CN102511386A - 60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line - Google Patents
60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line Download PDFInfo
- Publication number
- CN102511386A CN102511386A CN2011103884123A CN201110388412A CN102511386A CN 102511386 A CN102511386 A CN 102511386A CN 2011103884123 A CN2011103884123 A CN 2011103884123A CN 201110388412 A CN201110388412 A CN 201110388412A CN 102511386 A CN102511386 A CN 102511386A
- Authority
- CN
- China
- Prior art keywords
- wheat
- gram
- electrophoresis
- add
- molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention belongs to a wheat radiation breeding method, in particular to a 60Co-Gamma radiation method for breeding a new wheat high-molecular-weight glutenin subunit line. Reports on the 60Co-Gamma ray breeding of a new wheat high-molecular-weight glutenin subunit mutant line in the prior art are not seen. The method includes the following steps: wheat seeds are irradiated by 60Co-Gamma rays for ten minutes, and the dosage rate is 200Gy; a first mutagenized generation is produced; seeds of a second mutagenized generation are produced; individuals of the M2 generation are grown and harvested; the wheat glutenin of the M2 generation is extracted by the half-seed method, and protein electrophoresis is carried out; half seeds are grown, and a third mutagenized generation is harvested; the wheat glutenin of the M3 generation is extracted; M3 protein electrophoresis is carried out; and the new mutant line with different number of high-molecular-weight glutenin subunit bands or high-molecular-weight glutenin subunit bands at different positions is determined by comparing with the parents. The method has the advantages that: the method breeds the new wheat high-molecular-weight glutenin subunit mutant line by combining 60Co-Gamma ray radiation with the electrophoresis and combining the field planting with indoor electrophoresis screening; and the result is accurate, scientific and reliable.
Description
Technical field
The invention belongs to the method for wheat radiation breeding, specifically
60Co-γ radiation method seed selection wheat high-molecular-weight glutelin subunit new lines.
Background technology
The wheat seed storage protein is a kind of important food plant albumen, and its output accounts for 1/3 of all cereal food plant albumen.Wheat preservation protein is made up of 70% alcohol soluble protein and 30% glutelin.Glutelin mainly exists with the form of the big polymer GMP of glutelin, and GMP is connected through disulfide bond S-S with low-molecular-weight glutenin subunit LMW-GS by high-molecular-weight glutelin subunit HMW-GS and is polymerized.The content of the big polymer GMP of glutelin and the quality of character and wheat flour are closely related, the formation of the composition of high-molecular-weight glutelin subunit HMW-GS and quality influence GMP, and then influence the quality of flour.
High-molecular-weight glutelin subunit HMW-GS is encoded by 3 Glu-1 sites that are positioned at the wheat first homologous chromosome group (1A, 1B and 1D), 2 closely linked genes of each site coding.Because allelic variation and gene silencing, most of hexaploid wheats have 3-5 high-molecular-weight glutelin subunit.Though high-molecular-weight glutelin approximately only accounts for 10% of wheat endosperm storage protein, it is most important to the processing quality of wheat.Research shows that the allelic variation of high-molecular-weight glutelin subunit and wheat processing quality are closely related, and each site is followed successively by Glu-D1＞Glu-A1＞Glu-B1 to the size that influences of quality.The 5+10 that express in the Glu-D1 site, 1 and the 2* that express in the Glu-A1 site, the subunits such as 7+8,13+16,14+15 and 17+18 that express in the Glu-B1 site are obvious to the forward effect of wheat processing quality, are called as high quality subunit.High-molecular-weight glutelin subunit is used as the important indicator of weighing bread wheat, can be used as the Wheat Breeding for Quality index of generation selection early, is widely used in Wheat Breeding for Quality.
HMW-GS mainly shows both ways the effect of wheat quality: one of which, and the HMW-GS subunit content is different with number, and is obviously different to the contribution of baked wheat quality; Its two, allelic variation is different to the effect of baking properties on the different loci.The main cause of Wheat Cultivars baking quality of bread variation is formed variation by HMW-GS and is caused.In Glu-A1, Glu-B1 and three gene locis of Glu-D1, the effect of Glu-D1 is apparently higher than the effect of Glu-A1 and Glu-B1.Chromosome of wheat 1D goes up the HMW-GS 1Dx5 and 1Dy10 subunit the having the greatest impact to flour strength of Glu-D1 site coding.The subunit composition is Glu-1D5+10, Glu-1A1 preferably, 2, Glu-1B17+18, and 7+8.And some subunit, like Null, 2+12 etc., then with relatively poor correlation of attributes.Proposed the quality points-scoring system of HMW-GS according to single subunit or subunit pair and the relation of SDS lauryl sodium sulfate precipitation number, its standards of grading are followed successively by 5+10 (score 4 minutes)＞1=2*=17+18=7+8=13+16 (3 minutes)＞7+9=2+12=3+12 (2 minutes)＞7=6+8=4+12=Null (1 minute).
High-molecular-weight glutelin subunit HMW-GS content is low to be the lower main cause of the whole gluten strength of China's bread wheat.China's wheat has the of less types of high-quality HMW-GS and combination 1Dx5+1Dy10,1A2*, 1Bx17+1By18, and the kind with subunits inferior such as 1Dx2+1Dy12 is then more.For example the gene carrying rate of the hard red winter wheat Dx5+Dy10 of the U.S. is 62%, and the gene carrying rate of hard red spring wheat Dx5+Dy10 is 91%; The gene carrying rate of Canada wheat Dx5+Dy10 is 80%; And the gene carrying rate of China wheat Dx5+Dy10 is merely 11.9%.Therefore the wheat breed of seed selection with high-quality HMW-GS subunit gene such as Dx5+Dy10 become one of main path of current domestic Wheat Quality Improvement.
