CN109362573A - A kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method - Google Patents
A kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method Download PDFInfo
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Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to technical field of tissue culture, especially a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method.It the following steps are included: the selection of explant, the disinfection of explant, initial Fiber differentiation, the quick breeding of callus, leaf induction differentiation culture, the induction of root differentiation culture and hardening and transplanting.The present invention is using prairie milk vetch seed as explant, pass through the different phase in Shoots of Prairie Milkvetch Under Tissue culture, match the culture medium with different formulations and ratio, mate-assist is carried out using needle-plate high voltage electric field, prairie milk vetch callus is set to carry out quickly breeding differentiation, the leaf differentiation rate and Furcation defects rate of prairie milk vetch callus are improved simultaneously, to finally improve the survival rate of Shoots of Prairie Milkvetch Under Tissue regeneration culture, it is formed using husky Moindawang seed as explant, the prairie milk vetch tissue culture regeneration that quickly breeding differentiation is carried out using needle-plate high voltage electric field auxiliary, improves the quality and yield of prairie milk vetch.
Description
Technical field
The present invention relates to technical field of tissue culture, especially a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method.
Background technique
Prairie milk vetch (Astragalus adsurgens Pall.), the upright Radix Astragali of alias are pulse family (Leguminosae) yellow
Stilbene category herbaceos perennial, also known as upright Radix Astragali, numb beans seedling.The nutritive value that prairie milk vetch makees feed is higher, can directly make horse,
The sizes livestock greenfeed such as ox, sheep, camel, pig, rabbit, may be made as ensiling, hay and fermented feed.Prairie milk vetch can also be direct
Base manure is made in green manuring, and top dressing is made in strange land green manuring, or makes heap with its stalk, makes compost.In addition, prairie milk vetch is checked winds and fixed drifting sand, ability is strong,
The serious area of the hazardss of sand storms such as the ancient riverbed of the Yellow River, plantation prairie milk vetch can reduce hazards of sand storms, protection fruit-bearing forest, prevent erosion and
Improve soil.Prairie milk vetch is the distinctive tame forage grass in China, green manure, adaptable, the spies such as has drought resisting, impoverishment tolerant, fixes the sand
Point is the important species of water and soil conservation, restorated sandlot wasteland.
In recent years, the demand of the improvement of the ecological environment in the Inner Mongol or even the whole nation and poultry grassland industry development to prairie milk vetch constantly increases
Greatly, but since the breeding time of husky Moindawang is long and has the habit of white flower pollination and cross-pollination concurrently, its florescence is caused not concentrated, finally
The poor quality of seed is caused, yield is lower, directly affects it and promotes and apply.Callus is raw in traditional tissue culture mode
Long speed is slower, easy browning, and differentiation rate is lower.
Therefore, it is necessary to propose improvement project to existing husky Moindawang tissue cultures mode, foundation is with husky Moindawang seed
Explant carries out the prairie milk vetch tissue culture regeneration of fast numerous differentiation using needle-plate high voltage electric field auxiliary.
Summary of the invention
In view of the deficiency of the prior art, the purpose of the present invention is to provide one kind using seed as explant, benefit
The Shoots of Prairie Milkvetch Under Tissue culture regeneration method of fast numerous differentiation is carried out, with needle-plate high voltage electric field auxiliary to improve the numerous of Shoots of Prairie Milkvetch Under Tissue
It grows speed and yield, it is promoted to play a greater role in national improvement of the ecological environment and poultry grassland industry development and provide technical support.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method, it the following steps are included:
1) selection of explant: seed of the selection without small holes caused by worms mature and plump is explant；
2) it the disinfection of explant: is rinsed with water and is placed in the triangular flask by disinfection, alcohol is added in triangular flask
It carries out low-speed oscillation and sterilizes 10s or more, be then filtered dry alcohol, be subsequently added into mercuric chloride solution low-speed oscillation disinfection 5min or more, with
After filter mercuric chloride solution, then with aseptic water washing 3-5 times；
3) initial Fiber differentiation: initial medium are as follows: containing 0.5-2.0mg/L 2,4-D, 0.5-2.0mg/L6-BA and
The MS culture medium of 0.1-0.5mg/L NAA is placed 48 hours or more after sterilizing initial medium, then the initial training after disinfection
Feeding base is placed in triangular flask, then the seed after rigid disinfection is inoculated in initial medium, and the bottleneck of triangular flask is used fungi-proofing
Ventilated membrane is closed, and 25-28 degrees Celsius of cultivation temperature, using dark culture, until seed sprouts sprouting out；
4) the quick breeding of callus: by callus stripping and slicing and moving into culture dish, and culture dish is put into height after removing lid
In piezoelectric field, the average field-strength of high voltage electric field is 0.9-1.1kV/cm, and the processing time is 30min or more；
It is transferred to after high voltage electric field is handled on callus tissue culture base and carries out squamous subculture, 25-28 degrees Celsius of cultivation temperature,
It is cultivated using light dark interaction, periodicity of illumination 12-16h/d, intensity of illumination 2500-3000lx, callus tissue culture base are as follows:
Containing 0.3-1.0mg/L 2, the MS culture medium of 4-D, 0.5-1.5mg/L 6-BA and 0.1-0.5mg/L NAA.
