CN105918128B - A kind of American red-maple rapid propagation method - Google Patents
A kind of American red-maple rapid propagation method Download PDFInfo
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- CN105918128B CN105918128B CN201610339561.3A CN201610339561A CN105918128B CN 105918128 B CN105918128 B CN 105918128B CN 201610339561 A CN201610339561 A CN 201610339561A CN 105918128 B CN105918128 B CN 105918128B
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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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Abstract
The present invention relates to a kind of American red-maple rapid propagation method, described method includes：1) selection of explant, 2) explant sterilization, 3) initial Fiber differentiation, 4) callus induction, 5) Multiplying culture, 6) culture of rootage, 7) hardening and transplanting.American red-maple is bred using this method, production cost can be reduced, improves reproduction speed and survival rate.
Description
Technical field
The present invention relates to micropropagation of plants technical field, especially a kind of American red-maple quick breeding technology field.
Background technology
American red-maple (scientific name：Acer rubrum L.)：East coast of the United States is originated in, northern US is mostly come from and adds
Put on airs most area, China was introduced before 2000.Liaoning, Shandong, the band of Anhui one are mainly distributed on, due to special geography
Position makes American red-maple fine in northern color changeable effect.It is fallen leaves megaphanerophyte.Growth is very fast, high tree 12-18 rice of growing up, hat width
12 meters, adapt to the soil types growth of a variety of scopes.Spring blooms, premium color.Because its autumn color is brilliant, tree crown is clean and tidy,
It is widely used in park, cell, street plant, not only with Landscape but also can does shade tree, it is very popular, be
One of the beautification introduced in recent years, the preferable rare tree species in green planted city gardens.And it can uniquely be used as the color leaf of shade tree
Seeds.
At present, American red-maple carries out production cultivation using three kinds of seminal propagation, propagation by grafiting and cutting propagation modess of reproduction more
Training, this raising technology improve the purity of kind, maintain breediness, but survival rate is relatively low and reproduction speed is slower, constrains
The development of American red-maple industry.And tissue cultivating and seedling technology can both keep the purity and characteristic of kind, production can also be reduced
Cost, improve reproduction speed and survival rate.
The report that American red-maple regeneration plant is obtained through callus approach is there is no in the prior art.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of American red-maple rapid propagation method, through callus way
Footpath obtains American red-maple Multiple Buds, root induction, a large amount of quick breedings of American red-maple tissue-cultured seedling can be achieved.
In order to solve the 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) selection of explant：It is explant to take and give birth to diameter 2-5mm American red-maple stem apex or the stem section with axillary bud then,
Cut off and explant is cut into 1-3cm length with standby after blade；
2) explant sterilizes：Above-mentioned stem section is put into keeping temperature in the 20-25KHz supersonic wave cleaning machines for fill running water
30-35 DEG C of processing 30-45min, then rinses 10-20min with running water；Then with 70-75% wine on superclean bench
The essence sterilization 25-35 seconds, 5-8 minutes are handled in 0.1% mercuric chloride solution, then with aseptic water washing 4-5 times, be placed in sterile petri dish
It is standby；
3) initial Fiber differentiation：Explant after sterilizing is respectively cut off into 2-3mm, explant is downwardly into initial induction by end
Cultivated in culture medium, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Cultivate 4-6d
Wherein untainted explant stem section is transferred in new initial inducing culture afterwards, and continues to cultivate 12-18d to sprouting length
Go out；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：The bud in initial Fiber differentiation is taken, is put into callus inducing medium and cultivates 15-
20d, condition are light culture, 23 DEG C ± 2 DEG C of temperature；The callus tissue culture base is：WPM+6-BA1.0-2.0mg/L+
NAA0.1-0.5mg/L+PVP1-5mg/L；
5) Multiplying culture：The callus lines of clear, colorless are put into proliferated culture medium and cultivate 20-25d, propagation produces
A large amount of tufted seedlings, condition of culture are light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；The propagation training
Support with culture medium and be：1/2MS+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP 1-5mg/L；
6) culture of rootage：5) tufted seedling in step is cut into single, is transferred in root media and cultivates 15-20d
After take root, condition of culture is light intensity 2500-3500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；It is described to take root with culture
Base is：1/2MS+IBA0.1-0.5mg/L+PVP1-5mg/L；
