CN104396742B - The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss - Google Patents
The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss Download PDFInfo
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Abstract
Aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss, with Lilium sulphureum Baker bulbil as explant, through transition culture, take free of contamination explant and be transferred to callus culture base, calluss are bred with proliferated culture medium, Multiple Buds are differentiated with calluss again, Multiple Buds is transferred in Rooting and hardening-off culture base and is cultivated.Often walking carries out preferably, to obtain free of contamination explant, can inducing calluss in 7d to different external source plant growth regulator species, concentration, blend proportion, inductivity is up to 100%, calluss subculture more than 10 times being made, Multiple Buds being induced in 15d, inductivity is up to 98%；14d can obtain healthy and strong band root aseptic seedling, and inductivity is up to 100%.With sowed using seed, compared with the Sterile culture such as bulbil, scale cuttage, cycle is short, low cost, breeding coefficient are high.
Description
Technical field
The invention belongs to the tissue culture of biological technical field, more particularly to healed with five-step approach induction Lilium sulphureum Baker bulbil
Injured tissue differentiates the production method of aseptic seedling again.
Background technology
Lilium sulphureum Baker (Li lium sulphureum Baker) is wild species in Liliaceae lilium, produces
In Guizhou Province, it is perennial herb plant, is medicine-food two-purpose type, with higher economic worth.But due to developing
Degree, has been in Critical Condition at present.Lilium sulphureum Baker is conventional to be bred with bulb separation, scale cuttage, seed etc., but breeding coefficient
Low, and long-term nutrition breeding easily causes virus infection so as to have a strong impact on quality and yield, and tissue culture quick breeding is to solve
The effective way of the problems referred to above.The report of different lily cultivar tissue cultures has been related to both at home and abroad, but to Lilium sulphureum Baker
Report is less.
Li Dai（2012）Show Deng adopting wild Lilium sulphureum Baker bulb scale that cutting propagation research is carried out for material, adopt
Flowerpot mouth is carried out moisturizing cuttage, survival rate to 86.66%, increment system by Lilium sulphureum Baker middle level scale with plastic sheeting sealing
Number is 1.85；Zhang Wene（2008）Deng, with late August take particle diameter more than or equal to 1 cm bulbil, peel off outer scale and be followed by
The inducing effect of the adventitious bud kind in the 1/2MS culture medium of additional 0.1 mgLIBA preferably, type of culture medium and Subculture Time
Enrichment culture is had a significant impact, with switching subculture in 40 d in the culture medium of 1/2MS+0.1 mg/LKT+0.1 mg/LIBA
Cultivation effect preferably, up to 97.3%, Bulblet induction rate is up to 100% to its callus induction rate, and adventitious bud growth is strong
Strong.Grow in 1/2MS+0.1 mg/LIBA culture medium and the situation of taking root is well；Zhou Hongying（2007）Etc. adopting fertile soil
For substrate, the cutting of " ten " font is sent out, and Lilium sulphureum Baker monolithic bulb breeding coefficient is up to more than 4；Yang Xueqing（2007）Deng with light
The scale of Hemerocallis citrina Baroni Bulbus Lilii, blade, petiole are explant, establish the fast asexual propagation system of Lilium sulphureum Baker, and scale is for most
Good explant ,+2. 0mg of MS/L+0. 3mg of 6-BA/L NAA be the optimal inducing culture of clove, inductivity highest
95. 0 % are reached, average clove growth coefficient reaches as high as 8. 6, and the optimal Bulblet induction culture medium of blade, petiole is MS
+2.0mg /L6-BA +0.5mg /L NA A；Li Dai（2005）Deng with MS as minimal medium, variable concentrations sucrose, 6- is studied
Impacts of the BA and NAA to Lilium sulphureum Baker bud propagation and callus growth, as a result shows：The optimal medium of bud 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. carries out Tube propagation with the scale of Lilium sulphureum Baker as explant, filters out each training
Suitable culture medium of foster stage is respectively：1) inducing clumping bud ,+3% sucrose of+0.2 mg/L NAA of MS+2.0 mg/L 6-BA；
2) subculture multiplication ,+3% sucrose of 1.5 mg/L 6-BA+0.1mg/L NAA of MS+；3) take root and clove growth, 1/2 MS+
+ 0.1% activated carbon of+3% sucrose of 0.5～1.0 mg/L NAA ,+0.1% activated carbon of 1/2+6% sucrose of MS+ 0.5mg/L NAA.