Preferably, the initial medium are as follows: containing 1.0mg/L 2,4-D, 1.0mg/L 6-BA and 0.2mg/L NAA's
MS culture medium.
Preferably, the callus tissue culture base are as follows: contain 0.5mg/L 2,4-D, 1.0mg/L 6-BA and 0.2mg/L
The MS culture medium of NAA.
Preferably, the high voltage electric field in the step 4) is needle-plate high voltage electric field, and the needle-plate high voltage electric field is using upper
Extremely connect the high pressure stylus of ac high-voltage and under be extremely grounded plate.
Preferably, it further includes the induction differentiation culture of step 5) leaf: the callus of above-mentioned squamous subculture is put into height
It is handled in piezoelectric field, average field-strength 1.4-1.6kV/cm, the processing time is 30min or more；It is transferred to after high voltage electric field is handled
It cultivates, 25-28 degrees Celsius of cultivation temperature, is cultivated using light dark interaction, periodicity of illumination 12-16h/d, light on leave culture medium
It is 2500-3000lx, leave culture medium are as follows: contain 1.5-2.5mg/L 6-BA, 0.5-1.0mg/L NAA, 0.5- according to intensity
The MS culture medium of 1.5mg/L KT and 0.1-0.5mg/L IBA.
Preferably, the leave culture medium are as follows: containing 2.0mg/L 6-BA, 0.5mg/L NAA, 1.0mg/L KT and
The MS culture medium of 0.2mg/L IBA, the high voltage electric field in the step 5) are needle-plate high voltage electric field.
Preferably, it further include step 6) root induction differentiation culture: by it is above-mentioned through leaf induction differentiation culture after more
Injured tissue is put into needle-plate high voltage electric field, average field-strength 1.4-1.6kV/cm, and the processing time is 30min or more；Through high-voltage electricity
It is transferred to root media culture after the processing of field, 25-28 degrees Celsius of cultivation temperature, is cultivated using light dark interaction, periodicity of illumination is
12-16h/d, intensity of illumination 2500-3000lx, root media are as follows: contain 0.5-1.0mg/L NAA and 0.5-1.5mg/L
The MS culture medium of BAT.
Preferably, the root media are as follows: the MS culture medium containing 0.5mg/L NAA and 1.0mg/L BAT.
Preferably, it further includes step 7) hardening and transplanting: the tissue-cultured seedling growth formed after the induction differentiation culture of root
When to 6-8cm, sterile ventilated membrane hardening 7 days or more on triangular flask are opened, is transplanted in Flower nutrient soil after hardening and normally trains
It supports, watering in every 6-7 days is primary after transplanting.
Preferably, it includes a superclean bench, and the high pressure stylus includes top crown and the following table that top crown is arranged in
It face and is all set in superclean bench in the electric discharge needle body that is spaced apart, the top crown and ground connection plate and in contraposition up and down
Distribution, the top crown are connected with an ac high-voltage module, and the ac high-voltage module is controlled by a central control module, described
Central control module for control ac high-voltage module to top crown convey ac high-voltage with electric discharge needle body and ground connection plate it
Between formed needle-plate high voltage electric field.
