CN108782245A - Tissue culture and rapid propagation method and culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple - Google Patents
Tissue culture and rapid propagation method and culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple Download PDFInfo
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- CN108782245A CN108782245A CN201810628813.3A CN201810628813A CN108782245A CN 108782245 A CN108782245 A CN 108782245A CN 201810628813 A CN201810628813 A CN 201810628813A CN 108782245 A CN108782245 A CN 108782245A
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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
A kind of tissue culture and rapid propagation method and culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple, includes primary culture medium, proliferated culture medium and root media, wherein：Primary culture medium component is set as MS+6-BA+IBA+NAA+ sucrose+agar, proliferated culture medium component is set as MS+6-BA+IBA+ sucrose+agar, root media component is set as 1/2MS+IAA+IBA+ sucrose+agar, pass through primary culture medium and proliferated culture medium, tissue culture is carried out to stem apex body to handle, and by root media, rooting treatment is carried out to tissue-cultured seedling, the no longer breeding of cuttage and press strip, therefore improve slow seedling survival rate.
Description
Technical field
The present invention relates to a kind of tissue culture and rapid propagation method and culture mediums, especially a kind of to be used for the anti-continuous cropping dwarfing rootstock of apple
The tissue culture and rapid propagation method and culture medium of G41.
Background technology
Short anvil intensive culture is to work as Main Patterns, and how Newly built orchard overcomes continuous cropping obstacle to have become apple industry urgently
Significant problem to be solved, and overcome continuous cropping in addition to crop rotation, plough deeply, other than the agronomic measures such as soil moved in improve the original, using anti-continuous cropping dwarfing rootstock
It is most easy and effective mode, there are two types of existing Dwarf Stocks For Apple Trees mode of reproduction：Cuttage and press strip, it is anti-for apple
For continuous cropping stock G41, cuttage is not easy to take root, difficult seedling, and cutting demand is big, and breeding coefficient is low；Although and press strip can be at
Seedling, but need repeatedly to cultivate in reproductive process is all required to cultivating height and material mixture ratio, also easily to nursery stock when plant division lifting
Root system damages, and influences slow seedling and survives, breeding coefficient is relatively low.
Based on applicant's technical problem existing in the Disclosure of invention on March 15th, 2018 and background technology, technology
Feature and technique effect make the application technical solution of the present invention.
Invention content
The object of the present invention is a kind of tissue culture and rapid propagation method for the anti-continuous cropping dwarfing rootstock G41 of apple,
The object of the present invention is a kind of tissue-culturing rapid propagation culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple.
In order to overcome the technical drawbacks described above, the object of the present invention is to provide one kind being used for the anti-continuous cropping dwarfing rootstock G41 of apple
Tissue culture and rapid propagation method and culture medium, therefore improve slow seedling survival rate.
In order to achieve the above objectives, the technical solution adopted by the present invention is that：It is a kind of to be used for the anti-continuous cropping dwarfing rootstock G41's of apple
Tissue-culturing rapid propagation culture medium, includes primary culture medium, proliferated culture medium and root media,
Wherein：Primary culture medium component is set as MS+6-BA+IBA+NAA+sucrose+agar, the setting of proliferated culture medium component
For MS+6-BA+IBA+sucrose+agar, root media component is set as 1/2MS+IAA+IBA+sucrose+agar.
Due to devising primary culture medium, proliferated culture medium and root media, pass through primary culture medium and Multiplying culture
Base carries out tissue culture to stem apex body and handles, and by root media, rooting treatment, no longer cuttage and pressure are carried out to tissue-cultured seedling
The breeding of item, therefore improve slow seedling survival rate.
The present invention devises, and tissue-culturing rapid propagation culture medium is formed so that MS and 1/2MS are as the main component.
The present invention devises, and the kind of the anti-continuous cropping dwarfing rootstock of apple is set as G41.
