CN107980632A - A kind of method of mongolian amygdalus seed tissue-culturing rapid propagation - Google Patents
A kind of method of mongolian amygdalus seed tissue-culturing rapid propagation Download PDFInfo
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- CN107980632A CN107980632A CN201711260542.2A CN201711260542A CN107980632A CN 107980632 A CN107980632 A CN 107980632A CN 201711260542 A CN201711260542 A CN 201711260542A CN 107980632 A CN107980632 A CN 107980632A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention provides a kind of method of mongolian amygdalus seed tissue-culturing rapid propagation, comprise the following steps：1) by Prunus Mongolica seeds successively by circulating water flushing, alcohol soaking disinfection, HgCl2Disinfection seed is obtained after solution soaking disinfection and aseptic water washing；2) culture of 1/2MS solid mediums is inoculated in after the disinfection seed is removed the peel and obtains aseptic seedling within 15~20 days；3) stem section with axil is obtained from the aseptic seedling to be inoculated in cultivate 15~20 days in axillary bud deriving culture medium and obtain axillary bud；4) axillary bud that step 3) obtains is inoculated in root media to cultivate 15~20 days and obtains tissue-cultured seedling.The method of the invention pollution rate is zero, and breeding efficiency is high, and the used time is short, and breeding coefficient is high.
Description
Technical field
The invention belongs to plant tissue culture technical field, and in particular to a kind of method of mongolian amygdalus seed tissue-culturing rapid propagation.
Background technology
Mongolian amygdalus seed (Amygdalusmongolica), also known as Ulan-cotton clothes Le Si, downy cherry fruit, Rosales rare species, belong to
It is 1 to 2 meter high in machaka.Mongolian amygdalus seed is light property seeds, and well developed root system is drought-enduring, cold-resistant, barren-resistant；Florescence is 4 to 5
Month, it is 7 to August that fruit is ripe.Mongolian amygdalus seed is the distinctive xerophytic deciduous shrubs in the Gobi desert region, is the scape of Desert Regions and desert steppe
Plant and soil-and-water conservation effect, and important woody oil tree species are seen, have been confirmed as Chinese Second Class Key Protected Plant.
The breeding main method of mongolian amygdalus seed includes seminal propagation, branch cutting, propagation by grafiting at present, and time-consuming, efficiency
It is low.And the Explants In Tissue Culture of mongolian amygdalus seed be using the tender stem segments of mongolian amygdalus seed seedling as explant, it is right in practical operation
Explant disinfection requires high and pollution rate, and higher (such as Geng Jun etc. is published in for 2007《Northwest agricultural journal》Article《Mongolia
The foundation of almond stem-segment with single bud cultivating system》China and foreign countries' implant body pollution rate is 18.75%), breeding coefficient is low, easily causes manpower, thing
The significant wastage of power and financial resources.
The content of the invention
In view of this, it is an object of the invention to provide the mongolian amygdalus seed tissue culture that a kind of pollution rate is low, breeding coefficient is high is fast
Numerous method.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：A kind of side of mongolian amygdalus seed tissue-culturing rapid propagation
Method, comprises the following steps：1) by Prunus Mongolica seeds successively by circulating water flushing, alcohol soaking disinfection, HgCl2Soaking disinfection
With disinfection seed is obtained after aseptic water washing；2) 1/2MS solid mediums culture 15 is inoculated in after the disinfection seed is removed the peel
Obtain aseptic seedling within~20 days；3) stem section of the acquisition with axil is inoculated in axillary bud deriving culture medium and cultivates 15 from the aseptic seedling
Obtain axillary bud within~20 days；4) axillary bud that step 3) obtains is inoculated in root media to cultivate 15~20 days and obtains tissue-cultured seedling.
Preferably, the time of step 1) the alcohol soaking disinfection is 5~15min.
Preferably, the step 1) HgCl2Mass fraction be 0.05~0.15%.
Preferably, the HgCl2The time of soaking disinfection is 8~12min.
Preferably, the 1/2MS solid mediums described in step 2) include sucrose and agar；The sucrose is in 1/2MS
Concentration in solid medium is 12~18g/L, and concentration of the agar in 1/2MS solid mediums is 5~10g/L；Institute
The pH value for stating 1/2MS solid mediums is 5.7~5.9.
