CN106171972A - A kind of cultural method of cabbage type rape Isolated microspore plant strain - Google Patents
A kind of cultural method of cabbage type rape Isolated microspore plant strain Download PDFInfo
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- CN106171972A CN106171972A CN201610505179.5A CN201610505179A CN106171972A CN 106171972 A CN106171972 A CN 106171972A CN 201610505179 A CN201610505179 A CN 201610505179A CN 106171972 A CN106171972 A CN 106171972A
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- hormone
- sucrose
- fluid medium
- mass concentration
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The present invention relates to the cultural method of a kind of cabbage type rape Isolated microspore plant strain, specifically include following steps: the preparation of (1) Isolated microspore, (2) sporidiole prepared is added the NLN 13 containing 16% sucrose without in hormone fluid medium, in 30 35 DEG C of light culture 8h, then it is added thereto to isopyknic contain 4% sucrose, 105mg/L Colchicine NLN 13 is without hormone fluid medium, light culture 24h is continued in 30 35 DEG C, remove Colchicine, add the NLN 13 containing 10% sucrose and stand light culture 5 days without 25 DEG C of constant temperature of hormone fluid medium, it is eventually adding the NLN 13 of 13% sucrose without hormone fluid medium, the density of regulation sporidiole is 1 alabastrum/mL, stand light culture at 25 DEG C of constant temperature to produce to embryoid；(3) cultivation of microspore plant.The inductivity using the inventive method not only sporidiole is high, and microspore plant is prone to regeneration.
Description
Technical field
The present invention relates to plant tissue culture technique, be specifically related to the cultivation of a kind of cabbage type rape Isolated microspore plant strain
Method.
Background technology
Utilize plant microspore culture can obtain monoploid (Hapiods) or dihaploid
(Doublehapiods) plant, this plant is the ideal material of genetic research and transgenic research, has in breeding work
Value greatly.And the low wide variety of problem of this technology that always limits of Brassica campestris L sporidiole embryoid inductivity,
Although the method for inducing and cultivating of sporidiole is a lot, wherein with Application No. 200810300457.9 Chinese patent application effect at present
Preferably, but its need in Induction Process by identify sporidiole rate of expanding carried out screening in advance, only to induced material
The rate of the expanding material more than 50% could produce embryo, and the rate of the expanding material less than 50% is then difficult to work and cannot produce embryo, therefore
From substantially still solving the problem that inductivity is low；Therefore, find the high cabbage type rape of a kind of inductivity and dissociate little
The cultural method of spore plant seems most important.
Summary of the invention
It is an object of the invention to overcome the defect of prior art, it is provided that a kind of cabbage type rape Isolated microspore plant strain
Cultural method, the inductivity of the method not only sporidiole is high, and microspore plant is prone to regeneration.
To achieve these goals, the technical scheme that the present invention takes is as follows:
The cultural method of a kind of cabbage type rape Isolated microspore plant strain, specifically includes following steps:
Step one, the preparation of Isolated microspore: choose the cabbage type rape inflorescence bloomed 3-7 days big Tanaka, select
The alabastrum of 3.0-4.0mm, puts it in filter cloth, aseptically with 70% alcohol disinfecting 1min, then uses 0.1% mercuric chloride
Sterilization 10min, aseptic water washing 3-5 time, cleaned with sterilizing after alabastrum put into pH be 5.8-6.0, containing mass concentration 13%
The B5 of sucrose without in hormone fluid medium, sporidiole of gently extruding out, in 300 mesh nylon wire membrane filtrations to centrifuge tube, in
800rpm is centrifuged 8min, removes supernatant；Add identical B5 and under same rotating speed, be centrifuged 5min without hormone fluid medium,
Remove supernatant, obtain free Microspore of Brassica napus；
Step 2, the induction of sporidiole embryoid: the Microspore of Brassica napus prepared is added containing mass concentration
