CN107047296A - A kind of Phoebe chekiangensis somatic embryo inducement method - Google Patents
A kind of Phoebe chekiangensis somatic embryo inducement method Download PDFInfo
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 157
- 230000000392 somatic effect Effects 0.000 title claims abstract description 88
- 241000371496 Phoebe chekiangensis Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 35
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 24
- 239000012869 germination medium Substances 0.000 claims abstract description 12
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 8
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 17
- 239000007787 solid Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 229960002523 mercuric chloride Drugs 0.000 claims description 9
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 7
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 229940032628 glutamine 500 mg Drugs 0.000 claims description 5
- 108010079058 casein hydrolysate Proteins 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 5
- 239000002609 medium Substances 0.000 abstract description 5
- 230000008859 change Effects 0.000 abstract description 3
- 230000035784 germination Effects 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 241000422846 Sequoiadendron giganteum Species 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 244000050510 Cunninghamia lanceolata Species 0.000 description 1
- 241000218691 Cupressaceae Species 0.000 description 1
- 241000218652 Larix Species 0.000 description 1
- 235000005590 Larix decidua Nutrition 0.000 description 1
- 241000604739 Phoebe Species 0.000 description 1
- 241000351396 Picea asperata Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
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- 230000035876 healing Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 230000008929 regeneration Effects 0.000 description 1
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Classifications
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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
- A01H4/001—Culture apparatus for tissue culture
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of Phoebe chekiangensis somatic embryo inducement method, it comprises the following steps：1) with Phoebe chekiangensis crude fruit actually explant, surface sterilizing is carried out；2) seed rataria is stripped, is inoculated on somatic embryo inducement culture medium, somatic embryo inducement is carried out；3) by step 2) somatic embryos are inoculated on somatic embryo proliferated culture medium, carry out the propagation of somatic embryo, produce Secondary embryos.The present invention establishes the direct method for generation of somatic embryo of Phoebe chekiangensis immature zygotic embryos first, in globular embryo or to heart-shape embryo change when zygotic embryo on the culture medium of the present invention, body embryo incidence is up to 65%.In optimum multiplication medium, growth coefficient is up to 14.3, and in optimal body embryo germination medium, the germination rate of somatic embryo is up to 68.5%.
Description
Technical field
The invention belongs to plant biotechnology field, and in particular to a kind of Phoebe chekiangensis somatic embryo inducement method.
Background technology
Phoebe chekiangensis (Phoebe chekiangensis) belongs to the high megaphanerophyte of Lauraceae Phoebe, is that the original seed of " silkwood " is planted
One of species, are the peculiar rare tree in Subtropical Zone of East area, NATURAL DISTRIBUTION is in Zhejiang, Fu Jianbei with very high economic value
Portion, Jiangxi northeast and Southern Anhui Province (Chinese Plants will editorial board of the Chinese Academy of Sciences, 1982；Anhui flora cooperative groups,
1986).Trunk is logical straight, and material is graceful, is spy's material that top-grade furniture makes；Tree body is tall and big, and the four seasons are emerald green, is also afforestation
Fine tree species.Although Phoebe chekiangensis economy and ornamental value are extra-high, available resources are few, protect with expand numerous quality germplasm into
The focus and emphasis studied for this kind.Research before focuses mostly in seed dormancy (Shi Xiaohua and Shi Zhongli, 1990), nursery stock training
Educate (Li Donglin and to its cypress, 2004,2006；Wang Yi etc., 2013), artificial forest cultivation (Peng Longfu, 2003；Wu carries screen-like mountain peak, 2005)
With ecology (Ma Mingdong etc., 2008；Wang Qi etc., 2013) in terms of, and somatic embryo is carried out to it using modern biotechnology
Induction etc. fast numerous have no research report.
Phoebe chekiangensis mainly row seminal propagation, but because its big tree resource is few, solid unstable, grain weight is few, causes its kind
The supply wretched insufficiency of seedling.Meanwhile, seedling offspring separation is serious, it is impossible to fixed excellent maternal merit.It can be seen that, need badly
Asexual manner is carried out with modern biotechnology and expands numerous superior genotypes, with this child care, Phoebe chekiangensis excellent germplasm is bred.It is general asexual
Breeding is main to be realized by cuttage and the aspect of tissue cultures two.But cutting propagation survival rate is low so far for Phoebe chekiangensis, and by cuttage
With fringe bar material and season limit, big tree can not survive because physiological age is big, it is impossible to realize propagation in scale application.Tissue training
Supporting has the advantages that reproduction speed is fast, can keep maternal excellent hereditary capacity, realizes germplasm amount reproduction, preserves for a long time
Important optional approach, in particular by body cell embryo callus subculture, somatic embryos occur, to realize the fast numerous Phoebe chekiangensis of body embryo
High quality seedling.Meanwhile, realize that body embryo culture is preserved and further gene transformation technology, excellent miscellaneous for quality germplasm
Hand over combination to expand and numerous etc. be respectively provided with significance.Therefore, build perfect somatic embryo and occur system, improve artificial propagation efficiency
It is to ensure that with merit Phoebe chekiangensis heredity can be stablized and expanding propagation meets the important of germplasm child care and production of forestry demand
Approach.