The present invention devises, and includes that primary culture medium component is set as MS+6-BA0.5-2.0mg/L+ IBA0.05-
0.20mg/L+NAA0.01-0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-
BA0.5-1.5mg/L+IBA0.05-0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component are set as 1/
2MS+IAA1.0-2.0mg/L+IBA0.5-1.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
The present invention devises, and includes that primary culture medium component is set as MS+6-BA1.0mg/L+ IBA0.1mg/L+
NAA0.05mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5-1mg/L+
IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component be set as 1/2MS+IAA1.5mg/L+
IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
The present invention devises, MS nutrient media components：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| CaCl2·2H2O | 440 |
| MgSO4·7H2O | 370 |
| KH2PO4 | 700 |
| Inorganic constituents trace element | |
| KI | 0.83 |
| H3BO3 | 6.2 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| Organic principle | |
| Inositol | 100 |
| Hydrochloric acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
1/2 MS nutrient media components：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 825 |
| KNO3 | 950 |
| CaCl2·2H2O | 220 |
| MgSO4·7H2O | 185 |
| KH2PO4 | 350 |
| Inorganic constituents trace element | |
| KI | 0.83 |
| H3BO3 | 6.2 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| Organic principle | |
| Inositol | 100 |
| Hydrochloric acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
6-BA（6-benzyl aminopurine）
IBA（Indolebutyric acid）
NAA（Methyl α-naphthyl acetate）
IAA（Heteroauxin）.
The present invention devises, a kind of tissue culture and rapid propagation method for the anti-continuous cropping dwarfing rootstock G41 of apple,
Its step is：Stem apex body is seeded in MS+6-BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L
The primary culture medium of+agar 6.0g/L obtains explant, explant be seeded in MS+6-BA0.5-1mg/L+IBA0.1mg/L+
The proliferated culture medium of sucrose 30.0g/L+ agar 6.0g/L obtains tissue-cultured seedling, tissue culture plant inoculation 1/2MS+IAA1.5mg/L+
The root media of IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L obtains tissue-cultured seedling of taking root.
The present invention devises, and step is：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA0.5-2.0mg/L+ IBA0.05-0.20mg/L+NAA0.01-0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, are placed in
Light application time is 16h/d, intensity of illumination 2000Lux, temperature are 24-26 DEG C, the culture environment of relative air humidity 50%-70%
Lower carry out Primary culture, obtains explant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1.5mg/L+IBA0.05-0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, it is 16h/d, intensity of illumination to be placed in light application time
It is 24-26 DEG C for 2000Lux, temperature, carries out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtains tissue culture
Seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.0-2.0mg/L+IBA0.5-1.5mg/L+ sucrose
30.0g/L+ agar 6.0g/L, first dark treatment is after seven days, then be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Culture of rootage is carried out under the culture environment that degree is 25 DEG C, obtains tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
The present invention devises, and step is：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d,
Intensity of illumination is 2000Lux, temperature is 24-26 DEG C, Primary culture is carried out under the culture environment of relative air humidity 50%-70%,
Explant is obtained,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1mg/L+IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L, is placed in that light application time is 16h/d, intensity of illumination is
2000Lux, temperature are 24-26 DEG C, carry out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtain tissue-cultured seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.5mg/L+IBA0.5mg/L+ sucrose 30.0g/L+
Agar 6.0g/L, first dark treatment is after seven days, then is placed in that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C
Culture environment under carry out culture of rootage, obtain tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
The present invention devises, and is sampled in April 10 current year, mercuric chloride processing time is set as 8 seconds, in Primary culture
In base：The concentration that the concentration of 6-BA is set as 1.0mg/L, IBA is set as the concentration of 0.10mg/L, NAA and is set as 0.0.05mg/
L, in proliferated culture medium：The concentration that the concentration of 6-BA is set as 1.0mg/L, IBA is set as 0.10mg/L, in root media
In：The concentration that the concentration of IAA is set as 1.5mg/L, IBA is set as 0.50mg/L.
The technical effects of the invention are that：Using MS+6-BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose
30.0g/L+ agar 6.0g/L is primary culture medium, and survival rate is up to 80% or more, can establish effective tissue-cultured seedling group, is used
MS+6-BA0.5-1mg/L+IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L is proliferated culture medium, and growth coefficient is up to
5.14；It uses 1/2MS+IAA1.5mg/L+IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L for root media, takes root
Rate is up to 100%, and for root long mostly more than 2cm, transplanting survival rate passes through continuous Primary culture, Multiplying culture, training of taking root in 90%-95%
It supports and transplants, it can be achieved that industrial seedling rearing, resource base is provided for the more new development of Apple Industry.