Radioinduction is the important means of seed selection wheat germplasm.
60The Co-gamma-rays is the high energy light that 60 radiation of radioactive metal cobalt are sent, and is plant mutation source the most common, that application is the widest, and it can make chromosome of wheat recombinate; Break gene linkage, promote genetic recombination, mutation rate is high; Mutation spectrum is broad; And the valuable mutant character inheritance stability of being led to, the breeding time limit is short, is the important channel of seed selection excellent germplasm.FAO's statistics, the ratio that the world utilizes gamma-rays radiation improved variety to account for radiation breeding is 52.12%; China then accounts for 85.4%.
60The Co-gamma-rays has the advantages that to produce the wheat quality sudden change, and many in view of the above countries have bred the good wheat breeds of nutritional quality such as high egg, high-quality lysine content.But do not see
60The Co-gamma-rays is used for the wheat processing quality, particularly through seed selection high-molecular-weight glutelin subunit HMW-GS sudden change new lines, and then the report of realization processing quality breeding.
Use
60Co-gamma-rays radiation seed selection HMW-GS sudden change new lines is the germplasm basis of improvement China wheat processing quality, and for Wheat Breeding for Quality provides new method and approach, therefore, this invention is significant.
Through extensively inquiring about patent documentation and countries in the world publication, all not seeing has use
60The report of Co-γ radiation seed selection wheat high-molecular-weight glutelin subunit HMW-GS sudden change new lines.
Summary of the invention
The objective of the invention is:
1. provide
60The method of Co-γ radiation seed selection wheat high-molecular-weight glutelin subunit HMW-GS sudden change new lines; 2. improve the efficient of Wheat Breeding for Quality; 3. shorten the Wheat Breeding for Quality time limit.
The objective of the invention is to realize like this:
A kind of
60Co-γ radiation method seed selection wheat high-molecular-weight glutelin subunit new lines, method is following:
(1) the drying wheat seed is placed
60Under the Co-gamma-rays, dose rate is the 200Gy energy level, radiation 10 minutes; Placed 48 hours; Produce wheat mutagenesis 1 generation M
1
(2) plantation M
1For seed: trench sowing, 15 centimetres of ditch depths, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows; Mix the results mature seed, production mutagenesis 2 generation M
2Plant sub-group;
(3) plantation M
2For seed, trench sowing, 15 centimetres of ditch depths, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows, ripe back individual plant results, every strain is got 5 at random;
(4) half methods are extracted M
2For wheat gluten;
(5) M
2Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis;
(6) get the half granule seed plantation that gluten subunit sudden change germplasm has the embryo end, the same step of method (2), ripe back individual plant results mutagenesis 3 generation M
3Seed, every strain are randomly drawed 5;
(7) extract M with half method
3For wheat gluten;
(8) M
3Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis;
(9) confirm wheat high-molecular-weight glutelin matter subunit sudden change new lines: with quantity and the position of contrast ratio to the subunit band, quantity does not wait or upper-lower position is different is the new lines of suddenling change.
(4) half methods of step are extracted wheat M
2(7) half methods of glutelin and step are extracted M
3Method for wheat gluten is:
(1) preparation 50% isopropyl alcohol: get the 50ml isopropyl alcohol, add 50ml distilled water, be mixed;
(2) preparation of sample extracting solution: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml; The 25ml isopropyl alcohol; Distilled water 18.75ml, 50ml is sequentially added into altogether, is mixed;
(3) preparation of sample buffer: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml, 2 gram SDS lauryl sodium sulfate, glycerine 12.5ml, water 33.75ml is settled to 50ml, takes by weighing 0.001 gram bromjophenol blue and adds wherein, is mixed;
(4) get no half rice grain wheat, the 1.5ml centrifuge tube is put in fragmentation; 50% isopropyl alcohol that adds 0.5ml is positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(5) in the centrifuge tube of step (4), add 50% isopropyl alcohol of 0.5ml, place 60 ℃ of vapour to bathe the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(6) in the centrifuge tube of step (5), add 50% isopropyl alcohol of 0.5ml, be positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(7) in the centrifuge tube of deposition is arranged, add the 0.2ml sample extracting solution, 60 ℃ of vapour was bathed in the constant-temperature shaking casees 60 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 10 minutes, stay supernatant, inhale 120 μ L;
(8) in supernatant, add the sample buffer of 120 μ L, process wheat gluten liquid behind the mixing;
Step (5) M
2Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis and step (8) M
3The method of protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis is:
(1) preparation electrode buffer: in the beaker of 1000ml, add 800ml water, add glycine 14.4 grams, amino alkane 3.03 grams of Tris trihydroxy methyl, SDS lauryl sodium sulfate 1 gram is transferred pH to 8.3 with the NaOH sodium hydroxide of 1M, is settled to 1000ml;
(2) the amino alkane-hydrochloric acid solution of the Tris-HcL trihydroxy methyl of the 0.5M of preparation pH6.8: the amino alkane of 6.05 gram Tris trihydroxy methyls is dissolved in the 80ml water, transfers pH to 6.8 with the Hcl hydrochloric acid of 1M, is settled to 100ml;
(3) preparation of the amino alkane of 3M Tris-Hcl trihydroxy methyl-hydrochloric acid pH8.5 solution: the amino alkane of 36.395 gram Tris trihydroxy methyls, be dissolved in the 60m1 water, transfer pH to 8.5 with the Hcl hydrochloric acid of 1M, be settled to 100ml;
(4) preparation of 10%SDS sodium dodecyl sulfate solution: 5 gram SDS lauryl sodium sulfate add water and are settled to 50ml;
The preparation of (5) 1.5% ammonium persulfate solutions: 0.15 gram ammonium persulfate adds water and is settled to 10ml, joins existing usefulness at present;