System occurs the seeds such as current larch, dragon spruce, China fir for preferable body embryo, but inhomogeneity seeds have specifically
Body embryo generation technique, especially endangered species have higher technical requirements.For endangered species Phoebe chekiangensis, using from difference
The immature embryo collection of stage of development is started with, and finds the time of special Fiber differentiation, screening is most fit embryo occurs culture medium and
Its condition of culture, creates perfect Phoebe chekiangensis somatic embryo and occurs system.
The content of the invention
In order to solve the above problems, the present invention provides a kind of Phoebe chekiangensis somatic embryo inducement method.
First, the present invention provides a kind of Phoebe chekiangensis somatic embryo inducement culture medium group, and it includes somatic embryo inducement culture
Base, somatic embryo proliferated culture medium and somatic embryo germination medium, described somatic embryo inducement culture medium are containing following
The MS solid mediums of composition：Glutamine 400-600mg/L, caseinhydrolysate 400-600mg/L, 2,4-D 0.4-0.6mg/
L, 6-BA0.8-1.2mg/L, described somatic embryo proliferated culture medium are the MS solid mediums containing following compositions：6-BA
0.1-1.0mg/L, NAA 0.1-1.0mg/L, described somatic embryo germination medium is the MS solids training containing following compositions
Support base：6-BA 1.0mg/L, IBA 0.1mg/L or NAA0.1mg/L.
Wherein, described somatic embryo inducement culture medium is the MS solid mediums containing following compositions：Glutamine
500mg/L, caseinhydrolysate 500mg/L, 2,4-D 0.5mg/L, 6-BA1.0mg/L, described somatic embryo proliferated culture medium
For the MS solid mediums containing following compositions：NAA 0.1-1.0mg/L, described somatic embryo germination medium is containing under
State the MS solid mediums of composition：6-BA 1.0mg/L、IBA 0.1mg/L.
The present invention also provides a kind of Phoebe chekiangensis somatic embryo inducement method, and it comprises the following steps：
1) with Phoebe chekiangensis crude fruit actually explant, surface sterilizing is carried out；
2) seed rataria is stripped, is inoculated on described somatic embryo inducement culture medium, somatic embryo inducement is carried out；
3) by step 2) somatic embryos are inoculated on described somatic embryo proliferated culture medium, carry out somatic embryo
Propagation, produces Secondary embryos.
In an embodiment of the invention, described Phoebe chekiangensis somatic embryo inducement method also includes：By step 3)
To Secondary embryos be inoculated in the sprouting that described somatic embryo germination medium carries out somatic embryo.
In one embodiment of the invention, in step 2) during somatic embryo inducement or in step 3) somatic embryo
In breeding, somatic embryo surface produces the embryo callus of yellow particle shape, and the embryo callus is inoculated in
On the somatic embryo proliferated culture medium, carry out the propagation of callus and luring for somatic embryo every two weeks subculture once
Lead.
Wherein, step 1) surface sterilizing method is：Seed is cleaned, 1-3h is rinsed in flowing water, 75% alcohol is then used
Handle after 20-40s, aseptic water washing 3~4 times, sterilized 8-15min with 0.1% mercuric chloride, then with aseptic water washing 5~7 times, wash
Remove remaining mercuric chloride.
In one embodiment of the invention, rataria is inoculated into before somatic embryo inducement culture medium, and rataria is placed in into 0.4-
In 0.6M sucrose, 4 DEG C of pretreatment 48-96h.
Wherein, the seed rataria embryo age is globular embryo between heart-shape embryo.
