CN107494262A - Tissue culture and rapid propagation method and application and culture medium and preparation method, the disinfection treatment method of apple rootstock bar for apple rootstock - Google Patents
Tissue culture and rapid propagation method and application and culture medium and preparation method, the disinfection treatment method of apple rootstock bar for apple rootstock Download PDFInfo
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- CN107494262A CN107494262A CN201710749390.6A CN201710749390A CN107494262A CN 107494262 A CN107494262 A CN 107494262A CN 201710749390 A CN201710749390 A CN 201710749390A CN 107494262 A CN107494262 A CN 107494262A
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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
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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
A kind of tissue culture and rapid propagation method and application and culture medium and preparation method, the disinfection treatment method of apple rootstock bar for apple rootstock, its step：In the bud primary culture medium containing MS, axillary bud sprouting Primary culture is carried out to bud section and obtains in vitro cuttings, in the first proliferated culture medium containing NM, after fast breeding being carried out in vitro cuttings, then in the second proliferated culture medium containing MS, in vitro cuttings are bred and elongation growth obtains breeding test tube seedling, in the root media containing MS, culture of rootage is carried out to propagation test tube seedling and obtains plantlet of taking root, pass through bud primary culture medium, realize axillary bud sprouting startup processing, pass through proliferated culture medium, realize fast breeding and propagation and the two-step pretreatment of elongation growth, pass through root media, realize culture of rootage processing, it is achieved that the tissue-culturing rapid propagation of apple rootstock.
Description
Technical field
It is especially a kind of to be used for apple the present invention relates to a kind of tissue culture and rapid propagation method and application and culture medium and preparation method
The tissue culture and rapid propagation method and application and culture medium and preparation method, the disinfection treatment method of apple rootstock bar of stock.
Background technology
The group culturation rapid propagating technology of plant, height heterozygosis species offspring caused by generative propagation can be overcome to separate, can be used for
Famous-brand and high-quality or frequency danger species redemption, Plantlet in vitro and quick breedings, the fast seedling growing available for nontoxic nursery stock etc., therefore plant
Tissue-culturing rapid propagation is the very strong technology of application, is applied particularly in terms of the breeding and nontoxic seedling-wood breeding of gardening plant at most,
The tissue cultures of plant start from the second half in 19th century, and the stem section and root segment of trecul culture of ex vivo in 1853 obtain callus group
Knit, but full plants body could not be gone out by callus regeneration.1902, the concept of Haberlandt proposition cell culture, 1934
Year, the tomato root of White culture of ex vivo obtains Success in Experiment first.The Steward in the U.S. in 1958 and the Reinert of Germany divide
Not by the carrot cell induced synthesis cultivated, embryoid, nineteen sixty-five Vasil and Hildebrandt cultivate the thin of single separation
Born of the same parents obtain the regeneration of whole plant.The Study on tissue culture of plant experienced about half from experiment is started to first time success
Century.Afterwards, as the constantly improve of tissue culture technique, increasing plant species obtain the success of tissue cultures, arrived
In the 1960s, Plant Tissue Breeding progressively develops to batch production, commercialization.In production of fruit trees, in multiple cultivations
The batch production of tissue culture is successfully realized in kind and stock.Kind such as banana, blue Berry etc. have been achieved with tissue culture industrial seedling rearing,
The dwarfing rootstock 5 of excellent stock such as gean, No. 6 etc., Dwarf Stocks For Apple Trees M26, M9 etc. have realized nontoxic seedling
Tissue culture test tube is seedling industrialized.But successfully the group culturation rapid propagating technology of these kinds is all fast numerous skill of corresponding respective certain species
Art, its tissue culture and rapid propagation method of kind difference is also different, currently there are no a kind of tissue culture and rapid propagation method for apple rootstock and answers
With with culture medium and preparation method, the disinfection treatment method of apple rootstock bar, be particularly suitable for the group to apple rootstock ' 62-396 '
Tissue culture method for fast propagation and application and culture medium and preparation method, are also all to use QL culture mediums as minimal medium.
Based on existing technical problem, technical characteristic and technique effect, application technical scheme of the invention is made.
The content of the invention
The object of the present invention is a kind of tissue culture and rapid propagation method for apple rootstock,
The object of the present invention is a kind of application of the tissue culture and rapid propagation method for apple rootstock,
The object of the present invention is a kind of tissue-culturing rapid propagation culture medium for apple rootstock,
The object of the present invention is a kind of preparation method of the tissue-culturing rapid propagation culture medium for apple rootstock,
The object of the present invention is a kind of disinfection treatment method of the apple rootstock bar of the tissue culture and rapid propagation method for apple rootstock.
In order to overcome above-mentioned technical disadvantages, it is an object of the invention to provide a kind of tissue culture and rapid propagation method for apple rootstock
And application and culture medium and preparation method, the disinfection treatment method of apple rootstock bar, it is achieved that the tissue culture of apple rootstock is fast
It is numerous.
To reach above-mentioned purpose, the present invention adopts the technical scheme that：A kind of tissue culture and rapid propagation method for apple rootstock,
Its step：
In the bud primary culture medium containing MS, to bud section carry out axillary bud sprouting Primary culture obtain in vitro cuttings, containing
In NM the first proliferated culture medium, after carrying out fast breeding in vitro cuttings, then in the second proliferated culture medium containing MS
In, in vitro cuttings are bred and elongation growth obtains breeding test tube seedling, in the root media containing MS, to propagation
Test tube seedling carries out culture of rootage and obtains plantlet of taking root.
