CN106561452B - A kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture - Google Patents
A kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to technical field of cell culture, are related to a kind of method of Chinese catalpa cells,primordial suspension culture.Wherein include to pass through embryonic callus induction, primary suspension cultured products induction, the foundation of Embryogenic Callus Suspension Culture, plant regeneration process successively using the embryo of Chinese catalpa immature fruit as explant.The primary suspension cultured products inducing culture is 1/2MS；The Chinese catalpa cells,primordial suspension system culture solution is to be added to polyethylene glycol on 1/2MS culture mediums（PEG）6000.The method of the present invention can establish stable Chinese catalpa Embryogenic Callus Suspension Culture, and somatic embryo regeneration plant planting percent is high, emerge neatly without deformity.The present invention can study for Chinese catalpa body embryonic development, somatic hybridization, genetic transformation and Preservation of plant germplasin and provide stable material source and platform, and carrying out industrialization breeding for the bioreactor technology based on suspension cell culture provides most important theories and experimental basis.
Description
Technical field
The invention belongs to technical field of cell culture, are related to a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture.
Background technology
Chinese catalpa (Catalpa bungeiC.A.Meyer) belong to Bignoniaceae (Bignoniaceae) Catalpa (Catalpa),
It is originating in China, it is the distinctive rare tree in China, is known as the title of " the wooden king ".About 13 kinds of the plant that aster section Chinese catalpa belongs in the world, it is 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 zone, in Shandong, river
Soviet Union, Henan, Anhui Province's distribution are more（Shi Xin etc., 2011）.
Chinese catalpa is Precious Timber Species and famous ornamental plantation seeds using with a long history（Tang Ling etc., 2007）.Chinese catalpa
Important commerical tree species are classified as by country, timber has the characteristics that not warp and fracture, corrosion-resistant, easy processing, beautiful texture, special to add
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 a variety of toxic gases in environment, chlorine and sulfur dioxide etc. have stronger absorbability, it is higher to be that China has
The important afforestation of the value of environmental protection and ornamental tree species.Since self-sterility for Chinese catalpa, often bloom and shaky, seed germination rate
It is very low, and difficulty of taking root is bred using cottage method, breeding and utilization and extention to Chinese catalpa excellent strain cause prodigious obstacle,
In addition the excellent utilization for exacerbating people of material, cause the shortage of Chinese catalpa resource.
Currently, about the research and few that Chinese catalpa somatic embryo occurs, it is concentrated mainly on Somatic Embryogenesis
The initial research that induces of cytomorphology research, Physiology and biochemistry and body embryo, there is not yet efficiently steady relating to how to establish
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 it is easy to control, it can be achieved that the anniversary grow, do not influenced by geographical factor；Furthermore, it is also possible to generate new, satisfaction
The compound that the bioactivity such as medicine and chemical industry need；A successful cell suspension cultures system is established to carrying out basic research
It is most important with relying on bioreactor to carry out industrialization development.
Most common problem is browning and the disappearance of suspension cell embryo in plant cell suspension culture.It can be effectively
Both of these problems are solved, are established in place of cell morphology and ultrastructure.The producing cause of browning is more, such as the genotype of explant
And condition of culture etc..Common anti-browning substance includes mainly activated carbon, ascorbic acid, silver nitrate, citric acid, PVPP etc., this
A little additives can inhibit influence of the browning to suspended culture cell to a certain extent.The decline disappearance of suspension cell embryo is another
An outer major issue, especially during monocotyledon (such as sugarcane, rice and corn) cell suspension cultures, embryo
The problems demand that fails solves.Therefore, it by selecting suitable explant, maintains blast is firm to exist, establishes stable embryo
Property cell suspension cultures system, can be body embryo occur morphological observation, somatic hybridization, genetic transformation and industrialization it is numerous
It grows and experiment material and important theoretical experimental basis is provided.
