CN109392706A - The method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm - Google Patents
The method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm Download PDFInfo
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- CN109392706A CN109392706A CN201811344672.9A CN201811344672A CN109392706A CN 109392706 A CN109392706 A CN 109392706A CN 201811344672 A CN201811344672 A CN 201811344672A CN 109392706 A CN109392706 A CN 109392706A
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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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 method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm of the invention, it is related to rice polyploid breeding technical field, it is using the mature embryo of weakly sensitivity Oryza alta new germ plasm as explant evoked callus and to carry out chromosome doubling acquisition autoallopolyploid, this method is practical, obtained material has further utility value, can provide important materials source to carry out rice polyploid breeding.
Description
Technical field
Homologous heterologous more times the present invention relates to rice polyploid breeding technical field, especially weakly sensitivity high―strong nurserystock
The method for creating of body new germ plasm.
Background technique
Oryza alta originates in South America torrid areas, the favorable genes such as resistance not having containing a large amount of cultivated rices,
It is the precious resources of rice breeding.However, the genome of Oryza alta is CCDD, belong to allotetraploid, is planted with AA group
There are serious reproduction isolation during training rice progress interspecific hybridization, it is difficult to directly progress breeding utilization.
Existing research shows that autopolyploid can overcome cross infertility.So the induction high stalk of autoallopolyploid
Wild rice germplasm is the key that utilize its beneficial gene.
Summary of the invention
It is an object of the invention to avoid shortcoming in the prior art and to provide weakly sensitivity Oryza alta homologous different
The method for creating of source polyploid, to overcome cross infertility.
The purpose of the present invention is achieved through the following technical solutions:
The present invention provides the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm,
It as explant evoked callus and carries out chromosome doubling using the mature embryo of weakly sensitivity Oryza alta new germ plasm and obtains
Obtain autoallopolyploid.
Preferably, the method for creating of the mature embryo of the weakly sensitivity Oryza alta new germ plasm, comprising the following steps:
(1) high dose gamma Rays mutagenesis Oryza alta mature seed is utilized, (2) pass through super large group kind in 2 generations of radiation
It plants, selects weakly sensitivity mutant, be continuously selfed 21 generations, per generation selection weakly sensitivity and high fertility using the weakly sensitivity mutant
Single plant, the final Oryza alta new germ plasm for obtaining weakly sensitivity.
Preferably, the method specifically comprises the following steps:
Step 1: using the mature embryo of weakly sensitivity Oryza alta new germ plasm as explant, mature embryo callus induction is carried out；
Step 2: callus examination differentiation；
Step 3: callus colchicin doubles to handle；
Step 4: callus renewal cultivation and differentiation；
Step 5: regeneration bud Rooting and hardening-off culture；
Step 6: seedling hardening；
Step 7: seedling replanting；
Step 8: plant forms observation；
Step 9: root tip chromosomes counts；
Step 10: ploidy analysis.
