CN110301353A - Method masson pine embryo callus subculture proliferation and maintain culture - Google Patents
Method masson pine embryo callus subculture proliferation and maintain culture Download PDFInfo
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- CN110301353A CN110301353A CN201910617233.9A CN201910617233A CN110301353A CN 110301353 A CN110301353 A CN 110301353A CN 201910617233 A CN201910617233 A CN 201910617233A CN 110301353 A CN110301353 A CN 110301353A
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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
Abstract
The invention discloses a kind of methods masson pine embryo callus subculture proliferation and maintain culture, conventional body embryo will be passed through and induce being placed in the culture dish equipped with culture medium I in shape Embryogenic cell masses are reeled off raw silk from cocoons with the isolated embryo callus of egagametophyte for acquisition in 3~4 weeks, dead cell and excess tissue in 4~6 weeks removal embryo callus, it is transferred in centrifuge tube and the mixing of culture medium II is added and shake up, then the Embryonal suspensor mass obtained after shaking up is placed in the culture dish equipped with culture medium III, it takes the embryo callus subculture of appropriate size to be directly transferred in culture medium IV after 6~8 weeks and carries out normal subculture, 10~14 d of subculture cycle, it is rapid finally to obtain growth, flush and can carry out the masson pine embryogenic cell line of long term subculture.For embryo callus subculture of the invention without obvious browning and water stainization phenomenon in long term subculture, embryo callus subculture growth is fast, and cultivation effect stablizes height, and growth coefficient has preferable economic benefit and social benefit up to 25 or more.
Description
Technical field
The invention belongs to forest tree propagation technical fields, are related to tissue cultures embryo callus subculture shoot proliferation culture technique, especially
A kind of method masson pine embryo callus subculture proliferation and maintain culture.
Background technique
Masson pine (Pinus massonianaLamb.) be south China ecological construction and afforestation material chief species.
The tree species high as a kind of comprehensive utilization value, promotion prospect is wide, masson pine not only can be used to produce three plates, papermaking and chemical fibre
Industry manufacture may further be used to the raw materials of industry such as production rosin, turpentine oil.Masson pine breeding cycle is long, in addition recently as kind
Sub- garden elite stand aging, hip number decline, breeding is deficient, leads to that southern Masson Pine Plantation genetic variation and genetic differentiation difference is big, stand quality
It is not high, productivity is low.To push the high-efficient development of masson pine industry, the breeding industrialization of the asexual nursery of excellent strain is realized especially
It is crucial.The use of plant tissue culture technology is the effective way for realizing breeding nursery industry.Relative to organ in tissue culture technology
For generation, it is plant in plant cell engineering that a kind of expression way as totipotency of plant cell, which occurs, for somatic embryo
Regenerated important means structural integrity, inheritance stability and is not limited etc. by natural conditions many excellent with reproductive efficiency height
Gesture all has major application valence in numerous areas such as extensive vegetative propagation, artificial seed preparation, preserving seed and genetic transformations
Value is the current main means for carrying out forest Clonal regeneration in the world.It is ground currently, existing correlation occurs for masson pine somatic embryo
Study carefully, but since embryo callus subculture induces difficult, and when Subculture Time is more than more than half a year, the embryo callus subculture in squamous subculture occurs bright
Aobvious browning, water stainization phenomenon, callus activity are remarkably decreased, and hinder the development of body embryo seedling-raising technique.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of embryo callus subcultures in long term subculture, callus
Without browning, water stainization phenomenon, callus quality is good, and the strong masson pine embryo callus subculture proliferation of cells,primordial activity is cultivated with maintenance
Method.