(6) 30%Acr-0.8%Bis30% acrylamide-0.8N, the preparation of N '-methylene-bisacrylamide solution: 30 gram acryloyl ammoniums, be dissolved in the water less than 60ml, add 0.8 gram N then, N '-methylene-bisacrylamide is settled to 100ml;
(7) component and the compound method of concentrated glue:
Compound method: be sequentially added into above solution, mix;
(8) component of separation gel and compound method:
Compound method: be sequentially added into above solution, mix;
(9) preparation of gel: the component by separation gel is taken a sample in order, adds immediately in the electrophoresis tank after mixing, and adds to 70%, is filling it up with distilled water, places polymerization in 1 hour under fluorescent light.Pour out the water in the electrophoresis tank, take a sample in order, add immediately in the electrophoresis tank after mixing, till filling it up with, add sample comb, place polymerization in 1 hour under fluorescent light by the component that concentrates glue;
(10) extract comb,, be fixed in the electrophoresis tank with the distillation washing; The point sample electrophoresis: radiation offspring and parent's protein liquid, every groove point sample 15 μ l, every plate current stabilization 13mA, voltage one hectovolt is special, and electrophoresis 9 hours goes out the base to bromjophenol blue, and electrophoresis is 1 hour again;
(11) dye: Coomassie brilliant blue is added in the ethanol dissolve; With 12.5% trichloroacetic acid+0.05% Coomassie brilliant blue preparation dyeing liquor, the gel film that electrophoresis is good is put into dyeing liquor, places 8 hours;
(12) decolouring, take pictures: outwell dyeing liquor, adding distil water, decolouring is 1-2 days on decolorization swinging table, with gel imaging system gel is taken pictures;
(13) radiation offspring film band and parent's film band comparison, the new lines of screening high-molecular-weight glutelin subunit sudden change.
Main points of the present invention are:
The present invention provides
60The method of Co-gamma-rays radiation seed selection wheat high-molecular-weight glutelin subunit HMW-GS sudden change new lines, the efficient of raising Wheat Breeding for Quality shortens the Wheat Breeding for Quality time limit.Used radiation source is a radioactive metal cobalt 60; The radiation object is a common wheat, radiated time 10 minutes, and dose rate is 200Gy, taking-up plantation in 48 hours after the radiation; M
1, M
2, M
3Planting patterns is program request, 15 centimetres of the soil trench digging degree of depth, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows, ripe back results seed, M
1The full receipts; M
2Receive individual plant, every strain is got 5 seeds and is extracted glutelin with half method, and no embryo end carries out the SDS-PAGE electrophoresis, and screening has the seed of HMW-GS variation, and the plantation of embryo end is arranged; M
3Receive individual plant, every strain is got 5 seeds and is extracted glutelin with half method, and no embryo end carries out the SDS-PAGE electrophoresis, screens to have the seed that HMW-GS makes a variation, and has the embryo end to reserve seed for planting, plant new lines.
The present invention compares with class methods with existing both at home and abroad, its creativeness be following some:
1. first will
60The Co-γ radiation seed selection wheat high-molecular-weight glutelin subunit HMW-GS sudden change new lines that combines with the SDS-PAGE electrophoresis detection.
2. first will
60The Co-gamma-rays is applied to the wheat processing breeding for quality.
3. breeding efficiency is high.
4. shortened the breeding time limit.
The present invention is with the difference of domestic and international prior art:
1. breeding direction is different: both at home and abroad prior art be with
60The Co-gamma-rays is applied to wheat yield, resistance, nutritional quality breeding, and the present invention will
60The Co-gamma-rays is applied to wheat processing breeding for quality field.
Both at home and abroad prior art be with
60Plant after the radiation of Co-gamma-rays, in the field or indoors artificial observation screening, often receive the influence of natural conditions, the breeding quality is undesirable.The present invention with radiation after the screening of field planting and indoor SDS-PAGE electrophoresis experiment combine, screen according to The results in electrophoresis, improved the breeding quality.
3. both at home and abroad prior art detects with simple survey tool such as ruler, balance, and precision is low, and the result is unreliable, and the SDS-PAGE electrophoresis detection method condition that the present invention adopts is controlled, and instrumental resolution is high, the result accurately, science, reliable, the raising of breeding quality.
Advantage of the present invention is:
1.
60The Co-gamma-rays radiation seed selection wheat high-molecular-weight glutelin subunit HMW-GS sudden change new lines that combines with the SDS-PAGE electrophoresis detection; The result is accurate, science, reliable.
2. the screening of field planting and indoor SDS-PAGE electrophoresis experiment combines, and the result is accurate, science, reliable.
3. widened
60The gamma-ray range of application of Co-makes it to be applied to wheat processing breeding for quality field.
4. breeding efficiency is high, shortening the breeding cycle.
Description of drawings
Fig. 1 is the present invention
60The method flow diagram of Co-γ radiation seed selection wheat high-molecular-weight glutelin subunit sudden change new lines.
Embodiment
Through embodiment the present invention is further specified below.
Embodiment 1:
(1) gets 100 of Chongqing bread wheat dry seedses, put into nylon net bag, place
60Under the Co-gamma-rays, radiation dose rate is adjusted to the 200Gy energy level, and radiation 10 minutes is placed after 48 hours and taken out.
(2) plantation M
1For seed.15 centimetres of the soil trench digging degree of depth, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows become seedling 43 strains, and 1 fringe seed is gathered in the crops in the ripe every strain in back, and totally 1204 seeds mix, and constitute mutagenesis 2 generation (M
2) the kind sub-group.