The present invention obtains the sterilization side of Phoebe chekiangensis immature fruit most preferably by the comparison of the different disinfection way of explant
Method；By periodically (interval one week) collection immature seed, and combine anatomic observation, the system research conjunction of different developmental phases
Influence of the sub- embryo to body embryo incidence, learn in globular embryo or to heart-shape embryo change when zygotic embryo body embryo incidence it is higher；
By screening influence of the culture medium of hormon proportioning to body embryo incidence, the optimal culture of Phoebe chekiangensis body embryo induction is obtained
Base, incidence is up to 65%；By the screening to proliferated culture medium, optimum multiplication medium is obtained, growth coefficient is reachable
14.3；By the screening to body embryo germination medium, optimal body embryo germination medium is obtained, germination rate is up to 68.5%.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The induction immature zygotic embryos body embryo of embodiment 1 occurs
Taken respectively at mid-July to late August on Pan Shan robust growths, the excellent Phoebe chekiangensis individual plant of no disease and pests harm not into
Ripe fruit.
First 2h is rinsed in the running water of flowing.With 75% ethanol postincubation 30s on superclean bench in desinfection chamber,
Sterile distilled water rinse 3~4 times after, respectively with 0.1% mercuric chloride sterilize 8min, 10min, 12min and 14min, then with it is sterile steaming
Distilled water is rinsed 5~7 times, washes away remaining mercuric chloride.After the mercuric chloride processing of different time, the influence to explant is dramatically different, such as table
1。
The influence that the different mercuric chloride processing times of table 1 sterilize to explant
| Mercuric chloride processing time | Disinfection Effect | Explant state |
| 8min | Not thoroughly, still have bacterium residual | Pericarp keeps green |
| 10min | It is good | Pericarp keeps green |
| 12min | It is good | Some brownings of pericarp |
| 14min | It is good | Pericarp browning is more serious |
The same time is taken, the basically identical Phoebe chekiangensis immature fruit of size aseptically, takes its rataria, is divided into
Five groups, it is respectively put into 0M, 0.5M, 1.0M sucrose, about 90 ratarias of each group of placement are placed in 4 DEG C of refrigerators and trained in advance
Support.It is inoculated into every 24h (be respectively 24h, 48h and 72h) every group of taking-up 30 in BIM culture mediums, is inoculated with 3 times altogether, every time 90
Individual rataria, determines its body embryo incidence after totally 270,1 month.
The influence of table 2 different sucrose pretreatment concentration and time to body embryo incidence
As can be seen that sucrose solution pretreatment can significantly improve the generation of the somatic embryo of Phoebe chekiangensis from the result of table 2
Rate.But different sucrose concentrations and different pretreatments time have a significant impact for body embryo.0.5M sucrose solution, pre- place
Reason 72h is optimal preprocess method.
Fruit is aseptically cut, rataria is taken out, rataria is placed in 0.5M sucrose, 4 DEG C of pretreatment 72h, by children
Embryo is inoculated in the MS+CH500mg/L+L- glutamine 500mg/L solid mediums containing hormon concentration combination and carried out
Body embryo is tested.The young tender embryo number each handled is 15, connects 5 per ware, totally 3 ware, 4 repetitions.25 DEG C of cultivation temperature
± 2 DEG C, light culture.
Influence of the different culture media of table 3 for Phoebe chekiangensis rataria somatic embryo inducement
The immature zygotic embryos of Phoebe chekiangensis are inoculated on somatic embryo inducement culture medium, in explant table after 4 weeks
The generation of somatic embryo can be observed in face.These somatic embryos are obtained by direct development ways, at the end of culture, from outer
Come off in implant in culture medium.
At the end of Fiber differentiation, well-developed primary embryo can be observed in the medium, the nascent embryo surface in part has Huang
Color, granular embryo callus are produced.Obtained most of somatic embryo form is normal, tool two panels cotyledon, and part body
The paramophia of blast, is usually expressed as 3 cotyledons of tool or multi-disc cotyledon while being born on an embryo.
Different somatic embryo inducement culture mediums is as shown in table 3 for the influence of inductivity.From table 3 it is observed that most
Suitable culture medium is MS+CH 500mg/L+L- glutamine 500mg/L+6-BA 1.0mg/L+2,4-D 0.5mg/L.
Influence of the rataria different developmental phases of embodiment 2 to body embryo incidence
Respectively on July 17th, 2016, July 24, July 31, August 7 days, August 14 days, the August 4 excellent strains of collection on the 21st
Each 150 of seed, wherein 135 are tested for body embryo, another 15 are used as anatomic observation, analyze under each acquisition time
Stage of development residing for zygotic embryo.The seed of 4 excellent strains is denoted as A, B, C, D respectively, and the rataria stripped is placed in 0.5M sucrose,
4 DEG C of pretreatment 72h, are inoculated in BIM culture mediums i.e. MS+CH500mg/L+ glutamine 500mg/L+6-BA 1.0mg/L+2,4-D
Carry out body embryo in 0.5mg/L to test, the measure different acquisition time is zygotic embryo different developmental phases to body embryo incidence
Influence.Concrete operation step be the same as Example 1.