To achieve the goals above, technical scheme is as follows:
A kind of method masson pine embryo callus subculture proliferation and maintain culture, including embryo callus subculture separation, Multiplying culture primary, subculture
Culture pretreatment and subculture maintain culture processes, are in the shape Embryogenic cell masses that reel off raw silk from cocoons by induce obtain within 3~4 weeks by conventional body embryo
Isolated embryo callus is placed in the culture dish equipped with culture medium I with egagametophyte, 4~6 weeks removal embryo callus subcultures
Dead cell and excess tissue in tissue, be transferred in centrifuge tube and be added culture medium II mixing shake up, obtained after then shaking up
Embryonal suspensor mass be placed in the embryo callus subculture of appropriate size taken directly to be transferred to training in the culture dish equipped with culture medium III after 6~8 weeks
Support in base IV and carry out normal subculture, 10~14 d of subculture cycle, finally obtain growth rapidly, flush and can carry out for a long time after
The masson pine embryogenic cell line for being commissioned to train feeding；Main operational steps are as follows:
(1) embryo callus subculture separates:
It is induced 3~4 weeks by conventional body embryo, it, directly will be colourless, transparent, water stain-free when embryo callus subculture rolls into a ball diameter >=1 cm
The shape Embryogenic cell masses that reel off raw silk from cocoons separated according to a conventional method with egagametophyte after as be proliferated and maintain culture embryo callus subculture group
It knits；
(2) Multiplying culture primary:
Isolated embryo callus is placed in the culture dish equipped with culture medium I, half-light culture 4~6 weeks, cultivation temperature
20±0.5℃；
(3) squamous subculture pre-processes:
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1~1.5 g embryo
Property callus be transferred in 5 mL centrifuge tubes, into centrifuge tube be added II 3~5 mL of culture medium, after firmly shaking up obtain embryo training
Object is supported, 0.8 mL Embryonal suspensor mass is drawn with suction pipe and is put on the culture dish for being lined with double-layer filter paper, be subsequently placed in superclean bench
Upper 5 min of exhausting, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred in the culture dish equipped with culture medium III, half-light culture 6
~8 weeks, 21 ± 0.5 DEG C of cultivation temperature；
(4) subculture maintains culture:
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred in the culture dish equipped with culture medium IV, every ware
Inoculation 5~6, half-light culture, 10~14 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature；It finally obtains and grows rapid, vigor
Masson pine embryogenic cell line vigorous and that long term subculture can be carried out.
Culture medium I described in above step (2) are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
2.0-4.0 mg·L-1 + Meta-Topolin(MT) 1.0-2.0 mgL-1 135 mgL of+sodium thiosulfate-1+ paddy Guang
Sweet 100 mgL of peptide-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mg of+acid hydrolyzed casein
L-1 20000 mgL of+sucrose-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Culture medium II described in above step (3) are as follows: improvement 0.3 mgL of DCR culture medium+2,4-D-1 + NAA
2.0-4.0 mg·L-1 + MT 0.5-1.0 mg·L-1 85 mgL of+sodium thiosulfate-1100 mgL of+glutathione-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose 30000
mg·L-1 + 250 mg·L-1 MES。
Culture medium III described in above step (3) are as follows: improvement 0.3 mgL of DCR culture medium+2,4-D-1 + NAA
2.0-4.0 mg·L-1 + MT 0.5-1.0 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 + day
100 mgL of winter amide-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
Culture medium IV described in above step (4) are as follows: improvement DCR culture medium+NAA 2.0-4.0 mgL-1 + MT
1.0-2.0 mg·L-1 1000 mgL of+inositol-1 1500 mgL of+glutamine-1 100 mgL of+asparagine-1 +
Pentaaza cytidine 3.0-5.0 mgL-1 20000 mgL of+sucrose-1 10000 mgL of+maltose-1 + agar 6000
mg·L-1 + 250 mg·L-1 MES。
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Culture dish diameter described in above step (2), step (3) and step (4) is 90 mm.
In contrast to the prior art, the present invention is had the advantage that and good effect is as follows:
1, the present invention is directed to the different growth conditions of masson pine embryo callus, has carried out the operation of different culture processes, especially
It is also to have carried out squamous subculture pretreatment after Multiplying culture primary, before subculture maintenance culture, by embryo callus subculture group
It knits suspend and rocks incubation, the dispersibility of embryo callus is enhanced, in favor of the proliferation of Embryogenic cell masses, into one
Step improves callus proliferative capacity.
2, the present invention is directed to different operating procedures, carries out to form, size or inoculum density of Embryonal suspensor mass etc.
Corresponding specific restriction, there is very strong operability and directiveness, is further ensured that embryo callus activity.