Due to devising bud primary culture medium, proliferated culture medium and root media, by bud primary culture medium, realize
Axillary bud sprouting startup is handled, and by proliferated culture medium, is realized fast breeding and propagation and the two-step pretreatment of elongation growth, is passed through
Root media, culture of rootage processing is realized, it is achieved that the tissue-culturing rapid propagation of apple rootstock.
The present invention is devised, and propagation is combined in the proliferated culture medium respectively containing NM and MS and is extended in bud startup
Culture processing and culture of rootage processing are principal character.
The present invention devises, and its step is：Condition of culture：Cultivation temperature is（25 ± 2）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.3 ~ 0.6 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water,
Then put running water flowing water and rinse and be more than 30 minutes, material is taken out in the sterile beaker being put on super-clean bench, pours into 70% alcohol
Sterilization 1 minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 3 ~ 5 times, fall
Go out sodium hypochlorite to be washed 4 ~ 6 times with sterile, the taking-up of bud section is put surface moisture is sucked on aseptic filter paper, bud section is inoculated into and is equipped with
In the blake bottle of the mg/L IBA+30 g/L sucrose+6g/L agar powders of mg/L BA+0.1 of bud primary culture medium MS+1 ~ 2 ~ 0.5,
Blake bottle is put culturing room and carries out axillary bud sprouting Primary culture, the green seedling that germination and growth obtains in primary culture medium is sterile test tube
Seedling；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+
The fast breeding of bud is completed on 0.01 ~ 0.1 mg/L TDZ+30 g/L sucrose+6g/L agar powders；It is then transferred into the second propagation
Culture medium：The increasing of bud is completed on the mg/L IBA+30 g/L sucrose+6g/L agar powders of mg/L BA+0.1 of MS+0.5 ~ 1.2 ~ 0.5
Grow and reach the propagation test tube seedling for the standard of taking root with elongation growth culture, acquisition；
3rd, rooting of vitro seedling：
Cut propagation test tube seedling of the growing height more than 1.5 centimetres and be transferred to root media:The mg/L of 1/2MS+ 0.1~2
Culture of rootage is carried out on IBA+20 g/L sucrose, condition of culture is first continuous light culture 5 days, then goes to daily illumination 16 hours
Periodicity of illumination condition of culture under cultivate, culture of rootage 5 ~ 6 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 3
~ 5 days, bottle cap is then opened, gravity-flow ventilation hardening 1 ~ 3 day, plantlet of taking root is taken out with tweezers, cleans the culture of root residual
Base, it is transplanted into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with more than 0.1% after cultivation
The spraying spray of bacterium spirit solution is permeable, then with the good covered rearing with plastic film moisturizing of translucency, sprays 0.1% carbendazim every 3 ~ 5 days
Solution, treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves seedling in good time
Enter the seedling in field.
The present invention devises, minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
NM culture mediums, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 800 |
| KNO3 | 950 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 370 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water
Culture medium solidifying agent：Agar powder.
The present invention devises, a kind of application of tissue culture and rapid propagation method for apple rootstock in apple rootstock ' 62-396 '.
The present invention devises, a kind of side of disinfecting of the apple rootstock bar of tissue culture and rapid propagation method for apple rootstock
Method, its step：Apple rootstock bar is put running water flowing water and rinsed and is more than 30 minutes, apple rootstock bar is taken out and is put on super-clean bench
Sterile beaker in, pour into 70% alcohol sterilize 1 minute, outwell alcohol add effective chlorine be 5% liquor natrii hypochloritis, sterilization
10 minutes, during which shake 3 ~ 5 times, pour out sodium hypochlorite and washed 4 ~ 6 times with sterile, apple rootstock bar is taken out and puts aseptic filter paper
On suck surface moisture.
The present invention devises, a kind of tissue-culturing rapid propagation culture medium for apple rootstock, includes bud primary culture medium, propagation
Culture medium and root media.
The present invention devises, and the mg/L IBA+30 g/L sucrose of mg/L BA+0.1 of bud primary culture medium MS+1 ~ 2 ~ 0.5+
In the blake bottle of 6g/L agar powders,
Proliferated culture medium:First proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+ 0.01~0.1
Mg/L TDZ+30 g/L sucrose+6g/L agar powders, the second proliferated culture medium：MS+0.5~1.2 mg/L BA+0.1~0.5 mg/L
G/L sucrose+6g/L the agar powders of IBA+ 30,
Root media:The mg/L IBA+20 g/L sucrose of 1/2MS+0.1~2.
The present invention devises, minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
NM culture mediums, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 800 |
| KNO3 | 950 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 370 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water
Culture medium solidifying agent：Agar powder.
The present invention devises, a kind of preparation method of tissue-culturing rapid propagation culture medium for apple rootstock, and its step is：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 45 ~ 55 ml bud primary culture medium, proliferated culture medium
And root media, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 divides under 121 DEG C, 1 atmospheric pressure
Clock.
In the technical program, QL culture mediums, form as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 400 |
| KNO3 | 900 |
| KH2PO4 | 130 |
| MgSO4·7H2O | 180 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| FeSO4·7H2O | 27.8 |
| Na2·EDTA | 37.3 |
| KI | 0.83 |
| H3BO3 | 12 |
| MnSO4·H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| Organic principle | |
| VB1 | 0.4 |
| VB2 | 1.0 |
| Inositol | 100 |
In the technical program, it is important technology that propagation and elongation are combined in the proliferated culture medium respectively containing NM and MS
Feature, the tissue culture and rapid propagation method for apple rootstock and application and culture medium and preparation method, apple rootstock bar sterilization at
In the technical field of reason method, there is novelty, creativeness and practicality, the term in the technical program is all that can use this
Patent document in technical field is explained and understood.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the design sketch of the present invention,
A figures are the design sketch of test tube seedling foundation and axillary bud sprouting, and B figures are the design sketch of the propagation of test tube seedling bud, and C figure test tube seedlings are stretched
The design sketch of long design sketch, D figure test tube seedling proliferations and elongation, the design sketch of E rooting of vitro seedling, the effect that F figures test tube seedling is moved
Fruit is schemed.
