CN103299900A - Wheat mutation breeding method - Google Patents
Wheat mutation breeding method Download PDFInfo
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- CN103299900A CN103299900A CN 201310160142 CN201310160142A CN103299900A CN 103299900 A CN103299900 A CN 103299900A CN 201310160142 CN201310160142 CN 201310160142 CN 201310160142 A CN201310160142 A CN 201310160142A CN 103299900 A CN103299900 A CN 103299900A
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Abstract
The invention provides a wheat mutation breeding method and aims to improve the chemical mutagenesis efficiency, generate a relative high point mutation frequency and have relative little chromosomal aberration. The wheat mutation breeding method disclosed by the invention adopts a 0.5% EMS (Ethylmethane Sulfonate)-phosphoric acid buffering solution to treat wheat pollen to induce andro gametes to have variation so as to obtain variant seeds; the 0.5% EMS-phosphoric acid buffering solution takes a phosphoric acid buffering solution as a solvent and a chemical mutagen ethylmethane sulfonate EMS is diluted to the volume ratio concentration of 0.5%; the mol concentration of the phosphoric acid buffering solution is 1/15mol/L; the dosage of the 0.5% EMS-phosphoric acid buffering solution is 25-50 microliters on each floret; and the wheat pollen is pollen which is about to be ripe or pollen grains which are primarily ripe.
Description
Technical field
The invention belongs to the wheat breeding technical field, be specifically related to wheat mutant library structure, germplasm innovation and genetic and breeding method.
Background technology
At present, output, quality, resistance that China produces the main wheat breed of going up establishing in large scale have obtained obvious improvement mostly, but owing to reasons such as ecotopes, for various places breeding man, certain 1 ~ 2 proterties of certain kind if can be improved, good economic and social benefits can be produced.Wheat mutation breeding is artificially to utilize physical mutagen (as X ray, gamma-rays, β ray, neutron, laser, electron beam, ion beam etc.) and mutagenesis (as alkylating agent, azide, base analogue etc.) to bring out the wheat genetic variation, obtain within a short period of time to have and utilize the mutant that is worth, according to the breeding objective requirement, seed selection becomes new varieties directly to produce utilization, or breeds new germ plasm and make the parent utilizes (being the indirect utilization of mutant) in breeding breeding approach.Physical mutagenesis causes because of high-energy ray, and chromosome aberrations rate height, structure variation are extensive, cause the chromosome set disorder, the sterile rate height of offspring, and type of separation is wide, and it is long from generation to generation to isozygoty; Mutagenesis is chemical agent and genetic material generation biochemical reaction, and mostly the result is gene mutations, and certain selectivity is arranged, and is an approach that solves orthomutation.Ethylmethane sulfonate (ethyl methane sulfonate is efficient, a stable chemical mutagen EMS), the mutation frequency height that produces after the mutagenesis, in the mutagenesis crops breeding most widely used general, effect is best, mainly brings out point mutation.Be used for seed treatment with EMS mutagenesis wheat more at present, because the seed embryo is the many cells tissues, have only a few cell to undergo mutation after the processing, thereby form mutant cell and Normocellular chimera, the mutant cell competitive ability a little less than, be difficult for growing up to new individuality, thereby mutation rate is lower.Therefore, how improving mutagenic frequency, is the direction that chemical mutation is made great efforts.
Summary of the invention
The present invention seeks to overcoming the deficiency of above-mentioned prior art, a kind of mutagenesis frequency that improves is provided, the frequency that produces point mutation is higher relatively, and chromosome aberration is less relatively, physiological damage is little, can be used to a certain specialized character of wheat is improved, and high yield appears easily, the wheat method for mutation breeding of mutant such as of fine quality.
The inventive method adopts 0.5% EMS-phosphate buffer to handle wheat pollen, induces andro gamete to produce variation, thus the seed that obtains making a variation; 0.5% EMS-phosphate buffer is to be solvent with the phosphate buffer, and (EMS) is diluted to volume by volume concentration 0.5% with the chemical mutagen ethylmethane sulfonate; The molar concentration of phosphate buffer is 1/15mol/L, and PH is 7; 0.5% EMS-phosphate buffer dosage is every Xiao Hua 25 ~ 50 uL(or 1 ~ 2); Wheat pollen is for being about to ripe pollen or first mature pollen.The concrete operations step is:
(1) preparation solvent: with ADSP (Na
2HPO
4) be diluted to 1/15mol/L Na with dissolved in distilled water
2HPO
4Solution; With AMSP (NaH
2PO
4) be diluted to 1/15mol/L NaH with dissolved in distilled water
2PO
4Solution; Get 1/15mol/L Na
2HPO
4Solution 611 mL, 1/15mol/L NaH
2PO
4Solution 389 mL mix, and are mixed with the 1/15mol/L phosphate buffer of PH=7, as solvent.