(3) plantation M
2For seed.The same M of planting patterns
1Generation, plant 1204 strains, become seedling 915 strains, ripe back individual plant results, totally 915 strains, every strain are got 5 at random and are used for lauryl sodium sulfate-PAM SDS-PAGE electrophoresis.
(4) half methods are extracted wheat M
2Glutelin:
1. prepare 50% isopropyl alcohol: get the 50ml isopropyl alcohol, add 50ml distilled water, be mixed.
2. the preparation of sample extracting solution: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml; The 25ml isopropyl alcohol; Distilled water 18.75ml, 50ml is sequentially added into altogether, mixes.
3. the preparation of sample buffer: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml, 2 gram SDS lauryl sodium sulfate, glycerine 12.5ml, water 33.75ml is settled to 50ml, takes by weighing 0.001 gram bromjophenol blue and adds wherein, mixes.
4. get no half rice grain wheat, the 1.5ml centrifuge tube is put in fragmentation; 50% isopropyl alcohol that adds 0.5ml is positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
5. 50% isopropyl alcohol that in the centrifuge tube of deposition is arranged, adds 0.5ml places 60 ℃ of vapour to bathe the constant-temperature shaking case 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, and 10000 left the heart 1 minute, abandoned supernatant, stayed deposition;
6. have deposition centrifuge tube in add 50% isopropyl alcohol of 0.5ml, place 60 ℃ of vapour to bathe the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, centrifugal 1 minute, abandon supernatant, stay deposition;
7. in the centrifuge tube of deposition is arranged, add the 0.2ml sample extracting solution, 60 ℃ of vapour was bathed in the constant-temperature shaking casees 60 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins centrifugal 10 minutes, stay supernatant, inhale 120 μ L;
8. in supernatant, add the sample buffer of 120 μ L, be wheat gluten liquid behind the mixing;
(5) M
2Protein electrophorese:
1. prepare electrode buffer: in the beaker of 1000ml, add 800ml water, add glycine 14.4 grams, amino alkane 3.03 grams of Tris trihydroxy methyl, SDS lauryl sodium sulfate 1 gram is transferred pH to 8.3 with the NaOH sodium hydroxide of 1M, is settled to 1000ml;
2. prepare the amino alkane-hydrochloric acid solution of Tris-HcL trihydroxy methyl of the 0.5M of pH6.8: the amino alkane of 6.05 gram Tris trihydroxy methyls is dissolved in the 80ml water, transfers pH to 6.8 with the Hcl hydrochloric acid of 1M, is settled to 100ml;
3. the preparation of the amino alkane of 3M Tris-Hcl trihydroxy methyl-hydrochloric acid pH8.5 solution: the amino alkane of 36.395 gram Tris trihydroxy methyls, be dissolved in the 60ml water, transfer pH to 8.5 with the Hcl hydrochloric acid of 1M, be settled to 100ml;
4. the preparation of 10%SDS sodium dodecyl sulfate solution: 5 gram SDS lauryl sodium sulfate add water and are settled to 50ml;
5. the preparation of 1.5% ammonium persulfate solution: 0.15 gram ammonium persulfate adds water and is settled to 10ml, joins existing usefulness at present;
6. 30%Acr-0.8%Bis 30% acrylamide-0.8N, the preparation of N '-methylene-bisacrylamide solution: 30 gram acryloyl ammoniums, be dissolved in the water less than 60ml, add 0.8 gram N then, N '-methylene-bisacrylamide is settled to 100ml;
7. concentrate the component and the compound method of glue:
Compound method: be sequentially added into above solution, mix.
8. the component of separation gel and compound method:
Compound method: be sequentially added into above solution, mix.
9. the preparation of gel.Component by separation gel is taken a sample in order, adds immediately in the electrophoresis tank after mixing, and adds to 70%, is filling it up with distilled water, places polymerization in 1 hour under fluorescent light.Pour out the water in the electrophoresis tank, take a sample in order, add immediately in the electrophoresis tank after mixing, till filling it up with, add sample comb, place polymerization in 1 hour under fluorescent light by the component that concentrates glue.
10. extract comb,, be fixed in the electrophoresis tank with the distillation washing; Point sample electrophoresis: M
2Generation and parent's protein liquid, every groove point sample 15 μ l, every plate current stabilization 13mA, voltage one hectovolt is special, and electrophoresis 9 hours goes out the base to bromjophenol blue, and electrophoresis is 1 hour again;
Dye: Coomassie brilliant blue is added in the ethanol dissolve; With 12.5% trichloroacetic acid+0.05% Coomassie brilliant blue preparation dyeing liquor, the gel film that electrophoresis is good is put into dyeing liquor, places 8 hours.
Decolouring, take pictures: outwell dyeing liquor, adding distil water, decolouring is 1-2 days on decolorization swinging table, with gel imaging system gel is taken pictures;
Radiation offspring film band and the comparison of parent's film band, the new lines of screening high-molecular-weight glutelin subunit sudden change.
Filter out 21 of the individual plants of high-molecular-weight glutelin subunit variation.
(6) variation of plantation high-molecular-weight glutelin subunit has embryo end M
3Half granule seed, the same M of method
1, become seedling 15 strains, ripe back individual plant results seed, totally 15 strains, every strain are randomly drawed 5 and are carried out electrophoresis detection.
(7) extract wheat M with half method
3Glutelin.Method is extracted wheat M with (4) half methods
2Glutelin.
(8) M
3Protein electrophorese: method is with (5) M
2Protein electrophorese.
(9) right with contrast ratio, confirm wheat high-molecular-weight glutelin matter subunit sudden change new lines.Does not wait the quantity and the position of main comparison subunit band, quantity or upper-lower position is different is the new lines of suddenling change.
