CN106489742B - A kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture - Google Patents
A kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture Download PDFInfo
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- CN106489742B CN106489742B CN201611108750.6A CN201611108750A CN106489742B CN 106489742 B CN106489742 B CN 106489742B CN 201611108750 A CN201611108750 A CN 201611108750A CN 106489742 B CN106489742 B CN 106489742B
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention discloses a kind of methods that Chinese tallow tree regeneration plant is obtained by Anther Culture, include the following steps：The selection of explant, the Cold pretreatment of explant, the surface sterilization of explant, induction of callus, the Multiplying culture of callus, the differentiation of callus, the elongation of adventitious bud, adventitious bud are taken root.The advantage of the invention is that：Providing a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture, and in particular to a kind of anther using energy tree species Chinese tallow tree is explant, by the induction of callus, proliferation and differentiation, the final method for obtaining Chinese tallow tree intact plant.The advantage of the invention is that：Establish simple, quick, the stable vitro anther regenerating system of a set of suitable Chinese tallow tree, the structure of haploid breeding, genetic map for Chinese tallow tree and the establishment of later stage germ plasm resource etc. provide technical support, meanwhile genetic improvement to Chinese tallow tree and the selection and breeding of new varieties are of great significance.
Description
Technical field
The present invention relates to plant biotechnology field more particularly to a kind of Chinese tallow tree regeneration plant is obtained by Anther Culture
Method.
Background technology
Chinese tallow tree (Sapium sebiferum Roxb), Euphorbiaceae sapium deciduous tree, be it is a kind of collection it is ornamental, medicinal and
Oil is for integrated multi-functional seeds.Chinese tallow tree is adaptable, and distribution is wide, and seed oil content is up to 55%, is manufacture candle, fertilizer
Soap, metallic soap, lubricating grease, synthetic detergent, softening agent and produce palmitic acid, stearic acid, fiberglass, advanced spray painting raw material.
Chinese tallow tree seed also becomes the important source material of preparing biological diesel oil in recent years, while is also the important original for preparing chocolate, cosmetics
Material.In addition, Chinese tallow tree because its leaf color is changeable, shows the colors such as red, orange, yellow, green, purple and brown in the fall, and it is that one kind has hair
The landscape plant of potentiality is opened up, there is wide application prospect.
Since Chinese tallow tree is perennial woody plant, and height heterozygosis is difficult to meet to educate by traditional crossbreeding mode
Kind demand, and haploid breeding can greatly shorten the breeding time limit, accelerate breeding process, in plant genetics and breeding theory and practice
It is with a wide range of applications in research.Plant anther culture technique is the most common method for obtaining haplobiont, is passed through
Anther Culture can fast and effeciently obtain homozygous doubled haploid, and avoid needs inbreeding of more generation just can be with by traditional breeding method means
Obtain the homozygous strain stablized.In addition, structure, the training of genetic map are used directly for by the monoploid that Anther Culture obtains
It educates new varieties or is used for breed improvement as Parents.Therefore, the present invention establishes a set of be suitble to using Chinese tallow tree anther as material
Simple, quick, the vitro anther regenerating system stablized of Chinese tallow tree, be the haploid breeding of Chinese tallow tree, genetic map structure and after
Establishment of phase germ plasm resource etc. provides technical support；Meanwhile genetic improvement to Chinese tallow tree and the selection and breeding of new varieties all have it is important
Meaning.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of the simple, quick, stabilizations of suitable Chinese tallow tree
The method that Chinese tallow tree regeneration plant is obtained by Anther Culture.
The present invention is achieved by the following technical solutions：A kind of side that Chinese tallow tree regeneration plant is obtained by Anther Culture
Method includes the following steps：
(1) selection of explant
It chooses and grows to the Chinese tallow tree inflorescence of suitable time for follow-up cultivation；
(2) Cold pretreatment of explant
Inflorescence is built in refrigerator loaded on self-sealing plastic bag and carries out Cold pretreatment；
(3) surface sterilization of explant
Inflorescence after Cold pretreatment is placed on through flowing water flushing in aseptic operating platform and carries out surface sterilization；
(4) induction of callus
Filter paper blots the moisture on inflorescence surface after disinfection in step (3), strips anther, is inoculated in induction of callus
In base, using the illumination cultivation of 3-4 weeks, callus is obtained；
(5) Multiplying culture of callus
The callus switching obtained in step (4) is subjected to Multiplying culture in proliferated culture medium；
(6) differentiation of callus
Callus after proliferation in step (5) is cut into stripping and slicing, transfers and callus is carried out in differential medium
Differentiation culture；
(7) elongation of adventitious bud
By in step (6) it is differentiated go out Multiple Buds callus, transferring, it is indefinite to be carried out in Elongation of adventitious bud culture medium
The elongation culture of bud simultaneously obtains corresponding Chinese tallow tree seedling；
(8) adventitious bud is taken root
The Chinese tallow tree seedling switching extended in step (7) in root media is subjected to culture of rootage, finally obtains Chinese tallow tree
Regeneration plant.
