CN108668899A - A method of improving masson pine body embryo inductivity - Google Patents
A method of improving masson pine body embryo inductivity Download PDFInfo
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- CN108668899A CN108668899A CN201810545301.0A CN201810545301A CN108668899A CN 108668899 A CN108668899 A CN 108668899A CN 201810545301 A CN201810545301 A CN 201810545301A CN 108668899 A CN108668899 A CN 108668899A
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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
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Abstract
The present invention provides a kind of method improving masson pine body embryo inductivity, process is induced including explant pretreatment, explant disinfection and body embryo, acquisition health, no disease and pests harm, zygotic embryo are in the Pinus massoniana Plus Trees prematurity cone of cleavage polyembryony phase as explant, after explant is pre-processed and is disinfected, the sterile egagametophyte stripped in explant cone, it is lain against to be placed in inducing culture in suitable environment and be cultivated, the embryo callus subculture Fiber differentiation period is：34 weeks, finally obtain the masson pine embryogenic cell line that growth is rapid, flushes.Explant processing and disinfection of the present invention by series optimization, the egagametophyte stripped again in cone is induced, form the embryogenic cell line largely to flush, strong physical resources and technical guarantee have been established further to carry out the nursery of masson pine body embryo, germplasm innovation and bioengineering breeding, there are good economic benefit, social benefit and ecological benefits.
Description
Technical field
The invention belongs to technical field of plant propagation, are related to tissue cultures body embryo generation technique, especially a kind of raising horse hair
The method of loose body embryo inductivity.
Background technology
Masson pine（Pinus massonianaLamb.）It is the chief species of south China ecological construction and afforestation material.
The seeds 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.Guangxi is as a big forestry province, according to autonomous region " ten
Three or five " forest development is planned, the development of hundred billion yuan of special advantage industries such as Gum Rosin Industry, Wood industry is built in Guangxi economy of forestry
Occupy very important status in if, it is significant to greatly develop masson pine industry.Since " six or five " National Key Research Programs, I
State achieves in masson pine fine-variety breeding and Fast-growing Cultivation of Cinnamomun etc. compared with quantum jump.Due to weather and geographical environment etc.
The advantage of natural conditions is collected, in masson pine germ plasm resource in terms of preservation and fine-variety breeding, and Guangxi is in a leading position ground at home
Position.Variety of Pinus massoniana is one kind of masson pine, originates in portiatree township of Ningming of Guangxi county.1984 through national southern 14 provinces（Area）Forestry
Expert's comprehensive assessment is examined（Recognize）It is set to the excellent geographical provenance of national masson pine.As Guangxi special advantage seeds, Variety of Pinus massoniana
Growth traits is good, Gum productivity is high, material is excellent, adaptable, in southern provinces such as China Guangxi, Guangdong, Fujian, Sichuan, Zhejiang
（Area）There is extensive introducing and planting.
Masson pine breeding cycle is long, and in addition recently as breeding garden elite stand aging, hip number declines, and breeding is deficient, leads
The southern Masson Pine Plantation genetic variation and genetic differentiation difference of cause is big, stand quality is not high, productivity is low.To push masson pine industry efficient
Development realizes that the breeding industrialization of the asexual nursery of excellent strain is particularly critical.The use of plant tissue culture technology is to realize that breeding is educated
The effective way of seedling industrialization.In tissue culture technology for organ generation, somatic embryo occurs to be used as plant cell
A kind of expression way of totipotency is the important means of plant regeneration in plant cell engineering, with reproductive efficiency height, structure
Completely, it inheritance stability and many advantages such as is not limited by natural conditions, is prepared in extensive vegetative propagation, artificial seed, germplasm
It preserves and the numerous areas such as genetic transformation all has major application value, be the main of the current forest Clonal regeneration of progress in the world
Means.Currently, masson pine somatic embryo occur have correlative study, but still have frequency of embryonic callus induction it is low, differentiation
It is difficult, germination rate is low, the problems such as being difficult to obtain regeneration plant.
Invention content
It is numerous that in view of the deficiencies of the prior art, the present invention provides a kind of with masson pine fine germplasm resources Variety of Pinus massoniana select tree
Material is grown, embryo callus subculture is high-quality, the method for easily forming the raising masson pine body embryo inductivity of embryogenic cell line.