The NLN-13 of the sucrose of 16%, without in hormone fluid medium, in 30-35 DEG C of light culture 8h, is then added thereto to isopyknic
Sucrose containing mass concentration 4%, the full filtration sterilization containing 105mg/L Colchicine NLN-13 without hormone fluid medium, in
30-35 DEG C is continued light culture 24h, then with the NLN-without the full filtration sterilization that Colchicine and sucrose mass concentration are 10%
13 is centrifugal without the cleaning of hormone fluid medium, removes Colchicine, is subsequently adding same medium i.e. without Colchicine and sugarcane
Sugar concentration is that the NLN-13 of the full filtration sterilization of 10% stands light culture 5 days without hormone fluid medium, 25 DEG C of constant temperature,
The rear addition NLN-13 without Colchicine and full filtration sterilization that sucrose mass concentration is 13%, without hormone fluid medium, adjusts
The density of joint sporidiole is 1 alabastrum/mL, stands light culture at 25 DEG C of constant temperature and produces to embryoid；
Step 3, the cultivation of microspore plant: by produce embryoid sporidiole be placed on the shaking table of 80rpm, 25 DEG C of bars
Under part, in light culture 1-2 week, then proceed to the agar containing 0.8% mass concentration, 3% mass concentration by the microspore embryoid of normal development
Sucrose, 0.5mg/L6-benzylaminopurine, in the B5 solid medium of 0.02mg/L NAA and pH5.8-6.0, be placed in 22
DEG C, cultivate in 16h photoperiod incubator, change culture medium 2-3 week becomes normal adventitious shoot, then until plant strain growth
Turn root media and potted plant domestication again, finally transplant land for growing field crops.
Further, it is characterised in that possibly together with 0.5% mass concentration in the B5 solid medium described in step 3 kind
Activated carbon.
Further, a kind of described alabastrum of step is 3.5mm.
Compared with prior art, having the beneficial effect that acquired by the present invention:
1, the present invention is in the Induction Process of sporidiole embryoid, first uses the NLN-13 of the sucrose containing 16% mass concentration
In 30-35 DEG C of light culture 8h in fluid medium without hormone, then it is added thereto to isopyknic sugarcane containing 4% mass concentration
Sugar, 105mg/L Colchicine the NLN-13 of full filtration sterilization without hormone fluid medium, continue light culture in 30-35 DEG C
24h, then with the NLN-13 without the full filtration sterilization that Colchicine and sucrose mass concentration are 10% without hormone liquid culture
Base cleans centrifugal, removes Colchicine, and being subsequently adding same medium is i.e. 10% without Colchicine and sucrose mass concentration
The NLN-13 of full filtration sterilization without hormone fluid medium, 25 DEG C of constant temperature stand light culture 5 days, are eventually adding without Colchicum autumnale
The NLN-13 of element and full filtration sterilization that sucrose mass concentration is 13% without hormone fluid medium, the density of regulation sporidiole is
1 alabastrum/mL, stands light culture at 25 DEG C of constant temperature and produces to embryoid；Use the method culture medium and condition of culture and cultivation
Step can improve the inductivity of sporidiole embryoid to a great extent without the rate of expanding identifying sporidiole.
2, the present invention is in the incubation of microspore plant, uses the agar containing 0.8% mass concentration, and 3% mass is dense
The sucrose of degree, 0.5mg/L 6-benzylaminopurine, the activated carbon of 0.5% mass concentration, 0.02mg/L NAA and pH5.8-6.0
B5 solid medium in, be placed in 22 DEG C, cultivate in 16h photoperiod incubator, this culture medium is more suitable for using above-mentioned induction
The somatic embryogenesis that method induction obtains is plant.
The present invention, by sporidiole embryoid inductive condition is groped research, has searched out high little of a kind of inductivity
Spore embryoid induction method, and searched out optimal by the condition of culture of this somatic embryogenesis plant is carried out research
Microspore plant regeneration condition.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is carried out further details of narration.