Preferably, the Chinese tallow tree inflorescence that suitable time is grown in the step (1) is through sediments microscope inspection, chooses microspore
Chinese tallow tree inflorescence in monokaryon mid-term or mid-late uninucleate stage.
Preferably, the method for Cold pretreatment is in the step (2)：Inflorescence is placed in 3-5 DEG C of refrigerator processing 6-24h.
Preferably, the specific method of the surface sterilization of explant is in the step (3)：Inflorescence is rinsed into 20- through flowing water
After 30min, aseptic water washing is used in aseptic operating platform 3-5 times；It is sterile again using the ethanol disinfection 15-20s of 70%-75%
Water rinses 3-5 times, is repeated 1 times；Then it, then with the mercuric chloride of 0.1% (w/v) added with 0.5-1.0% (v/v) Tween sterilizes
60-120s is finally rinsed 5-8 times again with sterile water.
Preferably, stripping for anther carries out in aseptic operating platform by tip tweezers in the step (4).
Preferably, callus inducing medium is added with 0.6-3.0mg/L TDZ and 0.6- in the step (4)
The DKW culture mediums of 3.0mg/L IAA.
Preferably, proliferated culture medium is added with 0.1-0.5mg/L TDZ and 1.0-2.0mg/L in the step (5)
The DKW culture mediums of IAA.
Preferably, differential medium is added with 0.2-0.5mg/L TDZ and 0.1-0.5mg/L ZT in the step (6)
DKW culture mediums.
Preferably, Elongation of adventitious bud culture medium is the 1/2DKW trainings added with 0.05-0.2mg/L ZT in the step (7)
Support base.
Preferably, root media is the 1/2DKW cultures added with 0.1-2.0mg/L spermidines in the step (8)
Base.
The advantage of the invention is that：A kind of side that Chinese tallow tree regeneration plant is obtained by Anther Culture provided by the present invention
Method, and in particular to a kind of anther using energy tree species Chinese tallow tree by the induction of callus, proliferation and divides as explant
Change, the final method for obtaining Chinese tallow tree intact plant；The present invention establishes simple, quick, the stable anther of a set of suitable Chinese tallow tree
Vitro Regeneration System, the structure of haploid breeding, genetic map for Chinese tallow tree and the establishment of later stage germ plasm resource etc. provide technology
Support, meanwhile, the selection and breeding of genetic improvement and new varieties to Chinese tallow tree are of great significance.
Description of the drawings
Fig. 1 is the stream of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that 1-3 of the embodiment of the present invention is provided
Cheng Tu；
Fig. 2 is the small spore of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
Son is in the anther figure of mid-late uninucleate stage；
Fig. 3 is the small spore of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
Inflorescence size figure corresponding to anther of the son in mid-late uninucleate stage；
Fig. 4 is the anther of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
The induced map of callus；
Fig. 5 is the callus of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
The vegetative map of tissue；
Fig. 6 is the callus of a kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
The differentiation figure of tissue；
Fig. 7 is a kind of the indefinite of method that Chinese tallow tree regeneration plant is obtained by Anther Culture that the embodiment of the present invention 1 provides
The figure of taking root of bud.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment is carried out lower based on the technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
A kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture, as shown in Figure 1, including the following steps：
(1) selection of explant
Through sediments microscope inspection, choose microspore and be in the inflorescence corresponding to the Chinese tallow tree anther of mid-late uninucleate stage for subsequently training
It supports, wherein, the inflorescence size figure that microspore is in corresponding to the anther and anther of mid-late uninucleate stage is distinguished as shown in Figure 2 and Figure 3；
(2) Cold pretreatment of explant
Inflorescence is built in 3 DEG C of refrigerators loaded on self-sealing plastic bag and carries out Cold pretreatment 6h；
(3) surface sterilization of explant
