CN106718908A - Preparation method of artificial seeds of cadmium-resistant reed seedlings - Google Patents
Preparation method of artificial seeds of cadmium-resistant reed seedlings Download PDFInfo
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- CN106718908A CN106718908A CN201611174860.2A CN201611174860A CN106718908A CN 106718908 A CN106718908 A CN 106718908A CN 201611174860 A CN201611174860 A CN 201611174860A CN 106718908 A CN106718908 A CN 106718908A
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- reed
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- cadmium
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- 235000014676 Phragmites communis Nutrition 0.000 title claims abstract description 106
- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 23
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims description 18
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 25
- 239000010802 sludge Substances 0.000 claims abstract description 23
- 239000000725 suspension Substances 0.000 claims abstract description 21
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- 230000001954 sterilising effect Effects 0.000 claims abstract description 14
- 239000001110 calcium chloride Substances 0.000 claims abstract description 13
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 13
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- 238000011534 incubation Methods 0.000 claims abstract description 8
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- 239000011159 matrix material Substances 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 30
- 241000605008 Spirillum Species 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 22
- 239000012530 fluid Substances 0.000 claims description 20
- 235000015097 nutrients Nutrition 0.000 claims description 20
- 241000196324 Embryophyta Species 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 11
- 238000012549 training Methods 0.000 claims description 11
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- 244000025254 Cannabis sativa Species 0.000 claims description 7
- 230000008929 regeneration Effects 0.000 claims description 7
- 238000011069 regeneration method Methods 0.000 claims description 7
- XEYINGFRHZCVKK-UHFFFAOYSA-N 2,2-dichloro-2-phenoxyacetic acid Chemical compound OC(=O)C(Cl)(Cl)OC1=CC=CC=C1 XEYINGFRHZCVKK-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
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- 239000008367 deionised water Substances 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 239000002002 slurry Substances 0.000 claims description 6
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 5
- 239000005648 plant growth regulator Substances 0.000 claims description 5
- 239000000661 sodium alginate Substances 0.000 claims description 5
- 235000010413 sodium alginate Nutrition 0.000 claims description 5
- 229940005550 sodium alginate Drugs 0.000 claims description 5
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- 239000005972 6-Benzyladenine Substances 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
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- 229910052799 carbon Inorganic materials 0.000 claims description 3
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- 150000001875 compounds Chemical class 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 235000013372 meat Nutrition 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 230000017105 transposition Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000010455 vermiculite Substances 0.000 claims description 2
- 235000019354 vermiculite Nutrition 0.000 claims description 2
- 229910052902 vermiculite Inorganic materials 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 4
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- LCPDWSOZIOUXRV-UHFFFAOYSA-N phenoxyacetic acid Chemical compound OC(=O)COC1=CC=CC=C1 LCPDWSOZIOUXRV-UHFFFAOYSA-N 0.000 description 2
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
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- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
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- 239000003094 microcapsule Substances 0.000 description 1
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Classifications
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
- A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds
-
- 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
-
- 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
Landscapes
- 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 invention discloses a method for preparing artificial seeds of cadmium-resistant reed seedlings, which is characterized in that reed seeds are used as explantsA body; sterilizing, and inducing embryonic callus on the inducing culture medium; then obtaining embryonic culture to generate a complete plant; obtaining regenerated reed seedlings; transferring the reed regenerated seedlings to an aseptic tissue culture bottle, adding OECD simulation wastewater and activated sludge for incubation, taking root tissues for disinfection and grinding, diluting and coating pulp on an MS flat plate for culture, collecting thalli, cleaning to prepare bacterial resuspension, taking reed embryogenic cultures, immersing the reed embryogenic cultures into the bacterial resuspension for incubation, and then culturing the reed embryogenic cultures. Preparing sol suspension and CaCl2Adding the reed embryo culture into the seed coat matrix, sucking one reed embryo culture by using a sterile pasteur pipette each time, and dripping CaCl2Taking out the solution, cleaning and airing to form artificial seeds; the artificial seeds prepared by the method have the advantages of cadmium resistance, seed vitality improvement, seedling quality improvement and the like, and are suitable for large-scale popularization.
Description
Technical field
The present invention relates to vegetative propagation field, specially a kind of preparation of the artificial seed of resistance to cadmium reed seedling.