To achieve the goals above, technical scheme is as follows：
A method of masson pine body embryo inductivity being improved, including explant pretreatment, explant disinfection and body embryo induce process,
Acquisition health, no disease and pests harm, zygotic embryo are in the Pinus massoniana Plus Trees prematurity cone of cleavage polyembryony phase as explant, to explant
After body is pre-processed and disinfected, the sterile egagametophyte stripped in explant cone is lain against inducing culture
In be placed in suitable environment and cultivate, the embryo callus subculture Fiber differentiation period is：3-4 weeks, finally acquisition growth was rapid, vigor is prosperous
The masson pine embryogenic cell line of Sheng；Main operational steps are as follows：
（1）Explant pre-processes：
Acquisition health, no disease and pests harm, zygotic embryo are in the Pinus massoniana Plus Trees prematurity cone of cleavage polyembryony phase as explant, use
+ 100 mgL of 45% alcohol of volumetric concentration+volumetric concentration, 0.03% streptomysin-1Niacin mixed liquor carries out explant surface wipes and disappears
After poison, it in explant body device plastics valve bag, will be placed in 5-8 DEG C of refrigerator and store 10-14 d；
（2）Explant sterilizes：
Pretreated explant is taken out after flowing water rinses 2 h, the wide-spectrum bactericide that volumetric concentration is 0.1-0.3 % is placed in
Middle immersion 1-2 h, it is 3-5 min to be impregnated in 60 % alcohol, then move into volumetric concentration to take out explant and place it in volumetric concentration
To impregnate 2-3 min in the liquor natrii hypochloritis of 8-10 %, it is 8 % hydrogen peroxide+volumetric concentration 0.2% that taking-up, which is put in volumetric concentration,
+ 300 mgL of polysorbas20-1After 30-40 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing is finally used 4 times, often
Secondary flushing 3-5 min；
（3）Body embryo induces：
It is sterile to strip through step on superclean bench（2）Egagametophyte after processing in explant cone, is lain against and is lured
It leads to be placed in culture medium in suitable environment and cultivate, the embryo callus subculture Fiber differentiation period is：3-4 weeks, it is rapid, living to obtain growth
The vigorous masson pine embryogenic cell line of power；The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1
+ NAA 5.0-9.0 mg·L-1 + rape element class ester 1.0-2.0 mgL-1 + ABA 0.5-1.0 mg·L-1 + multiple-effect
0.66 mgL of azoles-1+ silver nitrate 0.5-1.0 mgL-12000 mgL of+glutamine-1 + acid hydrolyzed casein
1000 mg·L-1 20000 mgL of+glucose-1 10000 mgL of+fructose-1 10000 mgL of+lactose-1 + agar
4500 mg·L-1 + activated carbon 3000-5000mgL-1 + 250 mg·L-1 MES。
Preferably, above step（1）The Pinus massoniana Plus Trees are Variety of Pinus massoniana select tree.
Preferably, above step（3）The group of the improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3
120 mg·L-1；KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；
MgSO4·7H2O 650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O
30.0 mg·L-1；ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；
Na2MoO4·2H2O 0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0
mg·L-1；Vitamin B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；Vitamin E 0.5
mg·L-1；Vitamin B12 0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Preferably, above step（1）The specification of the plastics valve bag is the mm of 20 mm × 30；It is a per 4-5 when pack
Explant is fitted into a plastics valve bag, and sack sealing 80% will be placed in refrigerator again after sack downward doubling.
Preferably, above step（2）The suitable environment is：Temperature：20 ± 0.5 DEG C, light culture.
In contrast to the prior art, advantages of the present invention and good effect are as follows：
1, the present invention is to select zygotic embryo to be in using the excellent geographical provenance Variety of Pinus massoniana select tree of national masson pine as propagating materials and split
Raw polyembryony phase prematurity cone is as explant, the explant processing by series optimization and disinfection, then strips female in cone
Gametophyte is induced, and the embryogenic cell line largely to flush is formd, further to carry out the nursery of masson pine body embryo, germplasm
Innovation with bioengineering breeding established strong physical resources and technical guarantee, have good economic benefit, social benefit and
Ecological benefits.