Filter out individual plant 6 strains of high-molecular-weight glutelin subunit variation.
Embodiment 2:
(1) gets 100 of No. 2 dry seedses in University of Science and Technology, river, put into nylon net bag, place
60Under the Co-gamma-rays, radiation dose rate is adjusted to 200Gy, and radiation 10 minutes is placed after 48 hours and taken out.
(2) plantation M
1For seed.15 centimetres of the soil trench digging degree of depth, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows become seedling 51 strains, and 1 fringe seed is gathered in the crops in the ripe every strain in back, and totally 1173 seeds mix, and constitute mutagenesis 2 generation M
2Plant sub-group.
(3) plantation M
2For seed.The same M of planting patterns
1Generation, plant 1173 strains, become seedling 903 strains, ripe back individual plant results, totally 903 strains, every strain are got 5 at random and are used for lauryl sodium sulfate-PAM SDS-PAGE electrophoresis.
(4) half methods are extracted wheat M
2Glutelin.
1. prepare 50% isopropyl alcohol: get the 50ml isopropyl alcohol, add 50ml distilled water, be mixed.
2. the preparation of sample extracting solution: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml; The 25ml isopropyl alcohol; Distilled water 18.75ml, 50ml is sequentially added into altogether, is mixed.
3. the preparation of sample buffer: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml, 2 gram SDS lauryl sodium sulfate, glycerine 12.5ml, water 33.75ml is settled to 50ml, takes by weighing 0.001 gram bromjophenol blue and adds wherein, is mixed.
4. get no half rice grain wheat, the 1.5ml centrifuge tube is put in fragmentation; 50% isopropyl alcohol that adds 0.5ml is positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
5. 50% isopropyl alcohol that in the centrifuge tube of deposition is arranged, adds 0.5ml places 60 ℃ of vapour to bathe the constant-temperature shaking case 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, and 10000 left the heart 1 minute, abandoned supernatant, stayed deposition;
6. have deposition centrifuge tube in add 50% isopropyl alcohol of 0.5ml, place 60 ℃ of vapour to bathe the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, centrifugal 1 minute, abandon supernatant, stay deposition;
7. in the centrifuge tube of deposition is arranged, add the 0.2ml sample extracting solution, 60 ℃ of vapour was bathed in the constant-temperature shaking casees 60 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins centrifugal 10 minutes, stay supernatant, inhale 120 μ L;
8. in supernatant, add the sample buffer of 120 μ L, be wheat gluten liquid behind the mixing;
(5) M
2Protein electrophorese:
1. prepare electrode buffer: in the beaker of 1000ml, add 800ml water, add glycine 14.4 grams, amino alkane 3.03 grams of Tris trihydroxy methyl, SDS lauryl sodium sulfate 1 gram is transferred pH to 8.3 with the NaOH sodium hydroxide of 1M, is settled to 1000ml;
2. prepare the amino alkane-hydrochloric acid solution of Tris-HcL trihydroxy methyl of the 0.5M of pH6.8: the amino alkane of 6.05 gram Tris trihydroxy methyls is dissolved in the 80ml water, transfers pH to 6.8 with the Hcl hydrochloric acid of 1M, is settled to 100ml;
3. the preparation of the amino alkane of 3M Tris-Hcl trihydroxy methyl-hydrochloric acid pH8.5 solution: the amino alkane of 36.395 gram Tris trihydroxy methyls, be dissolved in the 60ml water, transfer pH to 8.5 with the Hcl hydrochloric acid of 1M, be settled to 100ml;
4. the preparation of 10%SDS sodium dodecyl sulfate solution: 5 gram SDS lauryl sodium sulfate add water and are settled to 50ml;
5. the preparation of 1.5% ammonium persulfate solution: 0.15 gram ammonium persulfate adds water and is settled to 10ml, joins existing usefulness at present;
6. 30%Acr-0.8%Bis30% acrylamide-0.8N, the preparation of N '-methylene-bisacrylamide solution: 30 gram acryloyl ammoniums, be dissolved in the water less than 60ml, add 0.8 gram N then, N '-methylene-bisacrylamide is settled to 100ml;
7. concentrate the component and the compound method of glue:
Compound method: be sequentially added into above solution, mix.
8. the component of separation gel and compound method:
Compound method: be sequentially added into above solution, mix.
9. the preparation of gel.Component by separation gel is taken a sample in order, adds immediately in the electrophoresis tank after mixing, and adds to 70%, is filling it up with distilled water, places polymerization in 1 hour under fluorescent light.Pour out the water in the electrophoresis tank, take a sample in order, add immediately in the electrophoresis tank after mixing, till filling it up with, add sample comb, place polymerization in 1 hour under fluorescent light by the component that concentrates glue.
10. extract comb,, be fixed in the electrophoresis tank with the distillation washing; Point sample electrophoresis: M
2Generation and parent's protein liquid, every groove point sample 15 μ l, every plate current stabilization 13mA, voltage one hectovolt is special, and electrophoresis 9 hours goes out the base to bromjophenol blue, and electrophoresis is 1 hour again;
Dye: Coomassie brilliant blue is added in the ethanol dissolve; With 12.5% trichloroacetic acid+0.05% Coomassie brilliant blue preparation dyeing liquor, the gel film that electrophoresis is good is put into dyeing liquor, places 8 hours.
Decolouring, take pictures: outwell dyeing liquor, adding distil water, decolouring is 1-2 days on decolorization swinging table, with gel imaging system gel is taken pictures;
Radiation offspring film band and the comparison of parent's film band, the new lines of screening high-molecular-weight glutelin subunit sudden change.
