CN106561452A - Method for establishing catalpa bungei embryonal cell suspension system - Google Patents
Method for establishing catalpa bungei embryonal cell suspension system Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the technical field of cell culture, and relates to a catalpa bungei embryonal cell suspension culture method, wherein the method comprises a step of taking embryos of immature fruits of catalpa bungei as explants, and sequentially comprises the steps of inducing embryonal callus, inducing an initial suspension culture product, establishing an embryonal cell suspension system, and regenerating plants; the induction culture medium of the initial suspension culture product is 1/2MS; and, the culture solution of the catalpa bungei embryonal cell suspension system is that polyethylene glycol (PEG) 6000 is added based on the 1/2MS culture medium. By means of the method disclosed by the invention, the steady catalpa bungei embryonal cell suspension system can be established; furthermore, the plantlet survival rate of embryo regeneration plants is high; emerged seedlings are tidy and normal; and the method disclosed by the invention can provide a steady material source and a platform for catalpa bungei embryo development, somatic hybridization, genetic transformation, and storage and research of plant germplasm resources, and provide important theoretical and experimental basis for industrial breeding of a bioreactor technology based on suspension cell culture.
Description
Technical field
The invention belongs to technical field of cell culture, is related to a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture.
Background technology
Chinese catalpa (Catalpa bungeiC.A.Meyer.) category Bignoniaceae (Bignoniaceae) Catalpa (Catalpa),
China is originated in, is the distinctive rare tree of China, have the title of " wooden king ".In the world about 13 kinds of the plant of Radix Asteriss section Chinese catalpa category, main
It is distributed in America and East Asia（Guo Congjian etc., 1994）.In China, Chinese catalpa is distributed by warm temperate zone and subtropical zones, in Shandong, river
Soviet Union, Henan, Anhui Province's distribution are more（Shi Xin etc., 2011）.
Chinese catalpa, using with a long history, is Precious Timber Species and famous ornamental plantation seeds（Tang Ling etc., 2007）.Chinese catalpa
Important commerical tree species are classified as by country, its timber have do not warp and fracture, corrosion-resistant, easy processing, beautiful texture the features such as, it is special adding
Work high-grade goods and Special Products.The tree-like grace of Chinese catalpa, leaf is close by close hair, the rough branch of skin, is conducive to sound insulation, noise abatement, noise control, stagnant
Dirt, and to various toxic gas, chlorine and sulfur dioxide etc. in environment have stronger absorbability, be China have it is higher
The important afforestation of the value of environmental protection and ornamental tree species.Due to Chinese catalpa, self-sterility, and Jing is normally opened colored and shaky, seed germination rate
It is very low, and difficulty of taking root is bred using cottage method, the breeding and utilization and extention to Chinese catalpa excellent strain causes very big obstacle,
In addition the excellent exploitation for exacerbating people of material, cause the shortage of Chinese catalpa resource.
At present, the research with regard to the generation of Chinese catalpa somatic embryo is simultaneously few, is concentrated mainly on Somatic Embryogenesis
Morphocytology research, Physiology and biochemistry and body embryo there is the initial research of induction, there is not yet efficiently steady relating to how to set up
The report of fixed Chinese catalpa somatic embryo suspension system culture.Plant cell suspension culture has growth fraction complete individuals plant fast,
Nutrition and environmental requirement are easy to control, are capable of achieving anniversary growth, are not affected by geographical factor；Furthermore, it is also possible to produce new, satisfaction
The compound that the biological activity such as medicine and chemical industry needs；A successful cell suspension cultures system is set up to carrying out basic research
It is most important with relying on bioreactor to carry out industrialization development.