3, the present invention is aiming at the problem that embryo callus subculture vigor in embryo callus subculture squamous subculture declines, in different culture processes
In use different culture mediums, be added to different active constituents for different culture purposes: such as sodium thiosulfate, gluathione
Peptide can effectively inhibit to induce the browning of the Embryogenic cell masses just formed and water stainization problem；Asparagine can enhance cell activity,
Accelerate embryo callus subculture multiplication rate；Pentaaza cytidine as a kind of effective DNA methylation inhibitor, keep embryo callus subculture after
It is commissioned to train in supporting and keeps lower methylation level, reduce the degeneration of embryo callus subculture differentiation capability again.
4, the present invention improves DCR culture medium, and emphasis reduces ammonium nitrogen amount, improves nitrate nitrogen and ammonium nitrogen
Than, while also having adjusted and maintaining the microelements such as closely related boron, manganese, zinc, iodine with activity with masson pine embryo callus subculture proliferation,
So that culture medium is more scientific, it is more suitable for masson pine embryo callus subculture proliferation and maintains to cultivate, reduction embryo callus subculture is water stain, browning is asked
Topic, significant effect.
5, the proliferation that the present invention uses maintains culture medium and training method, and embryo callus subculture is in long term subculture without obvious
Browning and water stainization phenomenon, embryo callus subculture growth is fast, and cultivation effect stablizes height, and growth coefficient has preferable warp up to 25 or more
Benefit of helping and social benefit.
Detailed description of the invention
Fig. 1 is the masson pine embryo callus that frequent regulator embryonal induction is just formed.
Fig. 2 is through shoot proliferation of the invention and the masson pine embryo callus for maintaining culture to obtain.
Specific embodiment
The present invention will be further described combined with specific embodiments below.
Embodiment 1:
The prematurity cone egagametophyte for being in the cleavage polyembryony phase using masson pine zygotic embryo is induced as explant by conventional body embryo
3~4 weeks, when embryo callus subculture roll into a ball diameter >=1 cm when, directly by it is colourless, transparent, water stain-free reel off raw silk from cocoons shape Embryogenic cell masses with it is female
Gametophyte separate according to a conventional method after as be proliferated and maintain culture embryo callus.
Isolated embryo callus is placed in the 90 mm culture dish of diameter equipped with culture medium I, half-light culture 4
Week, 20 ± 0.5 DEG C of cultivation temperature.The culture medium I are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
2.0 mg·L-1 + Meta-Topolin(MT) 1.0 mgL-1 135 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sugarcane
20000 mgL of sugar-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1g embryo
Callus is transferred in 5 mL centrifuge tubes, and II 3 mL of culture medium is added into centrifuge tube, obtains Embryonal suspensor mass after firmly shaking up,
0.8 mL Embryonal suspensor mass is drawn with suction pipe to be put on the 90 mm culture dish of diameter for being lined with double-layer filter paper, is subsequently placed in ultra-clean work
Make 5 min of exhausting on platform, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred to the 90 mm culture dish of diameter equipped with culture medium III
In, half-light culture 6 weeks, 21 ± 0.5 DEG C of cultivation temperature.The culture medium II are as follows: improvement DCR culture medium+2,4-D 0.3
mg·L-1 + NAA 3.0 mg·L-1 + MT 0.5 mg·L-1 85 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose
30000 mg·L-1 + 250 mg·L-1 MES.The culture medium III are as follows: improvement 0.3 mg of DCR culture medium+2,4-D
L-1 + NAA 3.0 mg·L-1 + MT 0.5 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 +
100 mgL of asparagine-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred to 90 mm of diameter equipped with culture medium IV
In culture dish, every ware inoculation 5~6, half-light culture, 12 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature.The culture medium
IV are as follows: improvement 4.0 mgL of DCR culture medium+NAA-1 + MT 2.0 mg·L-1 1000 mgL of+inositol-1 + glutamy
1500 mgL of amine-1 100 mgL of+asparagine-1 5.0 mgL of+pentaaza cytidine-1 20000 mgL of+sucrose-1
10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES.Squamous subculture 30 times, embryo callus subculture
Growth is rapid, flushes, growth coefficient 25.7.