Inflorescence after Cold pretreatment is placed on through flowing water flushing 20min in aseptic operating platform and uses aseptic water washing
3 times；70% ethanol disinfection 15s is used again, and aseptic water washing 3 times is repeated 1 times；Then, then with added with 0.5% (v/v)
The mercuric chloride disinfection 60s of 0.1% (w/v) of Tween, is finally rinsed 5 times again with sterile water；
(4) induction of callus
Filter paper blots the moisture on inflorescence surface after disinfection in step (3), is stripped in aseptic operating platform by tip tweezers
Anther is inoculated in callus inducing medium, is positioned over periodicity of illumination as 8/16 (illumination/dark), intensity of illumination
(2000-2500lx), constant temperature incubation room of the temperature for (24 ± 1) DEG C, by the culture of 3 weeks, obtains light green as shown in Figure 4
Anther callus, the inductivity of callus is up to 100%；Wherein the inducing culture of callus be added with
The DKW culture mediums of 0.6mg/L TDZ and 0.6mg/L IAA；
(5) Multiplying culture of callus
The switching of the anther callus of a diameter of 4mm induced will be cured in proliferated culture medium in step (4)
The Multiplying culture of injured tissue, the proliferation efficiency of callus is up to 6.8 after culture 5 weeks, wherein proliferated culture medium be added with
The DKW culture mediums of 0.1mg/L TDZ and 1.0mg/L IAA；
(6) differentiation of callus
Callus after proliferation as shown in Figure 5 is cut into stripping and slicing, transfers on differential medium, is in periodicity of illumination
16/8 (illumination/dark), intensity of illumination (2500-3000lx), temperature is in the constant temperature incubation room of (26 ± 1) DEG C, carries out callus
The differentiation of tissue；By the illumination cultivation of 3 weeks, 93.2% callus differentiated indefinite bud point as shown in Figure 6, wherein dividing
Change culture medium is the DKW culture mediums added with 0.2mg/L TDZ and 0.1mg/L ZT；
(7) elongation of adventitious bud
After adventitious bud length to 0.5cm, it will transfer in Elongation of adventitious bud culture medium with the callus of adventitious bud clump
It carries out the elongation culture of adventitious bud and obtains corresponding Chinese tallow tree seedling, wherein Elongation of adventitious bud culture medium is added with 0.05mg/L
The 1/2DKW culture mediums of ZT；
(8) adventitious bud is taken root
After illumination cultivation 2 weeks, the Chinese tallow tree adventitious bud of elongation is transferred, culture of rootage is carried out in root media, 1 month
The Chinese tallow tree regeneration plant of root system stalwartness as shown in Figure 7 is obtained afterwards, and wherein root media is added with 0.1mg/L spermidines
1/2DKW culture mediums.
Embodiment 2
A kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture, as shown in Figure 1, including the following steps：
(1) selection of explant
Through sediments microscope inspection, choose microspore and be in the inflorescence corresponding to the Chinese tallow tree anther of monokaryon mid-term for subsequently training
It supports；
(2) Cold pretreatment of explant
Inflorescence is built in 5 DEG C of refrigerators loaded on self-sealing plastic bag and carries out Cold pretreatment for 24 hours；
(3) surface sterilization of explant
Inflorescence after Cold pretreatment is placed on through flowing water flushing 30min in aseptic operating platform and uses aseptic water washing
5 times；75% ethanol disinfection 20s is used again, and aseptic water washing 5 times is repeated 1 times；Then, then with added with 1.0% (v/v)
The mercuric chloride disinfection 120s of 0.1% (w/v) of Tween, is finally rinsed 8 times again with sterile water；
(4) induction of callus
Filter paper blots the moisture on inflorescence surface after disinfection in step (3), is stripped in aseptic operating platform by tip tweezers
Anther is inoculated in callus inducing medium, is positioned over periodicity of illumination as 8/16 (illumination/dark), intensity of illumination
(2000-2500lx), constant temperature incubation room of the temperature for (24 ± 1) DEG C, by the culture of 4 weeks, obtains absinthe-green antherderived callus
Tissue, the inductivity of callus are up to 100%；Wherein the inducing culture of callus be added with 3.0mg/L TDZ and