Through finding to the retrieval of existing Bulbus Lilii relevant technical literature, number of patent application：201110071332.5, Shen Qing Publication
Number：CN102210266A, denomination of invention：A kind of culture medium for lilium pumilum tissues culture, these culture medium be respectively by
MS+1.0mg/L6-BA+0.1mg/LNAA compositions are made up of just for inducing culture MS+0.2mg/L6-BA+0.01mg/LNAA
Subculture multiplication medium, the root media being made up of 1/2MS+0.5mg/LIBA, by the sugar of 1/2MS+0.5mg/LIBA+ 2%
The balling culture medium of composition.Culture medium described in the patent is different from the culture medium that this technology is used, and subculture described in the patent increases
It is 60% to grow culture medium induction differentiation rate, and this technology is using NAA, TDZ, KT, GA of variable concentrations3Carry out matched combined to be formed
Culture medium, its inductivity is up to 98%, and the root media root induction rate described in the patent is 88.89%, and this technology makes
With the NAA of variable concentrations, IBA, bananas juice combination, its rooting rate up to 100%, and this technology adopt for MS minimal mediums, with
It is 1/2MS minimal mediums described in the patent.At present, for oriental hybrid lily, new PE curriculum, Huzhou Bulbus Lilii, Lilium tenuifolium, orchid
The existing more research of the aspect such as state Bulbus Lilii, Herba Crotalariae sessiliflorae tissue culture, and different Bulbus Liliies are because of differences such as its genotype, growing environments,
Technical conditions difference needed for which is larger, the basic training particularly during tissue-culturing quick-propagation is carried out, to using
The species of the external source plant growth regulator of foster base type and interpolation, concentration etc. have great difference, and at present to yellowish
The systematic study of flower Bulbus Lilii group culturation rapid propagating technology is also less.Through finding to the Literature Consult of Lilium sulphureum Baker tissue culture, culture used
Base has significantly different with the culture medium described in this technology, more carries out Lilium sulphureum Baker group without described in this technology using five-step approach
The relevant report of Seedling is cultivated, the transition culture described in this technology is showed no in the document about Bulbus Lilii tissue culture and refers to, and transition
The use of cultural method, can greatly reduce callus induction, callus proliferation culture, inducing clumping bud, strong plantlets and rootage
The stability of the pollution rate in stage and the materials such as culture, and add the NAA of variable concentrations, IBA, perfume (or spice) in Rooting and hardening-off culture base
After any of several broadleaf plants juice, its rooting rate reaches 100%, and root system is sturdy, Gen Maofada, and growth is vigorous, greatly improves the survival rate of transplanting.
Method of the present invention, can meet that Lilium sulphureum Baker is extensive, the demand of standardized planting, with certain originality and new
Newness.
Content of the invention
The purpose of the present invention is set up to differentiate aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil calluss,
The cropping pattern that is cultivated not against simple seminal propagation or bulbil breeding is built, bulbil is carried out callus induction again
The mode of differentiation and seedling emergence carries out extensive nursery, can greatly improve the breeding coefficient of the proliferative speed and bulbil of bulbil, and
The stability of plant can be ensured to greatest extent, be difficult degradation phenomena to be produced as seminal propagation, while can obtain in a short time
A large amount of character are consistent, the Lilium sulphureum Baker seedling of detoxification, provide seedling for extensive, standardized planting Lilium sulphureum Baker and protect
Card.
The present invention can be realized by following technology
1）Explant is obtained
The bulbil tweezers that Lilium sulphureum Baker blade base is grown are picked and are put in preprepared beaker, pearl
The collection of bud explant is suitable for the 8 of fine day:00~10:00 or the 10 of the cloudy day:00~12:00 is carried out.