As the above scheme is adopted, the present invention is using prairie milk vetch seed as explant, by Shoots of Prairie Milkvetch Under Tissue culture
Different phase matches the culture medium with different formulations and ratio, carries out mate-assist using needle-plate high voltage electric field, makes husky beat
Prosperous callus carries out quickly breeding differentiation, while improving the leaf differentiation rate and Furcation defects rate of prairie milk vetch callus, thus most
The survival rate for improving Shoots of Prairie Milkvetch Under Tissue regeneration culture eventually is formed using husky Moindawang seed as explant, auxiliary using needle-plate high voltage electric field
The prairie milk vetch tissue culture regeneration for carrying out quickly breeding differentiation is helped, the reproduction speed and yield of prairie milk vetch are improved, is had very strong
Practical value and market popularization value.
Detailed description of the invention
Fig. 1 is needle-plate high voltage electric field structural schematic diagram of the embodiment of the present invention；
Fig. 2 be the embodiment of the present invention different electric field treatments under prairie milk vetch callus growth rate；
Fig. 3 be the embodiment of the present invention different electric field treatments under prairie milk vetch callus leaf differentiation rate；
Fig. 4 be the embodiment of the present invention different electric field treatments under prairie milk vetch callus Furcation defects rate.
Specific embodiment
The embodiment of the present invention is described in detail below in conjunction with attached drawing, but the present invention can be defined by the claims
Implement with the multitude of different ways of covering.
As shown in Figures 1 to 4, a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method provided in an embodiment of the present invention, it include with
Lower step:
1) selection of explant: choosing (about 100), seed without small holes caused by worms mature and plump is explant.
2) disinfection of explant: being rinsed 3 times with tap water, is rinsed and is placed on what passing through in superclean bench 3 sterilized
In the triangular flask of 50ml, in triangular flask be added 70% alcohol, and on compact scroll vortex mixer low-speed oscillation disinfection 10s with
On, it is filtered dry alcohol with gauze, is subsequently added into 0.1% mercuric chloride solution, and low-speed oscillation sterilizes on compact scroll vortex mixer
5min or more, this low-speed oscillation disinfection, which can utterly destroy bacterium of seed coat etc., influences the substance of tissue culture, and can contract
Short disinfecting time reduces neutron absorption moisture；After filter mercuric chloride solution, and low speed shakes on compact scroll vortex mixer with sterile water
Swing flushing 3-5 times；For prairie milk vetch seed, centainly imbibition cannot be impregnated with sterile water deionized water, otherwise seed can be inhibited to send out
Bud, leading to the inductivity of callus is 0.
3) initial Fiber differentiation: the seed after rigid disinfection is inoculated in the initial medium for placing 48h or more after disinfection,
25-28 degrees Celsius of cultivation temperature, using dark culture, until seed sprouts sprouting out；Initial medium are as follows: contain 0.5-2.0mg/
The 2 of L, the MS culture medium of the NAA of the 6-BA and 0.1-0.5mg/L of 4-D, 0.5-2.0mg/L, preferably, initial medium are as follows:
The MS culture medium of the NAA of the 6-BA and 0.2mg/L of 2,4-D, 1.0mg/L containing 1.0mg/L；
Initial medium is placed in 50ml triangular flask after disinfection, and 3-5 seeds are inoculated in each triangular flask, and a batch connects
About 20 bottles of kind, the bottleneck of each triangular flask is closed with fungi-proofing ventilated membrane, and fungi-proofing ventilated membrane can guarantee in sterile air duct slats
It is conducive to the sprouting of seed under part, after seed is sprouted, hypocotyl starts gradually to expand, and 14d can form callus, and healing rate is
97%, and can be used for squamous subculture；
48h or more wherein is placed after initial medium disinfection, moisture is evaporated in the initial medium after can making disinfection,
If there are also excessive moistures on the surface of initial medium, can directly be outwelled in inoculation, initial medium moisture content height can inhibit
Prairie milk vetch germination；2,4-D (i.e. 2,4- dichlorphenoxyacetic acids) belong to auxins plant growth regulator substance, 6-BA
(i.e. 6- benzyl aminoadenine) belongs to cytokinin-like substance, and NAA (α-naphthylacetic acid) belongs to auxin plant growth regulator class object
Matter, 2,4-D are conducive to the induction of embryo callus, but high concentration 2 is used alone, and 4-D can promote callus browning, and
Callus is caused to be unable to normal growth and differentiation, the present invention utilizes 2,4-D, 6-BA, NAA3 kind auxins and cell division
Plain substance various concentration is used cooperatively, and faint yellow, graininess callus ratio (90% or more) occurs than being used alone
The ratio (48%) of 2,4-D is high.