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Embodiment 2:
The prematurity cone egagametophyte for being in the cleavage polyembryony phase using masson pine zygotic embryo is induced as explant by conventional body embryo
3~4 weeks, when embryo callus subculture roll into a ball diameter >=1 cm when, directly by it is colourless, transparent, water stain-free reel off raw silk from cocoons shape Embryogenic cell masses with it is female
Gametophyte separate according to a conventional method after as be proliferated and maintain culture embryo callus.
Isolated embryo callus is placed in the 90 mm culture dish of diameter equipped with culture medium I, half-light culture 6
Week, 20 ± 0.5 DEG C of cultivation temperature.The culture medium I are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
2.0 mg·L-1 + Meta-Topolin(MT) 2.0 mgL-1 135 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sugarcane
20000 mgL of sugar-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1 g embryo
Property callus be transferred in 5 mL centrifuge tubes, into centrifuge tube be added II 3 mL of culture medium, after firmly shaking up obtain embryo culture
Object is drawn 0.8 mL Embryonal suspensor mass with suction pipe and is put on the 90 mm culture dish of diameter for being lined with double-layer filter paper, is subsequently placed in ultra-clean
5 min of exhausting on workbench, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred to the culture of 90 mm of diameter equipped with culture medium III
In ware, half-light culture 6 weeks, 21 ± 0.5 DEG C of cultivation temperature.The culture medium II are as follows: improvement DCR culture medium+2,4-D 0.3
mg·L-1 + NAA 2.0 mg·L-1 + MT 1.0 mg·L-1 85 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose
30000 mg·L-1 + 250 mg·L-1 MES.The culture medium III are as follows: improvement 0.3 mg of DCR culture medium+2,4-D
L-1 + NAA 2.0 mg·L-1 + MT 1.0 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 +
100 mgL of asparagine-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred to 90 mm of diameter equipped with culture medium IV
In culture dish, every ware inoculation 5~6, half-light culture, 14 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature.The culture medium
IV are as follows: improvement 2.0 mgL of DCR culture medium+NAA-1 + MT 2.0 mg·L-1 1000 mgL of+inositol-1 + glutamy
1500 mgL of amine-1 100 mgL of+asparagine-1 4.0 mgL of+pentaaza cytidine-1 20000 mgL of+sucrose-1
10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES.Squamous subculture 50 times, embryo callus subculture
Growth is rapid, flushes, growth coefficient 28.9.
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Embodiment 3:
The prematurity cone egagametophyte for being in the cleavage polyembryony phase using masson pine zygotic embryo is induced as explant by conventional body embryo
3~4 weeks, when embryo callus subculture roll into a ball diameter >=1 cm when, directly by it is colourless, transparent, water stain-free reel off raw silk from cocoons shape Embryogenic cell masses with it is female
Gametophyte separate according to a conventional method after as be proliferated and maintain culture embryo callus.
Isolated embryo callus is placed in the 90 mm culture dish of diameter equipped with culture medium I, half-light culture 5
Week, 20 ± 0.5 DEG C of cultivation temperature.The culture medium I are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
4.0 mg·L-1 + Meta-Topolin(MT) 2.0 mgL-1 135 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sugarcane
20000 mgL of sugar-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1.5 g
Embryo callus is transferred in 5 mL centrifuge tubes, and II 5 mL of culture medium is added into centrifuge tube, and embryo training is obtained after firmly shaking up
Object is supported, 0.8 mL Embryonal suspensor mass is drawn with suction pipe and is put on the 90 mm culture dish of diameter for being lined with double-layer filter paper, be subsequently placed in super
5 min of exhausting on net workbench, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred to the training of 90 mm of diameter equipped with culture medium III
It supports in ware, half-light culture 8 weeks, 21 ± 0.5 DEG C of cultivation temperature.The culture medium II are as follows: improvement DCR culture medium+2,4-D
0.3 mg·L-1 + NAA 2.0 mg·L-1 + MT 1.0 mg·L-1 85 mgL of+sodium thiosulfate-1+ glutathione
100 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose
30000 mg·L-1 + 250 mg·L-1 MES.The culture medium III are as follows: improvement 0.3 mg of DCR culture medium+2,4-D
L-1 + NAA 2.0 mg·L-1 + MT 1.0 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 +
100 mgL of asparagine-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred to 90 mm of diameter equipped with culture medium IV
In culture dish, every ware inoculation 5~6, half-light culture, 12 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature.The culture medium
IV are as follows: improvement 2.0 mgL of DCR culture medium+NAA-1 + MT 2.0 mg·L-1 1000 mgL of+inositol-1 + glutamy
1500 mgL of amine-1 100 mgL of+asparagine-1 5.0 mgL of+pentaaza cytidine-1 20000 mgL of+sucrose-1
10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES.Squamous subculture 60 times, embryo callus subculture
Growth is rapid, flushes, growth coefficient 31.5.