The influence that the different developmental phases of the rataria of table 4 occur for body embryo
Table 4 show influence of the Phoebe chekiangensis zygotic embryo of different developmental phases to body embryo incidence, it can be seen that in ball
Shape embryo or to heart-shape embryo change when zygotic embryo body embryo incidence it is higher.
The propagation of the somatic embryo of embodiment 3
Somatic embryo obtained by induction is placed in proliferated culture medium, bred.The body embryo number each handled is 25,
5 are connect per ware, totally 5 ware, each processing sets 3 repetitions.25 DEG C ± 2 DEG C of cultivation temperature, light culture.
The influence that the different proliferated culture mediums of table 5 are bred for somatic embryo
| 1 | 2 | 3 | 4 | 5 | 6 | |
| 6-BA | 0 | 0 | 0 | 1.0 | 1.0 | 1.0 |
| NAA | 0.1 | 0.5 | 1.0 | 0.1 | 0.5 | 1.0 |
| Growth coefficient | 4.8±1.3 | 14.3±2.4 | 7.9±2.0 | 5.2±1.9 | 6.4±2.3 | 4.0±1.4 |
Primary embryo obtained by induction is cultivated in proliferated culture medium, observes that nascent embryo surface produces similar mother after 3 weeks
The white embryo shape structure of body, this is the Secondary embryos obtained by Secondary embryos occurring mode.Extend the time of Multiplying culture, observable
There is the generation of embryo callus to secondary embryo surface.The Secondary embryos of nanmu are easy to occur, and Multiplying culture can be obtained for one month
Secondary embryos to more than 10 times, even and in the somatic embryo of ripe greening, or even the somatic embryo sprouted
On, it can also be observed that the generation of Secondary embryos.Secondary embryos occur once can be produced new time on obtained Secondary embryos again
Raw embryo, this Secondary embryos constantly circulated repeat to occur for a long time continue on proliferated culture medium, make Secondary embryos quantity quick
Increase.
Counted by 4 weeks, optimum multiplication medium is MS+NAA 0.5mg/L.
The acquisition of the Phoebe chekiangensis embryo callus of embodiment 4
Embryo callus can be produced in the induction period of body cell, but the frequency occurred is very low.And institute will be induced
Somatic embryo be placed in when being cultivated in proliferated culture medium, after 1-2 months, somatic embryo surface generation Secondary embryos, and companion
With the formation for having embryo callus.
The embryo callus of Phoebe chekiangensis can be preserved for a long time on proliferated culture medium, be found in experiment, every two weeks
Subculture is carried out for keeping the state of embryo callus extremely important, subculture can not cause part callus brown for a long time
Become, and part callus is then developed for somatic embryo.And these by healing tissue development into somatic embryo can also pass through
Regular subculture is maintained, and extends or shorten Subculture Time interval, embryo callus can be formed again.Therefore in Phoebe chekiangensis
Each embryo system incubation in, often it can be seen that the phenomenon that exists jointly of embryo callus and somatic embryo.
The sprouting of the Phoebe chekiangensis somatic embryo of embodiment 5
Secondary embryos are transferred to germination medium and carry out sprouting test, and TDZ, 6-BA, NAA, IBA growth are added in MS culture mediums
Conditioning agent, is specifically shown in Table 6.Germination rate and observation body embryo phenotype and situation of taking root are counted after 2 months.Phoebe chekiangensis body embryo is learnt in experiment
The optimal medium of sprouting is MS+6-BA 1.0mg/L+IBA 0.1mg/L.Reduce after culture being found in experimentation 2 months
IBA concentration, sprouting obtained regeneration plant can preferably grow, and culture can find eugonic seedling after 3 months.
The influence that the different hormone combinations of table 6 are sprouted to body embryo
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (8)
1. a kind of Phoebe chekiangensis somatic embryo inducement culture medium group, it includes somatic embryo inducement culture medium, somatic embryo propagation training
Base and somatic embryo germination medium are supported, described somatic embryo inducement culture medium is the MS solid cultures containing following compositions
Base：Glutamine 400-600mg/L, caseinhydrolysate 400-600mg/L, 2,4-D 0.4-0.6mg/L, 6-BA0.8-1.2mg/
L, described somatic embryo proliferated culture medium is the MS solid mediums containing following compositions：6-BA 0.1-1.0mg/L、NAA
0.1-1.0mg/L, described somatic embryo germination medium is the MS solid mediums containing following compositions：6-BA 1.0mg/
L, IBA 0.1mg/L or NAA0.1mg/L.