Background technology
Reed be distributed widely in all over the world, well differentiated grassy marshland and muskeg constructive species in form, with weight
The ecology and economic value wanted.Reed is in natural environment based on root-like stock breeding, it is difficult to carry out seminal propagation.For a long time
Substantial amounts of division propagation can frequently result in kind of a degeneration for property, so as to influence the viability of plant.Simultaneously because mankind's activity opposite
The severe jamming of state environment, Partial Wetland heavy metal pollution degree allows of no optimist.It is chronically in Heavy-metal Polluted Environment, plant
Rhizome survival and breeding suffer from suppressing, and cause population depression.So, it is to avoid take reed from reed Proterozoic and protect
While shield Proterozoic ecological environment, how about light, quickly breeding has the seedling of heavy metal tolerance, as recovery reed
The bottleneck of population study.
Plant can be bred by two sexual and asexual life growth cycles, for it is difficult to be carried out with seed sexual
The plant species of breeding, the artificial seed being prepared from by microcapsule embedded propagulum, alternative natural seed carries out asexual
Breeding.Conventional artificial seed development always is that foundation on the theory of plant individualism, i.e., is trained by vitro tissue
Support and produce somatic embryo or adventitious bud etc. as embedding brood body, and take plant and microbial consortia as the research for cultivating material
Seldom.Vitro Plant tissue cultures typically set up and maintain aseptically, to reduce microorgranic contaminant in vitro culture
Vegetable material do harmful competition.And have the endophytic bacterium of ecological uniqueness, can not only with host plant coevolution,
And the metabolite with bioactivity can be produced and plant is improved to biotic or the resistivity of abiotic stress.Thus
Introduce the endogenetic bacteria of specific function to strengthen artificial seed vigor and quality, will there are the potentiality of improvement plant phenotype；Therefore
The present invention prepares a kind of artificial seed of resistance to cadmium reed seedling under isolated culture condition.
The content of the invention
It is an object of the invention to provide a kind of preparation method of the artificial seed of resistance to cadmium reed seedling, to solve the above-mentioned back of the body
The problem proposed in scape technology.
To achieve the above object, the present invention provides following technical scheme：Step is as follows：
Step one：The ripe reed small ear of collection, removes flower glume, and the reed seed with surface nondestructive is as explant；
Step 2：After by the explant sterilization described in step one, it is placed on inducing culture and induces sticky callus group
Knit；
Step 3：The sticky callus described in step 2 is stripped, by the sticky callus transposition in fresh
On inducing culture, embryo callus are further induced；
Step 4：By the embryo callus described in step 3 by Multiplying culture, Embryonal suspensor mass is obtained；
Step 5：Embryonal suspensor mass described in step 4 is gone to and generated on the regeneration culture medium without plant growth regulator
Whole plant；
Step 6：The whole plant that will be generated in step 5 is cultivated, and obtains reed regrowth；
Step 7：Cultivated during reed regrowth described in step 6 is gone into aseptic tissue culture bottle, to described aseptic group
Aseptic OECD simulated wastewaters are added in training bottle；Collection activated sludge, will add the tissue culture after activated sludge centrifugation, cleaning
In bottle, bottleneck is covered with tissue culture film, the tissue culture bottle is incubated in growth cabinet；
Step 8：The root tissue of the reed regrowth of step 7 of learning from else's experience treatment, the surface of the root tissue is carried out disinfection
After grind to form slurry, then the slurry standard dilution of the root tissue is coated on MS flat boards, the MS flat boards are placed on constant temperature training
Culture in case is supported, treats that bacterium colony grows；
Step 9：Careless spirillum single bacterium colony is selected from the MS flat boards described in step 8, training in NB fluid nutrient mediums is seeded to
Support；It is then centrifuged for processing the NB fluid nutrient mediums with collects thalline, cleans the thalline collected, uses MS fluid nutrient mediums
Again the thalline that suspends is made bacterium re-suspension liquid, adjusts the OD of the bacterium re-suspension liquid medium-height grass spirillum600nmIt is 0.01-1 to be worth；Take
At least one reed Embryonal suspensor mass is immersed in the bacterium re-suspension liquid and is incubated, and the reed Embryonal suspensor mass is gone into aseptic filter
After removing surface thalline on paper, then the reed Embryonal suspensor mass is placed on MS flat boards is cultivated；
Step 10：Prepare artificial endosperm；To sodium alginate is added in the artificial endosperm solution, colloidal sol suspension work is configured to
It is kind of a scytoblastema matter, while preparing CaCl using deionized water2Solution as complexing agent, by the colloidal sol suspension and CaCl2Solution point
Tiao Jie not sterilization treatment after pH value；
Step 11：By described in step 9 reed Embryonal suspensor mass add step 10 described in kind scytoblastema matter in, uniformly
After mixing, then suck a reed Embryonal suspensor mass every time with sterile Pasteur pipet, instill the CaCl described in step 102It is molten
In liquid, reed Embryonal suspensor mass cleaning is dried after taking-up, that is, form artificial seed；
Step 12：Artificial seed described in step 11 is placed on carries out sprouting culture on MS culture mediums.