2）Explant sterilizes
The bulbil returned will be adopted and first ramentum is peeled with tweezers, then ramentum is placed in clean beaker, instill 2-3 drops
Common liquid detergent or appropriate detergent are poured on preprepared gauze after cleaning 2 ~ 3 times, will wrap the gauze of ramentum
Be bundled on faucet 1 ~ 3h is rinsed with tap water；Explant material after by flushing uses 75% ethanol on superclean bench, first
Immersion 30s, period are constantly stirred with tweezers so that ethanol can submergence explant, then with aseptic water washing 3 times, then will
Explant after flushing is poured into, aseptic water washing 6 times, and each 2min, finally by explant
Pour in preprepared sterile petri dish, with the moisture remained on aseptic filter paper blotting material；
3）Transition culture
Explant after by sterilizing is inoculated in transitional culture medium, during transition 7 ~ 15d of culture；
4）Callus induction
Free of contamination explant in transitional culture medium is transferred in callus inducing medium, by ramentum during inoculation
Be disposed across in culture medium, and scale is gently pushed with tweezers makes which be fully contacted with culture medium；Culture room temperature is daytime（25±
1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx, relative humidity 80%, training
After foster 7d, visible light green Lax callus are grown from ramentum base portion, treat that calluss grow to the wide fritters of 3.5cm or so
When, proceeded to callus proliferation medium；
5）Callus proliferation culture
The calluss blade for inducing is cut into the wide fritters of 0.5cm or so, then fritter is transferred to enrichment culture
Cultivate in base, be transferred to when calluss grow to 2.5cm or so in inducing clumping bud culture medium；
6）Inducing clumping bud and propagation
Loose, peak green, the calluss for growing fine are transferred in inducing clumping bud culture medium, with tweezers, gently pressure heals
Injured tissue is allowed to be fully contacted with culture medium, and culture room temperature is daytime（25±1）DEG C, in evening (20 ± 1) DEG C, replace per 12h
Cultivated, intensity of illumination：（1500~2500）Lx, relative humidity 80%, after culture 10d, it is seen that a large amount of calluss are divided into
Cells,primordial, is further cultured for the i.e. visible Multiple Buds of 5d or so and grows in a large number, continues culture 10d, can obtain well differentiated, and growing way is vigorous
Multiple Buds；
7) Rooting and hardening-off culture
The Multiple Buds that high 3 ~ 5cm is intercepted with blade, are inoculated on Rooting and hardening-off culture base, and culture room temperature is daytime（25
±1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx, relative humidity 80%, training
Healthy and strong Lilium sulphureum Baker aseptic seedling can be obtained after foster 14d.
The present invention as bulbil is made up of scale from level to level, is grown in the wild in inoculation operating process again, it is easy to
Antibacterial is carried secretly in the middle of scale, therefore before explant sterilizing is carried out, bulbil first ramentum need to be peeled with tweezers, so may be used
Avoid the pollution thoroughly not caused using the sterilizing of whole bulbil；When induction of callus inoculation is carried out, need to be by ramentum
Be disposed across in culture medium, and scale is gently pushed with tweezers makes which be fully contacted with culture medium；When inducing clumping bud is carried out, more
Injured tissue is transferred in inducing clumping bud culture medium, needs gently to press calluss to be allowed to be fully contacted with culture medium with tweezers.Keep away
Inoculation material caused growth insufficient contact and caused with culture medium in irregular shape is exempted from bad or even dead.
Technical scheme, relative to directly being sowed using prior seed sowing or bulbil, with following benefit：Numerous
Grow speed fast, the time of 3 months or so is only needed from explant to seedling；Breeding coefficient is high, and Rhizoma Trillii Tschonoskii, bud constantly can be healed
Injured tissue propagation is broken up again, and breeding coefficient is the hundreds of times of single bulbil；Do not affected by region, external environment and time, can
Seedling breeding is carried out according to cultivated area and cultivation demand；Seedling good stability, the situation generation of degeneration are less；Seedling passes through
Tissue culture, all detoxic seedlings, the phene of Seedling are basically identical, and beneficial to high yield, few pest and disease damage, the lepisphere stem size of production are basic
Unanimously, it is not required to carry out staged care；Small investment, only needs a small amount of primary bulbil explant to be achieved with substantial amounts of aseptic seedling, passes through
Ji high efficiency, can implantation in large scale, solve merely using seed or bulbil breeding be slow and coefficient this problem low, the method is produced
Tissue cultured seedling be not only advantageous on a large scale, standardized planting, be simultaneously also beneficial to the recovery of wild Lilium sulphureum Baker population, contain
The excavation of wild resource, improves the ecological environment, and can produce good economic, social and ecological benefits.
