CN107821168B - A kind of propagation method of Ammopiptanthus mongolicus tissue-cultured seedling - Google Patents
A kind of propagation method of Ammopiptanthus mongolicus tissue-cultured seedling Download PDFInfo
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- CN107821168B CN107821168B CN201711297833.9A CN201711297833A CN107821168B CN 107821168 B CN107821168 B CN 107821168B CN 201711297833 A CN201711297833 A CN 201711297833A CN 107821168 B CN107821168 B CN 107821168B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present invention provides a kind of propagation methods of Ammopiptanthus mongolicus tissue-cultured seedling, comprising the following steps: 1) Ammopiptanthus mongolicus seed is successively passed through circulating water flushing, alcohol soaking disinfection, HgCl2Disinfection seed is obtained after solution soaking disinfection and aseptic water washing；2) disinfection seed is inoculated in 10~20 days acquisition aseptic seedlings of 1/2MS solid medium culture；3) it is inoculated in cultivate 15~20 days in axillary bud deriving culture medium from acquisition stem-segment with node in the aseptic seedling and obtains axillary bud；4) axillary bud that step 3) obtains is inoculated in root media and cultivates 10~15 days acquisition tissue-cultured seedling.The propagation method of Ammopiptanthus mongolicus tissue-cultured seedling of the present invention acquires the breeding that explant carries out tissue-cultured seedling from aseptic seedling, and easy to operate, the used time is short, pollution rate zero, and emergence rate is high.
Description
Technical field
The invention belongs to Ammopiptanthus mongolicus seedling-raising technique fields, and in particular to a kind of propagation method of Ammopiptanthus mongolicus tissue-cultured seedling.
Background technique
Ammopiptanthus mongolicus (Ammopiptanthus mongolicus) is Central Asia Desert Area endemic species, is China's Precious, Rare, Endangered
Plant is protected, the sand in the area such as Inner Mongol Alashan, Wuhai, Ih Ju League's Hangjin Banner, Jingtai Regions of Gansu, Ningxia Lingwu is mainly distributed on
On ground, gobi or stone matter hillside.The Origin of Species is ancient, is the deleted species of the damp and hot plant xerophilization type in Tethys, in
It is gradually evolved into drought resisting in the western very long Environment Change in sub- and China and northwest arid climate band forming process, resists cold, is resistance to
The psammophyte type of heat has special Morphological plasticity feature, physiological adaptability mechanism and resistance Molecular and genetic basis, because
And there is extremely important conservative genetics value.The typical xerophyte of Ammopiptanthus Genus, can anti-blown sand, the season of growth cyclopentadienyl
It is close, dark green, be good nectariferous plant since Ammopiptanthus mongolicus is the only evergreen shrubs in the north, even more meagrely-populated desert and
It is difficult to the fine tree species that the bare hills and mountains managed and protected build forests for water and soil conservation.The Propogation and culture of Ammopiptanthus mongolicus utilizes sand more in the prior art
Chinese ilex seed carries out container nursery, and complicated for operation, emergence rate is low, and growing-seedling period is long.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of Ammopiptanthus mongolicus tissue cultures easy to operate, emergence rate is high, the used time is short
Seedling breeding method.
In order to achieve the above-mentioned object of the invention, the present invention is the following technical schemes are provided: a kind of breeding side of Ammopiptanthus mongolicus tissue-cultured seedling
Method, comprising the following steps: 1) Ammopiptanthus mongolicus seed is successively passed through into circulating water flushing, alcohol soaking disinfection, HgCl2Solution immersion disappears
Disinfection seed is obtained after poison and aseptic water washing；2) disinfection seed is inoculated in 1/2MS solid medium culture 10~20
It obtains aseptic seedling；3) it is inoculated in axillary bud deriving culture medium and cultivates 15~20 days from the stem section for obtaining belt segment in the aseptic seedling
Obtain axillary bud；4) axillary bud that step 3) obtains is inoculated in root media and cultivates 10~15 days acquisition tissue-cultured seedling；The step
It is rapid 2), 3) He 4) in cultivation temperature stand alone as 24~26 DEG C.