Preferably, the step 1 is specifically: by the mature seed of Oryza alta new germ plasm slough shell disinfection after
It is seeded in MB+2, on the culture medium of 4-D 2.5mg/L+6-BA 1.0mg/L+Sucrose 3%+ Phytagel 0.4%, control
Temperature processed is 25 DEG C, and evoked callus generates under dark condition；
The step 2 is specifically: the embryo callus that induction generates is seeded in MS+KT 2.0mg/L+NAA 0.2mg/L
On the culture medium of+Sucrose 3%+ Phytagel 0.4%, temperature is controlled at 27 DEG C, callus is carried out under illumination condition
Examination differentiation；
Preferably, the step 3 is specifically: callus is placed in the NB+Sucrose 3%+ of the colchicin containing 200mg/L
In the fluid nutrient medium of DMSO 1.5%, temperature is controlled at 25 DEG C, the shaking in 150 turns per minute of 150rpm() under dark condition
Culture 120 hours is shaken in bed；
The step 4 is specifically: drying surface moisture after processed callus is washed away colchicin, is inoculated into later
On the culture medium of MS+KT 2.0mg/L+NAA 0.2mg/L+Sucrose 3%+Phytagel 0.4%, controlled at 25 DEG C,
Renewal cultivation 48 hours under dark condition return again under the illumination condition that temperature is 27 DEG C and carry out differentiation culture；
Preferably, the step 5 is specifically: the young shoot differentiated is transferred to MS+KT 2.0mg/L+6-BA 2.0mg/L+
On the Rooting and hardening-off culture base of NAA 0.5mg/L+IAA 0.5mg/L+ active carbon 0.5g/L, temperature is controlled at 27 DEG C, in illumination
Under the conditions of carry out Rooting and hardening-off culture；
The step 6 is specifically: the seedling for growing into 10 to 15cm being washed away to the culture medium of root attachment, is transplanted to bottom dress
Have in the plastic culture box of 1/3 to 1/2 mud, frequently with watering can towards water several times are sprayed on plant leaf during transplanting, has transplanted
Finish and cover culture box with one layer of plastic fresh-keeping membrane, surrounding is fixed, is placed in 28 DEG C of illumination cultivation room and cultivates 5 to 7 days,
After obvious growth situation occurs in seedling, plastic fresh-keeping membrane is scratched into thin mouth together with knife, it is allowed slowly to contact with external environment,
Preservative film can be fully opened after continued growth 7 and moves it to outdoor shady place, hereafter, day illumination in 6 to 8 hours is given once daily
Duration is penetrated, but noon strong light direct beam should be avoided, this process is 6 to 8 days；
Preferably, the step 7 is specifically: in the dusk or cloudy day, the plant crossed through hardening treatment band root soil being transplanted
To crop field；
The step 8 is specifically: its upgrowth situation of routine observation during plant field growing, including plant plant type, blade-shaped
State, the material for notable difference occur to phenotype observe preliminary screening variation plant in turn to its Stoma of Leaves
Preferably, the step 9 includes pretreatment, fixes and save three sub-steps.
Preferably, the pretreatment is specifically: the method for drawing material of the control material tip of a root are as follows: by Oryza alta new germ plasm
Mature seed peels off shell, is placed in clear water and impregnates 48 hours, a water was changed every 24 hours, is sorted out after seed shows money or valuables one carries unintentionally
It is placed in the culture dish for being covered with wetting filter paper, puts into 33 DEG C of germinating boxs after covering culture ware lid and carry out culture of rootage, until tender
When being about 1-2cm, the tip of a root about 0.5cm during clip is in mitosis under the conditions of that morning 10-11 point room temperature is set
It is pre-processed 3 hours in the 8-hydroxyquinoline of 0.002mol/L,
Double the sampling of the material tip of a root using living body sampling method, specifically: before 9 points of that morning, the material that will be grown in the field
Plant is dug out, and delicate tip of a root 1-2cm is selected between 10-11 point and is first rinsed well with clear water, is then placed at room temperature
It is pre-processed 3 hours in the 8-hydroxyquinoline of 0.002mol/L, the fixing step is specifically: pretreated two kinds of tips of a root is equal
It 3 times wash with distilled water, stops 10 minutes every time, places into the fixer of Kano and fix 24 hours, wash away fixation with distilled water
The tip of a root is put into 1N hydrochloric acid after liquid and is dissociated, hydrochloric acid need to not cross the tip of a root, dissociate 25 minutes in 60 DEG C of water-baths, after the completion of dissociation
3 times wash with distilled water, cleaning in 5 minutes is stopped every time and takes 1 tip of a root to be placed on glass slide after the completion, it will with tweezers and dissecting needle
The tip of a root is smashed to pieces, and carbolfuchsin is dripped, and is exposed in air and is dyed 3 minutes, the side of coverslip is contacted with dyeing liquor, wait contaminate
After color liquid is uniformly spread along contact surface, then coverslip is slowly put down, extra dyeing liquor is sucked with blotting paper, with thumb with appropriate
Dynamics pressing, notice that hand not slide, coverslip a few minutes gently beaten with rubber tip, the slide pressed is placed in microscope
Lower inspection, capture when finding good split coil method.