Preferably, the preparation method of the inducing culture is：To the dichloro-benzenes that 1mg is added in every liter of MS fluid nutrient medium
Fluoroacetic acid, the methyl α-naphthyl acetate of 1mg, the 6-benzyladenine of 0.5mg, the sucrose of 30g and 0.5mgL- glutamine；The propagation
The compound method of culture medium be to added in every liter of MS fluid nutrient medium the dichlorphenoxyacetic acid of 2.5mg, the sucrose of 30g and
The agar of 16g；The collocation method of the regeneration culture medium is：To the Dichlorophenoxy that 2.5mg is added in every liter of MS fluid nutrient medium
The agar of acetic acid, the sucrose of 30g and 16g.
Preferably, the sterilization described in step 2 is to carry out surface sterilization, step 2 with the sodium hypochlorite that mass concentration is 5%
Described sticky callus is white, translucent, the glossy tissue in plumule end；Embryo callus subculture described in step 3
Being organized as surface has the tissue of tiny knob and green point；Embryo callus described in step 4 are by mass concentration
2.5mgL-12,4-D carry out Multiplying culture.
Preferably, the height of the whole plant described in step 6 is 5cm~10cm, and training method is to go to 1/2MS liquid
Water planting is carried out in culture medium, or is carried out by cultivation matrix of vermiculite.
Preferably, a height of 20cm of the aseptic tissue culture bottle described in step 7, a diameter of 5cm, the OECD simulated wastewaters
Formula is the K to the peptone of addition 160mg, the meat extract of 110mg, the urea of 30mg, 28mg in 0.5L deionized waters2HPO4、
The CaCl of NaCl, 4mg of 7mg2·2H2The MgSO of O and 2mg4·7H2O, is configured to mixed liquor, then will be described mixed with deionized water
Close liquid and be settled to 1L, then sterilized with the membrane filtration that specification is 0.22 μm, that is, be made OECD simulated wastewaters；It is described to add aseptic group
The volume of the OECD simulated wastewaters in training bottle is 150ml；The activated sludge is gathered from sewage treatment plant, and the centrifugal condition is
In 5min is centrifuged under 12000rpm, the cleaning is to be cleaned 3 times with the phosphate buffer that pH value is 7.4, in the tissue culture bottle
The final concentration of 2.5g VSS L of solution-1, the size of the tissue culture film is 16cm × 16cm, the condition in the growth cabinet
It is 24 DEG C of temperature, light application time 16h d-1, 60 μm of ol.m of intensity of illumination-2.s-1, the incubation time is 14d.
Preferably, the weight of the root tissue that step 8 is taken is 1g, and the grinding is carried out in sterile mortar, the constant temperature
Temperature in incubator is 30 DEG C, and incubation time is 2d~3d.
Preferably, the condition of culture described in step 9 is 150rpm shaken cultivations 24h at 30 DEG C；Centrifugal condition is
5min is centrifuged under 12000rpm；The cleaning is to be cleaned with phosphate buffer 3 times, adjusts the bacterium re-suspension liquid medium-height grass spirillum
OD600nmBe worth is 0.01；The reed Embryonal suspensor mass number is 10, and the volume of the bacterium re-suspension liquid is 20mL, described to incubate
The time educated is 30min.
Preferably, artificial endosperm described in step 10 contains with MS culture mediums as minimal medium in the minimal medium
Mass concentration be 3% sucrose as carbon source；The mass concentration of the sodium alginate is 3%, the CaCl2The concentration of solution is
100mM, the colloidal sol suspension and CaCl2PH value after solution regulation is 5.8, the sterilizing at 121 DEG C HTHP go out
Bacterium 20min.
Preferably, there is a green point on the reed Embryonal suspensor mass surface described in step 11, the pasteur pipet it is a diameter of
7mm, instills described CaCl2Time in solution is 30min, after the reed Embryonal suspensor mass is cleaned into 5 times with sterilized water
Dried on aseptic filter paper.