7) hardening and transplanting：The culture medium bottle cap for having the American red-maple seedling to take root is opened, after room temperature lower refining seedling 3-5d,
Seedling is taken out, washes away root culture medium, transplanting is equipped with the seedbed of river sand matrix, 20-25 DEG C of keeping temperature, humidity 70%-
85%, appropriate shade；
Described WPM culture medium prescriptions are：A great number of elements：Ammonium nitrate NH4NO3400mg/L, potassium sulfate K2SO4900mg/
L, potassium dihydrogen phosphate KH2PO3170mg/L, magnesium sulfate MgSO47H2O370mg/L；Calcium salt：Calcium chloride CaCl2
2H2O96mg/L；Calcium nitrate tetrahydrate Ca (NO3) 24H2O556mg/L；Trace element：Boric acid H3BO36.2mg/L, manganese sulfate
MnSO44H2O22.5mg/L, zinc sulfate ZnSO47H2O8.6mg/L, sodium molybdate Na2MoO42H2O0.25mg/L, sulfuric acid
Copper CuSO45H2O0.25mg/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 prescriptions are：A great number of elements：Potassium nitrate KNO31900mg/L, ammonium nitrate NH4NO31650mg/
L, potassium dihydrogen phosphate KH2PO3170mg/L, magnesium sulfate MgSO47H2O370mg/L；Calcium salt：Calcium chloride CaCl2
2H2O440mg/L；Trace element：KI KI0.83mg/L, boric acid H3BO36.2mg/L, manganese sulfate MnSO4
4H2O22.3mg/L, zinc sulfate ZnSO47H2O8.6mg/L, sodium molybdate Na2MoO42H2O0.25mg/L, copper sulphate
CuSO45H2O0.025mg/L, cobalt chloride CoCl26H2O0.025mg/L；Molysite：Disodium ethylene diamine tetraacetate Na2-
EDTA37.25mg/L, ferrous sulfate FeSO47H2O27.85mg/L；Organic acid：Inositol 100mg/L, glycine 2mg/L, salt
Allithiamine element 0.5mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L；Sucrose 30g/L, agar powder 7g/L, pH 5.7；
Described 1/2MS culture medium prescriptions are：A great number of elements：Potassium nitrate KNO3950mg/L, ammonium nitrate NH4NO3825mg/
L, potassium dihydrogen phosphate KH2PO385mg/L, magnesium sulfate MgSO47H2O185mg/L；It is remaining with above-mentioned MS culture mediums；
Described 6-BA is 6-benzyl aminopurine；
Described NAA is α-naphthylacetic acid；
Described IBA is indole -3-butyric acid；
Described PVP is polyvinylpyrrolidone.
Preferably, initial inducing culture composition is in step 3)：WPM+6-BA0.8mg/L+NAA0.2mg/L+
PVP2mg/L。
Preferably, callus tissue culture base is in step 4)：WPM+6-BA1.6mg/L+NAA0.3mg/L+PVP3mg/L.
Preferably, Multiplying culture is with culture medium in step 5)：1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP
3mg/L。
Preferably, taken root in step 6) and be with culture medium：1/2MS+IBA0.3mg/L+PVP2mg/L.
American red-maple is bred using the present invention, a large amount of quick breedings of American red-maple tissue-cultured seedling can be achieved, be American red-maple
Production cultivation and breed improvement lay the foundation.
In order to preferably illustrate technical scheme, with reference to embodiment, the present invention is further illustrated, but
Protection domain of the presently claimed invention is not limited to the following example.
Embodiment
Embodiment 1
1) selection of explant：It is explant to take and give birth to diameter 2-5mm American red-maple stem apex or the stem section with axillary bud then,
Cut off and explant is cut into 1-3cm length with standby after blade；；
2) explant sterilizes：Above-mentioned stem section is put into keeping temperature 30 in the 20KHz supersonic wave cleaning machines for fill running water
DEG C processing 30min, then with running water rinse 10min；Then on superclean bench with 70% alcohol disinfecting 25 seconds,
Handle 5 minutes in 0.1% mercuric chloride solution, then with aseptic water washing 4 times, it is standby to be placed in sterile petri dish；
3) initial Fiber differentiation：Explant after sterilizing is respectively cut off into 2-3mm, explant is downwardly into initial induction by end
Cultivated in culture medium, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；After cultivating 6d
Wherein untainted explant stem section is transferred in new initial inducing culture, and continues to cultivate 18d and grows to sprouting；Institute
The initial inducing culture composition stated is divided into：WPM+6-BA0.5mg/L+NAA0.1mg/L+PVP1mg/L；
4) callus induction：The bud in initial Fiber differentiation is taken, is put into callus inducing medium and cultivates 20d,
Condition is light culture, 23 DEG C ± 2 DEG C of temperature；The callus tissue culture base is：WPM+6-BA1.0mg/L+NAA0.1mg/L+
PVP1mg/L；
5) Multiplying culture：The callus lines of clear, colorless are put into proliferated culture medium and cultivate 25d, propagation produces a large amount of
Tufted seedling, condition of culture are light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；The Multiplying culture is used
Culture medium is：1/2MS+6-BA0.5mg/L+NAA0.1mg/L+PVP 1mg/L；
6) culture of rootage：5) tufted seedling in step is cut into it is single, be transferred in root media cultivate it is raw after 20d
Root, condition of culture are light intensity 2500-3500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Described take root be with culture medium：
1/2MS+IBA0.1mg/L+PVP1mg/L；
7) hardening and transplanting：The culture medium bottle cap for having the American red-maple seedling to take root is opened, after room temperature lower refining seedling 3d, taken
Going out seedling, wash away root culture medium, transplanting is equipped with the seedbed of river sand matrix, 20-25 DEG C of keeping temperature, humidity 70%-85%,
Appropriate shade.