2, the present invention uses broad spectrum antimicrobial agent, alcohol, sodium hypochlorite, the dioxygen of certain concentration and disinfecting time successively
Water, tween and the matched mode disinfected of anti-oxidation reducing agent, Brown is few, and aseptic explant acquisition rate is high,
Reach 95% or more, solves the problems such as explant disinfection is difficult, browning is serious in the generation of masson pine body embryo, significant effect.
3, the present invention for the easy browning of explant in the in vitro tissue culture of masson pine, physiological ageing is apparent the problems such as, from cytology,
Physiology and biochemistry and molecular biology angle, have carried out a large amount of analysis and test, according to test result, on the basis of preferred,
Clear stipulaties mineral element in the inducing culture that the present invention uses, hormone, vitamin, organic nitrogen isoreactivity material composition
Proportioning and condition of culture, embryo callus subculture inductivity are up to 92.1%, and grow rapid, subculture and maintain normal proliferation, under can meeting
The needs of the Multiplying culture of one step adapt to demand of the forestry industrialization development to masson pine breeding seedling.
Description of the drawings
Fig. 1 is the present invention with the embryo callus of Variety of Pinus massoniana select tree prematurity cone zygote embryonal induction.
Fig. 2 is the Variety of Pinus massoniana embryogenic cell line to flush that the present invention is built.
Specific implementation mode
The present invention is further explained in the light of specific embodiments.
Embodiment 1：
On June 25th, 2015 sends in the Variety of Pinus massoniana superior stand of Yangshan forest farm in Guangxi is state-run, acquisition health, no disease and pests harm, zygote
Embryo is in the Variety of Pinus massoniana select tree prematurity cone of cleavage polyembryony phase as explant, with 45% alcohol of volumetric concentration+volumetric concentration
+ 100 mgL of 0.03% streptomysin-1After niacin mixed liquor carries out explant surface wipes disinfection, one is packed into per 4-5 explant
A specification is in the plastics valve bag of the mm of 20 mm × 30, and sack sealing 80% will be placed in 8 DEG C of refrigerators again after sack downward doubling
Middle storage 14d.
Pretreated explant is taken out from refrigerator after flowing water rinses 2 h, it is 0.1-0.3 % to be placed in volumetric concentration
Wide-spectrum bactericide in impregnate 1 h, it is 3-5 min to be impregnated in 60 % alcohol, then move to take out explant and place it in volumetric concentration
Enter and impregnate 2-3 min in the liquor natrii hypochloritis that volumetric concentration is 8 %, it is that 8 % hydrogen peroxide+volume is dense that taking-up, which is put in volumetric concentration,
Spend+300 mgL of 0.2% polysorbas20-1After 30 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing 4 is finally used
It is secondary, 3-5 min are rinsed every time.
On superclean bench, the sterile egagametophyte stripped in sterile-processed rear explant cone is lain against
It is placed in 20 ± 0.5 DEG C of temperature in inducing culture, is cultivated in the environment of light culture, the embryo callus subculture Fiber differentiation period is：3-4
Week.The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1 + NAA 5.0 mg·L-1 + oil
1.0 mgL of dish element class ester-1 + ABA 0.5 mg·L-1 0.66 mgL of+paclobutrazol-10.5 mgL of+silver nitrate-1
2000 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 20000 mgL of+glucose-1 + fructose
10000 mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + activated carbon 3000mgL-1 + 250
mg·L-1 MES.The Variety of Pinus massoniana embryogenic cell line that growth is rapid, flushes finally is obtained, body embryo inductivity is 92.1%.
The group of above-described improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；
KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O
650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；
ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O
0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin
B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12
0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Embodiment 2：
On July 15th, 2016 sends in the Variety of Pinus massoniana superior stand of Yangshan forest farm in Guangxi is state-run, acquisition health, no disease and pests harm, zygote
Embryo is in the Variety of Pinus massoniana select tree prematurity cone of cleavage polyembryony phase as explant, with 45% alcohol of volumetric concentration+volumetric concentration
+ 100 mgL of 0.03% streptomysin-1After niacin mixed liquor carries out explant surface wipes disinfection, one is packed into per 4-5 explant
A specification is in the plastics valve bag of the mm of 20 mm × 30, and sack sealing 80% will be placed in 6 DEG C of refrigerators again after sack downward doubling
12 d of middle storage.
