CN105432465B - A kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate - Google Patents
A kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate Download PDFInfo
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- CN105432465B CN105432465B CN201510771278.3A CN201510771278A CN105432465B CN 105432465 B CN105432465 B CN 105432465B CN 201510771278 A CN201510771278 A CN 201510771278A CN 105432465 B CN105432465 B CN 105432465B
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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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- 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
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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
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate, the present invention relates to a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate.The present invention is to solve existing Korean pine when carrying out somatic embryo generation, frequency of embryonic callus induction and turn the problem of embryo rate is low, method is：First, the sterilizing of material；2nd, embryo callus subculture is induced；3rd, the subculture of callus is kept and propagation；4th, the induction of proembryo；5th, the developing of somatic embryo, ripe and plant regeneration, that is, complete.Rational proportion and suitable condition of culture that the present invention passes through each component in culture medium, make Korean pine when carrying out somatic embryo generation, frequency of embryonic callus induction and turn embryo rate and be obtained for raising, frequency of embryonic callus induction can reach 33.3%, the present invention makes the reduction of embryo callus subculture browning degree by adding inositol, and the conversion of globular embryo is more conducive to, it is be not added with inositol 68 times to turn embryo rate.The present invention is applied to field of forestry.
Description
Technical field
The present invention relates to a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate.
Background technology
Korean pine (Pinus koraiensis) is the extremely important high quality timber seeds in the Northeast of China, before being also development
The huge nut nonwood forest trees of scape.Somatic embryo is the basis that quick breeding and artificial seed are developed, and heredity is changed
It is good significant.Carry out Korean pine somatic embryo to occur to study with adventitious bud, can both be provided for its biological study
Effective tissue culture system, is used directly for the asexual propagation of elite germplasm material again.But when carrying out the generation of Korean pine body cell now,
Frequency of embryonic callus induction and turn that embryo rate is all relatively low, seriously constrain the research in terms of the somatic embryo generation of Korean pine.
The content of the invention
The present invention is to solve existing Korean pine when carrying out somatic embryo generation, frequency of embryonic callus induction and turn embryo
There is provided a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate for the problem of rate is low.
A kind of raising Korean pine frequency of embryonic callus induction of the present invention is carried out according to the following steps with the method for turning embryo rate：
First, the sterilizing of material
After the cones of Pinus koraiensis sterilizing in the cleavage polyembryony phase, kind of a skin is peelled off, is sterilized again, the zygote after being sterilized
Embryo；
2nd, embryo callus subculture is induced
Zygotic embryo after the sterilizing that step one is obtained is inoculated in culture medium, under conditions of 23~25 DEG C, light culture
58~65d, obtains embryo callus, wherein culture medium is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+
Sucrose 35g/L+NAA2mg/L+6-BA 1.5mg/L+ acid hydrolyzed caseins 0.8g/L；Medium pH=5.8；
3rd, the subculture of callus is kept and propagation
The embryo callus that step 2 is obtained carries out squamous subculture, the callus after being bred；
4th, the induction of proembryo
Callus after propagation is inoculated into the induction that proembryo is carried out in culture medium, under conditions of 23~25 DEG C, secretly
7~15d is cultivated, the callus with globular embryo is obtained；Wherein culture medium is DCR culture mediums+acid hydrolyzed casein 500mg/L
+ Glu 500mg/+ sucrose 20g/L+ agar 6.5g/L+ activated carbon 2g/L+ inositols 4g/L；PH=5.8；
5th, the development of somatic embryo and plant regeneration
Callus with globular embryo is inoculated into culture medium and carries out body embryonic development, completion turns embryo process, obtains son
Leaf embryo；Maturation induction is carried out to the cotyledonary embryos of acquisition simultaneously, mature embryo is obtained, is then sprouted, plant regeneration is with transplanting into
Seedling.