Preferably, the time of step 1) the alcohol soaking disinfection is 15~25min.
Preferably, the step 1) HgCl2Mass fraction be 0.05~0.15%.
Preferably, the HgCl2The time of soaking disinfection is 8~12min.
It preferably, include sucrose and agar in 1/2MS solid medium described in step 2)；The sucrose is in 1/2MS
Concentration in solid medium is 12~18g/L, and concentration of the agar in 1/2MS solid medium is 6~9g/L；It is described
The pH value of 1/2MS solid medium is 5.7~5.9.
Preferably, step 3) the axillary bud deriving culture medium is the MS culture medium for adding 6-BA and IBA.
Preferably, concentration of the 6-BA in axillary bud deriving culture medium is 1~2mg/L；The IBA is trained in axillary bud deriving
Supporting the concentration in base is 0.5~1.5mg/L.
It preferably, further include sucrose and agar in the axillary bud deriving culture medium；The sucrose is in axillary bud deriving culture medium
In concentration be 25~35g/L, concentration of the agar in axillary bud deriving culture medium be 6~9g/L；The axillary bud deriving training
The pH value for supporting base is 5.7~5.9.
Preferably, root media described in step 4) is the 1/2MS culture medium for adding IBA；The IBA is in training of taking root
Supporting the concentration in base is 0.8~1.2mg/L.
Beneficial effects of the present invention: the method for the breeding of Ammopiptanthus mongolicus tissue-cultured seedling of the present invention acquires outer from aseptic seedling
Implant carries out the breeding of tissue-cultured seedling, easy to operate, overcomes the situation high using seedling explant pollution rate, pollution rate is
Zero, emergence rate is high.Short according to the embodiment record method used time of the present invention, breeding coefficient is high, can breed one within 35~45 days
Criticize regeneration tissue-cultured seedling.
Specific embodiment
The present invention provides a kind of propagation methods of Ammopiptanthus mongolicus tissue-cultured seedling, comprising the following steps: 1) by Ammopiptanthus mongolicus seed according to
It is secondary to pass through circulating water flushing, alcohol soaking disinfection, HgCl2Disinfection seed is obtained after solution soaking disinfection and aseptic water washing；2)
The disinfection seed is inoculated in 10~20 days acquisition aseptic seedlings of 1/2MS solid medium culture；3) it is obtained from the aseptic seedling
It takes the stem section with axillary bud to be inoculated in axillary bud deriving culture medium and cultivates 15~20 days acquisition axillary buds；4) axillary bud for obtaining step 3)
It is inoculated in root media and cultivates 10~15 days acquisition tissue-cultured seedling；The step 2), 3) and 4) in cultivation temperature stand alone as
24~26 DEG C.
In the present invention, Ammopiptanthus mongolicus seed is stripped out from the pod of Ammopiptanthus mongolicus, by the Ammopiptanthus mongolicus seed successively through overcurrent
Dynamic water flushing, alcohol soaking disinfection, HgCl2Disinfection seed is obtained after solution soaking disinfection and aseptic water washing.In the present invention, institute
The time for stating circulating water flushing is preferably 15~25min, more preferably 20min；The purpose that the circulating water rinses is
Except the surface of the seed sundries.The specific method that the circulating water rinses in the present invention is preferably: seed is put into beaker, rim of a cup
3 layers of gauze of lid, then connect a silicone tube on water tap, and 3/4 height of beaker height is penetrated from beaker mouth, is finally used
Gauze and silicone tube are bound at beaker cup edge by cotton rope, open tap continual rinsing, flowing water speed is rush gauze
Fall to be advisable, grasp according to the actual situation.
Ammopiptanthus mongolicus seed of the present invention is after circulating water rinses, with alcohol soaking disinfection；The volumetric concentration of the alcohol
Preferably 70~80%, more preferably 75%；The time of the alcohol soaking disinfection is preferably 15~25min, more excellent
Choosing is 18~22min, most preferably 20min.