Pretreated explant is taken out from refrigerator after flowing water rinses 2 h, it is 0.1-0.3 % to be placed in volumetric concentration
Wide-spectrum bactericide in impregnate 1.5 h, take out explant place it in volumetric concentration be 3-5 min are impregnated in 60 % alcohol, then
It moves into the liquor natrii hypochloritis that volumetric concentration is 8 % and impregnates 2-3 min, it is 8 % hydrogen peroxide+volume that taking-up, which is put in volumetric concentration,
+ 300 mgL of 0.2% polysorbas20 of concentration-1After 300 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing is finally used
4 times, 3-5 min are rinsed every time.
On superclean bench, the sterile egagametophyte stripped in sterile-processed rear explant cone is lain against
It is placed in 20 ± 0.5 DEG C of temperature in inducing culture, is cultivated in the environment of light culture, the embryo callus subculture Fiber differentiation period is：3-4
Week.The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1 + NAA 6.0 mg·L-1 + oil
2.0 mgL of dish element class ester-1 + ABA 1.0 mg·L-1 0.66 mgL of+paclobutrazol-10.5 mgL of+silver nitrate-1
2000 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 20000 mgL of+glucose-1 + fructose
10000 mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + activated carbon 3500mgL-1 + 250
mg·L-1 MES.The Variety of Pinus massoniana embryogenic cell line that growth is rapid, flushes finally is obtained, body embryo inductivity is 89.2%.
The group of above-described improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；
KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O
650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；
ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O
0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin
B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12
0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Embodiment 3：
On July 8th, 2017 sends in the Variety of Pinus massoniana superior stand of Yangshan forest farm in Guangxi is state-run, acquisition health, no disease and pests harm, zygotic embryo
Variety of Pinus massoniana select tree prematurity cone in the cleavage polyembryony phase is as explant, with 45% alcohol of volumetric concentration+volumetric concentration
+ 100 mgL of 0.03% streptomysin-1After niacin mixed liquor carries out explant surface wipes disinfection, one is packed into per 4-5 explant
A specification is in the plastics valve bag of the mm of 20 mm × 30, and sack sealing 80% will be placed in 7 DEG C of refrigerators again after sack downward doubling
14 d of middle storage.
Pretreated explant is taken out from refrigerator after flowing water rinses 2 h, it is 0.1-0.3 % to be placed in volumetric concentration
Wide-spectrum bactericide in impregnate 1 h, it is 3-5 min to be impregnated in 60 % alcohol, then move to take out explant and place it in volumetric concentration
Enter and impregnate 2-3 min in the liquor natrii hypochloritis that volumetric concentration is 9 %, it is that 8 % hydrogen peroxide+volume is dense that taking-up, which is put in volumetric concentration,
Spend+300 mgL of 0.2% polysorbas20-1After 35 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing 4 is finally used
It is secondary, 3-5 min are rinsed every time.
On superclean bench, the sterile egagametophyte stripped in sterile-processed rear explant cone is lain against
It is placed in 20 ± 0.5 DEG C of temperature in inducing culture, is cultivated in the environment of light culture, the embryo callus subculture Fiber differentiation period is：3-4
Week.The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1 + NAA 7.0 mg·L-1 + oil
1.5 mgL of dish element class ester-1 + ABA 0.5 mg·L-1 0.66 mgL of+paclobutrazol-11.0 mgL of+silver nitrate-1
2000 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 20000 mgL of+glucose-1 + fructose
10000 mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + activated carbon 4000mgL-1 + 250
mg·L-1 MES.The Variety of Pinus massoniana embryogenic cell line that growth is rapid, flushes finally is obtained, body embryo inductivity is 90.1%.