Embodiment 1
The cultural method of a kind of cabbage type rape Isolated microspore plant strain, specifically includes following steps:
Step one, the preparation of Isolated microspore: choose the cabbage type rape inflorescence bloomed 3-7 days big Tanaka, select
The alabastrum of 3.2mm, puts it in filter cloth, aseptically with the alcohol disinfecting 1min of 70% mass concentration, then uses
The mercuric chloride sterilization 10min of 0.1% mass concentration, aseptic water washing 3-5 time, cleaned with sterilizing after alabastrum put into pH be 5.9,
The B5 of the sucrose containing 13% mass concentration is without in hormone fluid medium, and sporidiole of gently extruding out, through 300 mesh nylon wire membrane filtrations
In centrifuge tube, it is centrifuged 8min in 800rpm, removes supernatant；Add identical B5 to turn same without hormone fluid medium
The lower centrifugal 5min of speed, removes supernatant, obtains Isolated microspore；
Step 2, the induction of sporidiole embryoid: the Microspore of Brassica napus prepared is added containing 16% mass dense
The NLN-13 of the sucrose of degree without in hormone fluid medium in 33 DEG C of light culture 8h, be then added thereto to isopyknic contain 4%
The sucrose of mass concentration, 105mg/L Colchicine the NLN-13 of full filtration sterilization without hormone fluid medium (pH5.9), in
33 DEG C are continued light culture 24h, then with the NLN-13 without the full filtration sterilization that Colchicine and sucrose mass concentration are 10%
Fluid medium without hormone (pH5.9) cleans centrifugal, removes Colchicine, is subsequently adding same medium i.e. without Colchicine
And the NLN-13 of the full filtration sterilization that sucrose mass concentration is 10% stands without hormone fluid medium (pH5.9), 25 DEG C of constant temperature
Light culture 5 days, is eventually adding the NLN-13 without the full filtration sterilization that Colchicine and sucrose mass concentration are 13% without hormone
Fluid medium, the density of regulation sporidiole is 1 alabastrum/mL, stands light culture at 25 DEG C of constant temperature and produces to embryoid；
Step 3, the cultivation of microspore plant: by produce embryoid sporidiole be placed on the shaking table of 80rpm, 25 DEG C of bars
Light culture 10 days under part, then proceed to the agar containing 0.8% mass concentration, 3% mass concentration by the microspore embryoid of normal development
Sucrose, 0.5mg/L6-benzylaminopurine, in the B5 solid medium of 0.02mg/L NAA and pH5.9, be placed in 22 DEG C, 16h
Cultivating in photoperiod incubator, culture medium of conversion in 2 weeks is until plant strain growth becomes normal adventitious shoot, the most again reincarnation root
Culture medium and potted plant domestication, finally transplant land for growing field crops.
Embodiment 2
The cultural method of a kind of cabbage type rape Isolated microspore plant strain, specifically includes following steps:
Step one, the preparation of Isolated microspore: choose the cabbage type rape inflorescence bloomed 3-7 days big Tanaka, select
The alabastrum of 3.5mm, puts it in filter cloth, aseptically with the alcohol disinfecting 1min of 70% mass concentration, then uses
The mercuric chloride sterilization 10min of 0.1% mass concentration, aseptic water washing 3-5 time, cleaned with sterilizing after alabastrum put into pH be 5.9,
The B5 of the sucrose containing 13% mass concentration is without in hormone fluid medium, and sporidiole of gently extruding out, through 300 mesh nylon wire membrane filtrations
In centrifuge tube, it is centrifuged 8min in 800rpm, removes supernatant；Add identical B5 to turn same without hormone fluid medium
The lower centrifugal 5min of speed, removes supernatant, obtains Isolated microspore；
Step 2, the induction of sporidiole embryoid: the Microspore of Brassica napus prepared is added containing 16% mass dense
Degree sucrose NLN-13 without in hormone fluid medium (pH5.9) in 35 DEG C of light culture 8h, be then added thereto to equal-volume
The sucrose containing 4% mass concentration, 105mg/L Colchicine the NLN-13 of full filtration sterilization without hormone fluid medium
(pH5.9), continue light culture 24h in 35 DEG C, then go out with without full filtration that Colchicine and sucrose mass concentration are 10%