After Ammopiptanthus mongolicus seed alcohol soaking disinfection of the present invention, HgCl is used2Solution soaking disinfection, the HgCl2Solution
Mass fraction is preferably 0.07~0.12%, more preferably 0.1%；The HgCl2The time of solution soaking disinfection is preferred
It is 8~12min, more preferably 10min；The HgCl2The purpose of solution soaking disinfection is to carry out depth disinfection to seed,
Guarantee the state of the surface of the seed integral asepsis.
Ammopiptanthus mongolicus seed of the present invention is in the HgCl2After solution soaking disinfection, it is rinsed with sterile water；The nothing
The number that bacterium water rinses is preferably 4~6 times, more preferably 5 times, and the time of each aseptic water washing is preferably 8~
12min, more preferably 10min；The purpose of aseptic water washing of the present invention is that removal Ammopiptanthus mongolicus the surface of the seed is remaining
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 culture 10~20 after obtaining disinfection seed, by the disinfection seed
It obtains aseptic seedling.The 1/2MS solid medium is to halve the concentration of component in regular MS media to obtain in the present invention
?；It preferably further include sucrose and agar in the 1/2MS solid medium；The sucrose is in 1/2MS solid medium
Concentration is preferably 12~18g/L, more preferably 15g/L；Concentration of the agar in 1/2MS solid medium is preferred
For 6~9g/L, more preferably 8g/L；The pH value of the 1/2MS solid medium is preferably 5.7~5.9 in the present invention,
More preferably 5.8.
In the present invention, the kind nose end of Ammopiptanthus mongolicus seed is preferably submerged into downwards culture medium 0.1 in the seeded process
~0.5cm, more preferably 0.3cm.The time for cultivating the aseptic seedling on 1/2MS solid medium in the present invention is 10
~20 days, preferably 12~18 days；The temperature that the aseptic seedling is cultivated in the present invention is preferably 24~26 DEG C, more preferably
It is 25 DEG C；The illumination condition for cultivating the aseptic seedling is preferably the offer of tissue culture dedicated spectral lamp.
The present invention obtains stem-segment with node from the aseptic seedling and is inoculated in axillary bud deriving culture after culture obtains aseptic seedling
15~20 days acquisition axillary buds are cultivated in base.In the present invention preferably from the stem section of clip belt segment in the aseptic seedling；The band
Stem segment length preferred 1~2cm, more preferably 1.5cm.
The axillary bud deriving culture medium is the MS culture medium for adding 6-BA and IBA in the present invention.The MS culture medium is
Conventional minimal medium, in the specific implementation process using commercially available or homemade MS culture medium；The 6- in the present invention
Concentration of the BA in axillary bud deriving culture medium is preferably 1~2mg/L, more preferably 1.2~1.8mg/L, most preferably
1.5mg/L；Concentration of the IBA in axillary bud deriving culture medium is preferably 0.5~1.5mg/L, more preferably 0.8~
1.2mg/L, most preferably 1.0mg/L.
It preferably further include in the present invention sucrose and agar in the axillary bud deriving culture medium；The sucrose is lured in axillary bud
It leads the concentration in culture medium and is preferably 25~35g/L, more preferably 30g/L；The agar is in axillary bud deriving culture medium
Concentration be preferably 7~9g/L, more preferably 8g/L.
In the present invention, the pH value of the axillary bud deriving culture medium is preferably 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
It is that tissue culture dedicated spectral lamp provides.After axillary bud deriving culture of the present invention, eustipes part can induce and grow multiple Multiple Buds, average long
Degree is 1~5cm, and upgrowth situation is good.
The axillary bud of acquisition is inoculated in root media after obtaining axillary bud and cultivates 10~15 days acquisition tissue cultures by the present invention
Seedling.In the present invention, the axillary bud is preferably the Ammopiptanthus mongolicus axillary bud of robust growth；The root media is addition IBA
1/2MS culture medium；In the present invention, the 1/2MS culture medium is that conventional MS nutrient media components concentration is halved acquisition；It is described
Concentration of the IBA in root media is preferably 0.8~1.2mg/L, more preferably 1.0mg/L.