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Embodiment 4:
The prematurity cone egagametophyte for being in the cleavage polyembryony phase using masson pine zygotic embryo is induced as explant by conventional body embryo
3~4 weeks, when embryo callus subculture roll into a ball diameter >=1 cm when, directly by it is colourless, transparent, water stain-free reel off raw silk from cocoons shape Embryogenic cell masses with it is female
Gametophyte separate according to a conventional method after as be proliferated and maintain culture embryo callus.
Isolated embryo callus is placed in the 90 mm culture dish of diameter equipped with culture medium I, half-light culture 6
Week, 20 ± 0.5 DEG C of cultivation temperature.The culture medium I are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
4.0 mg·L-1 + Meta-Topolin(MT) 2.0 mgL-1 135 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sugarcane
20000 mgL of sugar-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1.5 g
Embryo callus is transferred in 5 mL centrifuge tubes, and II 5 mL of culture medium is added into centrifuge tube, and embryo training is obtained after firmly shaking up
Object is supported, 0.8 mL Embryonal suspensor mass is drawn with suction pipe and is put on the 90 mm culture dish of diameter for being lined with double-layer filter paper, be subsequently placed in super
5 min of exhausting on net workbench, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred to the training of 90 mm of diameter equipped with culture medium III
It supports in ware, half-light culture 6 weeks, 21 ± 0.5 DEG C of cultivation temperature.The culture medium II are as follows: improvement DCR culture medium+2,4-D
0.3 mg·L-1 + NAA 4.0 mg·L-1 + MT 1.0 mg·L-1 85 mgL of+sodium thiosulfate-1+ glutathione
100 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose
30000 mg·L-1 + 250 mg·L-1 MES.The culture medium III are as follows: improvement 0.3 mg of DCR culture medium+2,4-D
L-1 + NAA 4.0 mg·L-1 + MT 1.0 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 +
100 mgL of asparagine-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred to 90 mm of diameter equipped with culture medium IV
In culture dish, every ware inoculation 5~6, half-light culture, 10 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature.The culture medium
IV are as follows: improvement 4.0 mgL of DCR culture medium+NAA-1 + MT 1.5 mg·L-1 1000 mgL of+inositol-1 + glutamy
1500 mgL of amine-1 100 mgL of+asparagine-1 5.0 mgL of+pentaaza cytidine-1 20000 mgL of+sucrose-1
10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES.Squamous subculture 70 times, embryo callus subculture
Growth is rapid, flushes, growth coefficient 29.6.
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Embodiment 5:
The prematurity cone egagametophyte for being in the cleavage polyembryony phase using masson pine zygotic embryo is induced as explant by conventional body embryo
3~4 weeks, when embryo callus subculture roll into a ball diameter >=1 cm when, directly by it is colourless, transparent, water stain-free reel off raw silk from cocoons shape Embryogenic cell masses with it is female
Gametophyte separate according to a conventional method after as be proliferated and maintain culture embryo callus.