4) the quick breeding of callus: by callus stripping and slicing, size is about 0.8cm × 0.8cm × 0.8cm small
Block moves into the disposable plastic sterile petri dish that diameter is 9cm, and each culture dish can lay flat about 30 pieces of callus, culture
Ware is put into high voltage electric field after removing lid, average field-strength 1kV/cm, and the processing time is 30min；
It is transferred to after high voltage electric field is handled on callus tissue culture base and carries out squamous subculture, callus tissue culture base are as follows: contain
There are 2 of 0.3-1.0mg/L, the MS culture medium of the NAA of the 6-BA and 0.1-0.5mg/L of 4-D, 0.5-1.5mg/L, preferably,
Callus tissue culture base are as follows: 2 containing 0.5mg/L, the MS culture medium of the NAA of the 6-BA and 0.2mg/L of 4-D, 1.0mg/L；Training
26 degrees Celsius of temperature is supported, is cultivated using light dark interaction, periodicity of illumination 14h/d, intensity of illumination 2500-3000lx, to promote
The quick division and reproduction of callus；
High voltage electric field includes ordinary high pressure electric field and needle-plate high voltage electric field, and wherein needle-plate high voltage electric field is extremely connect using upper
The high pressure stylus of ac high-voltage, under be extremely grounded the ac high-voltage electric field that plate, pole span 2cm, average field-strength are 1kV/cm, such as
Shown in Fig. 2, by the processing of high voltage electric field, the growth rate of callus can be obviously increased, after ordinary high pressure electric field treatment
Growth rate improves 20% than the control group of electric field treatment is not added, and growth rate is than not powered after needle-plate high voltage electric field processing
The control group of field processing improves 48%, and the control group than ordinary high pressure electric field treatment improves 26%, and high voltage electric field processing can
Mitigate callus browning degree.
5) culture is broken up in the induction of leaf:
The callus of above-mentioned squamous subculture is put into high voltage electric field and is handled, average field-strength 1.5kV/cm, when processing
Between be 30min or more；It is transferred to after high voltage electric field is handled on leave culture medium and carries out leave differentiation culture, leave culture medium are as follows:
The MS of the IBA of the KT and 0.1-0.5mg/L of NAA, 0.5-1.5mg/L of 6-BA, 0.5-1.0mg/L containing 1.5-2.5mg/L
Culture medium, preferably, leave culture medium are as follows: the KT of NAA, 1.0mg/L of 6-BA, 0.5mg/L containing 2.0mg/L and
On the MS culture medium of the IBA of 0.2mg/L, 26 degrees Celsius of cultivation temperature, cultivated using light dark interaction, periodicity of illumination 14h/d,
Intensity of illumination is 2500-3000lx, to carry out evoked callus budding, promotes the differential growth of leaf, can grow sand after about 20d
Beat prosperous blade；Wherein, KT (kinetin, 6- furfuryl group adenine) belongs to cytokinin-like substance, and IBA (indolebutyric acid) belongs to auxin
Plant growth regulator substance, as shown in figure 3, the differential growth that can promote leaf improves leaf by the processing of high voltage electric field
Differentiation rate, control group of the differentiation rate of leaf than electric field treatment is not added is passed through compared to improving about 15% after ordinary high pressure electric field treatment
The differentiation rate of leaf improves about 40% than the control group of electric field treatment is not added after needle-plate high voltage electric field processing, than ordinary high pressure electric field
The control group of processing improves about 22%.
6) culture is broken up in the induction of root:
The above-mentioned callus after the induction differentiation culture of leaf is put into needle-plate high voltage electric field, average field-strength 1.5kV/
Cm, processing time are 30min or more；It is transferred to root media after high voltage electric field is handled and carries out differentiation culture of taking root, training of taking root
Support base are as follows: the MS culture medium of the BAT (root-inducing powder) of NAA and 0.5-1.5mg/L containing 0.5-1.0mg/L, preferably, taking root
Culture medium are as follows: the MS culture medium of the BAT of NAA and 1.0mg/L containing 0.5mg/L, 26 degrees Celsius of cultivation temperature, using light dark
Interaction culture, periodicity of illumination 14h/d, intensity of illumination 2500-3000lx, to promote the differential growth of root；As shown in figure 4,
The differentiation rate of root reduces about 7% than the control group of electric field treatment is not added instead after ordinary high pressure electric field treatment, high through needle-plate
The differentiation rate of root improves 50% than the control group of electric field treatment is not added after piezoelectric field processing, than the control of ordinary high pressure electric field treatment
Group improves about 57%.