Through experiment, the present embodiment is used to educate the seedbed transplanting survival rate of test tube seedling for 87.3%.
Embodiment 2
1) selection of explant：It is explant to take and give birth to diameter 2-5mm American red-maple stem apex or the stem section with axillary bud then,
Cut off and explant is cut into 1-3cm length with standby after blade；；
2) explant sterilizes：Above-mentioned stem section is put into keeping temperature 35 in the 25KHz supersonic wave cleaning machines for fill running water
DEG C processing 45min, then with running water rinse 20min；Then on superclean bench with 75% alcohol disinfecting 35 seconds,
Handle 8 minutes in 0.1% mercuric chloride solution, then with aseptic water washing 5 times, it is standby to be placed in sterile petri dish；
3) initial Fiber differentiation：Explant after sterilizing is respectively cut off into 2-3mm, explant is downwardly into initial induction by end
Cultivated in culture medium, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；After cultivating 4d
Wherein untainted explant stem section is transferred in new initial inducing culture, and continues to cultivate 12d and grows to sprouting；Institute
The initial inducing culture composition stated is divided into：WPM+6-BA1.0mg/L+NAA0.5mg/L+PVP5mg/L；
4) callus induction：The bud in initial Fiber differentiation is taken, is put into callus inducing medium and cultivates 15d,
Condition is light culture, 23 DEG C ± 2 DEG C of temperature；The callus tissue culture base is：WPM+6-BA2.0mg/L+NAA0.5mg/L+
PVP5mg/L；
5) Multiplying culture：The callus lines of clear, colorless are put into proliferated culture medium and cultivate 20d, propagation produces a large amount of
Tufted seedling, condition of culture are light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；The Multiplying culture is used
Culture medium is：1/2MS+6-BA1.0mg/L+NAA0.5mg/L+PVP 5mg/L；
6) culture of rootage：5) tufted seedling in step is cut into it is single, be transferred in root media cultivate it is raw after 15d
Root, condition of culture are light intensity 2500-3500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Described take root be with culture medium：
1/2MS+IBA0.5mg/L+PVP5mg/L；
7) hardening and transplanting：The culture medium bottle cap for having the American red-maple seedling to take root is opened, after room temperature lower refining seedling 5d, taken
Going out seedling, wash away root culture medium, transplanting is equipped with the seedbed of river sand matrix, 20-25 DEG C of keeping temperature, humidity 70%-85%,
Appropriate shade.
Through experiment, the present embodiment is used to educate the seedbed transplanting survival rate of test tube seedling for 90.1%.
Embodiment 3
1) selection of explant：It is explant to take and give birth to diameter 2-5mm American red-maple stem apex or the stem section with axillary bud then,
Cut off and explant is cut into 1-3cm length with standby after blade；；
2) explant sterilizes：Above-mentioned stem section is put into keeping temperature 35 in the 20KHz supersonic wave cleaning machines for fill running water
DEG C processing 35min, then with running water rinse 15min；Then on superclean bench with 75% alcohol disinfecting 30 seconds,
Handle 7 minutes in 0.1% mercuric chloride solution, then with aseptic water washing 5 times, it is standby to be placed in sterile petri dish；
3) initial Fiber differentiation：Explant after sterilizing is respectively cut off into 2-3mm, explant is downwardly into initial induction by end
Cultivated in culture medium, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；After cultivating 5d
Wherein untainted explant stem section is transferred in new initial inducing culture, and continues to cultivate 15d and grows to sprouting；Institute
The initial inducing culture composition stated is divided into：WPM+6-BA0.8mg/L+NAA0.2mg/L+PVP2mg/L；
4) callus induction：The bud in initial Fiber differentiation is taken, is put into callus inducing medium and cultivates 16d,
Condition is light culture, 23 DEG C ± 2 DEG C of temperature；The callus tissue culture base is：WPM+6-BA1.6mg/L+NAA0.3mg/L+
PVP3mg/L；
5) Multiplying culture：The callus lines of clear, colorless are put into proliferated culture medium and cultivate 22d, propagation produces a large amount of
Tufted seedling, condition of culture are light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；The Multiplying culture is used
Culture medium is：1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP 3mg/L；
6) culture of rootage：5) tufted seedling in step is cut into it is single, be transferred in root media cultivate it is raw after 16d
Root, condition of culture are light intensity 2500-3500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Described take root be with culture medium：
1/2MS+IBA0.3mg/L+PVP2mg/L；
7) hardening and transplanting：The culture medium bottle cap for having the American red-maple seedling to take root is opened, after room temperature lower refining seedling 5d, taken
Going out seedling, wash away root culture medium, transplanting is equipped with the seedbed of river sand matrix, 20-25 DEG C of keeping temperature, humidity 70%-85%,
Appropriate shade.