Invention content
The present invention provides a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, is established 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, includes the following steps：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus：The immature seed of Chinese catalpa is taken, after sterilized, is stripped
The tender embryo of children induces the explant as explant, obtains embryo callus；
2）Primary suspension cultured products induction：By step 1）The embryo callus of acquisition be transferred in primary culture solution into
Row culture, obtains Chinese catalpa primary suspension cultured products；
3）Cells,primordial, which suspends, cultivates screening：By step 2）The primary culture amplification product that suspends of acquisition turns after crossing cell sieve
It is connected in cells,primordial suspending nutrient solution and cultivates, obtain cells,primordial suspension system；
Complete the foundation of Chinese catalpa Embryogenic Callus Suspension Culture.
Preferably, the step 1）Sterilizing methods are that the liquor natrii hypochloritis successively through 75% alcohol, 2% sterilizes.
Preferably, the step 2）Primary culture solution is 1/2MS liquid minimal mediums.
Preferably, the step 2）Condition of culture is 100 ~ 120 r/min of rotating speed, and 23 ~ 27 DEG C of cultivation temperature, illumination is strong
900 ~ 1100Lx is spent, is cultivated under conditions of 14 ~ 16h/d of light application time, 16-20 days squamous subculture period, squamous subculture 2 times, after
It is sieved through sieve processing with 60 mesh, 80 aim cells when being commissioned to train foster, liquid abandons supernatant after standing 1h after sieving, fills into the fresh training of equivalent
Base is supported, and will continue to cultivate in the cell mass access fresh liquid culture medium on sieve.
Preferably, the step 3）Condition of culture is 100 ~ 120 r/min of rotating speed, and 23 ~ 27 DEG C of cultivation temperature, illumination is strong
900 ~ 1100Lx is spent, the CMC model of 14 ~ 16h/d of light application time, in 16-20 days squamous subculture period, squamous subculture was 60 mesh
Cell sieve processing.
It is further preferred that the step 3）It is that 1/2MS is added to poly- second two that cells,primordial, which suspends and cultivates screening and culturing liquid,
Alcohol 6000, Macrogol 6000 mass concentration is 0%-20% in the culture solution
It is further preferred that Macrogol 6000 mass concentration is 10% in the culture solution.
Advantageous effect of the present invention：
1, the present invention provides a kind of method for building up of Embryogenic Callus Suspension Culture of Precious Timber Species Chinese catalpa.This method with
The tender embryo of children of Chinese catalpa immature seed induces embryo callus, and stable embryo is established by the method for cell suspension cultures
Property cell morphology and ultrastructure.Cultured in vitro advantage is carried out with the tender seed of children to be to induce differentiation capability strong, long-term subculture process is not easy to move back
Change, the cells,primordial suspension culture system of the stabilization of acquisition, can be used for secondary metabolite Commercial cultivation, Study on Genetic Transformation and
Bioreactor large-scale production etc., it may also be used for screen ideal mutant and suspension cell line physiological and biochemical index
Research.
2, the present invention is selected by culture solution, can be filtered out suitable culture formula of liquid and effective additive ingredient, be lured
It leads suspension cultured products to convert to embryogenic cell line, promotes cell Proliferation, avoid browning dead.By establishing liquid suspension culture
System can get the uniform consistent Embryogenic cell masses or unicellular of size, establish suspension culture system, be bioreactor scale
Metaplasia production, somatic embryo develop research of marching into the arena, somatic hybridization and Study on Genetic Transformation and provide experiment material and important reason
By experimental basis.
Description of the drawings
The invention will be further described with reference to the accompanying drawings and examples：
Photo of Fig. 1 Chinese catalpa primary suspension cultured products under stereomicroscope；
Fig. 2 is not added with more subculture suspension cultured products of PEG；
Fig. 3 adds the cells,primordial suspension culture amplification product of PEG；
Fig. 4 adds the suspension cultured products solid culture proliferation of PEG；
Fig. 5 adds the plant regeneration of the suspension cultured products of PEG.
Specific implementation mode
It is further illustrated the present invention with reference to embodiment, but the scope of protection of present invention is not limited to implement
The range of example statement.
Embodiment 1
MS minimal mediums：It 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）It prepares.
A kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, comprises the steps of：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus：
Explant sterilize, take the immature seed of Chinese catalpa, successively through 75% alcohol, 2% liquor natrii hypochloritis sterilizing after, stripping
Take the tender embryo of children as explant；The explant is placed in a series of solid mediums and is induced, faint yellow embryo is obtained
Callus.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of callus：With sterilized Chinese catalpa
The tender embryo of tree children is explant, places it in evoked callus on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is added on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. callus
The proliferation of tissue：Callus is placed on proliferated culture medium and is proliferated；Culture medium is added on the basis of 1/2MS minimal mediums
The α-naphthylacetic acid of 6-benzyladenine (6-BA) and a concentration of 0.1mg/L of a concentration of 2.0 mg/L are added（NAA）；3. embryo is cured
The induction of injured tissue：Callus after proliferation is placed in induced embryonic callus on induction culture medium, induction
Culture medium is that the 6- benzyl purines (6-BA) of a concentration of 1.0 mg/L and dense are added on the basis of 1/2MS minimal mediums
Degree is the α-naphthylacetic acid of 0-0.1 mg/L（NAA））
2）Primary suspension cultured products induction：By step 1）The embryo callus of acquisition be transferred in primary culture solution into
Row is cultivated, and the amount of culture solution is 100ml culture solutions in the triangular flask of 250ml, in rotating speed 110r/min, 25 DEG C of cultivation temperature, light
It according to intensity 1000Lx, is cultivated under conditions of light application time 16h/d, the Fiber differentiation period is 18 days, with 60 mesh, 80 when squamous subculture
Aim cell is sieved through sieve processing, and liquid abandons supernatant after standing 1h after sieving, fills into equivalent fresh culture, and will be thin on sieve
Continue to cultivate in born of the same parents group's access fresh liquid culture medium.The suspending nutrient solution contains：1/2MS obtains the suspension culture of Chinese catalpa primary
Product；
As shown in Figure 1, being observed under product suspension to stereomicroscope after culture, it is seen that largely dissociate transparent strip
Cell and sphaerocyst, cell mass made of being assembled based on sphaerocyst on a small quantity, and cell is well-illuminated, content is thin.It is secondary
Continue to use 1/2MS culture solution subcultures after subculture, the phenomenon of cell browning death easily occurs in incubation, suspension muddiness has greatly
Measure browning cultured products（Fig. 2）.
3）Cells,primordial, which suspends, cultivates screening：By step 2）It is inoculated in after the Chinese catalpa primary suspension cultured products sieving of acquisition
Cells,primordial suspends to cultivate and be cultivated in screening and culturing liquid, in rotating speed 110r/min, 25 DEG C ± 2 of cultivation temperature, intensity of illumination
It under conditions of 1000Lx, light application time 16h/d, cultivates 9 days, starts white or lurid globular embryo condensate occur（Such as figure
3）, squamous subculture 3 times, culture total time is 36 days, obtains the culture amplification product that suspends, establishes Embryogenic Callus Suspension Culture, finds
Suspension cultured products growth rate is very fast, size is uniform, and suspension is limpider, no browning；The cells,primordial suspends
Culture screening and culturing liquid is that 1/2MS is added to Macrogol 6000, and Macrogol 6000 mass concentration is in the culture solution
10%。
4）Plant regeneration
By step 3）The embryo materials that liquid suspension culture obtains are inoculated into addition 0.8%（W/V）The 1/2MS solids of agar
It is cultivated in culture medium, embryonal connective tissue keeps stablizing（Fig. 4）, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums of agar
Induction differentiation, embryonal connective tissue break up synchronism height, and body embryo sprout time is more consistent, and it is neat to emerge, and is not observed in form abnormal
Shape（Fig. 5）.Condition of culture：25 ± 2 DEG C of temperature, light application time 14h/ days, 2000 Lx of intensity of illumination.
Embodiment 2
MS minimal mediums：It 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）It prepares.