Filter out individual plant 7 strains of high-molecular-weight glutelin subunit variation.
Embodiment 3:
(1) gets 100 of No. 2 dry seedses in University of Science and Technology, river, put into nylon net bag, place
60Under the Co-gamma-rays, radiation dose rate is adjusted to 200Gy, and radiation 10 minutes is placed after 48 hours and taken out.
(2) plantation M
1For seed.15 centimetres of the soil trench digging degree of depth, 20 centimetres of line-spacings, the program request seed, 10 centimetres of spacing in the rows become seedling 51 strains, and 1 fringe seed is gathered in the crops in the ripe every strain in back, and totally 1173 seeds mix, and constitute mutagenesis 2 generation M
2Plant sub-group.
(3) plantation M
2For seed.The same M of planting patterns
1Generation, plant 1173 strains, become seedling 903 strains, ripe back individual plant results, totally 903 strains.
(4) plantation M
3For seed.17 have half rice grain seed (M
3), the same M of planting patterns
1
Embodiment 4:
Measure the step and the result of HMW-GS component with half method.
Lauryl sodium sulfate-PAM SDS-PAGE electrophoresis.
(1) half method is extracted wheat M
2Glutelin.
1. prepare 50% isopropyl alcohol: get the 50ml isopropyl alcohol, add 50ml distilled water, be mixed.
2. the preparation of sample extracting solution: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml; The 25ml isopropyl alcohol; Distilled water 18.75ml, 50ml is sequentially added into altogether, is mixed.
3. the preparation of sample buffer: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml, 2 gram SDS lauryl sodium sulfate, glycerine 12.5ml, water 33.75ml is settled to 50ml, takes by weighing 0.001 gram bromjophenol blue and adds wherein, is mixed.
4. get no half rice grain wheat, the 1.5ml centrifuge tube is put in fragmentation; 50% isopropyl alcohol that adds 0.5ml is positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
5. 50% isopropyl alcohol that in the centrifuge tube of deposition is arranged, adds 0.5ml places 60 ℃ of vapour to bathe the constant-temperature shaking case 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, and 10000 left the heart 1 minute, abandoned supernatant, stayed deposition;
6. have deposition centrifuge tube in add 50% isopropyl alcohol of 0.5ml, place 60 ℃ of vapour to bathe the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, centrifugal 1 minute, abandon supernatant, stay deposition;
7. in the centrifuge tube of deposition is arranged, add the 0.2ml sample extracting solution, 60 ℃ of vapour was bathed in the constant-temperature shaking casees 60 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins centrifugal 10 minutes, stay supernatant, inhale 120 μ L;
8. in supernatant, add the sample buffer of 120 μ L, be wheat gluten liquid behind the mixing;
(2) protein electrophorese:
1. prepare electrode buffer: in the beaker of 1000ml, add 800ml water, add glycine 14.4 grams, amino alkane 3.03 grams of Tris trihydroxy methyl, SDS lauryl sodium sulfate 1 gram is transferred pH to 8.3 with the NaOH sodium hydroxide of 1M, is settled to 1000ml;
2. prepare the amino alkane-hydrochloric acid solution of Tris-HcL trihydroxy methyl of the 0.5M of pH6.8: the amino alkane of 6.05 gram Tris trihydroxy methyls is dissolved in the 80ml water, transfers pH to 6.8 with the Hcl hydrochloric acid of 1M, is settled to 100ml;
3. the preparation of the amino alkane of 3M Tris-Hcl trihydroxy methyl-hydrochloric acid pH8.5 solution: the amino alkane of 36.395 gram Tris trihydroxy methyls, be dissolved in the 60ml water, transfer pH to 8.5 with the Hcl hydrochloric acid of 1M, be settled to 100ml;
4. the preparation of 10%SDS sodium dodecyl sulfate solution: 5 gram SDS lauryl sodium sulfate add water and are settled to 50ml;
5. the preparation of 1.5% ammonium persulfate solution: 0.15 gram ammonium persulfate adds water and is settled to 10ml, joins existing usefulness at present;
6. 30%Acr-0.8%Bis30% acrylamide-0.8N, the preparation of N '-methylene-bisacrylamide solution: 30 gram acryloyl ammoniums, be dissolved in the water less than 60ml, add 0.8 gram N then, N '-methylene-bisacrylamide is settled to 100ml;
7. concentrate the component and the compound method of glue:
Compound method: be sequentially added into above solution, mix.
8. the component of separation gel and compound method:
Compound method: be sequentially added into above solution, mix.
9. the preparation of gel.Component by separation gel is taken a sample in order, adds immediately in the electrophoresis tank after mixing, and adds to 70%, is filling it up with distilled water, places polymerization in 1 hour under fluorescent light.Pour out the water in the electrophoresis tank, take a sample in order, add immediately in the electrophoresis tank after mixing, till filling it up with, add sample comb, place polymerization in 1 hour under fluorescent light by the component that concentrates glue.
10. extract comb,, be fixed in the electrophoresis tank with the distillation washing; Point sample electrophoresis: M
2Generation and parent's protein liquid, every groove point sample 15 μ l, every plate current stabilization 13mA, voltage one hectovolt is special, and electrophoresis 9 hours goes out the base to bromjophenol blue, and electrophoresis is 1 hour again;
Dye: Coomassie brilliant blue is added in the ethanol dissolve; With 12.5% trichloroacetic acid+0.05% Coomassie brilliant blue preparation dyeing liquor, the gel film that electrophoresis is good is put into dyeing liquor, places 8 hours.
Decolouring, take pictures: outwell dyeing liquor, adding distil water, decolouring is 1-2 days on decolorization swinging table, with gel imaging system gel is taken pictures;
Radiation offspring film band and the comparison of parent's film band, the new lines of screening high-molecular-weight glutelin subunit sudden change.