The DKW culture mediums of 3.0mg/L IAA；
(5) Multiplying culture of callus
The switching of the anther callus of a diameter of 6mm induced will be cured in proliferated culture medium in step (4)
The Multiplying culture of injured tissue, the proliferation efficiency of callus is up to 6.8 after culture 5 weeks, wherein proliferated culture medium be added with
The DKW culture mediums of 0.5mg/L TDZ and 2.0mg/L IAA；
(6) differentiation of callus
Callus after proliferation is cut into stripping and slicing, is transferred on differential medium, in periodicity of illumination for 16/8 (illumination/
It is dark), intensity of illumination (2500-3000lx), temperature is in the constant temperature incubation room of (26 ± 1) DEG C, carries out the differentiation of callus；
By the illumination cultivation of 3 weeks, 93.2% callus differentiated indefinite bud point, and wherein differential medium is added with 0.5mg/
The DKW culture mediums of L TDZ and 0.5mg/L ZT；
(7) elongation of adventitious bud
After adventitious bud length to 1.0cm, it will transfer in Elongation of adventitious bud culture medium with the callus of adventitious bud clump
It carries out the elongation culture of adventitious bud and obtains corresponding Chinese tallow tree seedling, wherein Elongation of adventitious bud culture medium is added with 0.2mg/L ZT
1/2DKW culture mediums；
(8) adventitious bud is taken root
After illumination cultivation 2 weeks, the Chinese tallow tree adventitious bud of elongation is transferred, culture of rootage is carried out in root media, 1 month
The Chinese tallow tree regeneration plant of root system stalwartness is obtained afterwards, and wherein root media is the 1/2DKW cultures added with 2.0mg/L spermidines
Base.
Embodiment 3
A kind of method that Chinese tallow tree regeneration plant is obtained by Anther Culture, as shown in Figure 1, including the following steps：
(1) selection of explant
Through sediments microscope inspection, choose microspore and be in the inflorescence corresponding to the Chinese tallow tree anther of monokaryon mid-term for subsequently training
It supports；
(2) Cold pretreatment of explant
Inflorescence is built in 4 DEG C of refrigerators loaded on self-sealing plastic bag and carries out Cold pretreatment 12h；
(3) surface sterilization of explant
Inflorescence after Cold pretreatment is placed on through flowing water flushing 25min in aseptic operating platform and uses aseptic water washing
4 times；73% ethanol disinfection 18s is used again, and aseptic water washing 4 times is repeated 1 times；Then, then with added with 0.8% (v/v)
The mercuric chloride disinfection 100s of 0.1% (w/v) of Tween, is finally rinsed 6 times again with sterile water；
(4) induction of callus
Filter paper blots the moisture on inflorescence surface after disinfection in step (3), is stripped in aseptic operating platform by tip tweezers
Anther is inoculated in callus inducing medium, wherein the inducing culture of callus be added with 1.0mg/L TDZ and
The DKW culture mediums of 2.0mg/L IAA；
(5) Multiplying culture of callus
The anther callus induced switching will be carried out to the proliferation of callus in proliferated culture medium in step (4)
Culture, wherein proliferated culture medium are the DKW culture mediums added with 0.3mg/L TDZ and 1.5mg/L IAA；
(6) differentiation of callus
Callus after proliferation is cut into stripping and slicing, is transferred on differential medium, wherein differential medium be added with
The DKW culture mediums of 0.3mg/L TDZ and 0.3mg/L ZT；
(7) elongation of adventitious bud
After adventitious bud length to 0.7cm, it will transfer in Elongation of adventitious bud culture medium with the callus of adventitious bud clump
It carries out the elongation culture of adventitious bud and obtains corresponding Chinese tallow tree seedling, wherein Elongation of adventitious bud culture medium is added with 0.1mg/L ZT
1/2DKW culture mediums；
(8) adventitious bud is taken root
After illumination cultivation 1.5 weeks, the Chinese tallow tree adventitious bud of elongation is transferred, culture of rootage is carried out in root media, 1
The Chinese tallow tree regeneration plant of root system stalwartness is obtained after month, wherein root media is the 1/2DKW trainings added with 1.0mg/L spermidines
Support base.