Isolated embryo callus is placed in the 90 mm culture dish of diameter equipped with culture medium I, half-light culture 4
Week, 20 ± 0.5 DEG C of cultivation temperature.The culture medium I are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA
3.0 mg·L-1 + Meta-Topolin(MT) 1.5 mgL-1 135 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1100 mgL of+inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sugarcane
20000 mgL of sugar-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1 g embryo
Property callus be transferred in 5 mL centrifuge tubes, into centrifuge tube be added II 4 mL of culture medium, after firmly shaking up obtain embryo culture
Object is drawn 0.8 mL Embryonal suspensor mass with suction pipe and is put on the 90 mm culture dish of diameter for being lined with double-layer filter paper, is subsequently placed in ultra-clean
5 min of exhausting on workbench, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred to the culture of 90 mm of diameter equipped with culture medium III
In ware, half-light culture 7 weeks, 21 ± 0.5 DEG C of cultivation temperature.The culture medium II are as follows: improvement DCR culture medium+2,4-D 0.3
mg·L-1 + NAA 3.0 mg·L-1 + MT 0.5 mg·L-1 85 mgL of+sodium thiosulfate-1+ glutathione 100
mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 + sucrose
30000 mg·L-1 + 250 mg·L-1 MES.The culture medium III are as follows: improvement 0.3 mg of DCR culture medium+2,4-D
L-1 + NAA 3.0 mg·L-1 + MT 0.5 mg·L-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 +
100 mgL of asparagine-1 30000 mgL of+sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred to 90 mm of diameter equipped with culture medium IV
In culture dish, every ware inoculation 5~6, half-light culture, 14 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature.The culture medium
IV are as follows: improvement 3.0 mgL of DCR culture medium+NAA-1 + MT 1.0 mg·L-1 1000 mgL of+inositol-1 + glutamy
1500 mgL of amine-1 100 mgL of+asparagine-1 3.0 mgL of+pentaaza cytidine-1 20000 mgL of+sucrose-1
10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES.Squamous subculture 80 times, embryo callus subculture
Growth is rapid, flushes, growth coefficient 27.5.
The composition of above-described improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；
KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·
7H2O 17.5 mg·L-1；Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0
mg·L-1；CuSO4·5H2O 0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；Cigarette
1.0 mgL of acid-1。
Claims (7)
1. a kind of method masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: separated including embryo callus subculture, is primary
Multiplying culture, squamous subculture pretreatment and subculture maintenance culture processes, are in take out by induce obtain within 3~4 weeks by conventional body embryo
Filamentous Embryogenic cell masses and the isolated embryo callus of egagametophyte are placed in the culture dish equipped with culture medium I, and 4~6
Week removal embryo callus in dead cell and excess tissue, be transferred in centrifuge tube and be added culture medium II mixing shake up, so
The Embryonal suspensor mass obtained after shaking up afterwards is placed in the culture dish equipped with culture medium III, and the embryo of appropriate size is taken after 6~8 weeks
Callus, which is directly transferred in culture medium IV, carries out normal subculture, 10~14 d of subculture cycle, and finally acquisition growth is rapid, flushes
And the masson pine embryogenic cell line of long term subculture can be carried out；Main operational steps are as follows:
(1) embryo callus subculture separates:
It is induced 3~4 weeks by conventional body embryo, it, directly will be colourless, transparent, water stain-free when embryo callus subculture rolls into a ball diameter >=1 cm
The shape Embryogenic cell masses that reel off raw silk from cocoons separated according to a conventional method with egagametophyte after as be proliferated and maintain culture embryo callus subculture group
It knits；
(2) Multiplying culture primary:
Isolated embryo callus is placed in the culture dish equipped with culture medium I, half-light culture 4~6 weeks, cultivation temperature
20±0.5℃；
(3) squamous subculture pre-processes:
Remove dead cell and excess tissue after Multiplying culture primary in embryo callus, remove it is miscellaneous after 1~1.5 g embryo
Property callus be transferred in 5 mL centrifuge tubes, into centrifuge tube be added II 3~5 mL of culture medium, after firmly shaking up obtain embryo training
Object is supported, 0.8 mL Embryonal suspensor mass is drawn with suction pipe and is put on the culture dish for being lined with double-layer filter paper, be subsequently placed in superclean bench
Upper 5 min of exhausting, then the upper layer filter paper equipped with Embryonal suspensor mass is transferred in the culture dish equipped with culture medium III, half-light culture 6
~8 weeks, 21 ± 0.5 DEG C of cultivation temperature；
(4) subculture maintains culture:
The Embryogenic cell masses for 3~5 mm of diameter that acquisition is cultivated on filter paper are transferred in the culture dish equipped with culture medium IV, every ware
Inoculation 5~6, half-light culture, 10~14 d of subculture cycle, 22 ± 0.5 DEG C of cultivation temperature；It finally obtains and grows rapid, vigor
Masson pine embryogenic cell line vigorous and that long term subculture can be carried out.