Preferably, described in step 12 20 artificial seeds are placed in each MS culture medium, condition of culture is 24 DEG C, light
It is 16hd according to the time-1, 60 μm of ol.m of intensity of illumination-2.s-1, repeat culture 3 times.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention adjusts reed embryo under isolated culture condition
The affinity of the internal sward spirillum of culture, then with Embryonal suspensor mass and careless spirillum homobium as propagating materials, through hydrogel
Embedding is prepared into reed artificial seed, and then induces the reed children for producing resistance to cadmium by artificial seed vertical transmission grass spirillum
Seedling, is adapted to large-scale promotion.
Brief description of the drawings
Fig. 1 is that reed callus induction of the present invention regenerates with reed seedling；
Fig. 2 is colonized for endogenetic bacteria root of the present invention；
Fig. 3 is careless spirillum Function Identification；
Fig. 4 is that careless spirillum co-cultures with reed Embryonal suspensor mass；
Fig. 5 is that reed seed plumular axis rots to scheme；
Fig. 6 is the resistance to cadmium performance detection comparison diagram of reed artificial seed.
Wherein, A is reed small ear in Fig. 1；B is reed seed；C is sticky callus；D, E are embryo callus；
F, G, H regenerate for reed seedling；I is the reed vegetative seedling that water culture is cultivated；
A is the reed regrowth that inactive sludge is incubated in Fig. 2；B is the reed regrowth that activated sludge is incubated；C is work
Property the generation of sludge regeneration induction seedling lateral root；D is the contrast of the reed regrowth with normal reed vegetative seedling of activated sludge incubation；
E is the root tissue endogenetic bacteria denaturing gradient gel electrophoresis finger-print of reed regrowth；
A is that careless spirillum produces heteroauxin Function Identification in Fig. 3, and B is that careless spirillum produces siderophore Function Identification；
A is careless spirillum grass OD in Fig. 4600nmIt is worth the reed Embryonal suspensor mass and normal reed embryo being incubated for 0.01 Incubating Solution
Property culture comparison diagram；B is through careless spirillum grass OD600nmIt is worth the reed vegetative seedling being incubated for 0.01 Incubating Solution.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Referring to Fig. 1-6, it is as follows that the present invention provides a kind of technical scheme steps：
Step one：The ripe reed small ear of collection, removes flower glume, and the reed seed with surface nondestructive is as explant；
Step 2：After the explant in step one is carried out into surface sterilization with the sodium hypochlorite that mass concentration is 5%, will be outer
Implant is placed on inducing culture, and white, translucent, glossiness sticky callus group is induced in explant plumule end
Knit, the preparation method of inducing culture is：It is dichlorphenoxyacetic acid, the 1mg of 1mg to concentration is added in every liter of MS fluid nutrient medium
Methyl α-naphthyl acetate, the 6-benzyladenine of 0.5mg, the sucrose of 30g and 0.5mg Glus；
Step 3：The sticky callus described in step 2 is stripped, by the sticky callus transposition in fresh
On inducing culture, further inducing surface has the embryo callus of tiny knob and green point；
Step 4：It is 2.5mgL that embryo callus described in step 3 are passed through into mass concentration-12,4-D bred
Culture, obtains Embryonal suspensor mass, and the compound method of proliferated culture medium is to the dichloro that 2.5mg is added in every liter of MS fluid nutrient medium
The agar of phenoxy acetic acid, the sucrose of 30g and 16g；
Step 5：Embryonal suspensor mass described in step 4 is gone to and generated on the regeneration culture medium without plant growth regulator
Whole plant, the collocation method of regeneration culture medium is：To in every liter of MS fluid nutrient medium add 2.5mg dichlorphenoxyacetic acid,
The sucrose of 30g and the agar of 16g；
Step 6：The height that will be generated in step 5 enters water-filling in going to 1/2MS fluid nutrient mediums for the whole plant of 7cm
Training, obtains reed regrowth, and reed regrowth survival rate reaches 95%；
Step 7：Trained in the aseptic tissue culture bottle that the reed regrowth of step 6 is gone to a height of 20cm, a diameter of 5cm
Support, to the aseptic OECD simulated wastewaters that 150ml is added in aseptic tissue culture bottle, the formula of OECD simulated wastewaters is to 0.5L deionizations
Peptone, the meat extract of 110mg, the urea of 30mg, the K of 28mg of 160mg are added in water2HPO4, 7mg NaCl, 4mg
CaCl2·2H2The MgSO of O and 2mg4·7H2O, is configured to mixed liquor, then the mixed liquor is settled into 1L with deionized water, then