Modal problem is the disappearance of browning and suspension cell embryo in plant cell suspension culture.Can
The two problems are solved, is the crucial part for setting up cell morphology and ultrastructure.The producing cause of browning is more, such as the genotype of explant
And condition of culture etc..Conventional anti-browning material mainly includes activated carbon, ascorbic acid, silver nitrate, citric acid, PVPP etc., this
A little additives can impact of the Browning control to suspended culture cell to a certain extent.It is another that the decline of suspension cell embryo disappears
An outer major issue, particularly during monocotyledon (such as Caulis Sacchari sinensis, Oryza sativa L. and Semen Maydiss) cell suspension cultures, embryo
Decline problems demand is solved.Therefore, by selecting suitable explant, the firm presence of somatic embryo is maintained, sets up stable embryo
Sexual cell suspension culture system, can be that morphological observation, somatic hybridization, genetic transformation and industrialization that body embryo occurs are numerous
Offer experiment material and important theoretical experimental basis are provided.
The content of the invention
The present invention provides a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, is set up by the method for cell suspension cultures
Stable Embryogenic Callus Suspension Culture.
Technical solution of the present invention：
A kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, comprises the following steps：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus：Take the immature seed of Chinese catalpa, it is sterilized after, strip children tender
Embryo induces described explant as explant, obtains embryo callus；
2）Primary suspension culture product induction：By step 1）The embryo callus of acquisition are transferred in primary culture fluid and are trained
Support, obtain Chinese catalpa primary suspension culture product；
3）Cells,primordial suspension culture is screened：By step 2）The primary suspension culture amplification product of acquisition cross after cell sieve transfer in
Cultivate in cells,primordial suspending nutrient solution, obtain cells,primordial suspension system；
Complete the foundation of Chinese catalpa Embryogenic Callus Suspension Culture.
Preferably, the step 1）Sterilizing methods are the ethanol of Jing 75%, 2% liquor natrii hypochloritises sterilizing successively.
Preferably, the step 2）Primary culture fluid is 1/2MS liquid minimal mediums.
Preferably, the step 2）Condition of culture be the r/min of rotating speed 100 ~ 120,23 ~ 27 DEG C of cultivation temperature, illumination is strong
900 ~ 1100Lx of degree, cultivates, 16-20 days successive transfer culture cycle, successive transfer culture 2 times under conditions of 14 ~ 16h/d of light application time, after
It is sieved through sieve and is processed with 60 mesh, 80 aim cells during culture, liquid stands and abandon supernatant after 1h after sieving, and fills into the fresh training of equivalent
Foster base, and the cell mass on sieve is accessed into continuation culture in fresh liquid culture medium.
Preferably, the step 3）Condition of culture be the r/min of rotating speed 100 ~ 120,23 ~ 27 DEG C of cultivation temperature, illumination is strong
900 ~ 1100Lx of degree, the CMC model of 14 ~ 16h/d of light application time, 16-20 days successive transfer culture cycle, successive transfer culture was 60 mesh
Cell sieve process.
It is further preferred that the step 3）Cells,primordial suspension culture screening and culturing liquid with the addition of poly- second two for 1/2MS
Alcohol 6000, polyethylene glycol 6000 mass concentration is 0%-20% in the culture fluid
It is further preferred that polyethylene glycol 6000 mass concentration is 10% in the culture fluid.
Beneficial effect of the present invention：
1st, the invention provides a kind of method for building up of Embryogenic Callus Suspension Culture of Precious Timber Species Chinese catalpa.The method is with Chinese catalpa
The young tender embryo of immature seed induces embryo callus, sets up stable embryo by the method for cell suspension cultures thin
Born of the same parents' suspension system.It is that induction differentiation capability is strong to carry out isolated culture advantage with young tender seed, and long-term subculture process is difficult to degenerate, and obtains
The stable cells,primordial suspension culture system for obtaining, can be used for secondary metabolite Commercial cultivation, Study on Genetic Transformation and biology
The aspects such as reactor large-scale production, it may also be used for the preferable mutant of screening and suspension cell line physiological and biochemical index grind
Study carefully.