In the technical scheme, as the main component for important technical characteristic with MS and 1/2MS, short for the anti-continuous cropping of apple
Change in the tissue culture and rapid propagation method of stock G41 and the technical field of culture medium, there is novelty, creativeness and practicability, in this skill
Term in art scheme is all that can be explained and understood with patent document in the art.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is the Primary culture design sketch of the present invention,
Fig. 2 is the seedling separation design sketch of the present invention,
Fig. 3 is the Multiplying culture design sketch of the present invention,
Fig. 4 is the culture of rootage design sketch of the present invention,
Fig. 5 is the situation design sketch of taking root of the present invention,
Fig. 6 is the first time of the present invention to transplant design sketch,
Fig. 7 is second of transplanting design sketch of the present invention,
Fig. 8 is that nursery lot of the present invention transplants design sketch,
Fig. 9 is the seedling design sketch of the present invention.
Specific implementation mode
According to guidelines for examination, such as " having ", "comprising" and " comprising " term used in the present invention should understand that
Not allot the presence or addition of one or more of the other element or combinations thereof.
In the description of the present invention, it should be noted that term "center", "upper", "lower", "left", "right", "vertical",
The orientation or positional relationship of the instructions such as "horizontal", "inner", "outside" is the orientation or positional relationship of general expression, is merely for convenience of
The description present invention and simplified description, do not indicate or imply the indicated device or element must have a particular orientation, with spy
Fixed azimuth configuration and operation, therefore be not considered as limiting the invention.In addition, term " first ", " second ", " third "
It is used for description purposes only, is not understood to indicate or imply relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
Can also be electrical connection to be mechanical connection；It can be directly connected, can also indirectly connected through an intermediary, Ke Yishi
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood at this with concrete condition
Concrete meaning in invention.
As long as in addition, technical characteristic involved in invention described below different embodiments non-structure each other
It can be combined with each other at conflict.
With reference to embodiment, the present invention is further described, following embodiment is intended to illustrate invention rather than to this
Invention further limits.
A kind of tissue culture and rapid propagation method for the anti-continuous cropping dwarfing rootstock G41 of apple, one of embodiment of the present invention, step
It is：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA0.5-2.0mg/L+ IBA0.05-0.20mg/L+NAA0.01-0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, are placed in
Light application time is 16h/d, intensity of illumination 2000Lux, temperature are 24-26 DEG C, the culture environment of relative air humidity 50%-70%
Lower carry out Primary culture, obtains explant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1.5mg/L+IBA0.05-0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, it is 16h/d, intensity of illumination to be placed in light application time
It is 24-26 DEG C for 2000Lux, temperature, carries out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtains tissue culture
Seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.0-2.0mg/L+IBA0.5-1.5mg/L+ sucrose
30.0g/L+ agar 6.0g/L, first dark treatment is after seven days, then be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Culture of rootage is carried out under the culture environment that degree is 25 DEG C, obtains tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
The two of one embodiment, step are：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d,
Intensity of illumination is 2000Lux, temperature is 24-26 DEG C, Primary culture is carried out under the culture environment of relative air humidity 50%-70%,
Explant is obtained,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1mg/L+IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L, is placed in that light application time is 16h/d, intensity of illumination is
2000Lux, temperature are 24-26 DEG C, carry out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtain tissue-cultured seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.5mg/L+IBA0.5mg/L+ sucrose 30.0g/L+
Agar 6.0g/L, first dark treatment is after seven days, then is placed in that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C
Culture environment under carry out culture of rootage, obtain tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
In the present embodiment, it is sampled in April 10 current year, mercuric chloride processing time is set as 8 seconds, in Primary culture
In base：The concentration that the concentration of 6-BA is set as 1.0mg/L, IBA is set as the concentration of 0.10mg/L, NAA and is set as 0.0.05mg/
L, in proliferated culture medium：The concentration that the concentration of 6-BA is set as 1.0mg/L, IBA is set as 0.10mg/L, in root media
In：The concentration that the concentration of IAA is set as 1.5mg/L, IBA is set as 0.50mg/L.