In addition, the kinds of culture medium and the corresponding plant growth regulating additionally added that are used during above-mentioned Anther Culture
The type and concentration of agent are probed by certain experimental study, and main research process and result are as follows：
(1) influence of various concentration TDZ, IAA and type of culture medium to Chinese tallow tree induction of anther callus and proliferation
Explant after surface sterilization is inoculated in the culture medium added with various concentration TDZ and IAA and carries out callus
The induction of tissue and proliferation, condition of culture are：Temperature (24 ± 1 DEG C), intensity of illumination 2000lx, light application time 8h/d；Culture
After 30d, the inductivity of callus and the growing way (table 1) of callus are counted；
The influence to Chinese tallow tree induction of anther callus is used in combination with IAA by 1 TDZ of table
As known from Table 1, various concentration TDZ and IAA, which is applied in combination, has Chinese tallow tree induction of anther callus rate and growing way
Significant impact；Anther is inoculated on the DKW culture mediums added with 1.0mg/L TDZ and 2.0mg/L IAA, callus
Inductivity is up to 100%, and callus is green, and quality is loose, conducive to the proliferation in later stage and differentiation research；
(2) influence that variety classes minimal medium type breaks up Chinese tallow tree induction of anther callus
On the basis of above-mentioned callus induction, variety classes minimal medium type is further tested to Chinese tallow tree anther
Anther after above-mentioned disinfection is inoculated in added with 1.0mg/LTDZ and 2.0mg/L IAA's by the influence of callus induction
The induction of callus is carried out on different type minimal medium (MS, WPM, DKW and B5)；
Influence of 2 type of culture medium of table to Chinese tallow tree induction of anther callus
Research finds (table 2) that type of culture medium, which has the induction differentiation of Chinese tallow tree anther callus, to be significantly affected, in institute
In 4 type culture mediums of test, most beneficial for the induction of Chinese tallow tree anther callus, B5 is most disadvantageous in DKW minimal mediums
The induction of callus；
(3) influences of the various concentration TDZ and IAA to callus proliferation
On the basis of callus induction differentiation, shadow of the TDZ and IAA concentration to callus proliferation is further tested
It rings, research finds (table 3), and by adjusting the concentration of TDZ and IAA, growth coefficient and growing way to callus equally have bright
Aobvious influence；The anther for inducing callus is transferred on the culture medium added with 0.3mg/L TDZ and 1.5mg/L IAA
Squamous subculture is carried out, callus quality is loose, and color is crisp green, and conducive to the differentiation in later stage, and the growth coefficient of callus is high
Up to 6.8；
The influence that table 3 various concentration TDZ and IAA combined use are proliferated anther callus
(4) influence that various concentration TDZ and ZT breaks up Chinese tallow tree anther callus
Callus after proliferation is cut into 0.1cm2The stripping and slicing of size is inoculated in added with various concentration TDZ and ZT
The differentiation research of callus is carried out in culture medium；Condition of culture is：Temperature (26 ± 1 DEG C), intensity of illumination 3000lx, during illumination
Between be 16h/d；After cultivating 30d, the differentiation rate (table 4) of callus is counted；
The influence that 4 various concentration TDZ and ZT of table breaks up Chinese tallow tree anther callus
Result of study shows that TDZ and ZT have apparent influence to the differentiation of Chinese tallow tree anther callus；Low concentration
TDZ and ZT is more advantageous to the differentiation of callus, during added with 0.3mg/L TDZ and 0.3mg/LZT, the differentiation rate of callus
Up to 93.2%；
(5) influence for the Elongation of adventitious bud that various concentration ZT is differentiated to form Chinese tallow tree anther callus
The callus for differentiating indefinite bud point switching is subjected to adventitious bud in the culture medium added with various concentration ZT
Elongation research；Condition of culture is：Temperature (26 ± 1 DEG C), intensity of illumination 3000lx, light application time 16h/d.After cultivating 30d,
Count the elongation situation (table 5) of adventitious bud；
Influences of the 5 various concentration ZT of table to Chinese tallow tree anther Elongation of adventitious bud
| ZT(mg/L) | Bud elongation (%) | Average bud length (cm) |
| 0.05 | 36.8 | 1.8 |
| 0.1 | 91.7 | 3.3 |
| 0.2 | 51.1 | 0.9 |
Result of study shows that the ZT of low concentration has important shadow to the elongation of Chinese tallow tree anther adventitious bud and average bud length
It rings, when 0.1mg/L ZT are added in culture medium, Elongation of adventitious bud rate is up to 91.7%, a length of 3.3cm of average bud；