The NLN-13 of bacterium cleans centrifugal without hormone fluid medium (pH5.9), removes Colchicine, is subsequently adding same medium the most not
NLN-13 containing the full filtration sterilization that Colchicine and sucrose mass concentration are 10% without hormone fluid medium (pH5.9), 25
DEG C constant temperature stands light culture 5 days, is eventually adding without the full filtration sterilization that Colchicine and sucrose mass concentration are 13%
NLN-13 is without hormone fluid medium (pH5.9), and the density of regulation sporidiole is 1 alabastrum/mL, stands dark training at 25 DEG C of constant temperature
Support to embryoid generation；
Step 3, the cultivation of microspore plant: by produce embryoid sporidiole be placed on the shaking table of 80rpm, 25 DEG C of bars
Under part, in light culture 1-2 week, then proceed to the agar containing 0.8% mass concentration, 3% mass concentration by the microspore embryoid of normal development
Sucrose, 0.5mg/L 6-benzylaminopurine, in the B5 solid medium of 0.02mg/L NAA and pH5.8-6.0, be placed in 22
DEG C, cultivate in 16h photoperiod incubator, culture medium of conversion in 2 weeks becomes normal adventitious shoot, the most again until plant strain growth
Turn root media and potted plant domestication, finally transplant land for growing field crops.
Embodiment 3
The cultural method of a kind of cabbage type rape Isolated microspore plant strain, specifically includes following steps:
Step one, the preparation of Isolated microspore: choose the cabbage type rape inflorescence bloomed 3-7 days big Tanaka, select
The alabastrum of 3.5mm, puts it in filter cloth, aseptically with the alcohol disinfecting 1min of 70% mass concentration, then uses
The mercuric chloride sterilization 10min of 0.1% mass concentration, aseptic water washing 3-5 time, cleaned with sterilizing after alabastrum put into pH be 5.9,
The B5 of the sucrose containing 13% mass concentration is without in hormone fluid medium, and sporidiole of gently extruding out, through 300 mesh nylon wire membrane filtrations
In centrifuge tube, it is centrifuged 8min in 800rpm, removes supernatant；Add identical B5 to turn same without hormone fluid medium
The lower centrifugal 5min of speed, removes supernatant, obtains Isolated microspore；
Step 2, the induction of sporidiole embryoid: the Microspore of Brassica napus prepared is added containing 16% mass dense
Degree sucrose NLN-13 without in hormone fluid medium (pH5.9) in 35 DEG C of light culture 8h, be then added thereto to equal-volume
The sucrose containing 4% mass concentration, 105mg/L Colchicine the NLN-13 of full filtration sterilization without hormone fluid medium
(pH5.9), continue light culture 24h in 35 DEG C, then go out with without full filtration that Colchicine and sucrose mass concentration are 10%
The NLN-13 of bacterium cleans centrifugal without hormone fluid medium (pH5.9), removes Colchicine, is subsequently adding same medium the most not
NLN-13 containing the full filtration sterilization that Colchicine and sucrose mass concentration are 10% without hormone fluid medium (pH5.9), 25
DEG C constant temperature stands light culture 5 days, is eventually adding without the full filtration sterilization that Colchicine and sucrose mass concentration are 13%
NLN-13 is without hormone fluid medium (pH5.9), and the density of regulation sporidiole is 1 alabastrum/mL, stands dark training at 25 DEG C of constant temperature
Support to embryoid generation；
Step 3, the cultivation of microspore plant: by produce embryoid sporidiole be placed on the shaking table of 80rpm, 25 DEG C of bars
Under part, in light culture 1-2 week, then proceed to the agar containing 0.8% mass concentration, 3% mass concentration by the microspore embryoid of normal development
Sucrose, the activated carbon of 0.5% mass concentration, 0.5mg/L 6-benzylaminopurine, 0.02mg/L NAA and pH5.8-6.0
In B5 solid medium, being placed in 22 DEG C, the interior cultivation of 16h photoperiod incubator, culture medium of conversion in 2 weeks is until plant strain growth becomes
For normal adventitious shoot, turn root media and potted plant domestication the most again, finally transplant land for growing field crops.
