CN105918128A - Method for quickly reproducing acer rubrum - Google Patents
Method for quickly reproducing acer rubrum Download PDFInfo
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- CN105918128A CN105918128A CN201610339561.3A CN201610339561A CN105918128A CN 105918128 A CN105918128 A CN 105918128A CN 201610339561 A CN201610339561 A CN 201610339561A CN 105918128 A CN105918128 A CN 105918128A
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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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- 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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- Developmental Biology & Embryology (AREA)
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Abstract
The invention relates to a method for quickly reproducing acer rubrum. The method comprises the steps of 1 explant selecting; 2 explant disinfecting; 3 initial induction culturing; 4 cellus inducing; 5 multiplication culturing; 6 rooting culturing; 7 seedling exercising and transplanting. By adopting the method to reproduce the acer rubrum, the production cost can be reduced, and the reproducing speed and the survival rate can be increased.
Description
Technical field
The present invention relates to micropropagation of plants technical field, especially a kind of American red-maple quick breeding technology neck
Territory.
Background technology
American red-maple (formal name used at school: Acer rubrum L.): originate in east coast of the United States, mostlys come from U.S. north
Portion and Canada's most area, introduced China before 2000.It is mainly distributed on Liaoning, Shandong, peace
Emblem one carries, owing to special geographical position makes American red-maple fine at north color changeable effect.It is fallen leaves great Qiao
Wood.Growth is very fast, and height tree 12-18 rice of growing up, hat 12 meters, the soil types adapting to multiple scope is raw
Long.Spring blooms, premium look.Because color in its brilliant in autumn, tree crown is clean and tidy, be widely used in park,
Planting in community, street, not only can be very popular with Landscape but also shade tree can be done, and are the most several
Year introduce beautify, one of the preferable rare tree species in green planted city gardens.Also it is to be uniquely used as shade tree
Colored-leaf Plants.
At present, American red-maple many employings seminal propagation, propagation by grafiting and three kinds of modess of reproduction of cottage propagation are carried out
Producing cultivation, this raising technology improves the purity of kind, maintains breediness, but survival rate is relatively low and numerous
Grow speed relatively slow, constrain the development of American red-maple industry.And tissue cultivating and seedling technology both can keep planting
Purity and characteristic, it is also possible to reduce production cost, improve reproduction speed and survival rate.
Prior art there is no the report obtaining American red-maple regeneration plant through callus approach.
Summary of the invention
The technical problem to be solved is to provide a kind of American red-maple rapid propagation method, through callus
Institutional approach obtains American red-maple Multiple Buds, root induction, can realize a large amount of quick of American red-maple plantlet in vitro
Breeding.
In order to solve above-mentioned technical problem, the present invention proposes following technical proposal:
A kind of American red-maple rapid propagation method, it is characterised in that described method comprises the following steps:
1) the choosing of outer implant: the stem section taking the American red-maple stem apex or band axillalry bud of giving birth to diameter 2-5mm then is
Outer implant, is cut into 1-3cm length with standby by outer implant after cutting off blade；
2) outer implant sterilization: above-mentioned stem section is put in the 20-25KHz supersonic wave cleaning machine filling running water
Keep temperature 30-35 DEG C to process 30-45min, then rinse 10-20min with running water；Subsequently in ultra-clean work
With the alcohol disinfecting 25-35 second of 70-75% in station, in 0.1% mercuric chloride solution process 5-8 minute, then with
Aseptic water washing 4-5 time, is placed in sterile petri dish standby；
3) initial Fiber differentiation: implant outer after sterilizing is respectively cut off 2-3mm, at the beginning of outer implant is downwardly into as end