2nd, the present invention is selected by culture fluid, can filter out suitable culture formula of liquid and effective additive composition, is lured
Lead suspension culture product to convert to embryogenic cell line, promote cell propagation, it is to avoid browning is dead.By setting up fluid suspension culture
System, can obtain the homogeneous consistent Embryogenic cell masses or unicellular of size, set up suspension culture system, be bioreactor scale
Metaplasia is produced, somatic embryo development is marched into the arena, and research, somatic hybridization and Study on Genetic Transformation provide experiment material and important reason
By experimental basis.
Description of the drawings
With reference to the accompanying drawings and examples the invention will be further described：
Photo of Fig. 1 Chinese catalpas primary suspension culture product under stereomicroscope；
Fig. 2 is not added with many subculture suspension culture products of PEG；
Fig. 3 adds the cells,primordial suspension culture amplification product of PEG；
The suspension culture product solid culture propagation of Fig. 4 addition PEG；
The plant regeneration of the suspension culture product of Fig. 5 addition PEG.
Specific embodiment
The present invention is further illustrated with reference to embodiment, but the scope of protection of present invention is not limited to implement
The scope of example statement.
Embodiment 1
MS minimal mediums：Can refer to document（Murashige T, Skoog F. A revised medium for rapid
grouth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15:
473-497）Prepare.
A kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, comprises the steps of：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus：
Explant sterilizes, and takes the immature seed of Chinese catalpa, after ethanol, 2% liquor natrii hypochloritises sterilizing successively Jing 75%, strips children
Tender embryo is used as explant；Described explant is placed in a series of solid mediums and is induced, obtain faint yellow embryo callus subculture
Tissue.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of calluss：With sterilized Chinese catalpa
It is explant to set young tender embryo, is placed on callus induction on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is with the addition of on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. wound healing
The propagation of tissue：Calluss are placed on proliferated culture medium and are bred；Culture medium is added on the basis of 1/2MS minimal mediums
The 6-benzyladenine (6-BA) that concentration is 2.0 mg/L and the α-naphthaleneacetic acid that concentration is 0.1mg/L are added（NAA）；3. embryo is healed
The induction of injured tissue：Calluss after propagation are placed in into induced embryonic callus in induction culture medium, induction
Culture medium is that 6- benzyl purines (6-BA) that concentration is 1.0 mg/L and dense is with the addition of on the basis of 1/2MS minimal mediums
Spend for the α-naphthaleneacetic acid of 0-0.1 mg/L（NAA））
2）Primary suspension culture product induction：By step 1）The embryo callus of acquisition are transferred in primary culture fluid and are trained
Support, the amount of culture fluid is 100ml culture fluid in the triangular flask of 250ml, and in rotating speed 110r/min, 25 DEG C of cultivation temperature, illumination is strong
Degree 1000Lx, cultivates under conditions of light application time 16h/d, and the inducing culture cycle is 18 days, with 60 mesh, 80 purposes during successive transfer culture
Cell sieve is sieved process, and liquid to stand and abandon supernatant after 1h after sieving, and fills into equivalent fresh culture, and by the cell mass on sieve
To access continue in fresh liquid culture medium and cultivate.The suspending nutrient solution contains：1/2MS, obtains Chinese catalpa primary suspension culture product；
As shown in figure 1, the product suspension after culture is observed to stereomicroscope, it is seen that dissociate in a large number transparent strip cell
And sphaerocyst, the cell mass assembled based on sphaerocyst on a small quantity, and cell be well-illuminated, inclusions are thin.Secondary subculture
Continue afterwards to use 1/2MS culture fluid subcultures, easily occur the dead phenomenon of cell browning in incubation, suspension muddiness has a large amount of brown
Change cultured products（Fig. 2）.