Sterilized with the membrane filtration that specification is 0.22 μm, that is, be made OECD simulated wastewaters；Gathered from Wang Tang sewage treatment plants of Hefei City and lived
Property sludge, in view of activated sludge process is substantially the reinforcing of natural water body self cleaning, the present invention is incubated using activated sludge
The means of reed regrowth colonize endogenetic bacteria, and effective endophyte resource is excavated with from reed root tissue, by activated sludge in
Be centrifuged under 12000rpm 5min, with pH value be 7.4 phosphate buffer cleaning active sludge 3 times, activated sludge is added into group
To the final concentration of 2.5g VSS L of solution in tissue culture bottle in training bottle-1, it is that 16cm × 16cm tissue cultures film covers bottleneck with size, will
Tissue culture bottle is incubated 14d in growth cabinet, the regrowth contrast being incubated with the inactive sludge under same culture conditions, activity
The regrowth energy normal growth that sludge is incubated, and the inducible regrowth lateral root generation of activated sludge, its root growth speed are substantially fast
In the regrowth that inactive sludge is incubated；
Step 8：The root tissue of the reed regrowth that 1g is processed through step 7 is taken, after the surface of root tissue is carried out disinfection
Slurry is ground to form in sterile mortar, then the slurry standard dilution of the root tissue is coated on MS flat boards, the MS flat boards are put
Culture 2d~3d in 30 DEG C of constant incubators is put, treats that bacterium colony grows,；To just from the original of Wang Tang sewage treatment plants of Hefei City collection
Beginning activated sludge extracts STb gene with the symbiosis activated sludge for being incubated reed regrowth using Genome DNA extraction kit, to root group
Knit endogenetic bacteria and STb gene is extracted using CTAB methods；By primer 341F-GC-clamp (5'-
CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCCTACGGGAGGCAGCA G-3') and 518R (5'-
ATTACCGCGGCTGCTGG-3' the V3 hypervariable regions of microorganism 16S rRNA genes) are expanded；Use DCodeTMGeneral abrupt climatic change
System, carries out denatured gradient and coagulates through voltage 80V, electrophoresis time 14h, temperature 60 C, 1 × TAE of electrophoretic buffer, the μ L of applied sample amount 10
Gel electrophoresis, then dyeed with argentation；60 dominant bands on denaturing gradient gel electrophoresis collection of illustrative plates are carried out cutting the re-spread increasing of glue, gram
Grand sequencing；The characteristic sequence of correlation is chosen in gene pool, Clustal W softwares is reinstated with sequencing result one, according to most Datong District
The principle of source property carries out Multiple sequence alignments, and the result of detection shows there is abundant endophyte resource in regrowth root tissue；
The genomic DNA of bacterium in bacterium colony is extracted using DNA of bacteria kit, bacterial 16 S rRNA full length gene sequences, primer is expanded
Using 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3 '), will be pure
The amplified production of change is connected on pEASY-T3 cloning vectors, delivers to the sequencing of Shanghai Sheng Gong bioengineering limited company,
The characteristic sequence of correlation is chosen in gene pool, Clustal W softwares is reinstated with sequencing result one, according to the principle of maximum homology
Multiple sequence alignments are carried out, by the software kits of MEGA 5.0, the estimation of systematic evolution tree, channeled journey is carried out with ortho position connection method
Sequence inspection generation phylogenetic tree, and carries out tetraploid rice through gene pool server, the bacterium colony and careless spirillum GSF30 homologys
Up to 99.33%, thus identify that the bacterial strain is careless spirillum, its GenBank sequences accession number is KP713806；According to lattice Rickman with
Dessau method (Glickmann, E., Dessaux, Y.A., the Sol Coase base examination of the benzazolyl compounds that plant pathogenetic bacteria is produced
Agent examine, using with environmental microbiology, 1995,61:793-796.) the heteroauxin yield of quantitative determination grass spirillum is
60.93±1μg mL-1, according to Alexandria and Zhu Baier methods (Alexander, D.B., Zuberer, D.A. chrome azurol S
Reagent produces the evaluation of siderophore, soil biology and fertility, 1991,12 to rhizosphere bacteria:39-45.) its product iron of qualitative detection is carried
Body function, resistance to cadmium test detects that its minimal inhibitory concentration is 400 μM；
Step 9：Careless spirillum single bacterium colony is selected from the MS flat boards described in step 8, training in NB fluid nutrient mediums is seeded to