The foregoing description is merely preference of the present invention, is not used for limiting the present invention, and is all within principle of the present invention, made any be equal to alternative, revise and change, all within protection scope of the present invention.
Claims (3)
1. one kind
60Co-γ radiation method seed selection wheat high-molecular-weight glutelin subunit new lines, method is following:
(1) the drying wheat seed is placed
60Under the Co-gamma-rays, dose rate is the 200Gy energy level, radiation 10 minutes; Placed 48 hours; Produce wheat mutagenesis 1 generation M
1
(2) plantation M
1For seed: trench sowing, 15 centimetres of ditch depths, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows; Mix the results mature seed, production mutagenesis 2 generation M
2Plant sub-group;
(3) plantation M
2For seed, trench sowing, 15 centimetres of ditch depths, 20 centimetres of line-spacings, 10 centimetres of spacing in the rows, ripe back individual plant results, every strain is got 5 at random;
(4) half methods are extracted M
2For wheat gluten;
(5) M
2Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis;
(6) get the half granule seed plantation that gluten subunit sudden change seed has the embryo end, the same step of method (2), ripe back individual plant results mutagenesis 3 generation M
3Seed, every strain are randomly drawed 5;
(7) extract M with half method
3For wheat gluten;
(8) M
3Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis;
(9) confirm wheat high-molecular-weight glutelin subunit sudden change new lines: compare the quantity and the position of subunit band with the parent, quantity does not wait or upper-lower position is different is the new lines of suddenling change.
2. according to claim 1
60Co-γ radiation seed selection wheat high-molecular-weight glutelin subunit new lines is characterized in that: (4) half methods of step are extracted wheat M
2Glutelin and step (7) are extracted M with half method
3Method for wheat gluten is:
(1) preparation 50% isopropyl alcohol: get the 50ml isopropyl alcohol, add 50ml distilled water, be mixed;
(2) preparation of sample extracting solution: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml; The 25ml isopropyl alcohol; Distilled water 18.75ml, 50ml is sequentially added into altogether, is mixed;
(3) preparation of sample buffer: get 0.5 gram mercaptoethanol; The amino alkane of the Tris-Hcl trihydroxy methyl of the 0.5M of pH6.8-hydrochloric acid solution 6.25ml, 2 gram SDS lauryl sodium sulfate, glycerine 12.5ml, water 33.75ml is settled to 50ml, takes by weighing 0.001 gram bromjophenol blue and adds wherein, is mixed;
(4) get no half rice grain wheat, the 1.5ml centrifuge tube is put in fragmentation; 50% isopropyl alcohol that adds 0.5ml is positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(5) in the centrifuge tube of step (4), add 50% isopropyl alcohol of 0.5ml, place 60 ℃ of vapour to bathe the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(6) in the centrifuge tube of step (5), add 50% isopropyl alcohol of 0.5ml, be positioned over 60 ℃ of vapour and bathed in the constant-temperature shaking casees 30 minutes, hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 1 minute, abandon supernatant, stay deposition;
(7) in the centrifuge tube of deposition is arranged, add the 0.2ml sample extracting solution, 60 ℃ of vapour was bathed in the constant-temperature shaking casees 60 minutes, and hunting speed per minute 60 times changes in the centrifuge, 10000 rev/mins of rotating speeds, and centrifugal 10 minutes, stay supernatant, inhale 120 μ L;
(8) in supernatant, add the sample buffer of 120 μ L, process wheat gluten liquid behind the mixing;
3. according to claim 1
60Co-γ radiation method seed selection wheat high-molecular-weight glutelin subunit new lines is characterized in that: step (5) M
2Protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis and step (8) M
3The method of protein lauryl sodium sulfate-polyacrylamide gel SDS-PAGE electrophoresis is:
(1) preparation electrode buffer: in the beaker of 1000ml, add 800ml water, add glycine 14.4 grams, amino alkane 3.03 grams of Tris trihydroxy methyl, SDS lauryl sodium sulfate 1 gram is transferred pH to 8.3 with the NaOH sodium hydroxide of 1M, is settled to 1000ml;
(2) the amino alkane-hydrochloric acid solution of the Tris-HcL trihydroxy methyl of the 0.5M of preparation pH6.8: the amino alkane of 6.05 gram Tris trihydroxy methyls is dissolved in the 80ml water, transfers pH to 6.8 with the Hcl hydrochloric acid of 1M, is settled to 100ml;
(3) preparation of the amino alkane of 3M Tris-Hcl trihydroxy methyl-hydrochloric acid pH8.5 solution: the amino alkane of 36.395 gram Tris trihydroxy methyls, be dissolved in the 60ml water, transfer pH to 8.5 with the Hcl hydrochloric acid of 1M, be settled to 100ml;
(4) preparation of 10%SDS sodium dodecyl sulfate solution: 5 gram SDS lauryl sodium sulfate add water and are settled to 50ml;
The preparation of (5) 1.5% ammonium persulfate solutions: 0.15 gram ammonium persulfate adds water and is settled to 10ml, joins existing usefulness at present;
(6) 30%Acr-0.8%Bis 30% acrylamide-0.8N, the preparation of N '-methylene-bisacrylamide solution: 30 gram acryloyl ammoniums, be dissolved in the water less than 60ml, add 0.8 gram N then, N '-methylene-bisacrylamide is settled to 100ml;
(7) component and the compound method of concentrated glue:
Compound method: be sequentially added into above solution, mix;
(8) component of separation gel and compound method:
Compound method: be sequentially added into above solution, mix;