7) hardening and transplanting
When the tissue-cultured seedling formed after the induction differentiation culture of root grows to 6-8cm, open sterile ventilative on triangular flask
It film hardening 7 days or more, is transplanted in Flower nutrient soil after hardening and normally cultivates, watering in every 6-7 days is primary in transplanting, pays attention to pouring
It cannot be irrigated when water, survival rate is up to 95% after transplanting.
Further, as shown in Figure 1, the Shoots of Prairie Milkvetch Under Tissue culture regeneration method of the present embodiment further includes a superclean bench
3, high pressure stylus includes top crown 1 and the lower surface that top crown 1 is arranged in and is in the electric discharge needle body 11 being spaced apart, top crown 1
It is all set in superclean bench 3 with ground connection plate 2 and is connected with an ac high-voltage module in contraposition distribution, top crown 1 up and down
4, ground connection plate 2 is grounded by an ammeter 6, and ac high-voltage module 4 is controlled by a central control module 5, central control module 5
Ac high-voltage is conveyed to be formed between electric discharge needle body 11 and ground connection plate 2 to top crown 1 for controlling ac high-voltage module 4
Needle-plate high voltage electric field.Needle-plate high voltage electric field mesohigh electrode is needle, and needle spacing is adjustable, and needle spacing is 4cm in the present embodiment, is connect
Ground extremely plane copper plate, pole span is continuously adjustable, and pole span is 2cm in the present embodiment, and high voltage power supply exports 50Hz alternating current, voltage 0-
50kV is continuously adjustable.Needle used in the present embodiment-plate high voltage electric field and unconventional point discharge electric field, traditional point discharge
Electric field refers to a kind of inhomogeneous field that can generate localized corona electric discharge, and power line is most in inhomogeneous field, at the tip of electrode
It concentrates, electric field strength is also maximum, after adding high pressure, since free of air can generate localized corona electric discharge near electrode, this
Needle used in embodiment-plate high voltage electric field is by non-homogeneous high voltage electric field, corona discharge, plasma injection, secondary electron
A variety of physical factor synergistic effects such as transmitting generate facilitation to the growth and differentiation of callus.Used in the present embodiment
Needle-plate high voltage electric field is different from ordinary high pressure electric field, and bottom crown 2 is plate parallel to each other, and high pressure on ordinary high pressure electric field
Power supply is DC high-voltage power supply.The electric field that this common high voltage electric field device generates is uniform electric field, and will not corona put
Electricity does not have plasma active species, acts on for electric field single factors.So promoting prairie milk vetch callus growth and dividing
It is obvious not as good as needle plate high voltage electric field effect to change aspect.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
Equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content is applied directly or indirectly in other correlations
Technical field, be included within the scope of the present invention.
Claims (10)
1. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method, it is characterised in that: it the following steps are included:
1) selection of explant: seed of the selection without small holes caused by worms mature and plump is explant；
2) disinfection of explant: being rinsed with water and be placed in the triangular flask by disinfection, and alcohol is added in triangular flask and carries out
Low-speed oscillation sterilizes 10s or more, is then filtered dry alcohol, is subsequently added into mercuric chloride solution low-speed oscillation disinfection 5min or more, then filters
Fall mercuric chloride solution, then with aseptic water washing 3-5 times；
3) initial Fiber differentiation: initial medium are as follows: contain 0.5-2.0mg/L 2,4-D, 0.5-2.0mg/L 6-BA and 0.1-
The MS culture medium of 0.5mg/L NAA is placed 48 hours or more after sterilizing initial medium, then the initial medium after disinfection
It is placed in triangular flask, then the seed after rigid disinfection is inoculated in initial medium, the bottleneck of triangular flask is with fungi-proofing ventilative
Film is closed, and 25-28 degrees Celsius of cultivation temperature, using dark culture, until seed sprouts sprouting out；
4) the quick breeding of callus: by callus stripping and slicing and moving into culture dish, and culture dish is put into high-voltage electricity after removing lid
In, the average field-strength of high voltage electric field is 0.9-1.1kV/cm, and the processing time is 30min or more；
It is transferred to after high voltage electric field is handled on callus tissue culture base and carries out squamous subculture, 25-28 degrees Celsius of cultivation temperature, used
Light dark interaction culture, periodicity of illumination 12-16h/d, intensity of illumination 2500-3000lx, callus tissue culture base are as follows: contain
The MS culture medium of 0.3-1.0mg/L 2,4-D, 0.5-1.5mg/L 6-BA and 0.1-0.5mg/L NAA.
2. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as described in claim 1, it is characterised in that: the initial medium
Are as follows: the MS culture medium containing 1.0mg/L 2,4-D, 1.0mg/L 6-BA and 0.2mg/L NAA.
3. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as described in claim 1, it is characterised in that: the callus tissue culture
Base are as follows: the MS culture medium containing 0.5mg/L 2,4-D, 1.0mg/L 6-BA and 0.2mg/L NAA.
4. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as described in claim 1, it is characterised in that: the height in the step 4)
Piezoelectric field be needle-plate high voltage electric field, the needle-plate high voltage electric field using the upper high pressure stylus for extremely connecing ac high-voltage and under extremely
It is grounded plate.
5. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as claimed in claim 4, it is characterised in that: it further includes step 5) leaf
Induction break up culture: the callus of above-mentioned squamous subculture is put into high voltage electric field and is handled, average field-strength 1.4-
1.6kV/cm, processing time are 30min or more；It is transferred on leave culture medium and cultivates after high voltage electric field is handled, cultivation temperature 25-
It 28 degrees Celsius, is cultivated using light dark interaction, periodicity of illumination 12-16h/d, intensity of illumination 2500-3000lx, leave culture
Base are as follows: the MS containing 1.5-2.5mg/L 6-BA, 0.5-1.0mg/L NAA, 0.5-1.5mg/L KT and 0.1-0.5mg/L IBA
Culture medium.
6. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as claimed in claim 5, it is characterised in that: the leave culture medium
Are as follows: the MS culture medium containing 2.0mg/L 6-BA, 0.5mg/L NAA, 1.0mg/L KT and 0.2mg/L IBA, the step 5)
In high voltage electric field be needle-plate high voltage electric field.
7. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as claimed in claim 5, it is characterised in that: it further includes step 6) root
Induction break up culture: by it is above-mentioned through leaf induction differentiation culture after callus be put into needle-plate high voltage electric field, mean field
It is by force 1.4-1.6kV/cm, the processing time is 30min or more；It is transferred to root media culture after high voltage electric field is handled, cultivates
It 25-28 degrees Celsius of temperature, is cultivated using light dark interaction, periodicity of illumination 12-16h/d, intensity of illumination 2500-3000lx are raw
Root culture medium are as follows: the MS culture medium containing 0.5-1.0mg/L NAA and 0.5-1.5mg/L BAT.
8. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as claimed in claim 7, it is characterised in that: the root media
Are as follows: the MS culture medium containing 0.5mg/L NAA and 1.0mg/L BAT.
9. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as claimed in claim 7, it is characterised in that: it further includes step 7) refining
Seedling and transplanting: it when the tissue-cultured seedling formed after the induction differentiation culture of root grows to 6-8cm, opens sterile ventilative on triangular flask
It film hardening 7 days or more, is transplanted in Flower nutrient soil after hardening and normally cultivates, watering in every 6-7 days is primary after transplanting.
10. a kind of Shoots of Prairie Milkvetch Under Tissue culture regeneration method as described in any one of claim 4-7, it is characterised in that: it is wrapped
A superclean bench is included, the high pressure stylus includes top crown and the lower surface that top crown is arranged in and is in the electric discharge being spaced apart
Needle body, the top crown and ground connection plate are all set in superclean bench and in contraposition distribution up and down, the top crown connections
There is an ac high-voltage module, the ac high-voltage module is controlled by a central control module, and the central control module is for controlling
Ac high-voltage module processed conveys ac high-voltage to top crown to form needle-plate high-voltage electricity between electric discharge needle body and ground connection plate
?.
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| CN110583145A (en) * | 2019-09-29 | 2019-12-20 | 内蒙古工业大学 | Method for improving hydrophilicity of leguminous plant seeds |
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