Support；Then NB fluid nutrient mediums are centrifuged 5min with collects thalline under the conditions of 12000rpm, 3 institutes is cleaned with phosphate buffer
The thalline of collection, bacterium re-suspension liquid, the OD of the careless spirillum of regulation are made using MS fluid nutrient mediums again suspension thalline600nmIt is worth and is
0.01；Take and be incubated 30min in 10 reed Embryonal suspensor mass immersion 20mL bacterium re-suspension liquids, by reed Embryonal suspensor mass go in
On aseptic filter paper, surface thalline is removed, then reed Embryonal suspensor mass is placed on MS flat boards is cultivated, through careless spirillum OD600nm
It is worth the Embryonal suspensor mass being incubated for 0.01 bacterium solution, 60% can further break up and form whole seedlings, and scanning electron microscopy
Mirror can be observed careless spirillum and survive in Embryonal suspensor mass, show that careless spirillum is successfully colonized in regrowth root tissue, be counted as every
Contain 4.0 × 10 in gram root tissue5The careless spirillum of CFU；
Step 10：With MS culture mediums as minimal medium, the sucrose conduct that mass concentration is 3% is contained in minimal medium
Carbon source, prepares artificial endosperm, because reed Embryonal suspensor mass can regenerate plant on the culture medium without plant growth regulator,
Thus any plant growth regulator need not be added in artificial endosperm；It is 3% to mass concentration is added in the endosperm solution
Sodium alginate, is configured to colloidal sol suspension as kind of a scytoblastema matter, while preparing 100mM CaCl using deionized water2Solution is used as network
Mixture, by colloidal sol suspension and CaCl2Solution adjusts pH value for after 5.8 respectively, the autoclave sterilization 20min at 121 DEG C；
Step 11：Kind scytoblastema matter in thering is green reed Embryonal suspensor mass to add step 10 on the surface in step 9
In, uniform mixing；A reed Embryonal suspensor mass is sucked every time with a diameter of 7mm sterile Pasteur pipets again, instills step
CaCl in ten230min in solution, will dry after reed Embryonal suspensor mass 5 times after taking-up on aseptic filter paper, that is, form artificial
Seed；
Step 12：Artificial seed described in step 11 is placed on carries out sprouting culture on MS culture mediums, each MS trainings
Support and 20 artificial seeds are placed in base, condition of culture is 24 DEG C, and light application time is 16hd-1, 60 μm of ol.m of intensity of illumination-2.s-1,
Culture 3 times is repeated, statistics seed germination rate is 90%.Stored 3 months at 4 DEG C, germination rate is constant.And careless spirillum root colonizes and is still
Every gram of root tissue 4.0 × 105CFU, this shows that kind of scytoblastema matter does not produce influence on coculture with complexing agent.
To the cadmium concentration higher than 300 μM, reed seed occurs obvious plumular axis putrefactive phenomenon.Based on this, will just
Normal reed seed (RZS), embedding reed seed (RZSM), the reed artificial seed (SS) of hydrogel embedding, and through careless spirillum
Reed seed (ZSH), embedding reed seed (ZSMH), the class seed of artificial seed (SSH) 6 being incubated, are placed in containing 300 μM 1/
Resistance to cadmium performance detection is carried out in 2MS flat boards.Per 5 seeds of plate, it is repeated 10 times.Observed after 50 days and found：Normal reed seed
(RZS) can not normally sprout；Reed seed (RZSM), the reed artificial seed (SS) of hydrogel embedding) can sprout, but root is stretched
Length is suppressed；Reed seed (ZSH), embedding seed (ZSMH), the sprouting of artificial seed (SSH) being incubated through careless spirillum are not
It is impacted, and root can normally extend.Wherein there is artificial seed (SSH) most flourishing root system to unite, and can be multiplied into a large amount of childrens
Seedling.These results indicate that although Artificial Seed Coats can play effect that barrier metal absorbs and protects embryo, this reinforcing is made
With very limited to reed growth of seedling.The introducing of careless spirillum, hence it is evident that alleviate influence of the Cd stress to reed growth of seedling, and energy
Induce a large amount of resistance to cadmium seedling.It is breeding with the artificial seed containing careless spirillum in the case where reed genetic potential is not influenceed
Means, can improve seed vitality and seedling quality, and for artificial swamp construction provides high quality seedling, be adapted to large-scale promotion.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding can carry out various changes, modification, replacement to these embodiments without departing from the principles and spirit of the present invention
And modification, the scope of the present invention be defined by the appended.