Embodiment
According to guidelines for examination, such as " having ", "comprising" and " comprising " term used in the present invention should be understood
Not allot the presence or addition of one or more of the other element or its combination.
In the description of the invention, it is necessary to explanation, term " " center ", " on ", " under ", "left", "right", " vertical ",
The orientation or position relationship of the instruction such as " level ", " interior ", " outer " are the orientation or position relationship of general expression, are for only for ease of
Description is of the invention to be described with simplified, rather than the device or element of instruction or hint meaning must be with specific orientation, Yi Te
Fixed azimuth configuration and operation, therefore be not considered as limiting the invention.In addition, term " first ", " second ", " the 3rd "
It is only used for describing purpose, and it is not intended that instruction or hint relative importance.
In the description of the invention, it is necessary to illustrate, unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " should be interpreted broadly, for example, it may be being fixedly connected or being detachably connected, or be integrally connected；Can
To be mechanical connection or electrical connection；Can be joined directly together, can also be indirectly connected by intermediary, Ke Yishi
The connection of two element internals.For the ordinary skill in the art, with concrete condition above-mentioned term can be understood at this
Concrete meaning in invention.
As long as in addition, technical characteristic involved in invention described below different embodiments non-structure each other
It is be combined with each other into conflict can.
A kind of tissue culture and rapid propagation method for apple rootstock, with reference to embodiment, the present invention is further described, below
Embodiment is intended to illustrate invention rather than limitation of the invention further.One embodiment, its step are：Cultivate bar
Part：Cultivation temperature is（25,）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.3 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water, then
Put running water flowing water to rinse 30 minutes, material is taken out in the sterile beaker being put on super-clean bench, pour into 70% alcohol and sterilize 1 point
Clock, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 3 times, pour out hypochlorous acid
Sodium is washed 4 times with sterile, the taking-up of bud section is put surface moisture content is sucked on aseptic filter paper, bud section is inoculated into equipped with bud Primary culture
In the blake bottle of the mg/L IBA+30 g/L sucrose+6g/L agar powders of base MS+1 mg/L BA+ 0.1, blake bottle is put culturing room
Axillary bud sprouting Primary culture is carried out, the green seedling that germination and growth obtains in primary culture medium is in vitro cuttings；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.1 mg/L BA+0.01 mg/L IBA+ 0.01mg/L
TDZ+30 g/L sucrose+6g/L agar powders, the upper fast breeding for completing bud；It is then transferred into the second proliferated culture medium：MS+0.5
Propagation and the elongation growth culture of bud are completed on mg/L BA+0.1 mg/L IBA+30 g/L sucrose+6g/L agar powders, is reached
To the propagation test tube seedling for the standard of taking root；
3rd, rooting of vitro seedling：
The propagation test tube seedling that growing height is cut at 1.5 centimetres is transferred to root media:1/2MS+ 0.1 mg/L IBA+20
Culture of rootage is carried out on g/L sucrose, condition of culture is first continuous light culture 5 days, then goes to the illumination of daily illumination 16 hours
Cultivated under cycle condition of culture, culture of rootage 5 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 3
My god, bottle cap is then opened, gravity-flow ventilation hardening 1 day, plantlet of taking root is taken out with tweezers, cleans the culture medium of root residual, move
Plant into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with 0.1% carbendazim after cultivation
Solution spraying spray is permeable, molten every 0.1% carbendazim of spray in 3 ~ 5 days then with the good covered rearing with plastic film moisturizing of translucency
Liquid, treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves into seedling in good time
The seedling in field.
In the present embodiment,
Minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
NM culture mediums, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 800 |
| KNO3 | 950 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 370 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water
Culture medium solidifying agent：Agar powder.
Dwarf Stocks For Apple Trees ' 62-396 ' are tested, its experimental result is as follows：
1st, the germination and growth of bud
The bud section of Dwarf Stocks For Apple Trees ' 62-396 ' have successfully been obtained the green bud tip of axillary bud sprouting growth（Fig. 1, A）, illustrate culture
The mg/L IBA+30 g/L sucrose+6g/L agar powders of mg/L BA+0.1 of base MS+1 ~ 2 ~ 0.5 are effective to the Primary culture of axillary bud
's.
2nd, the propagation growth of test tube seedling
The sterile green seedling that axillary bud sprouting obtains is transferred to shoot proliferation on proliferated culture medium, the results showed that by two-steps tissue culture method,
I.e. in the culture medium 1. mg/L BA+0.01 of NM+0.1 ~ 0.5 ~ g/L of 0.1 mg/L IBA+, 0.01 ~ 0.1 mg/L TDZ+30
The fast breeding of bud is carried out on sucrose+6g/L agar powders（Fig. 1, B）；Be then transferred into 2. the mg/L BA+0.1 of MS+0.5 ~ 1.2 ~
Propagation and the elongation growth (Fig. 1, D) of bud are carried out on 0.5 mg/L IBA+30 g/L sucrose+6g/L agar powders, to fast breeding
It is most effective with strong sprout, and in the mg/L IBA+30 g/L sucrose+6g/L fine jades of mg/L BA+0.1 of culture medium QL+0.5 ~ 1.2 ~ 0.5
Although being also presented with preferable elongation growth on cosmetics, propagation is poor（Fig. 1, C）, it is not optimal subculture multiplication medium.