The method that Chinese catalpa suspension culture system of the present invention is established, comprises the steps of：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus
Explant sterilize, take the immature seed of Chinese catalpa, successively through 75% alcohol, 2% liquor natrii hypochloritis sterilizing after, stripping
Take the tender embryo of children as explant；The explant is placed in a series of solid mediums and is induced, faint yellow embryo is obtained
Callus.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of callus：With sterilized Chinese catalpa
The tender embryo of tree children is explant, places it in evoked callus on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is added on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. callus
The proliferation of tissue：Callus is placed on proliferated culture medium and is proliferated；Culture medium is added on the basis of 1/2MS minimal mediums
The α-naphthylacetic acid of 6-benzyladenine (6-BA) and a concentration of 0.05mg/L of a concentration of 1.0 mg/L are added（NAA）；3. embryo
The induction of callus：Callus after proliferation is placed in induced embryonic callus on induction culture medium, differentiation lures
Lead culture medium be added on the basis of 1/2MS minimal mediums a concentration of 0.9 mg/L 6- benzyl purines (6-BA) and
The α-naphthylacetic acid of a concentration of 0.05 mg/L（NAA））
2）Primary suspension cultured products induction
By step 1）The embryo callus of acquisition is transferred in primary culture solution and is cultivated, and is trained in 100ml triangular flasks
The amount of nutrient solution is 40ml, cultivates rotating speed 100r/min, 23 DEG C, intensity of illumination 1000Lx of cultivation temperature, the item of light application time 16h/d
Under part, 32 days shaken cultivation time, every 16 days subcultures are primary, handled with the nylon wire sieving of 60 mesh, 80 mesh when squamous subculture, mistake
Liquid abandons supernatant after standing 1h after sieve, fills into equivalent fresh culture, and the cell mass on sieve is accessed fresh liquid culture
Continue to cultivate in base.The suspending nutrient solution：1/2MS.
3）Cells,primordial, which suspends, cultivates screening：By step 2）It is inoculated in after the Chinese catalpa primary suspension cultured products sieving of acquisition
Cells,primordial suspends to cultivate and be cultivated in screening and culturing liquid,
The culture solution of cells,primordial suspension system is the PEG6000 of 1/2MS culture mediums addition 5%.
By switching after the primary culture amplification product sieving that suspends in embryonal suspension system culture solution, in rotating speed
100r/min is cultivated under the conditions of 25 DEG C, intensity of illumination 1000Lx, light application time 14h/d of temperature, 16 days squamous subculture periods, hair
Now suspension cultured products growth rate is very fast, size is uniform, and suspension is limpider, and no browning is thin to establish embryo
Born of the same parents' suspension system.
4）Plant regeneration
By step 3）The embryo materials that liquid suspension culture obtains are inoculated into addition 0.8%（W/V）The 1/2MS solids of agar
Cultivated in culture medium, embryonal connective tissue keep stablize, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums of agar induce
Differentiation, embryonal connective tissue break up synchronism height, and body embryo sprout time is more consistent, and it is neat to emerge, and does not observe deformity in form.Training
The condition of supporting：25 ± 2 DEG C of temperature, light application time 14h/ days, 2000 Lx of intensity of illumination.
Embodiment 3
MS minimal mediums：It 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）It prepares.
The method that Chinese catalpa suspension culture system of the present invention is established, comprises the steps of：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus
Explant sterilize, take the immature seed of Chinese catalpa, successively through 75% alcohol, 2% liquor natrii hypochloritis sterilizing after, stripping
Take the tender embryo of children as explant；The explant is placed in a series of solid mediums and is induced, faint yellow embryo is obtained
Callus.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of callus：With sterilized Chinese catalpa
The tender embryo of tree children is explant, places it in evoked callus on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is added on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. callus
The proliferation of tissue：Callus is placed on proliferated culture medium and is proliferated；Culture medium is added on the basis of 1/2MS minimal mediums
The α-naphthylacetic acid of 6-benzyladenine (6-BA) and a concentration of 0.06mg/L of a concentration of 0.8 mg/L are added（NAA）；3. embryo
The induction of callus：Callus after proliferation is placed in induced embryonic callus on induction culture medium, differentiation lures
Lead culture medium be added on the basis of 1/2MS minimal mediums a concentration of 0.07 mg/L 6- benzyl purines (6-BA) and
The α-naphthylacetic acid of a concentration of 0-0.1 mg/L（NAA））
2）Primary suspension cultured products induction
By step 1）The embryo callus of acquisition is transferred in primary culture solution and is cultivated, such as 200ml triangular flasks
The amount of middle culture solution is 80ml, cultivates rotating speed 90r/min, 25 ± 2 DEG C, intensity of illumination 1000Lx of cultivation temperature, light application time
Under conditions of 16h/d, 40 days shaken cultivation time, every 20 days subcultures are primary, are sieved through with 60 mesh, 80 aim cells when squamous subculture
Sieve processing, liquid abandons supernatant after standing 1h after sieving, fills into equivalent fresh culture, and the cell mass access on sieve is fresh
Continue to cultivate in fluid nutrient medium.The suspending nutrient solution contains：1/2MS0.