2. Phoebe chekiangensis somatic embryo inducement culture medium group as claimed in claim 1, it is characterised in that described somatic embryo is lured
Culture medium is led for the MS solid mediums containing following compositions：Glutamine 500mg/L, caseinhydrolysate 500mg/L, 2,4-D
0.5mg/L, 6-BA1.0mg/L, described somatic embryo proliferated culture medium are the MS solid mediums containing following compositions：NAA
0.1-1.0mg/L, described somatic embryo germination medium is the MS solid mediums containing following compositions：6-BA 1.0mg/
L、IBA 0.1mg/L。
3. a kind of Phoebe chekiangensis somatic embryo inducement method, it comprises the following steps：
1) with Phoebe chekiangensis crude fruit actually explant, surface sterilizing is carried out；
2) seed rataria is stripped, is inoculated on described somatic embryo inducement culture medium, somatic embryo inducement is carried out；
3) by step 2) somatic embryos are inoculated on described somatic embryo proliferated culture medium, carry out the increasing of somatic embryo
Grow, produce Secondary embryos.
4. Phoebe chekiangensis somatic embryo inducement method as claimed in claim 3, it is characterised in that also include：By step 3) obtain
Secondary embryos be inoculated in the sprouting that described somatic embryo germination medium carries out somatic embryo.
5. Phoebe chekiangensis somatic embryo inducement method as claimed in claim 3, it is characterised in that in step 2) somatic embryo inducement
During or in step 3) in somatic embryo breeding, somatic embryo surface produces the embryo callus subculture group of yellow particle shape
Knit, the embryo callus is inoculated on the somatic embryo proliferated culture medium, is cured every two weeks subculture once
The propagation of injured tissue and the induction of somatic embryo.
6. Phoebe chekiangensis somatic embryo inducement method as claimed in claim 3, it is characterised in that step 1) surface sterilizing method
For：Immature fruit is cleaned, 1-3h is rinsed in flowing water, then with 75% ethanol postincubation 20-40s, aseptic water washing 3~4
After secondary, sterilized 8-15min with 0.1% mercuric chloride, then with aseptic water washing 5~7 times, wash away remaining mercuric chloride.
7. the Phoebe chekiangensis somatic embryo inducement method as described in claim any one of 3-6, it is characterised in that rataria is inoculated into body
Before blast inducing culture, rataria is placed in 0.4-0.6M sucrose, 4 DEG C of pretreatment 48-96h.
8. the Phoebe chekiangensis somatic embryo inducement method as described in claim any one of 3-6, it is characterised in that the seed rataria
Embryo age is globular embryo between heart-shape embryo.
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| CN107372126A (en) * | 2017-09-15 | 2017-11-24 | 宁夏农林科学院农业生物技术研究中心 | A kind of method for inducing Chinese podophyllum root somatic embryo occur |
| CN108184631A (en) * | 2017-12-15 | 2018-06-22 | 浙江海洋大学 | A kind of ciltivating process of red nanmu |
| CN110833028A (en) * | 2019-12-13 | 2020-02-25 | 浙江农林大学 | Somatic embryogenesis and plant regeneration method for cinnamomum zhejiangense |
| CN112772416A (en) * | 2021-01-28 | 2021-05-11 | 宜宾金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN114651726A (en) * | 2022-05-05 | 2022-06-24 | 贵州大学 | Method for culturing nanmu seed embryo aseptic seedlings |
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| CN107372126A (en) * | 2017-09-15 | 2017-11-24 | 宁夏农林科学院农业生物技术研究中心 | A kind of method for inducing Chinese podophyllum root somatic embryo occur |
| CN107372126B (en) * | 2017-09-15 | 2019-03-15 | 宁夏农林科学院农业生物技术研究中心 | A method of induction Chinese podophyllum root somatic embryo occur |
| CN108184631A (en) * | 2017-12-15 | 2018-06-22 | 浙江海洋大学 | A kind of ciltivating process of red nanmu |
| CN110833028A (en) * | 2019-12-13 | 2020-02-25 | 浙江农林大学 | Somatic embryogenesis and plant regeneration method for cinnamomum zhejiangense |
| CN110833028B (en) * | 2019-12-13 | 2021-06-18 | 浙江农林大学 | Somatic embryogenesis and plant regeneration method for cinnamomum zhejiangense |
| CN112772416A (en) * | 2021-01-28 | 2021-05-11 | 宜宾金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN112772416B (en) * | 2021-01-28 | 2022-08-12 | 苏州金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN114651726A (en) * | 2022-05-05 | 2022-06-24 | 贵州大学 | Method for culturing nanmu seed embryo aseptic seedlings |
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