Cultivating in beginning inducing culture, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；After cultivating 4-6d, the most untainted outer implant stem section is transferred in new initial inducing culture,
And continue cultivate 12-18d grow to sprouting；Described initial inducing culture composition is divided into:
WPM+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP1-5mg/L；
4) callus induction: take the bud in initial Fiber differentiation, puts in callus inducing medium and trains
Supporting 15-20d, condition is light culture, temperature 23 DEG C ± 2 DEG C；Described callus tissue culture base is: WPM+6-BA
1.0-2.0mg/L+NAA0.1-0.5mg/L+PVP1-5mg/L；
5) Multiplying culture: the callus lines of clear, colorless is put into cultivation 20-25d in proliferated culture medium, increases
Growing a large amount of tufted seedling of generation, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described Multiplying culture culture medium is:
1/2MS+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP 1-5mg/L；
6) culture of rootage: by 5) tufted seedling in step cuts into single, it is transferred in root media cultivate
Taking root after 15-20d, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, temperature 25 DEG C ±
2℃；Described taking root with culture medium is: 1/2MS+IBA0.1-0.5mg/L+PVP1-5mg/L；
7) hardening and transplanting: the culture medium bottle cap having the American red-maple seedling taken root is opened, room temperature lower refining seedling
After 3-5d, take out seedling, wash away root culture medium, transplant on the seedbed equipped with river sand matrix, keep temperature
20-25 DEG C, humidity 70%-85%, suitably shade；
Described WPM culture medium prescription is: a great number of elements: ammonium nitrate NH4NO3400mg/L, potassium sulfate
K2SO4900mg/L, potassium dihydrogen phosphate KH2PO3170mg/L, magnesium sulfate MgSO4 7H2O370mg/L；Calcium
Salt: calcium chloride CaCl2 2H2O96mg/L；Calcium nitrate tetrahydrate Ca (NO3) 2 4H2O556mg/L；Micro-
Secondary element: boric acid H3BO36.2mg/L, manganese sulfate MnSO4 4H2O22.5mg/L, zinc sulfate ZnSO4
7H2O8.6mg/L, sodium molybdate Na2MoO4 2H2O0.25mg/L, copper sulphate CuSO4 5H2O0.25mg/L；
Molysite: disodium ethylene diamine tetraacetate Na2-EDTA37.3mg/L, ferrous sulfate FeSO4 7H2O27.8mg/L；
Organic acid: inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 1mg/L, puridoxine hydrochloride 0.5mg/L,
Nicotinic acid 0.5mg/L；
Described MS culture medium prescription is: a great number of elements: potassium nitrate KNO31900mg/L, ammonium nitrate
NH4NO31650mg/L, potassium dihydrogen phosphate KH2PO3170mg/L, magnesium sulfate MgSO4 7H2O370mg/L；
Calcium salt: calcium chloride CaCl2 2H2O440mg/L；Trace element: KI KI0.83mg/L, boric acid H3BO36.2
Mg/L, manganese sulfate MnSO4 4H2O22.3mg/L, zinc sulfate ZnSO4 7H2O8.6mg/L, sodium molybdate Na2MoO4
2H2O0.25mg/L, copper sulphate CuSO4 5H2O0.025mg/L, cobalt chloride CoCl2 6H2O0.025
mg/L；Molysite: disodium ethylene diamine tetraacetate Na2-EDTA37.25mg/L, ferrous sulfate FeSO4
7H2O27.85mg/L；Organic acid: inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.5mg/L,
Puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L；Sucrose 30g/L, agar powder 7g/L, pH are 5.7；
Described 1/2MS culture medium prescription is: a great number of elements: potassium nitrate KNO3950mg/L, ammonium nitrate
NH4NO3825mg/L, potassium dihydrogen phosphate KH2PO385mg/L, magnesium sulfate MgSO4 7H2O185mg/L；Remaining
With above-mentioned MS culture medium；
Described 6-BA is 6-benzyl aminopurine；
Described NAA is NAA；
Described IBA is indole-3-butyric acid；
Described PVP is polyvinylpyrrolidone.
Preferably, step 3) in initial inducing culture consist of:
WPM+6-BA0.8mg/L+NAA0.2mg/L+PVP2mg/L。
Preferably, step 4) in callus tissue culture base be: WPM+6-BA
1.6mg/L+NAA0.3mg/L+PVP3mg/L。
Preferably, step 5) in Multiplying culture culture medium be:
1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP 3mg/L。
Preferably, step 6) in take root with culture medium and be: 1/2MS+IBA0.3mg/L+PVP2mg/L.
Use the present invention to breed American red-maple, a large amount of Fast-propagations of American red-maple plantlet in vitro can be realized, for U.S.
The red maple of state produces cultivation and breed improvement lays the foundation.
In order to preferably illustrate technical scheme, below in conjunction with detailed description of the invention, the present invention is made further
Bright, but protection domain of the presently claimed invention is not limited to the following example.