The present invention makes Korean pine carry out body cell by the rational proportion and suitable condition of culture of each component in culture medium
When embryo occurs, frequency of embryonic callus induction and turn embryo rate and be obtained for raising, frequency of embryonic callus induction can reach
33.3%, present invention discover that induction and later stage body embryonic development of the inositol to globular embryo play an important role with maturation, by adding
Inositol makes the reduction of embryo callus subculture browning degree, and is more conducive to the conversion of globular embryo, and it is be not added with inositol 6-8 times to turn embryo rate.
Brief description of the drawings
Fig. 1 is the photo of embryo callus in embodiment one；
Fig. 2 is the photo of early stage proembryos in embodiment one；
Fig. 3 is the photo of globular embryo in embodiment one；
Fig. 4 and Fig. 5 is the photo of somatic embryo development in embodiment one.
Embodiment
Embodiment one：A kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate of present embodiment
Carry out according to the following steps：
First, the sterilizing of material
After the cones of Pinus koraiensis sterilizing in the cleavage polyembryony phase, kind of a skin is peelled off, is sterilized again, the zygote after being sterilized
Embryo；
2nd, embryo callus subculture is induced
Zygotic embryo after the sterilizing that step one is obtained is inoculated in culture medium, under conditions of 23~25 DEG C, light culture
58~65d, obtains embryo callus, wherein culture medium is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+
Sucrose 35g/L+NAA2mg/L+6-BA 1.5mg/L+ acid hydrolyzed caseins 0.8g/L；Medium pH=5.8；
3rd, the subculture of callus is kept and propagation
The embryo callus that step 2 is obtained carries out squamous subculture, the callus after being bred；
4th, the induction of proembryo
Callus after propagation is inoculated into the induction that proembryo is carried out in culture medium, under conditions of 23~25 DEG C, secretly
7~15d is cultivated, the callus with globular embryo is obtained；Wherein culture medium is DCR culture mediums+acid hydrolyzed casein 500mg/L
+ Glu 500mg/+ sucrose 20g/L+ agar 6.5g/L+ activated carbon 2g/L+ inositols 4g/L；PH=5.8；
5th, the development of somatic embryo and plant regeneration
Callus with globular embryo is inoculated into culture medium and carries out body embryonic development, completion turns embryo process, obtains son
Leaf embryo；Maturation induction is carried out to the cotyledonary embryos of acquisition simultaneously, mature embryo is obtained, is then sprouted, plant regeneration is with transplanting into
Seedling.
Cones of Pinus koraiensis in present embodiment is collected in Wei He forestry bureaus of Heilongjiang Province green hill tree seed orchard.
Present embodiment makes Korean pine carry out body by the rational proportion and suitable condition of culture of each component in culture medium
When cell stage occurs, frequency of embryonic callus induction and turn embryo rate and be obtained for raising, frequency of embryonic callus induction is reachable
To 33.3%, present embodiment finds that induction and later stage body embryo maturation of the inositol to globular embryo play an important role, by adding
Inositol makes the reduction of embryo callus subculture browning degree, and is more conducive to globular embryo and is converted to mature embryo, and it is the 6-8 for being not added with inositol to turn embryo rate
Times.
Embodiment two：Present embodiment from unlike embodiment one：It is many in schizogenesis in step one
Embryonic stage cones of Pinus koraiensis sterilizing method be：Cones of Pinus koraiensis is cleaned, fruit scale is then pruned, then with the wine that mass concentration is 70%
3~5min of precision processing, then with aseptic water washing 2~4 times, then with the sodium hypochlorite of mass concentration 10% soaks 20~30min.Its
It is identical with embodiment one.
Embodiment three：Present embodiment from unlike embodiment one or two：Gone out again in step one
The method of bacterium is：With the dioxygen water process 8min that mass concentration is 3%, aseptic water washing 2~4 times.Other and specific embodiment party
Formula one or two is identical.