The three of the embodiment of the present invention, step are：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and the April is chosen from elite stand and newly grows
Length about 1.0cm stem apex, with tap water rinse one hour after, the burning for filling 75% alcohol is first put on superclean bench
Cup in handle five seconds, immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilization seven minutes, during which weak vibrations three times, most
It is fully rinsed three times with aseptic deionized water afterwards, carry out disinfection sterilization cleaning treatment, obtains sterilization stem apex body,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA0.5mg/L+ IBA0.05mg/L+NAA0.01mg/L+ sucrose 30.0g/L+ agar 6.0g/L, it is 16h/ to be placed in light application time
D, intensity of illumination is 2000Lux, temperature is 24 DEG C, carries out Primary culture under the culture environment of relative air humidity 50%%, is obtained
Explant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5mg/L
+ IBA0.05mg/L+sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Degree is 24 DEG C, carries out Multiplying culture under the culture environment of relative air humidity 40%%, obtains tissue-cultured seedling,
Four, culture of rootage：It is 20 days tissue culture plant inoculations in root media robust growth, height 1.5cm, growth time
In, root media component is set as 1/2MS+IAA1.0mg/L+IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L, first
After dark treatment seven days, then be placed under the culture environment that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C into
Row culture of rootage, obtains tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to the shifting of culture plate or culture bag
Medium Culture is supported in cultivation, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/3 of 1/3 matrix is thick
River sand, spray moisture content is primary sooner or later daily, and it is 75%% to keep the water content of matrix and air humidity, is conducive to slow seedling, after seven days
Culture plate is moved to outside shade and starts second and transplants, when the rooted seedling young sprout of culture plate or the hardening of culture bag grows, has
When new root is pierced by from culture bag or culture pan bottom permeable hole, migration crop field plant school takes that wide row is 60cm, narrow row is
40cm, spacing in the rows are 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
The four of the embodiment of the present invention, step are：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and the August is chosen from elite stand and newly grows
Length about 1.5cm stem apex, with tap water rinse two hours after, the burning for filling 75% alcohol is first put on superclean bench
It handles seven seconds in cup, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized nine minutes, during which weak vibrations five times, most
It is fully rinsed five times with aseptic deionized water afterwards, carry out disinfection sterilization cleaning treatment, obtains sterilization stem apex body,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA2.0mg/L+ IBA0.20mg/L+NAA0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, it is 16h/ to be placed in light application time
D, intensity of illumination is 2000Lux, temperature is 26 DEG C, carries out Primary culture under the culture environment of relative air humidity 70%, is obtained outer
Implant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA1.5mg/L
+ IBA0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Degree is 26 DEG C, carries out Multiplying culture under the culture environment of relative air humidity 50%, obtains tissue-cultured seedling,
Four, culture of rootage：It is 25 days tissue culture plant inoculations in culture of rootage robust growth, height 1.9cm, growth time
In base, root media component is set as 1/2MS+IAA2.0mg/L+IBA-1.5mg/L+ sucrose 30.0g/L+ agar 6.0g/
L, first dark treatment is after seven days, then is placed in the culture environment that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C
Lower carry out culture of rootage, obtains tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to the shifting of culture plate or culture bag
Medium Culture is supported in cultivation, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/3 of 1/3 matrix is thick
River sand, spray moisture content is primary sooner or later daily, and it is 85% to keep the water content of matrix and air humidity, is conducive to slow seedling, will after seven days
Culture plate, which moves to outside shade, to be started second and transplants, when the rooted seedling young sprout of culture plate or the hardening of culture bag grows, there have to be new