Preferably, the step 10 is specifically: the blade of the fresh Oryza alta new germ plasm of clip is placed in clean glass
In ware；A certain amount of nucleus lysate submergence blade is added dropwise, blade is cut into pieces；A certain amount of dyeing liquor dyeing is added
Afterwards, with the screen to filtrate, upper machine measures its ploidy, obtains the data of cell granulations number, analyzes the data.
Beneficial effects of the present invention: the initiative of present invention offer weakly sensitivity Oryza alta autoallopolyploid new germ plasm
Method is to be explant evoked callus with the mature embryo of weakly sensitivity Oryza alta new germ plasm and carry out chromosome and add
Autoallopolyploid is obtained again, which can carry out interspecific hybridization with AA group cultivated rice, reach breeding utilization
Purpose.
Detailed description of the invention
Fig. 1 is at the co-cultivation of the new germ plasm Induction Process of weakly sensitivity Oryza alta autoallopolyploid of the invention
The case where managing callus differentiation budding；
Fig. 2 and Fig. 3 is the feelings of taking root of the new germ plasm Induction Process of weakly sensitivity Oryza alta autoallopolyploid of the invention
Condition；
Fig. 4 is the strong sprout situation of the new germ plasm Induction Process of weakly sensitivity Oryza alta autoallopolyploid of the invention；
Fig. 5 is the plant forms of the new germ plasm of weakly sensitivity Oryza alta autoallopolyploid of the invention；
Fig. 6 is the form of common Oryza alta plant;
Fig. 7 is the plant forms of the new germ plasm of weakly sensitivity Oryza alta autoallopolyploid of the invention；
Fig. 8 is the ploidy analyser testing result of common Oryza alta plant；
Fig. 9 is the ploidy analyser testing result of the new germ plasm of weakly sensitivity Oryza alta autoallopolyploid of the invention.
Specific embodiment
The invention will be further described with the following Examples.
The method for creating for present embodiments providing weakly sensitivity Oryza alta autoallopolyploid new germ plasm, is first formulated weak
Photonasty
The mature embryo of Oryza alta new germ plasm, then induced by explant of the mature embryo of weakly sensitivity Oryza alta new germ plasm
Callus simultaneously carries out chromosome doubling acquisition autoallopolyploid.
Preferably, the method for creating of the mature embryo of the weakly sensitivity Oryza alta new germ plasm, comprising the following steps:
(1) high dose gamma Rays mutagenesis Oryza alta mature seed is utilized；(2) pass through super large group kind in 2 generations of radiation
It plants, selects weakly sensitivity mutant, be continuously selfed 21 generations, per generation selection weakly sensitivity and high fertility using the weakly sensitivity mutant
Single plant, the final Oryza alta new germ plasm for obtaining weakly sensitivity.
Specifically includes the following steps:
A. the radioinduction of Oryza alta mature seed:
Pass through the high dose gamma Rays Oryza alta mature seed (Co of usual common rice of 320Gy dosage60Radiation
Dosage is usually 300 or less), plantation 1 generation of radiation and bagging harvest seed.
B. the radiation and screening of weakly sensitivity mutant:
In 2 generation of re-radiation, carries out screening weakly sensitivity mutant using super large group, and super large number of groups reaches 15750 plants.From
Weakly sensitivity mutant is selected in 15750 plants of materials, harvests the seed of mutant.