It preferably further include in the present invention sucrose and agar in the root media；The sucrose is in root media
In concentration be preferably 25~35g/L, more preferably 30g/L；Concentration of the agar in root media is preferred
For 6~9g/L, more preferably 8g/L；In the present invention, the pH value of the root media is preferably 5.7~5.9, more excellent
Choosing is 5.8.
The time of culture of rootage is 10~15 days, preferably 11~14 days in the present invention；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 tissue culture dedicated spectral lamp
It provides.Root long increases very fast in process of rooting culture of the present invention, and after culture, the adventitious root quantity of tissue-cultured seedling is 2~
4。
It is described in detail below with reference to propagation method of the embodiment to Ammopiptanthus mongolicus tissue-cultured seedling provided by the invention, but not
They can be interpreted as limiting the scope of the present invention.
Embodiment 1
(1) seed disinfection: Ammopiptanthus mongolicus seed flowing water is rinsed into 20min, then with 75% alcohol disinfecting 20min；It uses again
0.1%HgCl2Solution to seed depth sterilize 10min after use aseptic water washing 5 times, each 10min；Finally inhaled with aseptic filter paper
Dry seeds surface moisture.
(2) Aseptic seedling culture: the seed after disinfection is inoculated in 1/2MS solid culture primary surface, kind nose end submerges culture
Base 0.3cm is placed in 25 DEG C, cultivates 10 days under illumination condition；
(3) axillary bud deriving: clip Ammopiptanthus mongolicus aseptic seedling stem-segment with node, length 1.5cm are inoculated in axillary bud deriving culture
Base: MS+6-BA 1.5mg/L+IBA 1mg/L is placed in 25 DEG C, cultivates 15 days under illumination condition；Eustipes part can induce and grow 4-6
A Multiple Buds, average length about 3cm, upgrowth situation are good；
(4) culture of rootage: the Ammopiptanthus mongolicus axillary bud of robust growth in clip (3) is inoculated in root media 1/2MS+
IBA0.5mg/L is placed in 25 DEG C, cultivates 10 days under illumination condition, takes root very fast, rooting rate 100%.Breeding coefficient is 4.8.
Embodiment 2
(1) seed disinfection: Ammopiptanthus mongolicus seed flowing water is rinsed into 20min, then with 75% alcohol disinfecting 20min；It uses again
0.1%HgCl2Solution to seed depth sterilize 10min after use aseptic water washing 6 times, each 10min；Finally inhaled with aseptic filter paper
Dry seeds surface moisture.
(2) Aseptic seedling culture: the seed after disinfection is inoculated in 1/2MS solid culture primary surface, kind nose end submerges culture
Base 0.35cm is placed in 25 DEG C, cultivates 12 days under illumination condition；
(3) axillary bud deriving: clip Ammopiptanthus mongolicus aseptic seedling stem-segment with node, length 1.5cm are inoculated in axillary bud deriving culture
Base: MS+6-BA 1.5mg/L+IBA 1.5mg/L is placed in 25 DEG C, cultivates 15 days under illumination condition；Multiple Buds quantity is more, puts down
Length about 1.5cm is thin and delicate；
(4) culture of rootage: the Ammopiptanthus mongolicus axillary bud of robust growth in clip (3) is inoculated in root media 1/2MS+
IBA0.5mg/L is placed in 25 DEG C, cultivates 10 days under illumination condition, takes root very fast, adventitious root quantity 2~4；Rooting rate is 86%.
Breeding coefficient is 2.3.
Embodiment 3
(1) seed disinfection: Ammopiptanthus mongolicus seed flowing water is rinsed into 20min, then with 75% alcohol disinfecting 20min；It uses again
0.1%HgCl2Solution to seed depth sterilize 10min after use aseptic water washing 6 times, each 10min；Finally inhaled with aseptic filter paper
Dry seeds surface moisture.