When root is pierced by from culture bag or culture pan bottom permeable hole, migration crop field plant school takes that wide row is 60cm, narrow row is
40cm, spacing in the rows are 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
The five of the embodiment of the present invention, step are：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and June is chosen from elite stand and newly grows
Length about 1.25cm stem apex, with tap water rinse one hour after, the burning for filling 75% alcohol is first put on superclean bench
It handles six seconds in cup, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized eight minutes, during which weak vibrations four times, most
It is fully rinsed four times with aseptic deionized water afterwards, carry out disinfection sterilization cleaning treatment, obtains sterilization stem apex body,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA1.25mg/L+ IBA0.12mg/L+NAA0.04mg/L+ sucrose 30.0g/L+ agar 6.0g/L, it is 16h/ to be placed in light application time
D, intensity of illumination is 2000Lux, temperature is 25 DEG C, carries out Primary culture under the culture environment of relative air humidity 50%-70%, is obtained
To explant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA1.0mg/L
+ IBA0.10mg/L+sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Degree is 25 DEG C, carries out Multiplying culture under the culture environment of relative air humidity 45%, obtains tissue-cultured seedling,
Four, culture of rootage：It is 22 days tissue culture plant inoculations in culture of rootage robust growth, height 1.7cm, growth time
In base, root media component is set as 1/2MS+IAA1.5mg/L+IBA1.0mg/L+ sucrose 30.0g/L+ agar 6.0g/L,
After first dark treatment seven days, then it is placed under the culture environment that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C
Culture of rootage is carried out, tissue-cultured seedling of taking root is obtained,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 2.0cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to the shifting of culture plate or culture bag
Medium Culture is supported in cultivation, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/3 of 1/3 matrix is thick
River sand, spray moisture content is primary sooner or later daily, and it is 80% to keep the water content of matrix and air humidity, is conducive to slow seedling, will after seven days
Culture plate, which moves to outside shade, to be started second and transplants, when the rooted seedling young sprout of culture plate or the hardening of culture bag grows, there have to be new
When root is pierced by from culture bag or culture pan bottom permeable hole, migration crop field plant school takes that wide row is 60cm, narrow row is
40cm, spacing in the rows are 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
A kind of tissue-culturing rapid propagation culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple, one of one embodiment include
Primary culture medium component is set as MS+6-BA0.5-2.0mg/L+ IBA0.05-0.20mg/L+NAA0.01-0.07mg/L+ sugarcanes
Sugared 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5-1.5mg/L+IBA0.05-0.15mg/L
+ sucrose 30.0g/L+ agar 6.0g/L, root media component are set as 1/2MS+IAA1.0-2.0mg/L+IBA0.5-
1.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
In the present embodiment, MS nutrient media components：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| CaCl2·2H2O | 440 |
| MgSO4·7H2O | 370 |
| KH2PO4 | 700 |
| Inorganic constituents trace element | |
| KI | 0.83 |
| H3BO3 | 6.2 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| Organic principle | |
| Inositol | 100 |
| Hydrochloric acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
1/2 MS nutrient media components：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 825 |
| KNO3 | 950 |
| CaCl2·2H2O | 220 |
| MgSO4·7H2O | 185 |
| KH2PO4 | 350 |
| Inorganic constituents trace element | |
| KI | 0.83 |
| H3BO3 | 6.2 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| Organic principle | |
| Inositol | 100 |
| Hydrochloric acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
6-BA（6-benzyl aminopurine）
IBA（Indolebutyric acid）
NAA（Methyl α-naphthyl acetate）
IAA（Heteroauxin）.
The two of one embodiment include that primary culture medium component is set as MS+6-BA1.0mg/L+ IBA0.1mg/L
+ NAA0.05mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5-1mg/L+
IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component be set as 1/2MS+IAA1.5mg/L+
IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
The three of one embodiment include that primary culture medium component is set as MS+6-BA0.5mg/L+ IBA0.05mg/
L+NAA0.01mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5mg/L+
IBA0.05mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component be set as 1/2MS+IAA1.0mg/L+
IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