Specific embodiment
Embodiment：
The bulbil tweezers that Lilium sulphureum Baker blade base is grown are picked and are put into band in preprepared beaker
Return；First ramentum is peeled with tweezers first on testing stand, then ramentum is placed in clean beaker, instill 2 ~ 3 drops common
Liquid detergent or appropriate detergent are poured on preprepared gauze after cleaning 2 ~ 3 times, and the gauze for wrapping ramentum is bundled
1 ~ 3h is rinsed with tap water on faucet；Again by flushing after explant material on superclean bench, first with 75% ethanol soak
Bubble 30s, period are constantly stirred with tweezers so that ethanol can submergence explant, then with aseptic water washing 3 times, then will punching
Explant after washing is poured into, and explant is finally fallen by aseptic water washing 6 times, each 2min
Enter in preprepared sterile petri dish, with the moisture remained on aseptic filter paper blotting material；Explant after by sterilizing connects
Plant in transitional culture medium, when staying in 7 ~ 15d of culture in transitional culture medium, the free of contamination explant of picking is transferred to calluss
In inducing culture, ramentum is disposed across in culture medium during inoculation, and scale is gently pushed with tweezers makes which fill with culture medium
Tap is touched, and culture room temperature is daytime（25±1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：
（1500~2500）Lx, relative humidity 80%, after culture 7d, visible light green Lax callus are grown from ramentum base portion, are treated
When calluss grow to 3.5cm or so wide fritters, calluss are taken out, the wide fritters of 0.5cm or so are cut into blade, then
Fritter is transferred in proliferated culture medium, when calluss grow to 2.5cm or so, by loose, peak green, grow fine more
Injured tissue is transferred in inducing clumping bud culture medium, gently presses calluss to be allowed to be fully contacted with culture medium with tweezers, culturing room
Temperature is daytime（25±1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx,
Relative humidity 80%, after culture 10d, it is seen that a large amount of calluss are divided into cells,primordial, are further cultured for the i.e. visible Multiple Buds of 5d or so
Grow in a large number, continue culture 10d, well differentiated, the vigorous Multiple Buds of growing way can be obtained；The clump of high 3 ~ 5cm is finally intercepted with blade
Sprout, be inoculated on Rooting and hardening-off culture base, culture room temperature is daytime（25±1）DEG C, in evening (20 ± 1) DEG C, hand over per 12h
Replace and cultivated, intensity of illumination：（1500~2500）Lx, relative humidity 80%, culture 14d after can obtain healthy and strong Lilium sulphureum Baker without
Vaccine.
Scheme using culture medium 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 GA3+ 0.01 ~ 3.0mg/LTIBA+30g/L sucrose+5 ~ 7g/L agar.
Strong plantlets and rootage optimal medium：
The sucrose+4.5 of the appropriate bananas juice+30g/L of MS+0.01 ~ 3.0mg/LNAA+0.01mg/L ~ 2.5mg/LIBA+ ~
6.5g/L agar.
Claims (2)
1. the method for differentiating aseptic seedling again with five-step approach induction Lilium sulphureum Baker bulbil calluss, it is characterised in that following step
Suddenly：
1）Explant is obtained：The bulbil tweezers that Lilium sulphureum Baker petiole base is grown are picked and are put into preprepared
Take back in beaker；
Explant sterilizes：The bulbil returned will be adopted and first ramentum is peeled with tweezers, then ramentum is placed in clean beaker, drip
Enter after the common liquid detergent of 2-3 drops or appropriate detergent clean 2-3 time and pour on preprepared gauze, ramentum will be wrapped
Gauze be bundled on faucet with tap water rinse 1-3h；Explant material after by flushing is first used on superclean bench
75% alcohol-pickled 30s, period are constantly stirred with tweezers so that ethanol can submergence explant, then with aseptic water washing 3 times,
Then by flushing after explant pour into, aseptic water washing 6 times, each 2min finally will
Explant is poured in preprepared sterile petri dish, with the moisture remained on aseptic filter paper blotting material；
2）Transition culture：Explant after by sterilizing is inoculated in transitional culture medium, stays in 7 ~ 15d of culture in transitional culture medium
When, the free of contamination explant of picking carries out callus induction；