Embodiment four：Unlike one of present embodiment and embodiment one to three：It is dark in step 2
Cultivate 60d.It is other identical with one of embodiment one to three.
Embodiment five：Unlike one of present embodiment and embodiment one to four：Step 3 is relayed
Foster culture medium of being commissioned to train is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+ sucrose 30g/L+2,4-D0.5g/L+
6-BA 0.1g/L+ acid hydrolyzed caseins 0.5g/L；PH=5.8.It is other identical with one of embodiment one to four.
Embodiment six：Unlike one of present embodiment and embodiment one to five：Step 3 is relayed
It is the light culture 15d under conditions of 23~25 DEG C to be commissioned to train foster.It is other identical with one of embodiment one to five.
Embodiment seven：Unlike one of present embodiment and embodiment one to six：It is dark in step 4
Cultivate 12d.It is other identical with one of embodiment one to six.
Embodiment eight：Unlike one of present embodiment and embodiment one to seven：It is dark in step 5
Cultivate 70d.It is other identical with one of embodiment one to seven.
Embodiment nine：Unlike one of present embodiment and embodiment one to eight：Body in step 5
Embryo Maturation induction culture medium is DCR culture mediums+agar 7.1g/L+L- glutamine 500mg/L+ sucrose 60g/L+ abscisic acids
20mg/L+PEG400050g/L+IBA 0.2mg/L+ activated carbons 2g/L；PH=5.8.It is other with embodiment one to eight it
One is identical.
Embodiment ten：Unlike one of present embodiment and embodiment one to nine：Step 5 body embryo
Maturation induction is 60~80d of light culture under conditions of 23~25 DEG C, and every 3~4 weeks subcultures are once.Other and specific embodiment party
One of formula one to nine is identical.
The following examples will be further described to the present invention, but not thereby limiting the invention.
Embodiment 1：The present embodiment improves Korean pine frequency of embryonic callus induction and turns the method for embryo rate, enters according to the following steps
OK：
First, the sterilizing of material
Cones of Pinus koraiensis in the cleavage polyembryony phase is cleaned, fruit scale is then pruned, then with the alcohol that mass concentration is 70%
Handle 2min, then with aseptic water washing 3 times, then with the sodium hypochlorite of mass concentration 10% immersion 25min, after ultra-clean work
Kind of a skin is peelled off under platform, then with the dioxygen water process 8min that mass concentration is 3%, aseptic water washing 3 times, the conjunction after being sterilized
Sub- embryo；
2nd, embryo callus subculture is induced
Zygotic embryo after the sterilizing that step one is obtained is inoculated in culture medium, under conditions of 25 DEG C, light culture 60d,
Embryo callus is obtained, wherein culture medium is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+ sucrose 35g/L
+ NAA 2mg/L+6-BA 1.5mg/L+ acid hydrolyzed caseins 0.8g/L；Medium pH=5.8；The embryo callus of this step
Photo as shown in figure 1, frequency of embryonic callus induction can reach 33.3%.
3rd, the subculture of callus is kept and propagation
The embryo callus that step 2 is obtained carries out squamous subculture, and under conditions of 25 DEG C, light culture 15d is obtained
The culture medium of callus after propagation, wherein squamous subculture is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L
+ sucrose 30g/L+2,4-D 0.5g/L+6-BA 0.1g/L+ acid hydrolyzed caseins 0.5g/L；PH=5.8；
4th, the induction of proembryo
Callus after propagation is inoculated into the induction that proembryo is carried out in culture medium, under conditions of 25 DEG C, light culture
12d, obtains the callus with globular embryo；Wherein culture medium is DCR culture mediums+acid hydrolyzed casein 500mg/L+L- paddy ammonia
Acid amides 500mg/+ sucrose 20g/L+ agar 6.5g/L+ activated carbon 2g/L+ inositols 4g/L；PH=5.8；Generated in this step process
The photo of early stage proembryos is as shown in Fig. 2 the photo of globular embryo is as shown in Figure 3.