3. test tube seedling is taken root and transplanted
Healthy and strong green seedling cultivates 20 on the mg/L IBA+20 g/L sucrose+6g/L agar powders of root media 1/2MS+0.1~2
My god, rooting rate is more than 85%（Fig. 1, E）, root media selected by explanation can effectively induce apple rootstock ' 62-396 ' test tube
Seedling is taken root, and the g/L sucrose+6g/L agar powders of root media QL+0.1~2 mg/L IBA+ 20 and 1/2QL+0.1~
2 mg/L IBA+20 g/L sucrose+6g/L agar powders, rooting rate is all below 50%, it is impossible to meets the requirement of rapid seedling cultivation.
Then rooting tube plantlet opens bottle cap, gravity-flow ventilation hardening 1~3 by scattering natural lighting lower refining seedling 3~5 days
My god, it is transplanted into equipped with matrix：Peat soil：Perlite：It is permeable with the spraying spray of 0.1% carbendazim solution after cultivation in the hole tray of vermiculite,
Then with method for transplanting such as the good covered rearing with plastic film moisturizings of translucency, for survival rate more than 85%, the seedling survived is raw in basin
It is long good（Fig. 1, F）.
Wherein：QL culture mediums, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 400 |
| KNO3 | 900 |
| KH2PO4 | 130 |
| MgSO4·7H2O | 180 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| FeSO4·7H2O | 27.8 |
| Na2·EDTA | 37.3 |
| KI | 0.83 |
| H3BO3 | 12 |
| MnSO4·H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·6H2O | 0.025 |
| Organic principle | |
| VB1 | 0.4 |
| VB2 | 1.0 |
| Inositol | 100 |
Experiment shows：First proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+ 0.01~0.1
Mg/L TDZ+30 g/L sucrose+6g/L agar powders, the second proliferated culture medium：MS+0.5~1.2 mg/L BA+0.1~0.5 mg/L
In vitro cuttings are combined continuous processing by IBA+30 g/L sucrose+6g/L agar powders, and obtained propagation test tube seedling effect is most
It is good.
Second embodiment, its step are：Condition of culture：Cultivation temperature is（27）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.6 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water, then
Put running water flowing water to rinse 45 minutes, material is taken out in the sterile beaker being put on super-clean bench, pour into 70% alcohol and sterilize 1 point
Clock, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 5 times, pour out hypochlorous acid
Sodium is washed 6 times with sterile, the taking-up of bud section is put surface moisture content is sucked on aseptic filter paper, bud section is inoculated into equipped with bud Primary culture
In the blake bottle of the mg/L IBA+30 g/L sucrose+6g/L agar powders of base MS+2 mg/L BA+ 0.5, blake bottle is put culturing room
Axillary bud sprouting Primary culture is carried out, the green seedling that germination and growth obtains in primary culture medium is in vitro cuttings；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.5 mg/L BA+0.1 mg/L IBA+ 0.1 mg/L
TDZ+30 g/L sucrose+6g/L agar powders, the upper fast breeding for completing bud；It is then transferred into the second proliferated culture medium：MS+1.2
Propagation and the elongation growth culture of bud are completed on mg/L BA+0.5 mg/L IBA+30 g/L sucrose+6g/L agar powders, is reached
To the propagation test tube seedling for the standard of taking root；
3rd, rooting of vitro seedling：
The propagation test tube seedling that growing height is cut at 1.8 centimetres is transferred to root media:1/2MS+ 2 mg/L IBA+20 g/
Culture of rootage is carried out on L sucrose, condition of culture is first continuous light culture 5 days, then goes to the illumination week of daily illumination 16 hours
Cultivated under phase condition of culture, culture of rootage 6 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 5
My god, bottle cap is then opened, gravity-flow ventilation hardening 3 days, plantlet of taking root is taken out with tweezers, cleans the culture medium of root residual, move
Plant into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with 0.1% carbendazim after cultivation
Solution spraying spray is permeable, then with the good covered rearing with plastic film moisturizing of translucency, sprays 0.1% carbendazim solution every 5 days,
Treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves into field to seedling in good time
Between seedling.
3rd embodiment, its step are：Condition of culture：Cultivation temperature is（25 ± 2）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.3 ~ 0.6 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water,
Then put running water flowing water and rinse and be more than 30 minutes, material is taken out in the sterile beaker being put on super-clean bench, pours into 70% alcohol
Sterilization 1 minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 3 ~ 5 times, fall
Go out sodium hypochlorite to be washed 4 ~ 6 times with sterile, the taking-up of bud section is put surface moisture content is sucked on aseptic filter paper, bud section is inoculated into and is equipped with
In the blake bottle of the mg/L IBA+30 g/L sucrose+6g/L agar powders of bud primary culture medium MS+1 ~ 2 mg/L BA+ 0.1 ~ 0.5,
Blake bottle is put culturing room and carries out axillary bud sprouting Primary culture, the green seedling that germination and growth obtains in primary culture medium is sterile test tube
Seedling；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+
0.01 ~ 0.1 mg/L TDZ+30 g/L sucrose+6g/L agar powders, the upper fast breeding for completing bud；It is then transferred into the second propagation
Culture medium：The increasing of bud is completed on the mg/L IBA+30 g/L sucrose+6g/L agar powders of mg/L BA+0.1 of MS+0.5 ~ 1.2 ~ 0.5
Grow and reach the propagation test tube seedling for the standard of taking root with elongation growth culture, acquisition；
3rd, rooting of vitro seedling：
The propagation test tube seedling that growing height is cut at 2.0 centimetres is transferred to root media:The mg/L IBA of 1/2MS+ 0.1~2
Carry out culture of rootage on+20 g/L sucrose, condition of culture is first continuous light culture 5 days, then goes to daily illumination 16 hours
Cultivated under periodicity of illumination condition of culture, culture of rootage 5~6 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 3
~ 5 days, bottle cap is then opened, gravity-flow ventilation hardening 1 ~ 3 day, plantlet of taking root is taken out with tweezers, cleans the culture of root residual
Base, it is transplanted into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with more than 0.1% after cultivation
The spraying spray of bacterium spirit solution is permeable, then with the good covered rearing with plastic film moisturizing of translucency, sprays 0.1% carbendazim every 3 ~ 5 days
Solution, treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves seedling in good time
Enter the seedling in field.