Preferably, step 3) the axillary bud deriving culture medium is the MS culture mediums for including 6-BA and IBA.
Preferably, concentration of the 6-BA in axillary bud deriving culture medium is 0.8~1.2mg/L；The IBA is lured in axillary bud
It is 2.5~3.5mg/L to lead the concentration in culture medium.
Preferably, sucrose and agar are further included in the axillary bud deriving culture medium；The sucrose is in axillary bud deriving culture medium
In concentration be 12~18g/L, concentration of the agar in axillary bud deriving culture medium is 5~10g/L；The axillary bud deriving training
The pH value for supporting base is 5.7~5.9.
Preferably, the root media described in step 4) is the 1/3MS culture mediums of addition IBA；The IBA is in training of taking root
The concentration supported in base is 0.8~1.2mg/L.
Preferably, the step 2), 3) and 4) in cultivation temperature stand alone as 24~26 DEG C.
Beneficial effects of the present invention：The method of mongolian amygdalus seed tissue-culturing rapid propagation of the present invention gathers explant from aseptic seedling
Body, overcomes using the high situation of seedling explant pollution rate, pollution rate zero, so as to save seedling cost, improves
Breeding efficiency.Record that method used time of the present invention is short, and breeding coefficient is high according to embodiment, can breed within 45~55 days it is a collection of again
Raw tissue-cultured seedling, breeding coefficient are 3~5.
Embodiment
The present invention provides a kind of method of mongolian amygdalus seed tissue-culturing rapid propagation, comprise the following steps：1) by Prunus Mongolica seeds
Successively by circulating water flushing, alcohol soaking disinfection, HgCl2Disinfection seed is obtained after soaking disinfection and aseptic water washing；2) will
The culture of 1/2MS solid mediums, which is inoculated in, after the disinfection seed peeling obtains aseptic seedling within 15~20 days；3) from the aseptic seedling
Upper stem section of the acquisition with axil, which is inoculated in axillary bud deriving culture medium to cultivate 15~20 days, obtains axillary bud；4) step 3) is obtained
Axillary bud, which is inoculated in root media to cultivate 15~20 days, obtains tissue-cultured seedling.
In an embodiment of the present invention, the Prunus Mongolica seeds can be struck the kind shell of mongolian amygdalus seed using artificial method
Open, take out Prunus Mongolica seeds, ensure that Prunus Mongolica seeds stand intact.
After obtaining Prunus Mongolica seeds, the present invention rinses Prunus Mongolica seeds by circulating water successively, alcohol immersion disappears
Poison, HgCl2Soaking disinfection and aseptic water washing, obtain disinfection seed.In the present invention, the time that the circulating water rinses is preferable
For 8~12min, more preferably 10min；The purpose that the circulating water rinses is to remove seed coat surface debris.
Prunus Mongolica seeds of the present invention are after circulating water rinses, with alcohol soaking disinfection；The volume of the alcohol is dense
Degree preferably 70~80%, more preferably 75%；The time of the alcohol soaking disinfection is preferably 5~15min, more excellent
Choosing for 8~12min, most preferably 10min.
After Prunus Mongolica seeds alcohol soaking disinfection of the present invention, HgCl is used2Solution soaking disinfection, the HgCl2Matter
Amount fraction is preferably 0.05~0.15%, more preferably 0.08~0.12%, most preferably 0.1%；The HgCl2Leaching
The time of bubble disinfection is preferably 8~12min, more preferably 10min；The HgCl2The purpose of soaking disinfection is to seed
Depth disinfection is carried out, ensures the state of the surface of the seed integral asepsis.
Prunus Mongolica seeds of the present invention are in the HgCl2After soaking disinfection, it is rinsed with sterile water；It is described sterile
The number that water rinses is preferably 4~6 times, more preferably 5 times, and time of each aseptic water washing is preferably 0.5~
2min, more preferably 1min；The purpose of aseptic water washing of the present invention is to remove Prunus Mongolica seeds remained on surface
HgCl2.The present invention preferably blots the surface of the seed moisture with aseptic filter paper after the aseptic water washing and obtains disinfection seed.