The four of one embodiment include that primary culture medium component is set as MS+6-BA2.0mg/L+ IBA0.20mg/
L+NAA0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA1.5mg/L+
IBA0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component be set as 1/2MS+IAA2.0mg/L+
IBA1.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
The five of one embodiment include that primary culture medium component is set as MS+6-BA1.25mg/L+
IBA0.12mg/L+NAA0.04mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-
BA1.0mg/L+IBA00.10mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component are set as 1/2MS+
IAA1.5mg/L+IBA1.0mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
When being verified to the present invention：
Influence to nutrition to explant survival rate
| Nutrition/day the moon- | Inoculation number/ | Pollution number/ | Survive number/ | Pollution rate/% | Survival rate/% |
| 4-10 | 60 | 7 | 47 | 11.67 | 78.33 |
| 5-10 | 60 | 11 | 40 | 18.33 | 66.67 |
| 6-10 | 60 | 13 | 39 | 21.67 | 65.00 |
| 7-10 | 60 | 15 | 36 | 25.00 | 60.00 |
| 8-10 | 60 | 16 | 35 | 26.67 | 58.33 |
Influence of the mercuric chloride processing time to explant sterilization effect
| Processing time/s | Inoculation number/ | Pollution number/ | Survive number/ | Pollution rate/% | Survival rate/% |
| 6 | 30 | 7 | 19 | 23.33 | 63.33 |
| 8 | 30 | 2 | 24 | 6.67 | 80.00 |
| 10 | 30 | 1 | 21 | 3.33 | 70.00 |
Influence of the 6-BA concentration to explant inductivity
| 6-BA concentration/mg/L | Inoculation number/ | Rudiment number/ | Inductivity/% |
| 0.5 | 30 | 13 | 43.33 |
| 1.0 | 30 | 21 | 70.00 |
| 1.5 | 30 | 18 | 60.00 |
| 2.0 | 30 | 14 | 46.67 |
Influence of the IBA concentration to explant inductivity
| IBA/mg/L | 6-BA concentration/mg/L | Inoculation number/ | Rudiment number/ | Inductivity/% |
| 0.05 | 1.0 | 30 | 14 | 46.67 |
| 0.10 | 1.0 | 30 | 23 | 76.67 |
| 0.15 | 1.0 | 30 | 20 | 66.67 |
| 0.20 | 1.0 | 30 | 15 | 50.00 |
Influence of the NAA concentration to explant inductivity
| NAA concentration/mg/L | 6-BA concentration/mg/L | IBA concentration/mg/L | Inoculation number/ | Rudiment number/ | Inductivity/% |
| 0.01 | 1.0 | 0.1 | 30 | 15 | 50.00 |
| 0.03 | 1.0 | 0.1 | 30 | 23 | 76.67 |
| 0.05 | 1.0 | 0.1 | 30 | 25 | 83.33 |
| 0.07 | 1.0 | 0.1 | 30 | 16 | 53.33 |
Influence of the 6-BA and IBA concentration to G41 stocks growth coefficient and growth conditions
| 6-BA concentration/mg/L | IBA concentration/mg/L | Growth coefficient | Growth conditions |
| 0.5 | 0.05 | 3.92 | Green, Ye great |
| 0.5 | 0.10 | 4.03 | Green, Ye great |
| 0.5 | 0.15 | 4.15 | Green has leaf roll |
| 1.0 | 0.05 | 4.48 | Green, Ye great |
| 1.0 | 0.10 | 5.14 | Green, Ye great |
| 1.0 | 0.15 | 4.96 | Green has individually yellow leaf |
| 1.5 | 0.05 | 5.03 | Green has dead leaf |
| 1.5 | 0.10 | 4.97 | Yellow green has vitreous seedling |
| 1.5 | 0.15 | 5.13 | Green has vitreous seedling |
The influence that IAA and IBA concentration takes root to G41 stocks
| IAA concentration/mg/L | IBA concentration/mg/L | Rooting rate/% | It takes root number/item | Root long/cm |
| 1.0 | 0.5 | 93.33 | 6.25 | 2.12 |
| 1.0 | 1.0 | 94.86 | 6.78 | 1.97 |
| 1.0 | 1.5 | 92.58 | 6.86 | 2.07 |
| 1.5 | 0.5 | 100 | 8.26 | 2.26 |
| 1.5 | 1.0 | 95.24 | 8.07 | 2.31 |
| 1.5 | 1.5 | 93.27 | 7.99 | 2.25 |
| 2.0 | 0.5 | 91.02 | 7.16 | 2.18 |
| 2.0 | 1.0 | 90.57 | 7.35 | 2.20 |
| 2.0 | 1.5 | 90.03 | 7.48 | 2.21 |
Second embodiment of the present invention, tissue-culturing rapid propagation culture medium is formed so that MS and 1/2MS are as the main component.
In the present embodiment, the kind of the anti-continuous cropping dwarfing rootstock of apple is set as G41.
Second embodiment of the present invention is based on one embodiment.
The present invention has the following characteristics：
1, due to devising primary culture medium, proliferated culture medium and root media, by primary culture medium and proliferated culture medium,
Tissue culture is carried out to stem apex body to handle, by root media, rooting treatment carried out to tissue-cultured seedling, no longer cuttage and press strip
Breeding, therefore improve slow seedling survival rate.
2, as the main component due to devising MS, it is good that inducing effect is carried out to stem apex body.
3, as the main component due to devising 1/2MS, it is good that rooting efficiency is carried out to tissue-cultured seedling.
4, the restriction of numberical range has been carried out to planform due to devising, it is the technical side of the present invention to make numberical range
Technical characteristic in case is not to be calculated by formula or test the technical characteristic obtained by limited number of time, experiments have shown that the numerical value
The technical characteristic of range achieves good technique effect.