3）Cells,primordial, which suspends, cultivates screening：By step 2）It is inoculated in after the Chinese catalpa primary suspension cultured products sieving of acquisition
Cells,primordial suspends to cultivate and be cultivated in screening and culturing liquid, and the culture solution of cells,primordial suspension system is 1/2MS culture mediums addition 20%
PEG6000.
By switching after the primary culture amplification product sieving that suspends in embryonal suspension system culture solution, in rotating speed
100r/min is cultivated under the conditions of 27 DEG C, intensity of illumination 1000Lx, light application time 15h/d of temperature, 20 days squamous subculture periods, hair
Now suspension cultured products growth rate is very fast, size is uniform, and suspension is limpider, and no browning is thin to establish embryo
Born of the same parents' suspension system.
4）Plant regeneration
By step 3）The embryo materials that liquid suspension culture obtains are inoculated into addition 0.8%（W/V）The 1/2MS solids of agar
Cultivated in culture medium, embryonal connective tissue keep stablize, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums of agar induce
Differentiation, embryonal connective tissue break up synchronism height, and body embryo sprout time is more consistent, and it is neat to emerge, and does not observe deformity in form.Training
The condition of supporting：25 ± 2 DEG C of temperature, light application time 14h/ days, 2000 Lx of intensity of illumination.
Embodiment 4
MS minimal mediums：It 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）It prepares.
The method that Chinese catalpa suspension culture system of the present invention is established, comprises the steps of：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus
Explant sterilize, take the immature seed of Chinese catalpa, successively through 75% alcohol, 2% liquor natrii hypochloritis sterilizing after, stripping
Take the tender embryo of children as explant；The explant is placed in a series of solid mediums and is induced, faint yellow embryo is obtained
Callus.
The step 1）Chinese catalpa embryo callus preparation method includes：1. the induction of callus：With sterilized Chinese catalpa
The tender embryo of tree children is explant, places it in evoked callus on callus inducing medium；Inducing culture is 1/
6-benzyladenine (6-BA is added on the basis of 2MS minimal mediums）With 2,4 dichlorophenoxyacetic acid (2,4-D)；2. callus
The proliferation of tissue：Callus is placed on proliferated culture medium and is proliferated；Culture medium is added on the basis of 1/2MS minimal mediums
The α-naphthylacetic acid of 6-benzyladenine (6-BA) and a concentration of 0.08mg/L of a concentration of 1.5mg/L are added（NAA）；3. embryo is cured
The induction of injured tissue：Callus after proliferation is placed in induced embryonic callus on induction culture medium, induction
Culture medium is that the 6- benzyl purines (6-BA) of a concentration of 0.65 mg/L and dense are added on the basis of 1/2MS minimal mediums
Degree is the α-naphthylacetic acid of 0.01 mg/L（NAA））
2）Primary suspension cultured products induction
The Chinese catalpa callus of the acquisition is inoculated in suspending nutrient solution and carries out shake culture, such as 150ml triangles
The amount of culture solution is 60ml in bottle, cultivates rotating speed 120r/min, 24 DEG C, intensity of illumination 1000Lx of cultivation temperature, light application time
Under conditions of 14h/d, 36 days shaken cultivation time, every 18 days subcultures are primary, with the nylon wire mistake of 60 mesh, 80 mesh when squamous subculture
Sieve processing, liquid abandons supernatant after standing 1h after sieving, fills into equivalent fresh culture, and the cell mass access on sieve is fresh
Continue to cultivate in fluid nutrient medium.The suspending nutrient solution contains：1/2MS.
It is observed under the product suspension of culture to stereomicroscope, it is seen that largely dissociate transparent strip cell and spherical shape are thin
Born of the same parents, cell mass made of being assembled based on sphaerocyst on a small quantity, and cell is well-illuminated, content is thin.