2. the method according to claim 1 masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: step
(2) culture medium I described in are as follows: improvement 0.8 mgL of DCR culture medium+2,4-D-1 + NAA 2.0-4.0 mg·L-1 +
Meta-Topolin(MT) 1.0-2.0 mgL-1135 mgL of+sodium thiosulfate-1100 mgL of+glutathione-1 +
100 mgL of inositol-1 500 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 + sucrose 20000
mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + 250 mg·L-1 MES。
3. the method according to claim 1 masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: step
(3) culture medium II described in are as follows: improvement 0.3 mgL of DCR culture medium+2,4-D-1 + NAA 2.0-4.0 mg·L-1 +
MT 0.5-1.0 mg·L-185 mgL of+sodium thiosulfate-1100 mgL of+glutathione-1 1000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 30000 mgL of+sucrose-1 + 250 mg·L-1
MES。
4. the method according to claim 1 masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: step
(3) culture medium III described in are as follows: improvement 0.3 mgL of DCR culture medium+2,4-D-1 + NAA 2.0-4.0 mg·L-1 +
MT 0.5-1.0 mg·L-11000 mgL of+inositol-1 950 mgL of+glutamine-1 100 mgL of+asparagine-1 +
30000 mgL of sucrose-16500 mgL of+agar-1 + 250 mg·L-1 MES。
5. the method according to claim 1 masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: step
(4) culture medium IV described in are as follows: improvement DCR culture medium+NAA 2.0-4.0 mgL-1 + MT 1.0-2.0 mg·L-1+ flesh
1000 mgL of alcohol-1 1500 mgL of+glutamine-1 100 mgL of+asparagine-1 + pentaaza cytidine 3.0-5.0
mg·L-1 20000 mgL of+sucrose-1 10000 mgL of+maltose-1 6000 mgL of+agar-1 + 250 mg·L-1 MES。
6. according to claim 2-5 any the masson pine embryo callus subculture proliferation and the method for maintenance culture, it is characterised in that:
The composition of the improvement DCR culture medium are as follows: KNO3 570 mg·L-1；NH4NO3 80 mg·L-1；KH2PO4 150 mg·L-1；Ca(NO3)2·4H2O 500 mg·L-1；Mg(NO3)2·6H2O 430 mg·L-1；FeSO4·7H2O 17.5 mg·L-1；
Na2EDTA 20.0 mg·L-1；MnSO4·H2O 10.0 mg·L-1；ZnSO4·7H2O 21.0 mg·L-1；CuSO4·5H2O
0.2 mg·L-1；H3BO3 15.5 mg·L-1；Na2MoO4·2H2O 0.2 mg·L-1；KI 4.15 mg·L-1；CoCl2·
6H2O 0.2 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.1 mg·L-1；1.0 mgL of niacin-1。
7. the method according to claim 1 masson pine embryo callus subculture proliferation and maintain culture, it is characterised in that: step
(2), culture dish diameter described in step (3) and step (4) is 90 mm.
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| CN117106693A (en) * | 2023-09-18 | 2023-11-24 | 黑龙江省林业科学研究所 | Suspension culture method for proliferation of Korean pine embryogenic cells |
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| CN117106693A (en) * | 2023-09-18 | 2023-11-24 | 黑龙江省林业科学研究所 | Suspension culture method for proliferation of Korean pine embryogenic cells |
| CN117106693B (en) * | 2023-09-18 | 2024-03-15 | 黑龙江省林业科学研究所 | Suspension culture method for proliferation of Korean pine embryogenic cells |
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