The above embodiment is only the preferred embodiments of the present invention, and and the feasible enforcement of non-invention exhaustive.Right
For persons skilled in the art, to its done any showing on the premise of without departing substantially from the principle of the invention and spirit
The change being clear to, within all should being contemplated as falling with the claims of the present invention.
Claims (3)
1. the cultural method of a cabbage type rape Isolated microspore plant strain, it is characterised in that specifically include following steps:
Step one, the preparation of Isolated microspore: choose the cabbage type rape inflorescence bloomed 3-7 days big Tanaka, select 3.0-
The alabastrum of 4.0mm, puts it in filter cloth, aseptically with the alcohol disinfecting 1min of 70% mass concentration, then uses
0.1% mercuric chloride sterilization 10min, aseptic water washing 3-5 time, cleaned with sterilizing after alabastrum put into pH be 5.8-6.0, containing quality
The B5 of the sucrose of concentration 13% is without in hormone fluid medium, and sporidiole of gently extruding out, through 300 mesh nylon wire membrane filtrations to centrifugal
Guan Li, is centrifuged 8min in 800rpm, removes supernatant；Add identical B5 without hormone fluid medium under same rotating speed from
Heart 5min, removes supernatant, obtains free Microspore of Brassica napus；
Step 2, the induction of sporidiole embryoid: the Microspore of Brassica napus prepared is added containing mass concentration 16%
The NLN-13 of sucrose is without in hormone fluid medium, in 30-35 DEG C of light culture 8h, is then added thereto to isopyknic containing quality
The sucrose of concentration 4%, the NLN-13 of the full filtration sterilization containing 105mg/L Colchicine is without hormone fluid medium, in 30-35 DEG C
Continue light culture 24h, then with the NLN-13 without the full filtration sterilization that Colchicine and sucrose mass concentration are 10% without swashing
Element fluid medium cleans centrifugal, removes Colchicine, is subsequently adding same medium i.e. without Colchicine and sucrose quality
Concentration is that the NLN-13 of the full filtration sterilization of 10% stands light culture 5 days without hormone fluid medium, 25 DEG C of constant temperature, is eventually adding
NLN-13 without the full filtration sterilization that Colchicine and sucrose mass concentration are 13%, without hormone fluid medium, regulates little spore
The density of son is 1 alabastrum/mL, stands light culture at 25 DEG C of constant temperature and produces to embryoid；
Step 3, the cultivation of microspore plant: the sporidiole producing embryoid is placed on the shaking table of 80rpm, under the conditions of 25 DEG C
In light culture 1-2 week, then the microspore embryoid of normal development is proceeded to the agar containing 0.8% mass concentration, the sugarcane of 3% mass concentration
Sugar, 0.5mg/L 6-benzylaminopurine, in the B5 solid medium of 0.02mg/L NAA and pH5.8-6.0, be placed in 22 DEG C,
Cultivate in 16h photoperiod incubator, change a culture medium 2-3 week until plant strain growth becomes normal adventitious shoot, the most again
Turn root media and potted plant domestication, finally transplant land for growing field crops.
The cultural method of a kind of cabbage type rape Isolated microspore plant strain the most according to claim 1, it is characterised in that step
Possibly together with the activated carbon of 0.5% mass concentration in B5 solid medium described in rapid three.
The cultural method of a kind of cabbage type rape Isolated microspore plant strain the most according to claim 1, it is characterised in that step
Alabastrum described in rapid one is 3.5mm.
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| CN107410011A (en) * | 2017-06-08 | 2017-12-01 | 安徽省农业科学院作物研究所 | A kind of method of efficiently quick separating cabbage type rape A and C subgenome |
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| CN113337535A (en) * | 2021-07-06 | 2021-09-03 | 辽宁省农业科学院 | Method for inhibiting agrobacterium in liquid high-sugar culture medium |
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