3）Callus induction：Free of contamination explant in transitional culture medium is transferred in callus inducing medium, is connect
Ramentum is disposed across in culture medium when planting, and scale is gently pushed with tweezers makes which be fully contacted with culture medium；Culture room temperature
Spend for daytime（25±1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx, phase
To humidity 80%, after cultivating 7d, visible light green Lax callus are grown from ramentum base portion, treat that calluss grow to 3.5cm
During wide fritter, callus proliferation and subculture medium is proceeded to；
The callus inducing medium with MS as minimal medium, add 0.01mg/L ~ 1.5mg/L NAA, 0.01 ~
2, the 4-D of 2.0mg/L, the sucrose of 6-BA, 25g/L of 0.01 ~ 1.5mg/L, 5 ~ 7g/L agar, pH value adjust under room temperature to
6.0, autoclaving 20min；
Callus proliferation and successive transfer culture：The calluss blade for inducing is cut into the wide fritters of 0.5cm, then by fritter
Callus proliferation and culture in successive transfer culture is transferred to, and inducing clumping bud culture is transferred to when calluss grow to 2.5cm
In base；
The callus proliferation and successive transfer culture with MS as minimal medium, add 0.05 ~ 2.0mg/L NAA, 0.01 ~
The TDZ of 2,4-D, 0.01mg/L of 2.0mg/L ~ 1.2mg/L, the sucrose of KT, 25g/L of 0.01 ~ 1.5mg/L, the fine jade of 5 ~ 7g/L
Fat, pH value are adjusted to 6.0 under room temperature, autoclaving 20min；Calluss are gently pressed to be allowed to fill with culture medium with tweezers during inoculation
Tap is touched；
4）Inducing clumping bud：Loose, peak green, the calluss for growing fine are transferred in inducing clumping bud culture medium, are used
Tweezers gently press calluss to be allowed to be fully contacted with culture medium, and culture room temperature is daytime（25±1）DEG C, evening (20 ± 1)
DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx, relative humidity 80%, after culture 10d, it is seen that great Liang Yu
Injured tissue is divided into cells,primordial, is further cultured for the i.e. visible Multiple Buds of 5d and grows in a large number, continues culture 10d, can obtain well differentiated, long
The vigorous Multiple Buds of gesture；
The inducing clumping bud culture medium with MS as minimal medium, add 0.05 ~ 2.0mg/L NAA, 0.01mg/L ~
The TDZ of 2.0mg/L, the GA of KT, 0.01mg/L ~ 3.0mg/L of 0.01 ~ 2.5mg/L3, 0 ~ 3mg/L TIBA, 30g/L sugarcane
Sugar, the agar of 5 ~ 7g/L, pH value are adjusted to 6.0 under room temperature, autoclaving 20min；
5) Rooting and hardening-off culture：The Multiple Buds that high 3 ~ 5cm is intercepted with blade, are inoculated on Rooting and hardening-off culture base, cultivate room temperature
Spend for daytime（25±1）DEG C, in evening (20 ± 1) DEG C, alternately cultivate per 12h, intensity of illumination：（1500~2500）Lx, phase
To humidity 80%, after cultivating 14d, healthy and strong Lilium sulphureum Baker aseptic seedling can be obtained；
The Rooting and hardening-off culture base adds NAA, 0.01mg/L ~ 2.5mg/L of 0.01 ~ 3.0mg/L with MS as minimal medium
IBA, appropriate bananas juice, the sucrose of 30g/L, the agar of 4.5 ~ 6.5g/L, pH value adjusts to 6.0 under room temperature, autoclaving
20min.
2. the side that five-step approach according to claim 1 induces Lilium sulphureum Baker bulbil calluss to differentiate aseptic seedling again
Method, it is characterised in that：Step 2）Middle transitional culture medium with MS as minimal medium, without inositol and sucrose, add 4.5 ~
The agar of 6.5g/L, the NAA of 0.01 ~ 1.5mg/L, pH value are adjusted to 6.0 under room temperature, autoclaving 20min.
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| CN108450333A (en) * | 2018-04-17 | 2018-08-28 | 长江师范学院 | A kind of abductive approach of Lilium lancifo1ium Thunb callus |
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| 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 |
| CN112243631B (en) * | 2020-09-14 | 2022-03-22 | 云南省农业科学院花卉研究所 | Method for rapidly breaking dormancy of green flower lily seed bulbs |
| CN114128580B (en) * | 2021-11-26 | 2023-04-07 | 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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| CN102696483B (en) * | 2012-06-19 | 2013-09-04 | 西安文理学院 | Method for quickly propagating lilium fargesii |
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| CN108450333A (en) * | 2018-04-17 | 2018-08-28 | 长江师范学院 | A kind of abductive approach of Lilium lancifo1ium Thunb callus |
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