(2) preparation mutagen: (1/15mol/L, PH=7) middle ethylmethane sulfonate (EMS) 0.5 ml that adds is mixed with 0.5% EMS-phosphate buffer, as mutagen at the above-mentioned phosphate buffer of per 100 ml.
(3) select to physically well develop, healthy and strong, to have this kind feature and an anther development ripe or be about to ripe stem fringe or big tillering tassel, remove the tassel top and the bottom with tweezers earlier and grow bad small ear, only stay middle part 5-7 to big small ear, several Xiao Hua with staying small ear middle and upper part remove again, and every small ear only keeps two of base portions and grows best Xiao Hua.
(4) will keep the sterile lemma of Xiao Hua and interiorly coetoniumly cut off 1/3rd, suck above-mentioned mutagen with the 20mL injector for medical purpose, in syringe needle inserted from clip coetonium, every Xiao Hua injects 25 ~ 50 ul(1 ~ 2 droplet) soup, guarantee that soup is dipped into column cap and flower pesticide (pollen grain).
(5) put nearly 6 * 16 big or small sulfuric acid paper bags, the paper bag lower openings is amounted to along cob, pegs with staple, prevents from coming off.Write variety name, handle the date and handle the Xiao Hua number with pencil on the sulfuric acid paper bag.
(6) a wheat aging time results mutagenesis generation (M
1) seed, plantation M
1In generation, do not select, with individual plant, single fringe or the results M of the unit of being treated to
2For seed;
(7) plantation M
2Generation, M
2Generation is the maximum generation of variation, also is the critical period of selecting, and can select fine individual plant (fringe) according to breeding objective and character inheritance characteristics.
(8) M
3After generation, along with increase from generation to generation, proterties is separated minimizing, and some proterties is once obtaining and can stablizing rapidly.Selection through several generations just can obtain stable good mutantion line, and further new varieties are bred in test again.
Wheat is bloomed two peak periods in one day, for the morning 8 up to 11 o'clock and afternoon 2 up to 4 o'clock, thereby mutagenesis work generally the morning 8 up to 11 o'clock or between 4 o'clock, carry out afternoon 2.
The inventive method can obviously improve the mutagenesis frequency, and the frequency that produces point mutation is higher relatively, and chromosome aberration is less relatively, and physiological damage is little, can be used to a certain specialized character of wheat is improved, and high yield appears easily, mutant such as of fine quality.Handle the variation of wheat pollen induction with EMS, have easy and simple to handle, mutations in epithelial is high, can obtain advantage such as mass mutation body at short notice.Mutagenesis generation (M through the mutagenic treatment generation
1) germination rate, emergence rate, one-tenth strain rate, ripening rate generally lower, development delay, plant is downgraded or deformity, but these variations generally can not entail the offspring; Mutagenesis two generations (M
2) be the maximum generation of variation, except unfavorable variation, precocity, short bar, useful variation such as disease-resistant, degeneration-resistant, best in quality also can appear, and these variations are heritable, therefore from M
2In generation, rises and can carry out the pedigree selection.Because what EMS induced mostly is point mutation, the distortion of dye-free body and physiological damage are little, and selfing is isozygotied comparatively fast, but shortening the breeding cycle.Because wheat pollen is monoploid, the pollen grain (andro gamete) that produces mutagenesis can develop into the variation seed once finishing insemination, chimera can not occur, thereby variation frequency significantly improves than seed treatment (5～8%), generally can reach 8 ~ 10%.
Embodiment
Be example with No. 47 wheat breeds of Shanxi wheat.No. 47 wheat breeds of Shanxi wheat with characteristics such as its stable high yield are good, drought resistance is strong, have occupied leading position in the nonirrigated farmland always since the nineteen ninety-five authorization.But No. 47 plant of Shanxi wheat are higher, and stem stalk elasticity is relatively poor, easily lodge in abundance of water time or fertile nonirrigated farmland.Our test objective is on the basis that keeps No. 47 good characteristics of Shanxi wheat, improves its plant height and two proterties of stem stalk elasticity.