C. weakly sensitivity and the initiative of the Oryza alta new germ plasm of high fertility:
Radiating for 3 generations, by ultra-long time (10 days, common rice only needs 2-3 days), vernalization obtains seedling under the conditions of 30 DEG C, and
It sows at the beginning of Guangzhou 3 months, transplanting at the beginning of 4 months.Select the single plant that can be eared before local August part.To 10 generations since radiating for 4 generations,
It is carried out continuously selfing, per generation all carries out (- 8 days 5 days) 30 DEG C of vernalization, sowing and transplanting for a long time, and selection can be before July in Guangzhou
Heading, setting percentage are more than 50% single plant.To 13 generations since radiating for 11 generations, it is carried out continuously selfing, per generation selection setting percentage is more than
70% and seed holding difference single plant, breeding of reserving seed for planting finally obtains weakly sensitivity, high fertility and the Oryza alta novel species for being not easy shattering
Matter " China is No. 5 wild ".(referring to Fig. 1, Fig. 2, Fig. 3), moreover, Oryza alta new germ plasm " China is No. 5 wild " is longer in southern sunshine
Early season (the 6-8 month) also can normally bloom heading.
As shown in Figure 1, the initiative (induction) of the weakly sensitivity Oryza alta autoallopolyploid new germ plasm of the present embodiment
Method, specifically includes the following steps:
A. the mature embryo callus induction of " China is No. 5 wild ":
The mature seed of " China wild No. 5 " is sloughed into shell by hand, impregnates 90s with 75% ethyl alcohol, outwells ethyl alcohol, then with 0.1% mercuric chloride
It impregnates 10 minutes, was during which rocked several times every 2-3 minutes, use sterile water wash seed 6-8 times, be placed in sterile after outwelling thimerosal
The training inoculated in MB+2,4-D 2.5mg/L+6-BA 1.0mg/L+Sucrose 3%+Phytagel 0.4% is dried on filter paper
[wherein 2,4-D is a kind of artificial synthesized plant hormone, entitled (2, the 4-dichlorophenoxy) acetic of chemistry on feeding base
Acid], controlled at 25 DEG C, evoked callus is generated under dark condition.
B. callus examination differentiation:
The pale yellow that induction generates is selected, after the more hard embryo callus of quality removes the culture medium being attached to above
It is seeded on the culture medium of MS+KT 2.0mg/L+NAA 0.2mg/L+Sucrose 3%+Phytagel 0.4%, controls temperature
Callus examination differentiation is carried out at 27 DEG C, under illumination condition.
C. callus colchicin doubles to handle:
Select the liquid training that high-quality callus is placed in the NB+Sucrose 3%+ DMSO 1.5% of the colchicin containing 200mg/L
It supports in base, control temperature is at 25 DEG C, shake culture 120h in 150rpm shaking table under dark condition.
D. callus renewal cultivation and differentiation:
The total training liquid for outwelling processed callus, washes away remaining colchicin with sterile water, is placed on aseptic filter paper and dries
The culture medium of MS+KT 2.0mg/L+NAA 0.2mg/L+Sucrose 3%+Phytagel 0.4% is inoculated into after a period of time
On, 25 DEG C, renewal cultivation 48h under dark condition, 27 DEG C are returned again to, is broken up under illumination condition.
E. regeneration bud strong plantlets and rootage:
It is living that the young shoot differentiated is transferred to MS+KT 2.0mg/L+6-BA 2.0mg/L+NAA 0.5mg/L+IAA 0.5mg/L+
On the strong seedling culture base of property charcoal 0.5g/L, 27 DEG C, Rooting and hardening-off culture is carried out under illumination condition.
F. seedling hardening:
The culture medium that the seedling of height growth to 10-15cm is washed away to root attachment with tap water is transplanted to bottom equipped with 1/3-
In the plastic culture box of 1/2 mud, it is lauched with watering can towards plant leaf spray is several during transplanting, transplanting is finished to be protected with one layer of plastics
Fresh film covers culture box, and surrounding carries out sealing.28 DEG C are placed in, is cultivated 5-7 days in illumination cultivation room, seedling to be found has obviously
After growing situation, plastic fresh-keeping membrane can be cut open to thin mouth together contacts it slowly with external environment, can after continued growth a couple of days
It fully opens preservative film and is moved out culturing room to outdoor shady place and adapt to two days.Hereafter, it is transferred to outdoor and is given once daily one
The long solar radiation of timing, but noon strong light direct beam should be avoided.