The present invention is inoculated in 1/2MS solid medium cultures after disinfection seed is obtained after the disinfection seed is removed the peel
15~20 days, obtain aseptic seedling.The disinfection seed peeling uses artificial method in the present invention, specifically in implementation process
It is middle to be removed the kind skin of the disinfection seed with aseptic nipper.In the present invention after the disinfection seed peeling, by the peeling
Disinfection seed afterwards is inoculated in 1/2MS solid mediums and cultivates.In the present invention, the inoculation is preferably：Mongolia is flat
0.3~0.7cm of culture medium is submerged downwards in the kind hole of peach seeds, more preferably 0.5cm.
The 1/2MS solid mediums are that the concentration of component in regular MS media is halved acquisition in the present invention；Institute
State in 1/2MS solid mediums and further include sucrose and agar；Concentration of the sucrose in 1/2MS solid mediums is preferably
12~18g/L, more preferably 15g/L；Concentration of the agar in 1/2MS solid mediums is preferably 5~10g/L,
More preferably 8g/L；The pH value of the 1/2MS solid mediums is preferably 5.7~5.9 in the present invention, more preferably
5.8。
The time for cultivating the aseptic seedling on 1/2MS solid mediums in the present invention is 15~20 days, preferably
16~18 days；The temperature that the aseptic seedling is cultivated in the present invention is preferably 24~26 DEG C, more preferably 25 DEG C；Described in culture
The illumination condition of aseptic seedling is preferably provided by tissue culture dedicated spectral lamp.The aseptic seedling that present invention culture obtains is highly preferred
For 3~6cm, more preferably 5cm, the number of blade of the aseptic seedling are preferably 20~25, the axillary bud of the aseptic seedling
Do not grow.
The present invention is after culture obtains aseptic seedling, and stem section of the acquisition with axil is inoculated in axillary bud deriving from the aseptic seedling
Cultivated in culture medium 15~20 days and obtain axillary bud.In the present invention preferably from stem section of the clip with axil in the aseptic seedling；
Preferable 1.5~the 2.5cm of stem section length with axil, more preferably 2cm.The axillary bud deriving culture in the present invention
Base is preferably the MS culture mediums for adding 6-BA and IBA.The MS culture mediums are conventional minimal medium, are being embodied
Using commercially available or homemade MS culture mediums in journey；Concentration of the 6-BA in axillary bud deriving culture medium is preferable in the present invention
For 0.8~1.2mg/L, more preferably 1.0mg/L；Concentration of the IBA in axillary bud deriving culture medium is preferably 2.5~
3.5mg/L, more preferably 3.0mg/L.
In the present invention sucrose and agar are preferably further included in the axillary bud deriving culture medium；The sucrose is lured in axillary bud
The concentration led in culture medium is preferably 12~18g/L, more preferably 15g/L；The agar is in axillary bud deriving culture medium
Concentration be preferably 5~10g/L, more preferably 8g/L；In the present invention, the pH value of the axillary bud deriving culture medium is preferred
For 5.7~5.9, more preferably 5.8.
The time of axillary bud deriving culture in the present invention is 15~20 days, preferably 16~18 days；Axillary bud in the present invention
The temperature of Fiber differentiation is preferably 24~26 DEG C, more preferably 25 DEG C；The illumination condition of the axillary bud deriving culture is preferred
By tissue culture dedicated spectral lamp provide.Preferably grow 2~3 axillary buds in the present invention after axillary bud deriving culture at axil,
The length of the axillary bud is preferably 2~4cm, more preferably 3cm.
The axillary bud of acquisition is inoculated in root media after axillary bud is obtained and cultivates 15~20 days acquisition tissue cultures by the present invention
Seedling.The root media is the 1/3MS culture mediums of addition IBA in the present invention；In the present invention, the 1/3MS culture mediums
For conventional MS nutrient media components concentration is kept to 1/3 acquisition；Concentration of the IBA in root media is excellent in the present invention
Choosing for 0.8~1.2mg/L, more preferably 1.0mg/L.
In the present invention sucrose and agar are preferably further included in the root media；The sucrose is in root media
In concentration be preferably 15~25g/L, more preferably 20g/L；Concentration of the agar in root media is preferable
For 5~10g/L, more preferably 8g/L；In the present invention, the pH value of the root media is preferably 5.7~5.9, more
Preferably 5.8.