5th, the development of somatic embryo
Callus with globular embryo is inoculated into culture medium and carries out body embryonic development, completion turns embryo process；It is simultaneously right
The cotyledonary embryos of acquisition carry out Maturation induction, and under conditions of 25 DEG C, 60~80d of light culture, every 3~4 weeks subcultures once, are obtained into
Cooked flake, wherein culture medium are DCR culture mediums+agar 7.1g/L+L- glutamine 500mg/L+ sucrose 60g/L+ abscisic acids 20mg/
L+PEG400050g/L+IBA 0.2mg/L+ activated carbons 2g/L；PH=5.8；The photo of this step embryonic development such as Fig. 4 and Fig. 5 institutes
Show.
6th, the sprouting of somatic embryo and plant regeneration
The mature somatic embryo of induction is horizontally placed on germination medium DCR and sprouted, additional 20g/L sucrose, L-
Glutamine 500mg/L, acid hydrolyzed casein 2g/L, first light culture 6d, then in illuminance 2000lx, 16/8h light dark cultures
14d, sprouting culture is carried out to mature somatic embryo；Then WPM minimal mediums, additional 1.0mg/LIBA, 0.2mg/ are used
LNAA, 20g/L sucrose, 0.1g/L inositols and 6.5/L agar, illuminance 20001x, the brightness culture of 16/8 hour 1 month are entered
Row somatic embryo plant regeneration, obtains regeneration plant；
7th, the transplanting of regeneration plant
Transplanted after plant root elongation more than 2cm to be regenerated, 7d gradually opens sealed membrane before transplanting, and adapts to seedling
Extraneous environment, transplanting is using volume ratio for 1:3:1 perlite, Nutrition Soil and vermiculite are matrix, carry out transplanting culture, i.e., complete
Into.
Cones of Pinus koraiensis in the present embodiment is collected in Wei He forestry bureaus of Heilongjiang Province green hill tree seed orchard.
The present embodiment makes the reduction of embryo callus subculture browning degree by adding inositol, and is more conducive to globular embryo to mature embryo turn
Change, conversion ratio is be not added with inositol 6-8 times.
The rational proportion and suitable culture bar that each component in culture medium is passed through by above example, the present invention
Part, makes Korean pine when carrying out somatic embryo generation, frequency of embryonic callus induction and turns embryo rate and is obtained for raising, and embryo is cured
Injured tissue inductivity can reach 33.3%, present invention discover that induction and later stage body embryonic development and maturation of the inositol to globular embryo have
Important function, makes the reduction of embryo callus subculture browning degree by adding inositol, and is more conducive to the conversion of globular embryo, and conversion ratio is not
Plus 6-8 times of inositol.
It is described above, it is only the present invention preferably embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, the change or replacement that can be readily occurred in,
It should all be included within the scope of the present invention.