3）The foundation of cells,primordial suspension system
The culture solution of preferred Embryogenic suspension system is the PEG6000 of 1/2MS culture mediums addition 15%.
Switching is in suspension system culture solution after the primary culture amplification product that suspends is crossed cell sieve, in rotating speed
110r/min is cultivated under the conditions of 27 DEG C, intensity of illumination 1000Lx, light application time 14h/d of temperature, 18 days squamous subculture periods, hair
Now suspension cultured products growth rate is very fast, size is uniform, and suspension is limpider, and no browning is thin to establish embryo
Born of the same parents' suspension system.
4）Plant regeneration
The embryo materials that liquid suspension culture obtains are inoculated into addition 0.8%（W/V）In the 1/2MS solid mediums of agar
Culture, embryonal connective tissue keep stablize, after be seeded to addition 0.7%（W/V）The 1/2MS solid mediums induction differentiation of agar, embryo
Tissue differentiation synchronism is high, and body embryo sprout time is more consistent, and it is neat to emerge, and does not observe deformity in form.Condition of culture：Temperature
25 ± 2 DEG C of degree, light application time 14h/ days, 2000 Lx of intensity of illumination.
Module number and treatment scale described herein are the explanations for simplifying the present invention.To the present invention-Chinese catalpa embryo
The application of the method for building up of property cell morphology and ultrastructure, 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 limitation for the present invention, this Shen
Please in embodiment and embodiment in feature in the absence of conflict, mutually can arbitrarily combine.The protection model of the present invention
Enclose the equivalent replacement side of technical characteristic in the technical solution that should be recorded with claim, including the technical solution of claim record
Case is protection domain.Equivalent replacement i.e. within this range is improved, also within protection scope of the present invention.
Claims (3)
1. a kind of method for building up of Chinese catalpa Embryogenic Callus Suspension Culture, which is characterized in that include the following steps：
1）Explant sterilizes and the acquisition of Chinese catalpa embryo callus：The immature seed of Chinese catalpa is taken, after sterilized, it is tender to strip children
Embryo induces the explant as explant, obtains embryo callus；
2）Primary suspension cultured products induction：By step 1）The embryo callus of acquisition is transferred in primary culture solution and is trained
It supports, obtains Chinese catalpa primary suspension cultured products；
3）Cells,primordial, which suspends, cultivates screening：By step 3）The primary culture amplification product that suspends of acquisition cross after cell sieve switching in
It is cultivated in cells,primordial suspending nutrient solution, obtains cells,primordial suspension system；
Complete the foundation of Chinese catalpa Embryogenic Callus Suspension Culture；
The step 2）Primary culture solution is 1/2MS liquid minimal mediums；
The step 2）Condition of culture be 100 ~ 120 r/min of rotating speed, 23 ~ 27 DEG C of cultivation temperature, intensity of illumination 900 ~
It is cultivated under conditions of 1100Lx, 14 ~ 16h/d of light application time, the squamous subculture period is 16-20 days, squamous subculture 2 times, after being commissioned to train
It is sieved through sieve processing with 60 mesh, 80 aim cells when supporting, liquid abandons supernatant after standing 1h after sieving, fills into equivalent fresh cultured
Base, and will continue to cultivate in the cell mass access fresh liquid culture medium on sieve；
The step 3）Condition of culture be 100 ~ 120 r/min of rotating speed, 23 ~ 27 DEG C of cultivation temperature, intensity of illumination 900 ~
1100Lx, the CMC model of 14 ~ 16h/d of light application time, 16-20 days squamous subculture period, squamous subculture were 60 mesh cell sieves
Processing；
The step 3）It is that 1/2MS is added to Macrogol 6000, the culture solution that cells,primordial, which suspends and cultivates screening and culturing liquid,
Middle Macrogol 6000 mass concentration is 5%-20%.
2. according to the method described in claim 1, it is characterized in that：The step 1）Sterilizing methods be successively through 75% alcohol,
2% liquor natrii hypochloritis's sterilizing.
3. according to the method described in claim 1, it is characterized in that：Macrogol 6000 mass concentration is in the culture solution
10%。
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