3）Cells,primordial suspension culture is screened：By step 2）The Chinese catalpa primary suspension culture product of acquisition is inoculated in after sieving
Cultivate in cells,primordial suspension culture screening and culturing liquid, in rotating speed 110r/min, 25 DEG C ± 2 of cultivation temperature, intensity of illumination
1000Lx, under conditions of light application time 16h/d, cultivates 9 days, starts white or lurid globular embryo polymer occur（As schemed
3）, successive transfer culture 3 times, culture total time is 36 days, obtains suspension culture amplification product, sets up Embryogenic Callus Suspension Culture, is found
Suspension culture product growth rate is very fast, size is homogeneous, and suspension is limpider, without browning；The cells,primordial suspends
Culture screening and culturing liquid with the addition of polyethylene glycol 6000 for 1/2MS, and polyethylene glycol 6000 mass concentration is in the culture fluid
10%。
4）Plant regeneration
By step 3）The embryo materials that fluid suspension culture is obtained are inoculated into addition 0.8%（W/V）The 1/2MS solid culture of agar
Cultivate in base, embryonal connective tissue keeps stable（Fig. 4）, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums induction of agar
Differentiation, embryonal connective tissue differentiation synchronicity is high, and body embryo sprout time is more consistent, and it is neat to emerge, and deformity is not observed in form（Figure
5）.Condition of culture：25 ± 2 DEG C of temperature, light application time 14h/ days, the Lx of intensity of illumination 2000.
Embodiment 2
MS minimal mediums：Can refer to document（Murashige T, Skoog F. A revised medium for rapid
grouth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15:
473-497）Prepare.
The method that Chinese catalpa suspension culture system of the present invention is set up, comprises the steps of：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus
Explant sterilizes, and takes the immature seed of Chinese catalpa, after ethanol, 2% liquor natrii hypochloritises sterilizing successively Jing 75%, strips children
Tender embryo is used as explant；Described explant is placed in a series of solid mediums and is induced, obtain faint yellow embryo callus subculture
Tissue.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of calluss：With sterilized Chinese catalpa
It is explant to set young tender embryo, is placed on callus induction on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is with the addition of on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. wound healing
The propagation of tissue：Calluss are placed on proliferated culture medium and are bred；Culture medium is added on the basis of 1/2MS minimal mediums
The 6-benzyladenine (6-BA) that concentration is 1.0 mg/L and the α-naphthaleneacetic acid that concentration is 0.05mg/L are added（NAA）；3. embryo
The induction of calluss：Calluss after propagation are placed in into induced embryonic callus in induction culture medium, differentiation is lured
Lead culture medium be with the addition of on the basis of 1/2MS minimal mediums 6- benzyl purines (6-BA) that concentration is 0.9 mg/L and
Concentration is the α-naphthaleneacetic acid of 0.05 mg/L（NAA））
2）Primary suspension culture product induction
By step 1）The embryo callus of acquisition are transferred in primary culture fluid and are cultivated, culture fluid in 100ml triangular flasks
Amount be 40ml, cultivate rotating speed 100r/min, 23 DEG C of cultivation temperature, intensity of illumination 1000Lx, the condition of light application time 16h/d
Under, 32 days shaken cultivation time, sieved process with the nylon wire of 60 mesh, 80 mesh during successive transfer culture per 16 days subcultures once, sieve
Afterwards liquid to stand and abandon supernatant after 1h, fills into equivalent fresh culture, and the cell mass on sieve is accessed into fresh liquid culture medium
It is middle to continue to cultivate.The suspending nutrient solution：1/2MS.
3）Cells,primordial suspension culture is screened：By step 2）The Chinese catalpa primary suspension culture product of acquisition is inoculated in after sieving
Cultivate in cells,primordial suspension culture screening and culturing liquid,
The culture fluid of cells,primordial suspension system is the PEG6000 of 1/2MS culture medium addition 5%.