4th embodiment, its step are：Condition of culture：Cultivation temperature is（26）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.45 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water, and
After put running water flowing water and rinse 35 minutes, material is taken out in the sterile beaker that is put on super-clean bench, pours into 70% alcohol sterilization 1
Minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 4 times, pour out time chlorine
Sour sodium is washed 5 times with sterile, the taking-up of bud section is put surface moisture content is sucked on aseptic filter paper, and bud section is inoculated into equipped with bud to start and trained
In the blake bottle for supporting the mg/L IBA+30 g/L sucrose+6g/L agar powders of base MS+1.5 mg/L BA+ 0.3, blake bottle is put training
Support room and carry out axillary bud sprouting Primary culture, the green seedling that germination and growth obtains in primary culture medium is in vitro cuttings；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.3 mg/L BA+0.05 mg/L IBA+ 0.05 mg/L
TDZ+30 g/L sucrose+6g/L agar powders, the upper fast breeding for completing bud；It is then transferred into the second proliferated culture medium：MS+0.8
Propagation and the elongation growth culture of bud are completed on mg/L BA+0.3 mg/L IBA+30 g/L sucrose+6g/L agar powders, is reached
To the propagation test tube seedling for the standard of taking root；
3rd, rooting of vitro seedling：
The propagation test tube seedling that growing height is cut at 1.6 centimetres is transferred to root media:1/2MS+ 1 mg/L IBA+20
Culture of rootage is carried out on g/L sucrose, condition of culture is first continuous light culture 5 days, then goes to the illumination of daily illumination 16 hours
Cultivated under cycle condition of culture, culture of rootage 5 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 4
My god, bottle cap is then opened, gravity-flow ventilation hardening 2 days, plantlet of taking root is taken out with tweezers, cleans the culture medium of root residual, move
Plant into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with 0.1% carbendazim after cultivation
Solution spraying spray is permeable, then with the good covered rearing with plastic film moisturizing of translucency, sprays 0.1% carbendazim solution every 4 days,
Treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves into field to seedling in good time
Between seedling.
Application of a kind of tissue culture and rapid propagation method for apple rootstock in apple rootstock ' 62-396 ', due to apple rootstock
' 62-396 ' has the characteristic of cold-resistant, resistance to continuous cropping and dwarfing, has plantation performance well.
A kind of disinfection treatment method of the apple rootstock bar of tissue culture and rapid propagation method for apple rootstock, first implementation
Example, its step：Apple rootstock bar is put running water flowing water to rinse 30 minutes, apple rootstock bar is taken out the nothing being put on super-clean bench
In bacterium beaker, pour into 70% alcohol and sterilize 1 minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilization 10
Minute, during which shake 3 times, pour out sodium hypochlorite and washed 4 times with sterile, the taking-up of apple rootstock bar is put table is sucked on aseptic filter paper
Face moisture content.
Second embodiment, its step：Apple rootstock bar is put running water flowing water to rinse 45 minutes, apple rootstock bar is taken
Go out in the sterile beaker being put on super-clean bench, pour into 70% alcohol and sterilize 1 minute, outwell alcohol and add time that effective chlorine is 5%
Sodium chlorate solution, sterilize 10 minutes, during which shake 5 times, pour out sodium hypochlorite and washed 6 times with sterile, apple rootstock bar is taken out
Put and surface moisture content is sucked on aseptic filter paper.
3rd embodiment, its step：Apple rootstock bar is put running water flowing water and rinsed and is more than 30 minutes, apple rootstock
Bar takes out and is put into the sterile beaker on super-clean bench, pours into 70% alcohol and sterilizes 1 minute, outwells alcohol and adds effective chlorine as 5%
Liquor natrii hypochloritis, sterilize 10 minutes, during which shake 3 ~ 5 times, pour out sodium hypochlorite with it is sterile wash 4 ~ 6 times, apple anvil
Batten, which takes out to put, sucks surface moisture content on aseptic filter paper.
4th embodiment, its step：Apple rootstock bar is put running water flowing water to rinse 35 minutes, apple rootstock bar is taken
Go out in the sterile beaker being put on super-clean bench, pour into 70% alcohol and sterilize 1 minute, outwell alcohol and add time that effective chlorine is 5%
Sodium chlorate solution, sterilize 10 minutes, during which shake 4 times, pour out sodium hypochlorite and washed 5 times with sterile, apple rootstock bar is taken out
Put and surface moisture content is sucked on aseptic filter paper.
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely retouched
State, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made,
Belong to the scope of protection of the invention.
A kind of tissue-culturing rapid propagation culture medium for apple rootstock, the present embodiment, first implementation are illustrated with reference to accompanying drawing
Example, includes bud primary culture medium, proliferated culture medium and root media.