The time of culture of rootage is 15~20 days in the present invention, preferably 16~18 days；Culture of rootage in the present invention
Temperature be preferably 24~26 DEG C, more preferably 25 DEG C；The illumination condition of the culture of rootage is natural lighting.In this hair
In bright, the rooting rate of the culture of rootage is more than 80%, number preferably 2~4 of taking root, root long is preferably 0.8~
1.2cm。
A kind of method of mongolian amygdalus seed tissue-culturing rapid propagation provided by the invention is described in detail with reference to embodiment,
But they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
(1) seed treatment：Mongolian amygdalus seed kind shell is broken, carefully takes out seed, it is ensured that seed stands intact.
(2) seed disinfection：The Prunus Mongolica seeds flowing water for having removed shell is rinsed into 10min, then with 75% alcohol (volume
Fraction) disinfection 10min；0.1%HgCl is used again2(mass fraction) solution after seed depth sterilizing 10min to using aseptic water washing 5
It is secondary, 1min is rinsed every time；Finally the surface of the seed moisture is blotted with aseptic filter paper.
(3) Aseptic seedling culture：The kind skin of sterilized Prunus Mongolica seeds is carefully peelled off with tweezers, is inoculated in 1/2MS (often
Rule each Ingredient Amount of MS culture mediums halves) solid medium, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.Inoculation
When kind hole submerge culture medium 0.5cm downwards, be placed in 25 DEG C, cultivate 15 days, aseptic seedling height 5cm under illumination condition, the number of blade 22,
Axillary bud is not grown；
(4) axillary bud deriving culture：The stem section with axil of clip 2cm long, is inoculated in axillary bud deriving culture medium MS+6-
BA1mg/L+IBA3mg/L, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.It is placed in 25 DEG C, cultivates under illumination condition
15 days, axil director went out 2 axillary buds, length 3cm；
(5) culture of rootage：Clip axillary bud is inoculated in root media 1/3MS+IBA1mg/L, the culture medium sugarcane containing 20g/L
Sugar, 8g/L agar, pH 5.8.It is placed in 25 DEG C, cultivates 15 days under illumination condition, rooting rate 82%, radical 2~3, root long 1cm,
Breeding coefficient is 3.28.
Embodiment 2
(1) seed treatment：Mongolian amygdalus seed kind shell is broken, carefully takes out seed, it is ensured that seed stands intact.
(2) seed disinfection：The Prunus Mongolica seeds flowing water for having removed shell is rinsed into 9min, then with 75% alcohol (volume
Fraction) disinfection 12min；0.1%HgCl is used again2(mass fraction) solution after seed depth sterilizing 10min to using aseptic water washing 6
It is secondary, 1min is rinsed every time；Finally the surface of the seed moisture is blotted with aseptic filter paper.
(3) Aseptic seedling culture：The kind skin of sterilized Prunus Mongolica seeds is carefully peelled off with tweezers, is inoculated in 1/2MS (often
Rule each Ingredient Amount of MS culture mediums halves) solid medium, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.Inoculation
When kind hole submerge culture medium 0.5cm downwards, be placed in 25 DEG C, cultivate 18 days, aseptic seedling height 6cm under illumination condition, the number of blade 23,
Axillary bud is not grown；
(4) axillary bud deriving culture：The stem section with axil of clip 2.5cm long, is inoculated in axillary bud deriving culture medium MS+6-
BA0.9mg/L+IBA3.2mg/L, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.It is placed in 26 DEG C, under illumination condition
Culture 16 days, axil director goes out 3 axillary buds, length 4cm；
(5) culture of rootage：Clip axillary bud is inoculated in root media 1/3MS+IBA1mg/L, the culture medium sugarcane containing 20g/L
Sugar, 8g/L agar, pH 5.8.It is placed in 25 DEG C, cultivates 20 days under illumination condition, rooting rate 85%, radical 4, root long 1.2cm is numerous
Coefficient is grown for 5.1.
Embodiment 3
(1) seed treatment：Mongolian amygdalus seed kind shell is broken, carefully takes out seed, it is ensured that seed stands intact.
(2) seed disinfection：The Prunus Mongolica seeds flowing water for having removed shell is rinsed into 12min, then with 75% alcohol (volume
Fraction) disinfection 8min；0.1%HgCl is used again2(mass fraction) solution after seed depth sterilizing 8min to using aseptic water washing 5
It is secondary, 1min is rinsed every time；Finally the surface of the seed moisture is blotted with aseptic filter paper.