Test in beginning in 2009.No. 47 wheat breeds of Shanxi wheat are planted in the test site sowing, carry out pollen mutagenesis in the flowering initial stage with EMS.Mutagenesis is operated at 9 in the morning or mid-afternoon carries out.
The mutagenesis concrete steps are as follows:
(1) solvent preparation: preparation 1/15mol/L sodium hydrogen phosphate (Na
2HPO
4) solution and 1/15mol/L sodium dihydrogen phosphate (NaH
2PO
4) each 100 mL of solution; Get 1/15mol/L Na
2HPO
4Solution 61.1 mL, 1/15mol/L NaH
2PO
4Solution 38.9 mL put into 100 mL volumetric flasks, mix, and are mixed with 1/15mol/L phosphate buffer (PH=7).
(2) mutagen preparation: (1/15mol/L PH=7), adds ethylmethane sulfonate (EMS) 0.25 ml, is mixed with 0.5% EMS-phosphate buffer to get the above-mentioned phosphate buffer of 50 ml.
(3) select to physically well develop, healthy and strong, big fringe 30 fringes with No. 47 varietal characteristics of Shanxi wheat, require 2 ~ 3 Xiao Hua flower pesticide in fringe middle part to expose.Remove the tassel top and the bottom with tweezers and grow bad small ear, only stay the 5 pairs of small ears of growing basically identical in middle and lower part, several Xiao Hua with staying small ear middle and upper part remove again, and every small ear only keeps two of base portions and grows best Xiao Hua.
(4) will keep the sterile lemma of Xiao Hua and interiorly coetoniumly cut off 1/3rd, suck the 0.5% EMS soup for preparing in advance with the 20mL injector for medical purpose, syringe needle is inserted interior coetonium from clip, every Xiao Hua injects 25 ~ 50 uL(1 ~ 2 droplet) soup, guarantee that soup is dipped into column cap and flower pesticide (pollen grain).
(5) put sulfuric acid wheat hybridizing bag made of paper, the paper bag lower openings is amounted to along cob, pegs with staple, prevents from coming off.
(6) wheat aging time results M
1Seed obtains No. 47 mutagenesis kinds of 428 Shanxi wheats altogether.Bunch plating is planted M during (2009) autumn sowing in then
1In generation, do not select the present age, with the results M of the unit of being treated to
2For seed.
(7) M of chooser grain appearance quality better
2For 5000 in seed, program request plantation, line-spacing 25 cm, spacing in the rows 10 cm, No. 47 wheat breeds of Shanxi wheat in contrast, per 10 row M
2In generation, inserted No. 47 kinds of 2 row Shanxi wheats.M
2In generation, survive 4923 strains, therefrom filters out the dissimilar mutant of 458 strains, and aberration rate reaches 9.3%, and the mutant that individual plant is gathered in the crops the tool objective trait is pressed in objective trait mutant 25 strains such as wherein plant height reduction, stem stalk good springiness.
(8) M
3In generation, plants, select and gather in the crops by pedigree method later on.
At present, handle No. 47 wheat pollen inductions of Shanxi wheat andro gamete with 0.5% EMS and produce variation, its mutagenic progeny is planted to M
4In generation, it is that economical character meets breeding objective that 15 strains are arranged, and phenotypic character is stable, other has 2 strains to enter variety comparative test, its stem stalk good springiness, and lodging resistance is strong, mu Cheng Sui increases by 1.5 ten thousand fringes and 0.9 ten thousand fringes for No. 47 respectively than the Shanxi wheat, is expected to cultivate into new improved seeds.
Claims (3)
1. a grow wheat method for mutation breeding is characterized in that adopting 0.5% EMS-phosphate buffer to handle wheat pollen, induces andro gamete to produce variation, thus the seed that obtains making a variation; 0.5% EMS-phosphate buffer is to be solvent with the phosphate buffer, and EMS is diluted to volume by volume concentration 0.5% with the chemical mutagen ethylmethane sulfonate; The molar concentration of phosphate buffer is 1/15mol/L, and PH is 7; 0.5% EMS-phosphate buffer dosage is every Xiao Hua 25-50 uL; Wheat pollen is for being about to ripe pollen or first mature pollen.
