardening-off culture: the Multiple Buds intercepting high 3 ~ 5cm with blade, be inoculated on Rooting and hardening-off culture base, culturing room's temperature is daytime (25 ± 1) DEG C, evening (20 ± 1) DEG C, every 12h hockets cultivation, intensity of illumination: (1500 ~ 2500) lx, relative moisture 80%, can obtain healthy and strong Lilium sulphureum Baker aseptic seedling after cultivating 14d.
2. according to claims 1, differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, it is characterized in that: in step 3), transitional culture medium take MS as minimal medium, do not add inositol and sucrose, add the NAA of 4.5 ~ 6.5g/L agar, 0.01 ~ 1.5mg/L, pH value is adjusted to 6.0 at normal temperatures, autoclaving 20min.
3. according to claims 1, differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, it is characterized in that: in step 4), callus inducing medium take MS as minimal medium, add NAA, the 0.01 ~ 2.0mg/L2 of 0.01mg/L ~ 1.5mg/L, the sucrose of 4-D, 0.01 ~ 1.5mg/L6-BA, 25g/L, pH value are adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min, need press down scale gently with tweezers during inoculation and make it fully contact with medium.
4. according to claims 1, differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, it is characterized in that: in step 5) with proliferated culture medium with MS for minimal medium, add 0.05 ~ 2.0mg/LNAA, 0.01 ~ 2.0mg/L2,4-D, 0.01mg/L ~ 1.2mg/LTDZ, 0.01 ~ 1.5mg/LKT, 25g/L sucrose, pH value are adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min, gently press callus to make it fully to contact with medium during inoculation with tweezers.
5. according to claims 1, differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, it is characterized in that: in step 6), inducing clumping bud medium take MS as minimal medium, add 0.05 ~ 2.0mg/L NAA, 0.01mg/L ~ 2.0mg/LTDZ, 0.01 ~ 2.5mg/LKT, 0.01mg/L ~ 3.0mg/LGA
3, 0 ~ 3mg/LTIBA, 30g/L sucrose, pH value be adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min, gently presses callus to make it fully to contact with medium during inoculation with tweezers.
6. according to claims 1, differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil callus, it is characterized in that: in step 7), Rooting and hardening-off culture base take MS as minimal medium, add 0.01 ~ 3.0mg/LNAA, 0.01mg/L ~ 2.5mg/LIBA, appropriate bananas juice, 30g/L sucrose, pH value are adjusted to 6.0 under normal temperature, 4.5 ~ 6.5g/L agar, autoclaving 20min.
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| CN110268979A (en) * | 2019-06-03 | 2019-09-24 | 南京农业大学 | A kind of method and application of the foundation without pollen lily high-efficiency regeneration system |
| CN111280059A (en) * | 2020-03-26 | 2020-06-16 | 南京农业大学 | Efficient lily detoxification method |
| CN112243631A (en) * | 2020-09-14 | 2021-01-22 | 云南省农业科学院花卉研究所 | Method for rapidly breaking dormancy of green flower lily seed bulbs |
| CN114128580A (en) * | 2021-11-26 | 2022-03-04 | 北京市农林科学院 | Lily bulb breeding method |
| CN115152630A (en) * | 2022-07-28 | 2022-10-11 | 中国中医科学院中药研究所 | Lily callus culture method, lily test-tube plantlet and lily artificial seed culture method and plug culture medium |
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| CN108450333B (en) * | 2018-04-17 | 2020-02-07 | 长江师范学院 | Induction method of lilium tigrinum callus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110268979A (en) * | 2019-06-03 | 2019-09-24 | 南京农业大学 | A kind of method and application of the foundation without pollen lily high-efficiency regeneration system |
| CN110268979B (en) * | 2019-06-03 | 2022-03-11 | 南京农业大学 | Method for establishing efficient regeneration system of pollen-free lily and application |
| CN111280059A (en) * | 2020-03-26 | 2020-06-16 | 南京农业大学 | Efficient lily detoxification method |
| CN112243631A (en) * | 2020-09-14 | 2021-01-22 | 云南省农业科学院花卉研究所 | Method for rapidly breaking dormancy of green flower lily seed bulbs |
| CN114128580A (en) * | 2021-11-26 | 2022-03-04 | 北京市农林科学院 | Lily bulb breeding method |
| CN115152630A (en) * | 2022-07-28 | 2022-10-11 | 中国中医科学院中药研究所 | Lily callus culture method, lily test-tube plantlet and lily artificial seed culture method and plug culture medium |
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