(3) Aseptic seedling culture：The kind skin of sterilized Prunus Mongolica seeds is carefully peelled off with tweezers, is inoculated in 1/2MS (often
Rule each Ingredient Amount of MS culture mediums halves) solid medium, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.Inoculation
When kind hole submerge culture medium 0.4cm downwards, be placed in 26 DEG C, cultivate 18 days, aseptic seedling height 4cm under illumination condition, the number of blade 21,
Axillary bud is not grown；
(4) axillary bud deriving culture：The stem section with axil of clip 2cm long, is inoculated in axillary bud deriving culture medium MS+6-
BA1.2mg/L+IBA2.9mg/L, the culture medium sucrose containing 15g/L, 8g/L agar, pH 5.8.It is placed in 26 DEG C, under illumination condition
Culture 18 days, axil director goes out 3 axillary buds, length 4cm；
(5) culture of rootage：Clip axillary bud is inoculated in root media 1/3MS+IBA1mg/L, the culture medium sugarcane containing 20g/L
Sugar, 8g/L agar, pH 5.8.It is placed in 26 DEG C, cultivates 18 days under illumination condition, rooting rate 83%, radical 3, root long 1cm, breeding
Coefficient is 4.98.
From above-described embodiment, the method for mongolian amygdalus seed tissue-culturing rapid propagation of the present invention gathers explant from aseptic seedling
Body, overcomes using the high situation of seedling explant pollution rate, pollution rate zero, so as to save seedling cost, improves
Breeding efficiency.Used time is short, can breed within 45~55 days a collection of regeneration tissue-cultured seedling, and tissue-cultured seedling rooting rate is high, up to more than 80%, line of breeding
Number is high, and breeding coefficient is 3~5.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method of mongolian amygdalus seed tissue-culturing rapid propagation, comprises the following steps：
1) by Prunus Mongolica seeds successively by circulating water flushing, alcohol soaking disinfection, HgCl2Solution soaking disinfection and sterile water
Disinfection seed is obtained after flushing；
2) culture of 1/2MS solid mediums is inoculated in after the disinfection seed is removed the peel and obtains aseptic seedling within 15~20 days；
3) stem section with axil is obtained from the aseptic seedling to be inoculated in cultivate 15~20 days in axillary bud deriving culture medium and obtain armpit
Bud；
4) axillary bud that step 3) obtains is inoculated in root media to cultivate 15~20 days and obtains tissue-cultured seedling.
2. according to the method described in claim 1, it is characterized in that, the time of step 1) the alcohol soaking disinfection for 5~
15min。
3. the according to the method described in claim 1, it is characterized in that, step 1) HgCl2Mass fraction for 0.05~
0.15%.
4. the method according to claim 1 or 3, it is characterised in that the HgCl2The time of soaking disinfection is 8~12min.
5. according to the method described in claim 1, it is characterized in that, the 1/2MS solid mediums described in step 2) include
Sucrose and agar；
Concentration of the sucrose in 1/2MS solid mediums is 12~18g/L, and the agar is in 1/2MS solid mediums
Concentration be 5~10g/L；
The pH value of the 1/2MS solid mediums is 5.7~5.9.
6. according to the method described in claim 1, it is characterized in that, step 3) the axillary bud deriving culture medium be include 6-BA and
The MS culture mediums of IBA.
7. according to the method described in claim 6, it is characterized in that, concentration of the 6-BA in axillary bud deriving culture medium is
0.8~1.2mg/L；Concentration of the IBA in axillary bud deriving culture medium is 2.5~3.5mg/L.
8. the method according to claim 6 or 7, it is characterised in that further included in the axillary bud deriving culture medium sucrose and
Agar；
Concentration of the sucrose in axillary bud deriving culture medium is 12~18g/L, and the agar is in axillary bud deriving culture medium
Concentration is 5~10g/L；
The pH value of the axillary bud deriving culture medium is 5.7~5.9.
9. according to the method described in claim 1, it is characterized in that, the root media described in step 4) is addition IBA's
1/3MS culture mediums；Concentration of the IBA in root media is 0.8~1.2mg/L.
10. according to the method described in claim 1, it is characterized in that, the step 2), 3) and 4) in cultivation temperature stand alone as
24~26 DEG C.
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