The group of above-described improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；
KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O
650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；
ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O
0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin
B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12
0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Embodiment 4：
On July 15th, 2017 sends in the Variety of Pinus massoniana superior stand of Yangshan forest farm in Guangxi is state-run, acquisition health, no disease and pests harm, zygote
Embryo is in the Variety of Pinus massoniana select tree prematurity cone of cleavage polyembryony phase as explant, with 45% alcohol of volumetric concentration+volumetric concentration
+ 100 mgL of 0.03% streptomysin-1After niacin mixed liquor carries out explant surface wipes disinfection, one is packed into per 4-5 explant
A specification is in the plastics valve bag of the mm of 20 mm × 30, and sack sealing 80% will be placed in 7 DEG C of refrigerators again after sack downward doubling
12 d of middle storage.
Pretreated explant is taken out from refrigerator after flowing water rinses 2 h, it is 0.1-0.3 % to be placed in volumetric concentration
Wide-spectrum bactericide in impregnate 2 h, it is 3-5 min to be impregnated in 60 % alcohol, then move to take out explant and place it in volumetric concentration
Enter and impregnate 2-3 min in the liquor natrii hypochloritis that volumetric concentration is 10 %, it is 8 % hydrogen peroxide+volume that taking-up, which is put in volumetric concentration,
+ 300 mgL of 0.2% polysorbas20 of concentration-1After 40 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing 4 is finally used
It is secondary, 3-5 min are rinsed every time.
On superclean bench, the sterile egagametophyte stripped in sterile-processed rear explant cone is lain against
It is placed in 20 ± 0.5 DEG C of temperature in inducing culture, is cultivated in the environment of light culture, the embryo callus subculture Fiber differentiation period is：3-4
Week.The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1 + NAA 8.0 mg·L-1 + oil
1.0 mgL of dish element class ester-1 + ABA 1.0 mg·L-1 0.66 mgL of+paclobutrazol-11.0 mgL of+silver nitrate-1
2000 mgL of+glutamine-1 1000 mgL of+acid hydrolyzed casein-1 20000 mgL of+glucose-1 + fructose
10000 mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + activated carbon 4500mgL-1 + 250
mg·L-1 MES.The Variety of Pinus massoniana embryogenic cell line that growth is rapid, flushes finally is obtained, body embryo inductivity is 91.3%.
The group of above-described improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；
KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O
650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；
ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O
0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin
B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12
0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Embodiment 5：
On July 25th, 2017 sends in the Variety of Pinus massoniana superior stand of Yangshan forest farm in Guangxi is state-run, acquisition health, no disease and pests harm, zygote
Embryo is in the Variety of Pinus massoniana select tree prematurity cone of cleavage polyembryony phase as explant, with 45% alcohol of volumetric concentration+volumetric concentration
+ 100 mgL of 0.03% streptomysin-1After niacin mixed liquor carries out explant surface wipes disinfection, one is packed into per 4-5 explant
A specification is in the plastics valve bag of the mm of 20 mm × 30, and sack sealing 80% will be placed in 5 DEG C of refrigerators again after sack downward doubling
Middle storage 10d.
Pretreated explant is taken out from refrigerator after flowing water rinses 2 h, it is 0.1-0.3 % to be placed in volumetric concentration
Wide-spectrum bactericide in impregnate 2 h, it is 3-5 min to be impregnated in 60 % alcohol, then move to take out explant and place it in volumetric concentration
Enter and impregnate 2-3 min in the liquor natrii hypochloritis that volumetric concentration is 10 %, it is 8 % hydrogen peroxide+volume that taking-up, which is put in volumetric concentration,
+ 300 mgL of 0.2% polysorbas20 of concentration-1After 40 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing 4 is finally used
It is secondary, 3-5 min are rinsed every time.
On superclean bench, the sterile egagametophyte stripped in sterile-processed rear explant cone is lain against
It is placed in 20 ± 0.5 DEG C of temperature in inducing culture, is cultivated in the environment of light culture, the embryo callus subculture Fiber differentiation period is：3-4
Week.The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1 + NAA 9.0 mg·L-1 + oil
2.0 mgL of dish element class ester-1 + ABA 1.0 mg·L-1 0.66 mgL of+paclobutrazol-11.0 mgL of+silver nitrate-1 +
2000 mgL of glutamine-1 1000 mgL of+acid hydrolyzed casein-1 20000 mgL of+glucose-1 + fructose
10000 mg·L-1 10000 mgL of+lactose-1 4500 mgL of+agar-1 + activated carbon 5000mgL-1 + 250
mg·L-1 MES.The Variety of Pinus massoniana embryogenic cell line that growth is rapid, flushes finally is obtained, body embryo inductivity is 88.6%.