In the present embodiment, the mg/L IBA+30 g/L sucrose+6g/L fine jades of bud primary culture medium MS+1 mg/L BA+0.1
In the blake bottle of cosmetics,
Proliferated culture medium:First proliferated culture medium：NM+0.1 mg/L BA+0.01 mg/L IBA+ 0.01 mg/L TDZ +
30 g/L sucrose+6g/L agar powders, the second proliferated culture medium：The g/L sucrose of MS+0.5 mg/L BA+0.1 mg/L IBA+ 30+
6g/L agar powders,
Root media:The g/L sucrose of 1/2MS+0.1mg/L IBA+20.
In the present embodiment, minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
NM culture mediums, composition are as follows：
| Inorganic constituents a great number of elements | Working concentration（mg/L） |
| NH4NO3 | 800 |
| KNO3 | 950 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 370 |
| Ca(NO3)2·4H2O | 600 |
| Inorganic constituents trace element | |
| EDTA-NaFe.3H2O | 84.2 |
| 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 |
| Organic principle | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water
Culture medium solidifying agent：Agar powder.
Second embodiment, in the present embodiment, the mg/L IBA+30 of bud primary culture medium MS+2 mg/L BA+0.5
In the blake bottle of g/L sucrose+6g/L agar powders,
Proliferated culture medium:First proliferated culture medium：NM+0.5 mg/L BA+0.1 mg/L IBA+0.1 mg/L TDZ + 30
G/L sucrose+6g/L agar powders, the second proliferated culture medium：The g/L sucrose of MS+1.2 mg/L BA+0.5 mg/L IBA+ 30+
6g/L agar powders,
Root media:The g/L sucrose of the mg/L of 1/2MS+2 IBA+20.
3rd embodiment, in the present embodiment, the bud primary culture medium MS+1 ~ mg/L of 2 mg/L BA+0.1 ~ 0.5
In the blake bottle of IBA+30 g/L sucrose+6g/L agar powders,
Proliferated culture medium:First proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+ 0.01~0.1
G/L sucrose+6g/L the agar powders of mg/L TDZ+30, the second proliferated culture medium：MS+0.5~1.2 mg/L BA+0.1~0.5
G/L sucrose+6g/L the agar powders of mg/L IBA+ 30,
Root media:The g/L sucrose of 1/2MS+0.1~2 mg/L IBA+20.
4th embodiment, in the present embodiment, the mg/L IBA+30 of bud primary culture medium MS+1.5 mg/L BA+0.3
In the blake bottle of g/L sucrose+6g/L agar powders,
Proliferated culture medium:First proliferated culture medium：NM+0.3 mg/L BA+0.05mg/L IBA+ 0.05 mg/L TDZ +
30 g/L sucrose+6g/L agar powders, the second proliferated culture medium：The g/L sucrose of MS+0.8 mg/L BA+0.3 mg/L IBA+ 30+
6g/L agar powders,
Root media:The g/L sucrose of the mg/L of 1/2MS+1 IBA+20.
A kind of preparation method of tissue-culturing rapid propagation culture medium for apple rootstock, one embodiment, its step are：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 45 ml bud primary culture medium, proliferated culture medium and life
Root culture medium, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 minutes under 121 DEG C, 1 atmospheric pressure.
Second embodiment, its step are：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 55 ml bud primary culture medium, proliferated culture medium and life
Root culture medium, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 minutes under 121 DEG C, 1 atmospheric pressure.
3rd embodiment, its step are：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 45 ~ 55 ml bud primary culture medium, proliferated culture medium
And root media, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 divides under 121 DEG C, 1 atmospheric pressure
Clock.
4th embodiment, its step are：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 50 ml bud primary culture medium, proliferated culture medium and life
Root culture medium, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 minutes under 121 DEG C, 1 atmospheric pressure.
The present invention has lower feature：
1st, due to devising bud primary culture medium, proliferated culture medium and root media, by bud primary culture medium, armpit is realized
Bud sprouts startup processing, by proliferated culture medium, realizes fast breeding and propagation and the two-step pretreatment of elongation growth, passes through life
Root culture medium, culture of rootage processing is realized, it is achieved that the tissue-culturing rapid propagation of apple rootstock；
2nd, due to devising bud primary culture medium, proliferated culture medium and root media, apple rootstock ' 62-396 ' is realized
Tissue-culturing rapid propagation；
3rd, due to devising proliferated culture medium, the combination of propagation growth and elongation growth is realized；
4th, the restriction of number range has been carried out to planform due to devising, it is in technical scheme to make number range
Technical characteristic, be not to be calculated by formula or the number range is shown by the technical characteristic that draws of limited number of time experiment, experiment
Technical characteristic achieve good technique effect；
5th, due to devising technical characteristic of the invention, in the effect of the independent and mutual set of technical characteristic, pass through
Experiment shows that property indices of the invention are at least 1.7 times for existing property indices, have very by assessing
Good market value.
Also others in the proliferated culture medium respectively containing NM and MS with being combined propagation and extending identical or phase
Approximate technical characteristic is all one of embodiments of the invention, and each technical characteristic of embodiment described above can be appointed
The combination of meaning, to meet Patent Law, patent regulation and the requirement of guidelines for examination, no longer to each skill in above-described embodiment
The embodiment of all possible combination of art feature is all described.
Above-described embodiment is the tissue culture and rapid propagation method for apple rootstock and application and culture medium provided by the present invention
And a kind of way of realization of the disinfection treatment method of preparation method, apple rootstock bar, according to its of scheme provided by the present invention
He deforms, and increases and either reduces composition therein or step or the present invention is used for into other technologies close with the present invention
Field, belong to protection scope of the present invention.