(2) Aseptic seedling culture: the seed after disinfection is inoculated in 1/2MS solid culture primary surface, kind nose end submerges culture
Base 0.3cm is placed in 25 DEG C, cultivates 15 days under illumination condition；
(3) axillary bud deriving: clip Ammopiptanthus mongolicus aseptic seedling stem-segment with node, length 1.5cm are inoculated in axillary bud deriving culture
Base: MS+6-BA 1.5mg/L+IBA 0.5mg/L is placed in 25 DEG C, cultivates 15 days under illumination condition；It only can induce and grow simple bud,
Average length about 3cm, upgrowth situation are good；
(4) culture of rootage: the Ammopiptanthus mongolicus axillary bud of robust growth in clip (3) is inoculated in root media 1/2MS+
IBA0.5mg/L is placed in 25 DEG C, cultivates 15 days under illumination condition, takes root very fast, adventitious root quantity 2~4；Rooting rate is 100%.
Breeding coefficient is 1.
As can be seen from the above embodiments, the method for the breeding of Ammopiptanthus mongolicus tissue-cultured seedling of the present invention acquires outer from aseptic seedling
Implant carries out the breeding of tissue-cultured seedling, easy to operate, overcomes the situation high using seedling explant pollution rate, pollution rate is
Zero；Rooting rate is 100%, and breeding coefficient is 2~4, and emergence rate is high, and the used time is short, can breed within 35~45 days a collection of regeneration group
Train seedling.
The above is only a 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 are also answered
It is considered as protection scope of the present invention.
Claims (4)
1. a kind of propagation method of Ammopiptanthus mongolicus tissue-cultured seedling, comprising the following steps:
1) Ammopiptanthus mongolicus seed is successively passed through into circulating water flushing, alcohol soaking disinfection, HgCl2Solution soaking disinfection and sterile water punching
Disinfection seed is obtained after washing；
2) disinfection seed that the step 1) obtains is inoculated in 10~20 days acquisition aseptic seedlings of 1/2MS solid medium culture；
3) stem section that belt segment is obtained from the aseptic seedling that the step 2) obtains, is inoculated in axillary bud deriving culture medium for the stem section
15~20 days acquisition axillary buds of middle culture；
4) axillary bud that the step 3) obtains is inoculated in root media and cultivates 10~15 days acquisition tissue-cultured seedling；The step
2) cultivation temperature in, 3) He 4) stands alone as 24~26 DEG C；
Step 3) the axillary bud deriving culture medium is the MS culture medium for adding 6-BA and IBA；
Concentration of the 6-BA in axillary bud deriving culture medium is 1.5mg/L；Concentration of the IBA in axillary bud deriving culture medium
For 0.8~1.2mg/L；
Root media described in step 4) is the 1/2MS culture medium for adding IBA；The IBA is dense in root media
Degree is 0.5mg/L；
The time of step 1) the alcohol soaking disinfection is 15~25min；
Step 1) the HgCl2The mass fraction of solution is 0.05~0.15%；
The HgCl2The time of solution soaking disinfection is 8~12min.
2. the method according to claim 1, wherein also being wrapped in 1/2MS solid medium described in step 2)
Include sucrose and agar；Concentration of the sucrose in 1/2MS solid medium is 12~18g/L, and the agar is in 1/2MS solid
Concentration in culture medium is 6~9g/L；The pH value of the 1/2MS solid medium is 5.7~5.9.
3. the method according to claim 1, wherein further including sucrose and fine jade in the axillary bud deriving culture medium
Rouge；Concentration of the sucrose in axillary bud deriving culture medium is 25~35g/L, and the agar is dense in axillary bud deriving culture medium
Degree is 6~9g/L；The pH value of the axillary bud deriving culture medium is 5.7~5.9.
4. the method according to claim 1, wherein further including sucrose and agar in the root media；Institute
Stating concentration of the sucrose in root media is 25~35g/L, and concentration of the agar in root media is 6~9g/L；
The pH value of the root media is 5.7~5.9.
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| CN111011214B (en) * | 2019-12-30 | 2023-09-12 | 蒙草生态环境(集团)股份有限公司 | Tissue culture method of ammopiptanthus mongolicus |
| CN111011215B (en) * | 2019-12-30 | 2023-07-14 | 蒙草生态环境(集团)股份有限公司 | Culture medium for tissue culture of ammopiptanthus mongolicus |
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