5, due to devising the technical characteristic of the present invention, in the effect of the independent and mutual set of technical characteristic,
By experiments have shown that, property indices of the invention be existing property indices be at least 1.7 times, pass through assess tool
There is good market value.
It is also other and with MS and 1/2MS it is as the main component it is same or similar as technical characteristic be all the present invention reality
One of example is applied, and each technical characteristic of embodiment described above can be combined arbitrarily, it is real to meet Patent Law, patent
Apply the requirement of detailed rules and regulations and guidelines for examination, the no longer embodiment to all possible combination of each technical characteristic in above-described embodiment
All it is described.
Above-described embodiment be the tissue culture and rapid propagation method provided by the present invention for the anti-continuous cropping dwarfing rootstock G41 of apple and
A kind of way of realization of culture medium increases or reduces composition therein according to other deformations of scheme provided by the present invention
Or step, or the present invention is all belonged to the scope of protection of the present invention for other technical fields close with the present invention.
Claims (10)
1. a kind of tissue-culturing rapid propagation culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：It include Primary culture
Base, proliferated culture medium and root media,
Wherein：Primary culture medium component is set as MS+6-BA+IBA+NAA+sucrose+agar, the setting of proliferated culture medium component
For MS+6-BA+IBA+sucrose+agar, root media component is set as 1/2MS+IAA+IBA+sucrose+agar.
2. a kind of tissue-culturing rapid propagation culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：Based on MS and 1/2MS
Composition is wanted to form tissue-culturing rapid propagation culture medium.
3. the tissue-culturing rapid propagation culture medium according to claim 1 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：
The kind of the anti-continuous cropping dwarfing rootstock of apple is set as G41.
4. the tissue-culturing rapid propagation culture medium according to claim 1 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：
Include that primary culture medium component is set as MS+6-BA0.5-2.0mg/L+ IBA0.05-0.20mg/L+NAA0.01-
0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5-1.5mg/L+
IBA0.05-0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, root media component are set as 1/2MS+IAA1.0-
2.0mg/L+IBA0.5-1.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L.
5. the tissue-culturing rapid propagation culture medium according to claim 4 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：
MS nutrient media components：
1/2 MS nutrient media components：
6-BA（6-benzyl aminopurine）
IBA（Indolebutyric acid）
NAA（Methyl α-naphthyl acetate）
IAA（Heteroauxin）.
6. the tissue-culturing rapid propagation culture medium according to claim 4 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：
Include that primary culture medium component is set as MS+6-BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L+
Agar 6.0g/L, proliferated culture medium component are set as MS+6-BA0.5-1mg/L+IBA0.1mg/L+sucrose 30.0g/L+ agar
6.0g/L, root media component are set as 1/2MS+IAA1.5mg/L+IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/
L。
7. a kind of tissue culture and rapid propagation method for the anti-continuous cropping dwarfing rootstock G41 of apple,
Its step is：Stem apex body is seeded in MS+6-BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L
The primary culture medium of+agar 6.0g/L obtains explant, explant be seeded in MS+6-BA0.5-1mg/L+IBA0.1mg/L+
The proliferated culture medium of sucrose 30.0g/L+ agar 6.0g/L obtains tissue-cultured seedling, tissue culture plant inoculation 1/2MS+IAA1.5mg/L+
The root media of IBA0.5mg/L+ sucrose 30.0g/L+ agar 6.0g/L obtains tissue-cultured seedling of taking root.