Transfer in embryonal suspension system culture fluid, in rotating speed after the primary suspension culture amplification product is sieved
100r/min, 25 DEG C of temperature, intensity of illumination 1000Lx is cultivated, 16 days successive transfer culture cycles under the conditions of light application time 14h/d, is sent out
Existing suspension culture product growth rate is very fast, size is homogeneous, and suspension is limpider, thin so as to set up embryo without browning
Born of the same parents' suspension system.
4）Plant regeneration
By step 3）The embryo materials that fluid suspension culture is obtained are inoculated into addition 0.8%（W/V）The 1/2MS solid culture of agar
Cultivate in base, embryonal connective tissue keeps stable, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums induction differentiation of agar,
Embryonal connective tissue differentiation synchronicity is high, and body embryo sprout time is more consistent, and it is neat to emerge, and deformity is not observed in form.Culture bar
Part：25 ± 2 DEG C of temperature, light application time 14h/ days, the Lx of intensity of illumination 2000.
Embodiment 3
MS minimal mediums：Can refer to document（Murashige T, Skoog F. A revised medium for rapid
grouth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15:
473-497）Prepare.
The method that Chinese catalpa suspension culture system of the present invention is set up, comprises the steps of：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus
Explant sterilizes, and takes the immature seed of Chinese catalpa, after ethanol, 2% liquor natrii hypochloritises sterilizing successively Jing 75%, strips children
Tender embryo is used as explant；Described explant is placed in a series of solid mediums and is induced, obtain faint yellow embryo callus subculture
Tissue.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of calluss：With sterilized Chinese catalpa
It is explant to set young tender embryo, is placed on callus induction on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is with the addition of on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. wound healing
The propagation of tissue：Calluss are placed on proliferated culture medium and are bred；Culture medium is added on the basis of 1/2MS minimal mediums
The 6-benzyladenine (6-BA) that concentration is 0.8 mg/L and the α-naphthaleneacetic acid that concentration is 0.06mg/L are added（NAA）；3. embryo
The induction of calluss：Calluss after propagation are placed in into induced embryonic callus in induction culture medium, differentiation is lured
Lead culture medium be with the addition of on the basis of 1/2MS minimal mediums 6- benzyl purines (6-BA) that concentration is 0.07 mg/L and
Concentration is the α-naphthaleneacetic acid of 0-0.1 mg/L（NAA））
2）Primary suspension culture product induction
By step 1）The embryo callus of acquisition are transferred in primary culture fluid and are cultivated, such as train in 200ml triangular flasks
The amount of nutrient solution be 80ml, cultivate rotating speed 90r/min, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000Lx, light application time 16h/d
Under the conditions of, 40 days shaken cultivation time, it is sieved through sieve and is processed with 60 mesh, 80 aim cells during successive transfer culture per 20 days subcultures once,
Liquid to stand and abandon supernatant after 1h after sieving, and fills into equivalent fresh culture, and the cell mass on sieve is accessed into fresh liquid training
Continue to cultivate in foster base.The suspending nutrient solution contains：1/2MS0.
3）Cells,primordial suspension culture is screened：By step 2）The Chinese catalpa primary suspension culture product of acquisition is inoculated in after sieving
Cultivate in cells,primordial suspension culture screening and culturing liquid, the culture fluid of cells,primordial suspension system is 1/2MS culture medium addition 20%
PEG6000.
Transfer in embryonal suspension system culture fluid, in rotating speed after the primary suspension culture amplification product is sieved
100r/min, 27 DEG C of temperature, intensity of illumination 1000Lx is cultivated, 20 days successive transfer culture cycles under the conditions of light application time 15h/d, is sent out
Existing suspension culture product growth rate is very fast, size is homogeneous, and suspension is limpider, thin so as to set up embryo without browning
Born of the same parents' suspension system.