G. seedling replanting:
By the plant through hardening treatment at candlelight wait band root soil transplant to crop field.
H. plant forms are observed:
Its upgrowth situation of routine observation during plant field growing, including plant plant type, leaf morphology, after the plant that notes abnormalities
Further identified.
I. root tip chromosomes counts:
Decladding seed is impregnated into 48h and places it in the culture for being covered with wet filter paper after seed shows money or valuables one carries unintentionally every changing a water for 24 hours
In ware, vernalization is carried out in 33 DEG C of germinating boxs and is taken root.When extremely tender root long 1-2cm, the scissors clip tip of a root is used between Yu Shangwu 10-11 point
Position about 0.5cm, which is placed in the 8-hydroxyquinoline of 0.002mol/L, pre-processes 3h, and the above are the method for drawing material of the control material tip of a root；
The sampling of the material tip of a root is doubled using living body sampling method, i.e., 10 points of the morning or so, by the material part young root of field growing from mud
It is carefully dug out in soil, selects delicate tip of a root 1-2cm and first cleaned with clear water, then be placed in the 8-hydroxyquinoline of 0.002mol/L and locate in advance
Manage 3h.Treated the tip of a root 3 times wash with distilled water, stops 10 minutes every time, places into the fixer of Kano and fix 2h-24h.
It spends distilled water and washes away the tip of a root to be put into 1N hydrochloric acid after fixer and dissociate, hydrochloric acid need to not cross the tip of a root, dissociate in 60 DEG C of water-baths
25 minutes.Hydrochloric acid is cleaned 3 times with distilled water water after the completion of dissociation, is stopped 5 minutes every time.1 tip of a root is taken to put after the completion of cleaning
On glass slide, the tip of a root is smashed to pieces with tweezers and dissecting needle, drips carbolfuchsin, is exposed in air and dye 3 minutes.It will lid
The side of slide is contacted with dyeing liquor, after liquid to be dyed is uniformly spread along contact surface, then is slowly put down coverslip, is inhaled with blotting paper
Remove extra dyeing liquor.It is pressed with thumb with dynamics appropriate, notices that hand not slide, it is several that coverslip is gently beaten with rubber tip
Minute.The slide pressed is set into test under microscope, capture when finding good split coil method.
J. the ploidy analyser detects:
The fresh blade 0.5cm2 of clip, is placed in the clean glass dish of 6cm；400 μ L nucleus lysate (Sysmex are added dropwise
Cystain UV precise P Nuclei Extraction Buffer, #05-5002, Sysmex Partec GmbH, G
Rlitz, Germany), sample is submerged, is cut into pieces blade with sharp double-edged razor blade；1.6mL dyeing liquor (Sysmex is added
Cystain UV precise P Staining Buffer, #05-5002, Sysmex Partec GmbH, G rlitz,
Germany after) dyeing, 30 μm of the screen to filtrates, upper machine measures ploidy (Partec CyFlow Ploidy Analyzer, fluorescence
Source is 365 nm UV-LED, Sysmex Partec GmbH), measurement cell granulations number is 3000；Data use FCS
Express 5 (De Novo Software, Glendale, CA, USA) is analyzed.
The autoallopolyploid of method initiative through this embodiment can carry out interspecific hybridization with AA group cultivated rice, reach
The purpose of breeding utilization.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of range is protected, although explaining in detail referring to preferred embodiment to the present invention, those skilled in the art are answered
Work as understanding, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the reality of technical solution of the present invention
Matter and range.
Claims (10)
1. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm, it is characterised in that:
It as explant evoked callus and carries out chromosome doubling using the mature embryo of weakly sensitivity Oryza alta new germ plasm and obtains
Obtain autoallopolyploid.
2. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm as described in claim 1, feature
It is:
The method for creating of the mature embryo of the weakly sensitivity Oryza alta new germ plasm, comprising the following steps:
(1) high dose gamma Rays mutagenesis Oryza alta mature seed is utilized；
(2) it is planted in 2 generations of radiation by super large group, selects weakly sensitivity mutant, it is continuous using the weakly sensitivity mutant
It is selfed 21 generations, the single plant of per generation selection weakly sensitivity and high fertility, the final Oryza alta new germ plasm for obtaining weakly sensitivity.
3. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm as described in claim 1, feature
It is:
The method specifically comprises the following steps:
Step 1: using the mature embryo of weakly sensitivity Oryza alta new germ plasm as explant, mature embryo callus induction is carried out；
Step 2: callus examination differentiation；
Step 3: callus colchicin doubles to handle；
Step 4: callus renewal cultivation and differentiation；
Step 5: regeneration bud Rooting and hardening-off culture；
Step 6: seedling hardening；
Step 7: seedling replanting；
Step 8: plant forms observation；
Step 9: root tip chromosomes counts；
Step 10: ploidy analysis.
4. the initiative side of weakly sensitivity weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 2
Method, it is characterised in that: the step 1 is specifically: the mature seed of Oryza alta new germ plasm is sloughed into being followed by for shell disinfection
Kind is on MB+2, the culture medium of 4-D 2.5mg/L+6-BA 1.0mg/L+Sucrose 3%+ Phytagel 0.4%, control
Temperature is 25 DEG C, and evoked callus generates under dark condition；
The step 2 is specifically: the embryo callus that induction generates is seeded in MS+KT 2.0mg/L+NAA 0.2mg/L
On the culture medium of+Sucrose 3%+ Phytagel 0.4%, temperature is controlled at 27 DEG C, callus is carried out under illumination condition
Examination differentiation.
5. the initiative side of weakly sensitivity weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 4
Method, it is characterised in that: the step 3 is specifically: callus is placed in the NB+Sucrose 3% of the colchicin containing 200mg/L
In the fluid nutrient medium of+DMSO 1.5%, temperature is controlled at 25 DEG C, 150 turns per minute of 150rpm's) under dark condition
Culture 120 hours is shaken in shaking table；
The step 4 is specifically: drying surface moisture after processed callus is washed away colchicin, is inoculated into later
On the culture medium of MS+KT 2.0mg/L+NAA 0.2mg/L+Sucrose 3%+Phytagel 0.4%, controlled at 25 DEG C,
Renewal cultivation 48 hours under dark condition return again under the illumination condition that temperature is 27 DEG C and carry out differentiation culture.
6. the initiative side of weakly sensitivity weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 5
Method, it is characterised in that: the step 5 is specifically: the young shoot differentiated is transferred to MS+KT 2.0mg/L+6-BA 2.0mg/L
On the Rooting and hardening-off culture base of+NAA 0.5mg/L+IAA 0.5mg/L+ active carbon 0.5g/L, temperature is controlled at 27 DEG C, in illumination
Under the conditions of carry out Rooting and hardening-off culture；
The step 6 is specifically: the seedling for growing into 10 to 15cm being washed away to the culture medium of root attachment, is transplanted to bottom dress
Have in the plastic culture box of 1/3 to 1/2 mud, frequently with watering can towards water several times are sprayed on plant leaf during transplanting, has transplanted
Finish and cover culture box with one layer of plastic fresh-keeping membrane, surrounding is fixed, is placed in 28 DEG C of illumination cultivation room and cultivates 5 to 7 days,
After obvious growth situation occurs in seedling, plastic fresh-keeping membrane is scratched into thin mouth together with knife, it is allowed slowly to contact with external environment,
Preservative film can be fully opened after continued growth 7 and moves it to outdoor shady place, hereafter, day illumination in 6 to 8 hours is given once daily
Duration is penetrated, but noon strong light direct beam should be avoided, this process is 6 to 8 days.