(6) influence of the various concentration spermidine to Chinese tallow tree anther adventitious bud rooting
After illumination cultivation 2 weeks, the adventitious bud of elongation is transferred in the 1/2DKW culture mediums added with various concentration spermidine
The middle research of taking root for carrying out adventitious bud；Condition of culture is：Temperature (26 ± 1 DEG C), intensity of illumination 3000lx, light application time 16h/
d.After cultivating 30d, the elongation situation (table 6) of adventitious bud is counted；
Influence of the 6 various concentration spermidine of table to Chinese tallow tree anther adventitious bud rooting
Result of study shows to add rooting rate and shortening that spermidine is conducive to improve Chinese tallow tree anther adventitious bud in culture medium
The formation time of adventitious root；When adding 1.0mg/L spermidines in culture medium, adventitious bud rooting rate is up to 100%, average each
Explant generates 5.9 adventitious roots, and a length of 2.9cm of average root, the sturdy transplanting for being conducive to the later stage of root.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (5)
- A kind of 1. method that Chinese tallow tree regeneration plant is obtained by Anther Culture, which is characterized in that include the following steps：(1) selection of explantIt chooses and grows to the Chinese tallow tree inflorescence of suitable time for follow-up cultivation；(2) Cold pretreatment of explantInflorescence is built in refrigerator loaded on self-sealing plastic bag and carries out Cold pretreatment；(3) surface sterilization of explantInflorescence after Cold pretreatment is placed on through flowing water flushing in aseptic operating platform and carries out surface sterilization；(4) induction of callusFilter paper blots the moisture on inflorescence surface after disinfection in step (3), strips anther, is inoculated in callus inducing medium In, using the illumination cultivation of 3-4 weeks, obtain callus；Wherein, callus inducing medium is specially added with 0.6- The DKW culture mediums of 3.0mg/L TDZ and 0.6-3.0mg/L IAA；(5) Multiplying culture of callusThe callus switching obtained in step (4) is subjected to Multiplying culture in proliferated culture medium；Wherein, proliferated culture medium has Body is the DKW culture mediums added with 0.1-0.5mg/L TDZ and 1.0-2.0mg/L IAA；(6) differentiation of callusCallus after proliferation in step (5) is cut into stripping and slicing, transfers and the differentiation of callus is carried out in differential medium Culture；Wherein, differential medium is specially the DKW culture mediums added with 0.2-0.5mg/L TDZ and 0.1-0.5mg/L ZT；(7) elongation of adventitious budBy in step (6) it is differentiated go out Multiple Buds callus, transfer and adventitious bud carried out in Elongation of adventitious bud culture medium Elongation cultivates and obtains corresponding Chinese tallow tree seedling；Wherein, Elongation of adventitious bud culture medium is specially added with 0.05-0.2mg/L ZT 1/2DKW culture mediums；(8) adventitious bud is taken rootThe Chinese tallow tree seedling switching extended in step (7) is subjected to culture of rootage in root media, it is final to obtain Chinese tallow tree regeneration Plant；Wherein, root media is specially the 1/2DKW culture mediums added with 0.1-2.0mg/L spermidines.
- A kind of 2. method that Chinese tallow tree regeneration plant is obtained by Anther Culture according to claim 1, which is characterized in that institute The Chinese tallow tree inflorescence that suitable time is grown in step (1) is stated through sediments microscope inspection, to choose microspore and being in monokaryon mid-term or list Core keeps to the side the Chinese tallow tree inflorescence of phase.
- A kind of 3. method that Chinese tallow tree regeneration plant is obtained by Anther Culture according to claim 1, which is characterized in that institute The method for stating Cold pretreatment in step (2) is：Inflorescence is placed in 3-5 DEG C of refrigerator processing 6-24h.
- A kind of 4. method that Chinese tallow tree regeneration plant is obtained by Anther Culture according to claim 1, which is characterized in that institute The specific method for stating the surface sterilization of explant in step (3) is：By inflorescence after flowing water rinses 20-30min, in sterile working Aseptic water washing is used in platform 3-5 times；The ethanol disinfection 15-20s of 70%-75% is used again, and aseptic water washing 3-5 times repeats 1 It is secondary；Then 60-120s, then with 0.1% mercuric chloride added with 0.5-1.0%Tween is sterilized, finally rinses 5- again with sterile water 8 times.
- A kind of 5. method that Chinese tallow tree regeneration plant is obtained by Anther Culture according to claim 1, which is characterized in that institute Stripping for anther in step (4) is stated to carry out by tip tweezers in aseptic operating platform.
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