8. the tissue culture and rapid propagation method according to claim 7 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：Its
Step is：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA0.5-2.0mg/L+ IBA0.05-0.20mg/L+NAA0.01-0.07mg/L+ sucrose 30.0g/L+ agar 6.0g/L, are placed in
Light application time is 16h/d, intensity of illumination 2000Lux, temperature are 24-26 DEG C, the culture environment of relative air humidity 50%-70%
Lower carry out Primary culture, obtains explant,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1.5mg/L+IBA0.05-0.15mg/L+sucrose 30.0g/L+ agar 6.0g/L, it is 16h/d, intensity of illumination to be placed in light application time
It is 24-26 DEG C for 2000Lux, temperature, carries out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtains tissue culture
Seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.0-2.0mg/L+IBA0.5-1.5mg/L+ sucrose
30.0g/L+ agar 6.0g/L, first dark treatment is after seven days, then be placed in light application time be 16h/d, intensity of illumination 2000Lux, temperature
Culture of rootage is carried out under the culture environment that degree is 25 DEG C, obtains tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
9. the tissue culture and rapid propagation method according to claim 7 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：Its
Step is：
One, material selection：The G41 elite stands that robust growth is endangered without disease pest are selected first, and four to the August choose newly from elite stand
The stem apex of the length grown about 1.0-1.5cm is first put on superclean bench and fills after being rinsed one to two hour with tap water
It handles five to seven seconds in the beaker of 75% alcohol, is immersed again in the beaker for filling 0.1% mercuric chloride after taking-up, sterilized seven to nine minutes,
Period weak vibrations three to five times are finally fully rinsed three to five times with aseptic deionized water, and carry out disinfection sterilization cleaning treatment,
Sterilization stem apex body is obtained,
Two, Primary culture：Sterilization stem apex body is inoculated into primary culture medium, primary culture medium component is set as MS+6-
BA1.0mg/L+ IBA0.1mg/L+NAA0.05mg/L+ sucrose 30.0g/L+ agar 6.0g/L, be placed in light application time be 16h/d,
Intensity of illumination is 2000Lux, temperature is 24-26 DEG C, Primary culture is carried out under the culture environment of relative air humidity 50%-70%,
Explant is obtained,
Three, Multiplying culture：Explant is inoculated on proliferated culture medium, proliferated culture medium component is set as MS+6-BA0.5-
1mg/L+IBA0.1mg/L+sucrose 30.0g/L+ agar 6.0g/L, is placed in that light application time is 16h/d, intensity of illumination is
2000Lux, temperature are 24-26 DEG C, carry out Multiplying culture under the culture environment of relative air humidity 40%-50%, obtain tissue-cultured seedling,
Four, culture of rootage：Robust growth, highly more than 1.5cm, the tissue culture plant inoculation that growth time is 20 to 25 days
In root media, root media component is set as 1/2MS+IAA1.5mg/L+IBA0.5mg/L+ sucrose 30.0g/L+
Agar 6.0g/L, first dark treatment is after seven days, then is placed in that light application time is 16h/d, intensity of illumination 2000Lux, temperature are 25 DEG C
Culture environment under carry out culture of rootage, obtain tissue-cultured seedling of taking root,
Five, hardening：After tissue-cultured seedling length of taking root reaches 1.5cm, start hardening, intensity of illumination gradually enhance from 2000lux to
5000lux, hardening ten to 15 days, obtains the rooted seedling of hardening,
Six, it transplants：Start to transplant for the first time in shade, the rooted seedling of hardening is taken out out of culture bottle, chooses hardening
The root long degree of rooted seedling is 1.8-2.2cm, after the root culture medium of the rooted seedling of clean hardening, is transplanted to culture plate or culture bag
Transplanting culture substrate in, according to weight ratio：Transplanting culture substrate culture substrate is set as：+ 1/3 perlite+1/ of 1/3 matrix
3 thick river sands, spray moisture content is primary sooner or later daily, and it is 75%-85% to keep the water content of matrix and air humidity, is conducive to slow seedling,
Culture plate is moved to outside shade after seven days and starts second of transplanting, when the rooted seedling young sprout of culture plate or the hardening of culture bag is long
Go out, have new root from culture bag or culture pan bottom permeable hole be pierced by when, migration crop field plant school, take wide row be 60cm,
Narrow row is 40cm, spacing in the rows is 25cm and 10,000 plants of wide-and narrow-row nursery of nursery per acre, obtain it is intermediate at seedling body,
Seven, seedling：It emerges after centre is fallen leaves at seedling body, reduces the damage to the root system at seedling body and to dividing at seedling body
Grade preserves, and obtains into seedling body.
10. the tissue culture and rapid propagation method according to claim 9 for the anti-continuous cropping dwarfing rootstock G41 of apple, it is characterized in that：?
April 10 current year is sampled, and mercuric chloride processing time is set as 8 seconds, in primary culture medium：The concentration of 6-BA is set as
The concentration that the concentration of 1.0mg/L, IBA are set as 0.10mg/L, NAA is set as 0.0.05mg/L, in proliferated culture medium：6-BA
Concentration be set as the concentration of 1.0mg/L, IBA and be set as 0.10mg/L, in root media：The concentration of IAA is set as
The concentration of 1.5mg/L, IBA are set as 0.50mg/L.
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