2. wheat method for mutation breeding as claimed in claim 1 is characterized in that concrete steps are:
(1) preparation solvent: ADSP is diluted to 1/15mol/L Na with dissolved in distilled water
2HPO
4Solution; AMSP is diluted to 1/15mol/L NaH with dissolved in distilled water
2PO
4Solution; Get 1/15mol/L Na
2HPO
4Solution 611 mL, 1/15mol/L NaH
2PO
4Solution 389 mL mix, and are mixed with 1/15mol/L phosphate buffer 1000 mL of PH=7, as solvent;
(2) preparation mutagen: in the above-mentioned phosphate buffer of per 1000 ml, add ethylmethane sulfonate 5 mL, be mixed with concentration and be 0.5% EMS-phosphate buffer, as mutagen;
(3) select to physically well develop, healthy and strong, to have this kind feature and an anther development ripe or be about to ripe stem fringe or big tillering tassel, remove the tassel top and the bottom with tweezers earlier and grow bad small ear, only stay middle part 5-7 to big small ear, several Xiao Hua with staying small ear middle and upper part remove again, and every small ear only keeps two of base portions and grows best Xiao Hua;
(4) will keep the sterile lemma of Xiao Hua and interiorly coetoniumly cut off 1/3rd, and suck above-mentioned mutagen with the 20mL injector for medical purpose, in syringe needle is inserted from clip coetonium, every Xiao Hua injects 25-50 uL soup, guarantees that soup is dipped into column cap and flower pesticide;
(5) put the sulfuric acid paper bag, the paper bag lower openings is amounted to and is pegged along cob, prevents from coming off, and writes variety name, handles the date and handles the Xiao Hua number with pencil on the sulfuric acid paper bag;
(6) wheat aging time results mutagenesis generation seed, plantation mutagenesis generation seed is not selected, with individual plant, single fringe or the unit's of being treated to results mutagenesis two generations seed;
(7) plantation mutagenesis two generations seed, two generations of mutagenesis are maximum generations of variation, also are the critical periods of selecting, and can select fine individual plant or single fringe according to breeding objective and character inheritance characteristics;
(8) after the third generation, along with increase from generation to generation, proterties is separated minimizing, and some proterties can be stablized rapidly once acquisition, and the selection of several generations of process just can obtain stable good mutantion line, further tests and breeds new varieties.
3. wheat method for mutation breeding as claimed in claim 1 or 2 is characterized in that mutagenesis is operated in two peak periods that wheat is bloomed in a day and carries out, and namely the morning 8 is up to 11 o'clock or carried out between 4 o'clock afternoon 2.
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|---|---|---|---|
| CN 201310160142 CN103299900A (en) | 2013-05-03 | 2013-05-03 | Wheat mutation breeding method |
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|---|---|---|---|
| CN 201310160142 CN103299900A (en) | 2013-05-03 | 2013-05-03 | Wheat mutation breeding method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104642104A (en) * | 2015-02-10 | 2015-05-27 | 山东省林业科学研究院 | Method for constructing alfalfa EMS mutant library |
| CN107455257A (en) * | 2017-09-13 | 2017-12-12 | 安徽徽思远生态农业发展有限公司 | A kind of method of Chinese rose mutagenesis nursery |
| CN109729972A (en) * | 2019-01-15 | 2019-05-10 | 山西省农业科学院小麦研究所 | A method of improving wheat EMS induced mutation rate |
| CN109819889A (en) * | 2019-02-14 | 2019-05-31 | 河南省农业科学院植物营养与资源环境研究所 | A kind of method of the multiple mutation breeding of wheat |
| CN114223532A (en) * | 2021-10-21 | 2022-03-25 | 潘捷文 | Breeding method of new variety of big-ear wheat |
-
2013
- 2013-05-03 CN CN 201310160142 patent/CN103299900A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104642104A (en) * | 2015-02-10 | 2015-05-27 | 山东省林业科学研究院 | Method for constructing alfalfa EMS mutant library |
| CN107455257A (en) * | 2017-09-13 | 2017-12-12 | 安徽徽思远生态农业发展有限公司 | A kind of method of Chinese rose mutagenesis nursery |
| CN109729972A (en) * | 2019-01-15 | 2019-05-10 | 山西省农业科学院小麦研究所 | A method of improving wheat EMS induced mutation rate |
| CN109819889A (en) * | 2019-02-14 | 2019-05-31 | 河南省农业科学院植物营养与资源环境研究所 | A kind of method of the multiple mutation breeding of wheat |
| CN114223532A (en) * | 2021-10-21 | 2022-03-25 | 潘捷文 | Breeding method of new variety of big-ear wheat |
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Application publication date: 20130918 |