(9) preparation of gel: the component by separation gel is taken a sample in order, adds immediately in the electrophoresis tank after mixing, and adds to 70%, is filling it up with distilled water, places polymerization in 1 hour under fluorescent light.Pour out the water in the electrophoresis tank, take a sample in order, add immediately in the electrophoresis tank after mixing, till filling it up with, add sample comb, place polymerization in 1 hour under fluorescent light by the component that concentrates glue;
(10) extract comb,, be fixed in the electrophoresis tank with the distillation washing; The point sample electrophoresis: radiation offspring and parent's protein liquid, every groove point sample 15 μ l, every plate current stabilization 13mA, voltage one hectovolt is special, and electrophoresis 9 hours goes out the base to bromjophenol blue, and electrophoresis is 1 hour again;
(11) dye: Coomassie brilliant blue is added in the ethanol dissolve; With 12.5% trichloroacetic acid+0.05% Coomassie brilliant blue preparation dyeing liquor, the gel film that electrophoresis is good is put into dyeing liquor, places 8 hours;
(12) decolouring, take pictures: outwell dyeing liquor, adding distil water, decolouring is 1-2 days on decolorization swinging table, with gel imaging system gel is taken pictures;
(13) radiation offspring film band and parent's film band comparison, the new lines of screening high-molecular-weight glutelin subunit sudden change.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103884123A CN102511386A (en) | 2011-11-30 | 2011-11-30 | 60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103884123A CN102511386A (en) | 2011-11-30 | 2011-11-30 | 60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102511386A true CN102511386A (en) | 2012-06-27 |
Family
ID=46282694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103884123A Pending CN102511386A (en) | 2011-11-30 | 2011-11-30 | 60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102511386A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103798131A (en) * | 2014-02-14 | 2014-05-21 | 河北省农林科学院旱作农业研究所 | Method for breeding new germplasms of wheat including multi-ear wheat, large-ear wheat and large-grain wheat |
| CN104839008A (en) * | 2015-05-07 | 2015-08-19 | 新疆金天山农业科技有限责任公司 | Breeding method for high-quality strong gluten spring wheat |
| CN109757358A (en) * | 2019-03-15 | 2019-05-17 | 安徽普立米生物科技有限公司 | A kind of breeding method of high alcohol soluble protein rice and its application |
-
2011
- 2011-11-30 CN CN2011103884123A patent/CN102511386A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103798131A (en) * | 2014-02-14 | 2014-05-21 | 河北省农林科学院旱作农业研究所 | Method for breeding new germplasms of wheat including multi-ear wheat, large-ear wheat and large-grain wheat |
| CN104839008A (en) * | 2015-05-07 | 2015-08-19 | 新疆金天山农业科技有限责任公司 | Breeding method for high-quality strong gluten spring wheat |
| CN109757358A (en) * | 2019-03-15 | 2019-05-17 | 安徽普立米生物科技有限公司 | A kind of breeding method of high alcohol soluble protein rice and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107333643B (en) | A kind of resisting drought saving water strong gluten wheat directed cultivation method | |
| CN110089423B (en) | Breeding method of yellow-leaf red rice | |
| CN105052641B (en) | The screening technique of low cadmium rice and the method with the low cadmium routine Varieties In Late Rice of its seed selection | |
| CN104620969B (en) | Rapid breeding method of Sargassum fusiforme | |
| Terzić et al. | Screening wild sunflower species and F1 interspecific hybrids for resistance to broomrape | |
| CN102511386A (en) | 60Co-Gamma radiation method for breeding new wheat high-molecular-weight glutenin subunit line | |
| CN104719162A (en) | Method for screening dandelion salt-tolerance mutants | |
| CN102907317A (en) | Method for inducing euphorbia lathyris seeds and screening salt-tolerant mutants | |
| Shukla et al. | Influence of genetic variability on zinc response in wheat | |
| CN103493723A (en) | Breeding method of red glutinous rice | |
| CN102812803A (en) | Method for constructing pea EMS mutant library | |
| CN103283585B (en) | A kind of selection of black glutinous rice | |
| CN101361469B (en) | Common intertidal shellfish polymerization hybridization breeding method | |
| CN104938326A (en) | Rapid seed selection method of high-anthocyanin purple sweet potato variety | |
| CN107251834A (en) | A kind of selection of precocious intermediate keng rice kind | |
| CN102771386B (en) | Breeding method of rice | |
| CN1273002C (en) | Method for grafting solanum melongena L into solanum erianthum | |
| CN108849484A (en) | One kind using combine-head progeny row is the wheat breeding selection method of unit | |
| CN113317196A (en) | Creation method for processing special soybean protein germplasm | |
| CN109197578B (en) | Breeding method of P-group maize inbred line Lx286 | |
| Paul et al. | Correlation and path coefficient analysis in Giant Taro (Alocasia macrorrhiza L.) | |
| Fedoruk et al. | Analysis of potate quality: in vitro versus clonal propagation | |
| CN1903011A (en) | Method for cultivating photo-temp. sensitive type male nuclear sterile rice | |
| KR20130108235A (en) | A new plant variety of baemoochae xbrassicoraphanus having a stabilized high fertility | |
| KR102201400B1 (en) | New soybean variety having high content of functional compound and resistant to bacterial pustule and high productivity and breeding method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: ANHUI SCIENCE AND TECHNOLOGY COLLEGE Effective date: 20120816 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20120816 Address after: 233100 in Anhui Province in the eastern suburbs of Bengbu Fengyang County town of Donghua Road No. 9 Applicant after: Anhui Science and Technology University Address before: 233100 No. 2, unit 304, West 11, Anhui Science and Technology University, Bengbu, Anhui Applicant before: Zhang Congyu |
|
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120627 |