Claims (10)
1. a kind of tissue culture and rapid propagation method for apple rootstock, it is characterized in that：Its step：
In the bud primary culture medium containing MS, to bud section carry out axillary bud sprouting Primary culture obtain in vitro cuttings, containing
In NM the first proliferated culture medium, after carrying out fast breeding in vitro cuttings, then in the second proliferated culture medium containing MS
In, in vitro cuttings are bred and elongation growth obtains breeding test tube seedling, in the root media containing MS, to propagation
Test tube seedling carries out culture of rootage and obtains plantlet of taking root.
2. the tissue culture and rapid propagation method according to claim 1 for apple rootstock, it is characterized in that：Containing NM and MS respectively
Proliferated culture medium in be combined propagation and extend bud Primary culture processing and culture of rootage processing be principal character.
3. the tissue culture and rapid propagation method according to claim 1 for apple rootstock, it is characterized in that：Its step is：Cultivate bar
Part：Cultivation temperature is（25 ± 2）DEG C,
First, in vitro cuttings are established：
Clip gives birth to semi-lignified branch then in apple rootstock ' 62-396 ' scion, and branch is taken back experiment by polybag moisturizing
Room, blade is cut, petiole retains 0.3 ~ 0.6 cm, is cut into the bud section of one section of a bud, and bud section explant is first cleaned with washing powder water,
Then put running water flowing water and rinse and be more than 30 minutes, material is taken out in the sterile beaker being put on super-clean bench, pours into 70% alcohol
Sterilization 1 minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake 3 ~ 5 times, fall
Go out sodium hypochlorite to be washed 4 ~ 6 times with sterile, the taking-up of bud section is put surface moisture content is sucked on aseptic filter paper, bud section is inoculated into and is equipped with
The training of the g/L sucrose+6g/L agar powders of+0.1 ~ 0.5 mg/L IBA of bud primary culture medium+1 ~ 2 mg/L BA of MS+30
Support in bottle, blake bottle, which is put culturing room, carries out axillary bud sprouting Primary culture, and the green seedling that germination and growth obtains in primary culture medium is
In vitro cuttings；
2nd, test tube seedling proliferation：
In vitro cuttings are transferred to the first proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+
The fast breeding of bud is completed on the g/L sucrose+6g/L agar powders of 0.01 ~ 0.1 mg/L TDZ+30；It is then transferred into the second propagation
Culture medium：The increasing of bud is completed on the mg/L IBA+30 g/L sucrose+6g/L agar powders of mg/L BA+0.1 of MS+0.5 ~ 1.2 ~ 0.5
Grow and reach the propagation test tube seedling for the standard of taking root with elongation growth culture, acquisition；
3rd, rooting of vitro seedling：
Cut propagation test tube seedling of the growing height more than 1.5 centimetres and be transferred to root media:The mg/ of 1/2MS+0.1~2
Culture of rootage is carried out on L IBA+20 g/L sucrose, condition of culture is first continuous light culture 5 days, and it is small to then go to daily illumination 16
When periodicity of illumination condition of culture under cultivate, culture of rootage 5 ~ 6 weeks, obtain plantlet of being taken root in bottle；
4th, test tube transplantation of seedlings：
Before plantlet of taking root is removed outside bottle, blake bottle is removed from culturing room the place for being positioned over scattering natural lighting, hardening 3
~ 5 days, bottle cap is then opened, gravity-flow ventilation hardening 1 ~ 3 day, plantlet of taking root is taken out with tweezers, cleans the culture of root residual
Base, it is transplanted into equipped with seedling medium：According to part by weight, peat soil 4：Perlite 1：In the hole tray of vermiculite 1, with more than 0.1% after cultivation
The spraying spray of bacterium spirit solution is permeable, then with the good covered rearing with plastic film moisturizing of translucency, sprays 0.1% carbendazim every 3 ~ 5 days
Solution, treat that seedling has young leaves to grow and shows that seedling has grown new root, can now throw off film, survival and growth moves seedling in good time
Enter the seedling in field.
4. the tissue culture and rapid propagation method according to claim 3 for apple rootstock, it is characterized in that：Minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
NM culture mediums, composition are as follows：
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water culture medium solidifying agent：Agar powder.
5. a kind of tissue culture and rapid propagation method for apple rootstock is in apple rootstock ' 62-396 ' application.
6. a kind of disinfection treatment method of the apple rootstock bar of tissue culture and rapid propagation method for apple rootstock, its step：Apple
Stock bar, which is put running water flowing water and rinsed, to be more than 30 minutes, and apple rootstock bar is taken out in the sterile beaker being put on super-clean bench, fallen
Enter 70% alcohol to sterilize 1 minute, outwell alcohol and add the liquor natrii hypochloritis that effective chlorine is 5%, sterilize 10 minutes, during which shake
It is dynamic 3 ~ 5 times, pour out sodium hypochlorite and washed 4 ~ 6 times with sterile, the taking-up of apple rootstock bar is put surface moisture content is sucked on aseptic filter paper.
7. a kind of tissue-culturing rapid propagation culture medium for apple rootstock, include bud primary culture medium, proliferated culture medium and training of taking root
Support base.
8. the tissue-culturing rapid propagation culture medium according to claim 7 for apple rootstock, it is characterized in that：Bud primary culture medium MS
In the blake bottle of the mg/L IBA+30 g/L sucrose+6g/L agar powders of+1 ~ 2 mg/L BA+0.1 ~ 0.5,
Proliferated culture medium:First proliferated culture medium：NM+0.1~0.5 mg/L BA+0.01~0.1 mg/L IBA+ 0.01~0.1
Mg/L TDZ+30 g/L sucrose+6g/L agar powders, the second proliferated culture medium：MS+0.5~1.2 mg/L BA+0.1~0.5 mg/L
IBA+30 g/L sucrose+6g/L agar powders, root media:The mg/L IBA+20 g/L sucrose of 1/2MS+0.1~2.