7. the initiative side of weakly sensitivity weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 6
Method, it is characterised in that: the step 7 is specifically: in the dusk or cloudy day, by the plant crossed through hardening treatment band root soil
It transplants to crop field；
The step 8 is specifically: its upgrowth situation of routine observation during plant field growing, including plant plant type, blade-shaped
State, the material for notable difference occur to phenotype observe preliminary screening variation plant in turn to its Stoma of Leaves.
8. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 3, feature
Be: the step 9 includes pretreatment, fixes and save three sub-steps.
9. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 8, feature
Be: the pretreatment is specifically: the method for drawing material of the control material tip of a root are as follows: shells the mature seed of Oryza alta new germ plasm
Decapsidate is placed in clear water and impregnates 48 hours, a water was changed every 24 hours, is sorted out to be placed in after seed shows money or valuables one carries unintentionally and is covered with profit
In the culture dish of wet filter paper, puts into 33 DEG C of germinating boxs after covering culture ware lid and carry out culture of rootage, until tender root long about 1~2cm
When, the tip of a root about 0.5cm during clip is in mitosis under the conditions of that morning 10-11 point room temperature is placed in
It is pre-processed 3 hours in the 8-hydroxyquinoline of 0.002mol/L,
Double the sampling of the material tip of a root using living body sampling method, specifically: before 9 points of that morning, the material that will be grown in the field
Plant is dug out, and delicate 1~2cm of the tip of a root is selected between 10-11 point and is first rinsed well with clear water, is then placed at room temperature
It is pre-processed 3 hours in the 8-hydroxyquinoline of 0.002mol/L, the fixing step is specifically: pretreated two kinds of tips of a root is equal
It 3 times wash with distilled water, stops 10 minutes every time, places into the fixer of Kano and fix 24 hours, wash away fixation with distilled water
The tip of a root is put into 1N hydrochloric acid after liquid and is dissociated, hydrochloric acid need to not cross the tip of a root, dissociate 25 minutes in 60 DEG C of water-baths, after the completion of dissociation
3 times wash with distilled water, cleaning in 5 minutes is stopped every time and takes 1 tip of a root to be placed on glass slide after the completion, it will with tweezers and dissecting needle
The tip of a root is smashed to pieces, and carbolfuchsin is dripped, and is exposed in air and is dyed 3 minutes, the side of coverslip is contacted with dyeing liquor, wait contaminate
After color liquid is uniformly spread along contact surface, then coverslip is slowly put down, extra dyeing liquor is sucked with blotting paper, with thumb with appropriate
Dynamics pressing, notice that hand not slide, coverslip a few minutes gently beaten with rubber tip, the slide pressed is placed in microscope
Lower inspection, capture when finding good split coil method.
10. the method for creating of weakly sensitivity Oryza alta autoallopolyploid new germ plasm as claimed in claim 3, feature
Be: the step 10 is specifically: the blade of the fresh Oryza alta new germ plasm of clip is placed in clean glass dish；It is added dropwise
A certain amount of nucleus lysate submerges blade, and blade is cut into pieces；After a certain amount of dyeing liquor dyeing is added, with sieve mistake
Filter, upper machine measure ploidy, obtain the data of cell granulations number, analyze the data.
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| CN115226631A (en) * | 2022-08-22 | 2022-10-25 | 山西景致苗木有限公司 | Plant rooting and seedling strengthening culture medium |
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| CN1341350A (en) * | 2001-09-30 | 2002-03-27 | 湖北大学 | Method for high-efficiency breeding rice polyploid by combination of tissue culture and chemical induction |
| CN101044836A (en) * | 2007-04-28 | 2007-10-03 | 湖北大学 | Method for breeding autoallopolyploid rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115226631A (en) * | 2022-08-22 | 2022-10-25 | 山西景致苗木有限公司 | Plant rooting and seedling strengthening culture medium |
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