The group of above-described improvement DCR culture mediums becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；
KH2PO4 240 mg·L-1；CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O
650 mg·L-1；96.5 mgL of ferrous citrate-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；
ZnSO4·7H2O 43.0 mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O
0.25 mg·L-1；KI 4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin
B6 0.5 mg·L-1；0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12
0.5 mg·L-1；0.5 mgL of biotin-1；10000 mgL of inositol-1。
Claims (5)
1. a kind of method improving masson pine body embryo inductivity, it is characterised in that：Including explant pretreatment, explant disinfection and
Body embryo induces process, acquisition health, no disease and pests harm, zygotic embryo to be in the Pinus massoniana Plus Trees prematurity cone conduct of cleavage polyembryony phase
Explant, after explant is pre-processed and disinfected, the sterile egagametophyte stripped in explant cone is kept flat
It is placed in suitable environment and cultivates in inducing culture, the embryo callus subculture Fiber differentiation period is：It 3-4 weeks, is finally given birth to
The masson pine embryogenic cell line that length is rapid, flushes；Main operational steps are as follows：
（1）Explant pre-processes：
Acquisition health, no disease and pests harm, zygotic embryo are in the Pinus massoniana Plus Trees prematurity cone of cleavage polyembryony phase as explant, use
+ 100 mgL of 45% alcohol of volumetric concentration+volumetric concentration, 0.03% streptomysin-1Niacin mixed liquor carries out explant surface wipes and disappears
After poison, it in explant body device plastics valve bag, will be placed in 5-8 DEG C of refrigerator and store 10-14 d；
（2）Explant sterilizes：
Pretreated explant is taken out after flowing water rinses 2 h, the wide-spectrum bactericide that volumetric concentration is 0.1-0.3 % is placed in
Middle immersion 1-2 h, it is 3-5 min to be impregnated in 60 % alcohol, then move into volumetric concentration to take out explant and place it in volumetric concentration
To impregnate 2-3 min in the liquor natrii hypochloritis of 8-10 %, it is 8 % hydrogen peroxide+volumetric concentration 0.2% that taking-up, which is put in volumetric concentration,
+ 300 mgL of polysorbas20-1After 30-40 min are impregnated in stirring in the mixed liquor of ascorbic acid, aseptic water washing is finally used 4 times, often
Secondary flushing 3-5 min；
（3）Body embryo induces：
It is sterile to strip through step on superclean bench（2）Egagametophyte after processing in explant cone, is lain against and is lured
It leads to be placed in culture medium in suitable environment and cultivate, the embryo callus subculture Fiber differentiation period is：3-4 weeks, it is rapid, living to obtain growth
The vigorous masson pine embryogenic cell line of power；The inducing culture is：Improve 0.5 mgL of DCR culture mediums+2,4-D-1
+ NAA 5.0-9.0 mg·L-1 + rape element class ester 1.0-2.0 mgL-1+ ABA 0.5-1.0 mg·L-1 + paclobutrazol
0.66 mg·L-1+ silver nitrate 0.5-1.0 mgL-12000 mgL of+glutamine-1 + acid hydrolyzed casein 1000
mg·L-1 20000 mgL of+glucose-1 10000 mgL of+fructose-1 10000 mgL of+lactose-1 + agar 4500
mg·L-1 + activated carbon 3000-5000mgL-1 + 250 mg·L-1 MES。
2. a kind of method improving masson pine body embryo inductivity according to claim 1, it is characterised in that：Step（1）Institute
The Pinus massoniana Plus Trees stated are Variety of Pinus massoniana.