9. the tissue-culturing rapid propagation culture medium according to claim 8 for apple rootstock, it is characterized in that：Minimal medium：
MS（Murashige and Skoog）Culture medium, composition are as follows：
NM culture mediums, composition are as follows：
Plant growth regulating substance：6- benzylaminopurines（BA）, Thidiazuron（TDZ）, indolebutyric acid（IBA）, gibberellin
（GA3）,
Carbon source：Sucrose
Deionized water
Culture medium solidifying agent：Agar powder.
10. a kind of preparation method of tissue-culturing rapid propagation culture medium for apple rootstock, its step are：
Proportionally constituent is mixed respectively composition bud primary culture medium, proliferated culture medium and root media, bud is opened
Dynamic culture medium, proliferated culture medium and root media adjust pH value to 5.8 before sterilization, bud primary culture medium, proliferated culture medium
It is dispensed into root media in 100 ml bottles, each bottle is respectively provided with 45 ~ 55 ml bud primary culture medium, proliferated culture medium
And root media, bottleneck is sealed with high pressure resistant high temperature plastic film, then autoclaving 20 divides under 121 DEG C, 1 atmospheric pressure
Clock.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108094215A (en) * | 2018-01-31 | 2018-06-01 | 山西省农业科学院果树研究所 | Dwarf Stocks For Apple Trees are by tissue cultures obtained from the method for root anvil seedling |
| CN108184462A (en) * | 2018-01-05 | 2018-06-22 | 湖北乡香茄生态农业有限公司 | A kind of open country eggplant sapling stock breeding method and its method for grafting fruits and vegetables |
| CN108243756A (en) * | 2018-03-20 | 2018-07-06 | 山东农业大学 | A kind of Tissue-cultured apple seedling bottle grafts the micro-grafting method taken root outside |
| CN108401907A (en) * | 2018-04-25 | 2018-08-17 | 河南省农业科学院 | A kind of Apples Dwarf Stocks M9T337 tissue cultures volume production method |
| CN108496803A (en) * | 2018-06-19 | 2018-09-07 | 山东省果树研究所 | The efficient rapid propagation method of tissue cultures and culture medium for excellent Dwarf Stocks For Apple Trees ' M9T337 ' |
| CN108782245A (en) * | 2018-06-19 | 2018-11-13 | 山东省果树研究所 | Tissue culture and rapid propagation method and culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple |
| CN108935100A (en) * | 2018-07-20 | 2018-12-07 | 河南农业大学 | A kind of method of apple rootstock T337 tissue-cultured seedling rooting induction |
| CN109042319A (en) * | 2018-07-20 | 2018-12-21 | 河南农业大学 | A method of preventing the browning of apple tissue culture and adventitious bud inducing |
| CN109892224A (en) * | 2018-12-24 | 2019-06-18 | 江苏东郁植物科技有限公司 | Apple rootstock " Sprout Free " tissue culture of sprout is commercialized mating system |
| CN112167063A (en) * | 2020-10-28 | 2021-01-05 | 宁夏神聚农业科技开发有限公司 | Method for cultivating double-detoxified apple seedlings |
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Cited By (13)
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| CN108184462A (en) * | 2018-01-05 | 2018-06-22 | 湖北乡香茄生态农业有限公司 | A kind of open country eggplant sapling stock breeding method and its method for grafting fruits and vegetables |
| CN108094215A (en) * | 2018-01-31 | 2018-06-01 | 山西省农业科学院果树研究所 | Dwarf Stocks For Apple Trees are by tissue cultures obtained from the method for root anvil seedling |
| CN108243756B (en) * | 2018-03-20 | 2020-04-14 | 山东农业大学 | Micro-grafting method for grafting and rooting outside apple tissue culture seedling bottle |
| CN108243756A (en) * | 2018-03-20 | 2018-07-06 | 山东农业大学 | A kind of Tissue-cultured apple seedling bottle grafts the micro-grafting method taken root outside |
| CN108401907A (en) * | 2018-04-25 | 2018-08-17 | 河南省农业科学院 | A kind of Apples Dwarf Stocks M9T337 tissue cultures volume production method |
| CN108782245A (en) * | 2018-06-19 | 2018-11-13 | 山东省果树研究所 | Tissue culture and rapid propagation method and culture medium for the anti-continuous cropping dwarfing rootstock G41 of apple |
| CN108496803A (en) * | 2018-06-19 | 2018-09-07 | 山东省果树研究所 | The efficient rapid propagation method of tissue cultures and culture medium for excellent Dwarf Stocks For Apple Trees ' M9T337 ' |
| CN108935100A (en) * | 2018-07-20 | 2018-12-07 | 河南农业大学 | A kind of method of apple rootstock T337 tissue-cultured seedling rooting induction |
| CN109042319A (en) * | 2018-07-20 | 2018-12-21 | 河南农业大学 | A method of preventing the browning of apple tissue culture and adventitious bud inducing |
| CN108935100B (en) * | 2018-07-20 | 2021-09-28 | 河南农业大学 | Apple rootstock T337 tissue culture seedling rooting induction method |
| CN109042319B (en) * | 2018-07-20 | 2021-10-19 | 河南农业大学 | Method for preventing tissue culture browning and adventitious bud induction of apples |
| CN109892224A (en) * | 2018-12-24 | 2019-06-18 | 江苏东郁植物科技有限公司 | Apple rootstock " Sprout Free " tissue culture of sprout is commercialized mating system |
| CN112167063A (en) * | 2020-10-28 | 2021-01-05 | 宁夏神聚农业科技开发有限公司 | Method for cultivating double-detoxified apple seedlings |
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