Through experiment, the present embodiment is used to educate the seedbed transplanting survival rate of test tube seedling for 93.8%.
Claims (5)
1. a kind of American red-maple rapid propagation method, it is characterised in that described method comprises the following steps：
1) selection of explant：It is explant to take and give birth to diameter 2-5mm American red-maple stem apex or the stem section with axillary bud then, is cut off
Explant is cut into 1-3cm length with standby after blade；
2) explant sterilizes：Above-mentioned stem section is put into keeping temperature 30- in the 20-25KHz supersonic wave cleaning machines for fill running water
35 DEG C of processing 30-45min, then rinse 10-20min with running water；Then disappeared on superclean bench with 70-75% alcohol
The malicious 25-35 seconds, 5-8 minutes are handled in 0.1% mercuric chloride solution, then with aseptic water washing 4-5 times, it is standby to be placed in sterile petri dish
With；
3) initial Fiber differentiation：Explant after sterilizing is respectively cut off into 2-3mm, explant is downwardly into initial Fiber differentiation by end
Cultivated in base, condition of culture is light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Will after culture 4-6d
Wherein untainted explant stem section is transferred in new initial inducing culture, and is continued to cultivate 12-18d and grown 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：The bud in initial Fiber differentiation is taken, is put into callus inducing medium and cultivates 15-20d, bar
Part is light culture, 23 DEG C ± 2 DEG C of temperature；The callus tissue culture base is：WPM+6-BA1.0-2.0mg/L+NAA0.1-
0.5mg/L+PVP1-5mg/L；
5) Multiplying culture：The callus lines of clear, colorless are put into proliferated culture medium and cultivate 20-25d, propagation produces a large amount of
Tufted seedling, condition of culture are light intensity 1800-2500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；The Multiplying culture is used
Culture medium is：
1/2MS+6-BA0.5-1.0mg/L+NAA0.1-0.5mg/L+PVP 1-5mg/L；
6) culture of rootage：5) tufted seedling in step is cut into it is single, be transferred in root media cultivate it is raw after 15-20d
Root, condition of culture are light intensity 2500-3500Lx, photoperiod 10-14h/d, 25 DEG C ± 2 DEG C of temperature；Described take root be with culture medium：
1/2MS+IBA0.1-0.5mg/L+PVP1-5mg/L；
7) hardening and transplanting：The culture medium bottle cap for having the American red-maple seedling to take root is opened, after room temperature lower refining seedling 3-5d, taken out
Seedling, root culture medium is washed away, transplanting is equipped with the seedbed of river sand matrix, 20-25 DEG C of keeping temperature, humidity 70%-85%, is fitted
When shade；
Described 6-BA is 6-benzyl aminopurine；
Described NAA is α-naphthylacetic acid；
Described IBA is indole -3-butyric acid；
Described PVP is polyvinylpyrrolidone.
2. a kind of American red-maple rapid propagation method according to claim 1, it is characterised in that initial in the step 3)
Inducing culture forms：WPM+6-BA0.8mg/L+NAA0.2mg/L+PVP2mg/L.
A kind of 3. American red-maple rapid propagation method according to claim 1, it is characterised in that callus in the step 4)
Tissue culture medium (TCM) is：WPM+6-BA 1.6mg/L+NAA0.3mg/L+PVP3mg/L.
4. a kind of American red-maple rapid propagation method according to claim 1, it is characterised in that breed in the step 5)
Culture is with culture medium：1/2MS+6-BA0.8mg/L+NAA0.2mg/L+PVP 3mg/L.
5. a kind of American red-maple rapid propagation method according to claim 1, it is characterised in that taken root in the step 6)
It is with culture medium：1/2MS+IBA0.3mg/L+PVP2mg/L.
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| CN107278686A (en) * | 2017-06-06 | 2017-10-24 | 山东大丰园农业有限公司 | A kind of American red-maple tissue culture outside sprout-cultivating-bottle radication agent and method for culturing seedlings |
| 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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