3. a kind of method improving masson pine body embryo inductivity according to claim 1, it is characterised in that：Step（3）Institute
The group for the improvement DCR culture mediums stated becomes：KNO3 570 mg·L-1；NH4NO3 120 mg·L-1；KH2PO4 240 mg·L-1；
CaCl2·2H2O 75 mg·L-1；Ca(NO3)2·4H2O 100 mg·L-1；MgSO4·7H2O 650 mg·L-1；Citric acid
96.5 mgL of ferrous iron-1；Na2EDTA 40.0 mg·L-1；MnSO4·H2O 30.0 mg·L-1；ZnSO4·7H2O 43.0
mg·L-1；CuSO4·5H2O 0.25 mg·L-1；H3BO3 31.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；KI
4.15 mg·L-1；CoCl2·6H2O 0.25 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；
0.5 mgL of niacin-1；2.0 mgL of glycine-1；0.5 mgL of vitamin E-1；Vitamin B12 0.5 mg·L-1；It is raw
0.5 mgL of object element-1；10000 mgL of inositol-1。
4. a kind of method improving masson pine body embryo inductivity according to claim 1, it is characterised in that：Step（1）Institute
The specification for the plastics valve bag stated is the mm of 20 mm × 30；When pack, it is fitted into a plastics valve bag per 4-5 explant,
Sack sealing 80%, will be placed in refrigerator again after sack downward doubling.
5. a kind of method improving masson pine body embryo inductivity according to claim 1, it is characterised in that：Step（2）Institute
The suitable environment stated is：Temperature：20 ± 0.5 DEG C, light culture.
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|---|---|---|---|---|
| CN110305833A (en) * | 2019-07-10 | 2019-10-08 | 广西壮族自治区林业科学研究院 | A kind of suspension culture method of masson pine cells,primordial proliferation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731203A (en) * | 1996-06-14 | 1998-03-24 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis |
| CN101849505A (en) * | 2010-02-10 | 2010-10-06 | 南京林业大学 | Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana |
| EP2332405A1 (en) * | 2003-06-23 | 2011-06-15 | Weyerhaeuser Company | Media comprising gellan gum and methods for promoting maturation of conifer somatic embryos |
| KR101471923B1 (en) * | 2013-07-08 | 2014-12-11 | 대한민국 | Embryogenic tissue induction from immature seeds in loblolly pine (Pinus taeda) |
| CN105432465A (en) * | 2015-11-12 | 2016-03-30 | 东北林业大学 | Method for improving embryonic callus induction rate and embryo transfer rate of pinus koraiensis |
| CN107593454A (en) * | 2017-11-03 | 2018-01-19 | 南京林业大学 | A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method |
-
2018
- 2018-05-31 CN CN201810545301.0A patent/CN108668899B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731203A (en) * | 1996-06-14 | 1998-03-24 | Westvaco Corporation | Method for regeneration of coniferous plants by somatic embryogenesis |
| EP2332405A1 (en) * | 2003-06-23 | 2011-06-15 | Weyerhaeuser Company | Media comprising gellan gum and methods for promoting maturation of conifer somatic embryos |
| CN101849505A (en) * | 2010-02-10 | 2010-10-06 | 南京林业大学 | Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana |
| KR101471923B1 (en) * | 2013-07-08 | 2014-12-11 | 대한민국 | Embryogenic tissue induction from immature seeds in loblolly pine (Pinus taeda) |
| CN105432465A (en) * | 2015-11-12 | 2016-03-30 | 东北林业大学 | Method for improving embryonic callus induction rate and embryo transfer rate of pinus koraiensis |
| CN107593454A (en) * | 2017-11-03 | 2018-01-19 | 南京林业大学 | A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method |
Non-Patent Citations (4)
| Title |
|---|
| LI-HUA ZHU ET AL.: "Micropropagation of Pinus massoniana and mycorrhiza formation in vitro", 《PLANT CELL TISSUE CULT》 * |
| YU ZHANG ET AL.: "Direct organogenesis and plantlet regeneration from mature zygotic embryos of masson pine (Pinus massoniana L.)", 《PLANT CELL, TISSUE AND ORGAN CULTURE》 * |
| 李清清 等: "黑松未成熟胚的体细胞胚胎发生和植株再生", 《林业科学》 * |
| 杨模华 等: "马尾松幼胚体细胞胚胎发生研究", 《植物生理学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305833A (en) * | 2019-07-10 | 2019-10-08 | 广西壮族自治区林业科学研究院 | A kind of suspension culture method of masson pine cells,primordial proliferation |
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