Claims (3)
1. a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate, it is characterised in that this method is according to the following steps
Carry out：
First, the sterilizing of material
After the cones of Pinus koraiensis sterilizing in the cleavage polyembryony phase, kind of a skin is peelled off, is sterilized again, the zygotic embryo after being sterilized；
2nd, embryo callus subculture is induced
Zygotic embryo after the sterilizing that step one is obtained is inoculated in culture medium, under conditions of 23~25 DEG C, light culture 60d,
Embryo callus is obtained, wherein culture medium is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+ sucrose 35g/L
+ NAA 2mg/L+6-BA1.5mg/L+ acid hydrolyzed caseins 0.8g/L；Medium pH=5.8；
3rd, the subculture of callus is kept and propagation
The embryo callus that step 2 is obtained is under conditions of 23~25 DEG C, and light culture 15d carries out squamous subculture, is increased
Callus after growing；
4th, the induction of proembryo
Callus after propagation is inoculated into the induction that proembryo is carried out in culture medium, under conditions of 23~25 DEG C, light culture
12d, obtains the callus with globular embryo；Wherein culture medium is DCR culture mediums+acid hydrolyzed casein 500mg/L+L- paddy ammonia
Acid amides 500mg/+ sucrose 20g/L+ agar 6.5g/L+ activated carbon 2g/L+ inositols 4g/L；PH=5.8；
5th, the development of somatic embryo and plant regeneration
Callus with globular embryo is inoculated into culture medium and carries out body embryonic development, cotyledonary embryos are obtained；Simultaneously to acquisition
Cotyledonary embryos carry out Maturation induction, obtain mature embryo, then sprouted, plant regeneration with transplanting seedling；
The culture medium of squamous subculture is DCR culture mediums+agar 6.5g/L+L- glutamine 500mg/L+ sucrose wherein in step 3
30g/L+2,4-D 0.5g/L+6-BA0.1g/L+ acid hydrolyzed caseins 0.5g/L；PH=5.8；Body embryo Maturation induction in step 5
Culture medium is DCR culture mediums+agar 7.1g/L+L- glutamine 500mg/L+ sucrose 60g/L+ abscisic acids 20mg/L+PEG4000
50g/L+IBA0.2mg/L+ activated carbons 2g/L；PH=5.8；Step 5 body embryo Maturation induction is under conditions of 23~25 DEG C, secretly
60~80d is cultivated, every 3~4 weeks subcultures are once.
2. a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate according to claim 1, its feature
It is that the method that the cones of Pinus koraiensis in the cleavage polyembryony phase sterilizes in step one is：Cones of Pinus koraiensis is cleaned, fruit scale is then pruned,
Again with 3~5min of ethanol postincubation that mass concentration is 70%, then with aseptic water washing 2~4 times, then with mass concentration 10% time
Sodium chlorate soaks 20~30min.
3. a kind of method for improving Korean pine frequency of embryonic callus induction and turning embryo rate according to claim 1, its feature
It is that the method sterilized again in step one is：With the dioxygen water process 8min that mass concentration is 3%, aseptic water washing 2~4
It is secondary.
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| CN106135005A (en) * | 2016-08-26 | 2016-11-23 | 李军 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
| CN108668899B (en) * | 2018-05-31 | 2021-04-20 | 广西壮族自治区林业科学研究院 | Method for improving induction rate of masson pine somatic embryos |
| CN110004176B (en) * | 2019-04-12 | 2023-03-10 | 东北林业大学 | Construction method of hybrid larch genetic transformation system |
| CN110012835A (en) * | 2019-04-12 | 2019-07-16 | 江西省林业科学院 | A kind of method of wet-land pine tree embryonic callus induction and proliferation |
| CN110301353B (en) * | 2019-07-10 | 2022-03-01 | 广西壮族自治区林业科学研究院 | Method for multiplication and maintenance culture of pinus massoniana embryonic callus |
| CN112931215A (en) * | 2021-03-31 | 2021-06-11 | 东北林业大学 | Method for improving induction rate of embryonic callus of Korean pine |
| CN113068617A (en) * | 2021-05-14 | 2021-07-06 | 东北林业大学 | Method for improving germination rate of Korean pine somatic embryos through low-temperature treatment |
| CN113736821B (en) * | 2021-09-06 | 2023-04-11 | 东北林业大学 | Genetic transformation method for embryonic callus of Korean pine |
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| US4060933A (en) * | 1976-12-20 | 1977-12-06 | Gte Laboratories Incorporated | Tissue culture technique utilizing a specific light source |
| US7598073B2 (en) * | 2002-04-09 | 2009-10-06 | Weyerhaeuser Nr Company | Methods for producing high yields of zygotic-like cotyledonary pine embryos utilizing media that include a disaccharide and glucose |
| CN102577956A (en) * | 2012-02-21 | 2012-07-18 | 南京林业大学 | Pinus thunbergii cell embryogenesis and plant regeneration method |
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