4）Plant regeneration
By step 3）The embryo materials that fluid suspension culture is obtained are inoculated into addition 0.8%（W/V）The 1/2MS solid culture of agar
Cultivate in base, embryonal connective tissue keeps stable, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums induction differentiation of agar,
Embryonal connective tissue differentiation synchronicity is high, and body embryo sprout time is more consistent, and it is neat to emerge, and deformity is not observed in form.Culture bar
Part：25 ± 2 DEG C of temperature, light application time 14h/ days, the Lx of intensity of illumination 2000.
Embodiment 4
MS minimal mediums：Can refer to document（Murashige T, Skoog F. A revised medium for rapid
grouth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15:
473-497）Prepare.
The method that Chinese catalpa suspension culture system of the present invention is set up, comprises the steps of：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus
Explant sterilizes, and takes the immature seed of Chinese catalpa, after ethanol, 2% liquor natrii hypochloritises sterilizing successively Jing 75%, strips children
Tender embryo is used as explant；Described explant is placed in a series of solid mediums and is induced, obtain faint yellow embryo callus subculture
Tissue.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of calluss：With sterilized Chinese catalpa
It is explant to set young tender embryo, is placed on callus induction on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is with the addition of on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. wound healing
The propagation of tissue：Calluss are placed on proliferated culture medium and are bred；Culture medium is added on the basis of 1/2MS minimal mediums
The α-naphthaleneacetic acid that the 6-benzyladenine (6-BA) and concentration that concentration is 1.5mg/L is 0.08mg/L is added（NAA）；3. embryo is healed
The induction of injured tissue：Calluss after propagation are placed in into induced embryonic callus in induction culture medium, induction
Culture medium is that 6- benzyl purines (6-BA) that concentration is 0.65 mg/L and dense is with the addition of on the basis of 1/2MS minimal mediums
Spend for the α-naphthaleneacetic acid of 0.01 mg/L（NAA））
2）Primary suspension culture product induction
The Chinese catalpa calluss of the acquisition are inoculated in suspending nutrient solution carries out concussion and cultivate, such as in 150ml triangular flasks
The amount of culture fluid be 60ml, cultivate rotating speed 120r/min, 24 DEG C of cultivation temperature, intensity of illumination 1000Lx, light application time 14h/d
Under the conditions of, 36 days shaken cultivation time, sieved process with the nylon wire of 60 mesh, 80 mesh during successive transfer culture per 18 days subcultures once,
Liquid to stand and abandon supernatant after 1h after sieving, and fills into equivalent fresh culture, and the cell mass on sieve is accessed into fresh liquid training
Continue to cultivate in foster base.The suspending nutrient solution contains：1/2MS.
The product suspension of culture is observed to stereomicroscope, it is seen that dissociate in a large number transparent strip cell and spherical thin
Born of the same parents, the cell mass assembled based on sphaerocyst on a small quantity, and cell are well-illuminated, inclusions are thin.
3）The foundation of cells,primordial suspension system
The culture fluid of preferred Embryogenic suspension system is the PEG6000 of 1/2MS culture medium addition 15%.
The primary suspension culture amplification product is crossed after cell sieve and is transferred in suspension system culture fluid, in rotating speed
110r/min, 27 DEG C of temperature, intensity of illumination 1000Lx is cultivated, 18 days successive transfer culture cycles under the conditions of light application time 14h/d, is sent out
Existing suspension culture product growth rate is very fast, size is homogeneous, and suspension is limpider, thin so as to set up embryo without browning
Born of the same parents' suspension system.
4）Plant regeneration
The embryo materials that fluid suspension culture is obtained are inoculated into addition 0.8%（W/V）Cultivate in the 1/2MS solid mediums of agar,
Embryonal connective tissue keeps stable, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums induction differentiation of agar, embryonal connective tissue
Differentiation synchronicity is high, and body embryo sprout time is more consistent, and it is neat to emerge, and deformity is not observed in form.Condition of culture：Temperature 25
± 2 DEG C, light application time 14h/ days, the Lx of intensity of illumination 2000.