Claims (10)
1. a kind of preparation method of the artificial seed of resistance to cadmium reed seedling, it is characterised in that：Comprise the following steps：
Step one：The ripe reed small ear of collection, removes flower glume, and the reed seed with surface nondestructive is as explant；
Step 2：After by the explant sterilization described in step one, it is placed on inducing culture and induces sticky callus；
Step 3：The sticky callus described in step 2 is stripped, by the sticky callus transposition in fresh induction
On culture medium, embryo callus are further induced；
Step 4：By the embryo callus described in step 3 by Multiplying culture, Embryonal suspensor mass is obtained；
Step 5：Embryonal suspensor mass described in step 4 is gone to and generated completely on the regeneration culture medium without plant growth regulator
Plant；
Step 6：The whole plant that will be generated in step 5 is cultivated, and obtains reed regrowth；
Step 7：Cultivated during reed regrowth described in step 6 is gone into aseptic tissue culture bottle, to the aseptic tissue culture bottle
It is middle to add aseptic OECD simulated wastewaters；Collection activated sludge, will add the tissue culture bottle after activated sludge centrifugation, cleaning
In, bottleneck is covered with tissue culture film, the tissue culture bottle is incubated in growth cabinet；
Step 8：The root tissue of the reed regrowth of step 7 of learning from else's experience treatment, grinds after the surface of the root tissue is carried out disinfection
Slurry is worn into, then the slurry standard dilution of the root tissue is coated on MS flat boards, the MS flat boards are placed on constant incubator
Middle culture, treats that bacterium colony grows；
Step 9：Careless spirillum single bacterium colony is selected from the MS flat boards described in step 8, culture in NB fluid nutrient mediums is seeded to；So
NB fluid nutrient mediums described in centrifugal treating clean the thalline collected, using MS fluid nutrient mediums again with collects thalline afterwards
The thalline that suspends is made bacterium re-suspension liquid, adjusts the OD of the bacterium re-suspension liquid medium-height grass spirillum600nmIt is 0.01-1 to be worth；Take at least
One reed Embryonal suspensor mass is immersed in the bacterium re-suspension liquid and is incubated, and the reed Embryonal suspensor mass is gone on aseptic filter paper
After removing surface thalline, then the reed Embryonal suspensor mass is placed on MS flat boards is cultivated；
Step 10：Prepare artificial endosperm；To sodium alginate is added in the artificial endosperm solution, colloidal sol suspension is configured to as kind
Scytoblastema matter, while preparing CaCl using deionized water2Solution as complexing agent, by the colloidal sol suspension and CaCl2Solution is adjusted respectively
Sterilization treatment after section pH value；
Step 11：By in the kind scytoblastema matter described in the reed Embryonal suspensor mass addition step 10 described in step 9, uniformly mix
Afterwards, then with sterile Pasteur pipet a reed Embryonal suspensor mass is sucked every time, instills the CaCl described in step 102Solution
In, reed Embryonal suspensor mass cleaning is dried after taking-up, that is, form artificial seed；
Step 12：Artificial seed described in step 11 is placed on carries out sprouting culture on MS culture mediums.
2. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：It is described
The preparation method of inducing culture is：To added in every liter of MS fluid nutrient medium the dichlorphenoxyacetic acid of 1mg, the methyl α-naphthyl acetate of 1mg,
The 6-benzyladenine of 0.5mg, the sucrose of 30g and 0.5mg Glus；The compound method of the proliferated culture medium is
To the agar of the dichlorphenoxyacetic acid, the sucrose of 30g and 16g that 2.5mg is added in every liter of MS fluid nutrient medium；The regeneration training
Support base collocation method be：To the sucrose and 16g of the dichlorphenoxyacetic acid of addition 2.5mg, 30g in every liter of MS fluid nutrient medium
Agar.
3. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
Sterilization described in two is that surface sterilization, the sticky callus described in step 2 are carried out with the sodium hypochlorite that mass concentration is 5%
It is white, translucent, the glossy tissue in plumule end；Embryo callus described in step 3 are tiny for surface has
The tissue of knob and green point；Embryo callus described in step 4 are 2.5mgL by mass concentration-12,4-D increased
Grow culture.
4. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
The height of the whole plant described in six is 5cm~10cm, and training method carry out water planting in 1/2MS fluid nutrient mediums to go to, or
Carried out by cultivation matrix of vermiculite.
5. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
A height of 20cm of the aseptic tissue culture bottle described in seven, a diameter of 5cm, the formula of the OECD simulated wastewaters is to 0.5L deionized waters
Peptone, the meat extract of 110mg, the urea of 30mg, the K of 28mg of middle addition 160mg2HPO4, 7mg NaCl, 4mg CaCl2·
2H2The MgSO of O and 2mg4·7H2O, is configured to mixed liquor, then the mixed liquor is settled into 1L with deionized water, then uses specification
For 0.22 μm of membrane filtration sterilizes, that is, it is made OECD simulated wastewaters；The OECD simulated wastewaters added in aseptic tissue culture bottle
Volume be 150ml, the activated sludge from sewage treatment plant gather, the centrifugal condition be under 12000rpm be centrifuged
5min, the cleaning is to be cleaned 3 times with the phosphate buffer that pH value is 7.4, and solution is final concentration of in the tissue culture bottle
2.5g VSS L-1, the size of the tissue culture film is 16cm × 16cm, and the condition in the growth cabinet is 24 DEG C of temperature, light
According to time 16h d-1, 60 μm of ol.m of intensity of illumination-2.s-1, the incubation time is 14d.
6. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
The weight of eight root tissues for being taken is 1g, and the grinding is carried out in sterile mortar, and the temperature in the constant incubator is 30
DEG C, incubation time is 2d~3d.
7. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
The condition of the culture described in nine is 150rpm shaken cultivations 24h at 30 DEG C；Centrifugal condition be 12000rpm under 5min is centrifuged；It is described
Cleaning is to be cleaned with phosphate buffer 3 times, adjusts the OD600nmBe worth is 0.01；The reed Embryonal suspensor mass number is 10,
The volume of the bacterium re-suspension liquid is 20mL, and the time of the incubation is 30min.
8. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
Artificial endosperm described in ten contains the sucrose that mass concentration is 3% with MS culture mediums as minimal medium in the minimal medium
As carbon source；The mass concentration of the sodium alginate is 3%, the CaCl2The concentration of solution is 100mM, the colloidal sol suspension
And CaCl2PH value after solution regulation is 5.8, and the sterilizing is autoclave sterilization 20min at 121 DEG C.
9. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
There is green point on reed Embryonal suspensor mass surface described in 11, and a diameter of 7mm of the pasteur pipet instills described CaCl2
Time in solution is 30min, is dried on aseptic filter paper after the reed Embryonal suspensor mass is cleaned into 5 times with sterilized water.
10. the preparation method of the artificial seed of a kind of resistance to cadmium reed seedling according to claim 1, it is characterised in that：Step
20 artificial seeds are placed in each MS culture medium described in rapid 12, condition of culture is 24 DEG C of temperature, and light application time is 16hd-1, 60 μm of ol.m of intensity of illumination-2.s-1, repeat culture 3 times.
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| CN105850736A (en) * | 2016-04-11 | 2016-08-17 | 中南林业科技大学 | Production method of artificial seeds of Curculigo orchioides Gaertn. |
| CN105941146A (en) * | 2016-05-05 | 2016-09-21 | 青岛农业大学 | Preparation method of Echinodorus amazonicus artificial seed |
| CN106035091A (en) * | 2016-07-12 | 2016-10-26 | 成都东山兰韵农业有限公司 | Manufacturing method of cymbidium kanran artificial seeds |
| CN106134995A (en) * | 2016-06-30 | 2016-11-23 | 江苏大学 | A kind of Orychophragmus violaceus artificial seed preparation method of double-layer embedment |
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| CN105850736A (en) * | 2016-04-11 | 2016-08-17 | 中南林业科技大学 | Production method of artificial seeds of Curculigo orchioides Gaertn. |
| CN105941146A (en) * | 2016-05-05 | 2016-09-21 | 青岛农业大学 | Preparation method of Echinodorus amazonicus artificial seed |
| CN106134995A (en) * | 2016-06-30 | 2016-11-23 | 江苏大学 | A kind of Orychophragmus violaceus artificial seed preparation method of double-layer embedment |
| CN106035091A (en) * | 2016-07-12 | 2016-10-26 | 成都东山兰韵农业有限公司 | Manufacturing method of cymbidium kanran artificial seeds |
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