Detailed description of the invention
Embodiment 1
1) the choosing of outer implant: the stem section taking the American red-maple stem apex or band axillalry bud of giving birth to diameter 2-5mm then is
Outer implant, is cut into 1-3cm length with standby by outer implant after cutting off blade；；
2) outer implant sterilization: above-mentioned stem section is put in the 20KHz supersonic wave cleaning machine filling running water and keep
Temperature 30 DEG C processes 30min, then rinses 10min with running water；Subsequently with 70% on superclean bench
Alcohol disinfecting 25 seconds, processes in 0.1% mercuric chloride solution 5 minutes, then with aseptic water washing 4 times, is placed in
Sterile petri dish is standby；
3) initial Fiber differentiation: implant outer after sterilizing is respectively cut off 2-3mm, at the beginning of outer implant is downwardly into as end
Cultivating in beginning inducing culture, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；After cultivating 6d, the most untainted outer implant stem section is transferred in new initial inducing culture,
And continue cultivate 18d grow to sprouting；Described initial inducing culture composition is divided into:
WPM+6-BA0.5mg/L+NAA0.1mg/L+PVP1mg/L；
4) callus induction: take the bud in initial Fiber differentiation, puts in callus inducing medium and cultivates
20d, condition is light culture, temperature 23 DEG C ± 2 DEG C；Described callus tissue culture base is: WPM+6-BA
1.0mg/L+NAA0.1mg/L+PVP1mg/L；
5) Multiplying culture: the callus lines of clear, colorless is put into cultivation 25d in proliferated culture medium, propagation
Producing a large amount of tufted seedling, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described Multiplying culture culture medium is: 1/2MS+6-BA0.5mg/L+NAA0.1mg/L+PVP 1mg/L；
6) culture of rootage: by 5) tufted seedling in step cuts into single, it is transferred in root media cultivate
Taking root after 20d, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, temperature 25 DEG C ± 2 DEG C；
Described taking root with culture medium is: 1/2MS+IBA0.1mg/L+PVP1mg/L；
7) hardening and transplanting: the culture medium bottle cap having the American red-maple seedling taken root is opened, room temperature lower refining seedling
After 3d, take out seedling, wash away root culture medium, transplant on the seedbed equipped with river sand matrix, keep temperature
20-25 DEG C, humidity 70%-85%, suitably shade.
Through test, the seedbed transplanting survival rate using the present embodiment educated test tube seedling is 87.3%.
Embodiment 2
1) the choosing of outer implant: the stem section taking the American red-maple stem apex or band axillalry bud of giving birth to diameter 2-5mm then is
Outer implant, is cut into 1-3cm length with standby by outer implant after cutting off blade；；
2) outer implant sterilization: above-mentioned stem section is put in the 25KHz supersonic wave cleaning machine filling running water and keep
Temperature 35 DEG C processes 45min, then rinses 20min with running water；Subsequently with 75% on superclean bench
Alcohol disinfecting 35 seconds, processes in 0.1% mercuric chloride solution 8 minutes, then with aseptic water washing 5 times, is placed in
Sterile petri dish is standby；
3) initial Fiber differentiation: implant outer after sterilizing is respectively cut off 2-3mm, at the beginning of outer implant is downwardly into as end
Cultivating in beginning inducing culture, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；After cultivating 4d, the most untainted outer implant stem section is transferred in new initial inducing culture,
And continue cultivate 12d grow to sprouting；Described initial inducing culture composition is divided into:
WPM+6-BA1.0mg/L+NAA0.5mg/L+PVP5mg/L；
4) callus induction: take the bud in initial Fiber differentiation, puts in callus inducing medium and cultivates
15d, condition is light culture, temperature 23 DEG C ± 2 DEG C；Described callus tissue culture base is:
WPM+6-BA2.0mg/L+NAA0.5mg/L+PVP5mg/L；
5) Multiplying culture: the callus lines of clear, colorless is put into cultivation 20d in proliferated culture medium, propagation
Producing a large amount of tufted seedling, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described Multiplying culture culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.5mg/L+PVP 5mg/L；
6) culture of rootage: by 5) tufted seedling in step cuts into single, it is transferred in root media cultivate
Taking root after 15d, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, temperature 25 DEG C ± 2 DEG C；
Described taking root with culture medium is: 1/2MS+IBA0.5mg/L+PVP5mg/L；
7) hardening and transplanting: the culture medium bottle cap having the American red-maple seedling taken root is opened, room temperature lower refining seedling
After 5d, take out seedling, wash away root culture medium, transplant on the seedbed equipped with river sand matrix, keep temperature
20-25 DEG C, humidity 70%-85%, suitably shade.
Through test, the seedbed transplanting survival rate using the present embodiment educated test tube seedling is 90.1%.
Embodiment 3
1) the choosing of outer implant: the stem section taking the American red-maple stem apex or band axillalry bud of giving birth to diameter 2-5mm then is
Outer implant, is cut into 1-3cm length with standby by outer implant after cutting off blade；；
2) outer implant sterilization: above-mentioned stem section is put in the 20KHz supersonic wave cleaning machine filling running water and keep
Temperature 35 DEG C processes 35min, then rinses 15min with running water；Subsequently with 75% on superclean bench
Alcohol disinfecting 30 seconds, processes in 0.1% mercuric chloride solution 7 minutes, then with aseptic water washing 5 times, is placed in
Sterile petri dish is standby；
3) initial Fiber differentiation: implant outer after sterilizing is respectively cut off 2-3mm, at the beginning of outer implant is downwardly into as end
Cultivating in beginning inducing culture, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；After cultivating 5d, the most untainted outer implant stem section is transferred in new initial inducing culture,
And continue cultivate 15d grow to sprouting；Described initial inducing culture composition is divided into:
WPM+6-BA0.8mg/L+NAA0.2mg/L+PVP2mg/L；
4) callus induction: take the bud in initial Fiber differentiation, puts in callus inducing medium and cultivates
16d, condition is light culture, temperature 23 DEG C ± 2 DEG C；Described callus tissue culture base is: WPM+6-BA
1.6mg/L+NAA0.3mg/L+PVP3mg/L；
5) Multiplying culture: the callus lines of clear, colorless is put into cultivation 22d in proliferated culture medium, propagation
Producing a large amount of tufted seedling, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described Multiplying culture culture medium is: 1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP 3mg/L；
6) culture of rootage: by 5) tufted seedling in step cuts into single, it is transferred in root media cultivate
Taking root after 16d, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, temperature 25 DEG C ± 2 DEG C；
Described taking root with culture medium is: 1/2MS+IBA0.3mg/L+PVP2mg/L；
7) hardening and transplanting: the culture medium bottle cap having the American red-maple seedling taken root is opened, room temperature lower refining seedling
After 5d, take out seedling, wash away root culture medium, transplant on the seedbed equipped with river sand matrix, keep temperature
20-25 DEG C, humidity 70%-85%, suitably shade.