Module number described herein and treatment scale are the explanations for simplifying the present invention.To the present invention-Chinese catalpa embryo
The application of the method for building up of sexual cell suspension system, modifications and variations will be readily apparent to persons skilled in the art.
The above embodiments are only the preferred technical solution of the present invention, and are not construed as the restriction of the present invention, this Shen
Please in embodiment and the feature in embodiment in the case where not conflicting, can mutual combination in any.The protection model of the present invention
Enclose the equivalent side of technical characteristic in the technical scheme that should be recorded with claim, including the technical scheme of claim record
Case is protection domain.Equivalent i.e. within this range is improved, also within protection scope of the present invention.
Claims (7)
1. a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, it is characterised in that comprise the following steps：
1）Explant sterilizing and the acquisition of Chinese catalpa embryo callus：Take the immature seed of Chinese catalpa, it is sterilized after, strip children tender
Embryo induces described explant as explant, obtains embryo callus；
2）Primary suspension culture product induction：By step 1）The embryo callus of acquisition are transferred in primary culture fluid and are trained
Support, obtain Chinese catalpa primary suspension culture product；
3）Cells,primordial suspension culture is screened：By step 3）The primary suspension culture amplification product of acquisition cross after cell sieve transfer in
Cultivate in cells,primordial suspending nutrient solution, obtain cells,primordial suspension system；
Complete the foundation of Chinese catalpa Embryogenic Callus Suspension Culture.
2. method according to claim 1, it is characterised in that：The step 1）Sterilizing methods be successively the ethanol of Jing 75%,
2% liquor natrii hypochloritises' sterilizing.
3. method according to claim 1, it is characterised in that：The step 2）Primary culture fluid is that 1/2MS liquid is basic
Culture medium.
4. method according to claim 1, it is characterised in that：The step 2）Condition of culture is the r/ of rotating speed 100 ~ 120
Min, 23 ~ 27 DEG C of cultivation temperature, 900 ~ 1100Lx of intensity of illumination is cultivated, successive transfer culture under conditions of 14 ~ 16h/d of light application time
Cycle is 16-20 days, successive transfer culture 2 times, is sieved through sieve and is processed with 60 mesh, 80 aim cells during successive transfer culture, and liquid is quiet after sieving
Put and abandon after 1h supernatant, fill into equivalent fresh culture, and the cell mass on sieve is accessed into continuation in fresh liquid culture medium and train
Support.
5. method according to claim 1, it is characterised in that：The step 3）Condition of culture is the r/ of rotating speed 100 ~ 120
Min, 23 ~ 27 DEG C of cultivation temperature, 900 ~ 1100Lx of intensity of illumination, the CMC model of 14 ~ 16h/d of light application time, successive transfer culture week
16-20 days phase, successive transfer culture was that 60 mesh cell sieves are processed.
6. method according to claim 5, it is characterised in that：The step 3）Cells,primordial suspension culture screening and culturing liquid
Polyethylene glycol 6000 is with the addition of for 1/2MS, polyethylene glycol 6000 mass concentration is 0%-20% in the culture fluid.
7. method according to claim 6, it is characterised in that：Polyethylene glycol 6000 mass concentration is in the culture fluid
10%。
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| CN107058374A (en) * | 2017-05-11 | 2017-08-18 | 三峡大学 | A kind of Chinese catalpa loses the construction method of transformation system |
| CN115836647A (en) * | 2023-02-22 | 2023-03-24 | 中国林业科学研究院 | Method for regenerating catalpa bungei young embryo sterile induction plant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058374A (en) * | 2017-05-11 | 2017-08-18 | 三峡大学 | A kind of Chinese catalpa loses the construction method of transformation system |
| CN115836647A (en) * | 2023-02-22 | 2023-03-24 | 中国林业科学研究院 | Method for regenerating catalpa bungei young embryo sterile induction plant |
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| CN106561452B (en) | 2018-09-28 |
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