Through test, the seedbed transplanting survival rate using the present embodiment educated test tube seedling is 93.8%.
Claims (5)
1. an American red-maple rapid propagation method, it is characterised in that described method comprises the following steps:
1) the choosing of outer implant: the stem section taking the American red-maple stem apex or band axillalry bud of giving birth to diameter 2-5mm then is
Outer implant, is cut into 1-3cm length with standby by outer implant after cutting off blade；
2) outer implant sterilization: above-mentioned stem section is put in the 20-25KHz supersonic wave cleaning machine filling running water
Keep temperature 30-35 DEG C to process 30-45min, then rinse 10-20min with running water；Subsequently in ultra-clean work
With the alcohol disinfecting 25-35 second of 70-75% in station, in 0.1% mercuric chloride solution process 5-8 minute, then with
Aseptic water washing 4-5 time, is placed in sterile petri dish standby；
3) initial Fiber differentiation: implant outer after sterilizing is respectively cut off 2-3mm, at the beginning of outer implant is downwardly into as end
Cultivating in beginning inducing culture, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；After cultivating 4-6d, the most untainted outer implant stem section is transferred in new initial inducing culture,
And continue cultivate 12-18d grow to sprouting；Described initial inducing culture composition is divided into:
WPM+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP1-5mg/L；
4) callus induction: take the bud in initial Fiber differentiation, puts in callus inducing medium and cultivates
15-20d, condition is light culture, temperature 23 DEG C ± 2 DEG C；Described callus tissue culture base is: WPM+6-BA
1.0-2.0mg/L+NAA0.1-0.5mg/L+PVP1-5mg/L；
5) Multiplying culture: the callus lines of clear, colorless is put into cultivation 20-25d in proliferated culture medium, increases
Growing a large amount of tufted seedling of generation, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described Multiplying culture culture medium is:
1/2MS+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP 1-5mg/L；
6) culture of rootage: by 5) tufted seedling in step cuts into single, it is transferred in root media cultivate
Taking root after 15-20d, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, temperature 25 DEG C
±2℃；Described taking root with culture medium is: 1/2MS+IBA0.1-0.5mg/L+PVP1-5mg/L；
7) hardening and transplanting: the culture medium bottle cap having the American red-maple seedling taken root is opened, room temperature lower refining seedling
After 3-5d, take out seedling, wash away root culture medium, transplant on the seedbed equipped with river sand matrix, keep temperature
20-25 DEG C, humidity 70%-85%, suitably shade；
Described 6-BA is 6-benzyl aminopurine；
Described NAA is NAA；
Described IBA is indole-3-butyric acid；
Described PVP is polyvinylpyrrolidone.
A kind of American red-maple rapid propagation method, it is characterised in that described step 3)
In initial inducing culture consist of: WPM+6-BA0.8mg/L+NAA0.2mg/L+PVP2mg/L.
A kind of American red-maple rapid propagation method, it is characterised in that described step 4)
Middle callus tissue culture base is: WPM+6-BA 1.6mg/L+NAA0.3mg/L+PVP3mg/L.
A kind of American red-maple rapid propagation method, it is characterised in that described step 5)
Middle Multiplying culture culture medium is: 1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP 3mg/L.
A kind of American red-maple rapid propagation method, it is characterised in that described step 6)
In take root with culture medium and be: 1/2MS+IBA0.3mg/L+PVP2mg/L.
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| CN107333657B (en) * | 2017-09-06 | 2019-07-05 | 西南大学 | A kind of North America Acer palmatum ' Atropurpureum' radiance in October kind tissue culture and rapid propagation method |
| CN111053035A (en) * | 2020-01-10 | 2020-04-24 | 江苏农林职业技术学院 | Method for inducing formation of acer rubrum callus |
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