CN104642104A - Method for constructing alfalfa EMS mutant library - Google Patents
Method for constructing alfalfa EMS mutant library Download PDFInfo
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- CN104642104A CN104642104A CN201510070291.6A CN201510070291A CN104642104A CN 104642104 A CN104642104 A CN 104642104A CN 201510070291 A CN201510070291 A CN 201510070291A CN 104642104 A CN104642104 A CN 104642104A
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Abstract
The invention discloses a method for constructing an alfalfa EMS mutant library. The method comprises the following steps: taking the conventional alfalfa cultivated variety as a material, treating for a period of time by using an EMS solution of a certain concentration, performing mutant identification by utilizing each stage of seedling growth and development, and screening to obtain the alfalfa EMS mutant library. The method disclosed by the invention has the characteristics of simplicity and convenience in operation, high efficiency, stability, low cost and the like. By utilizing the method, the biological characters and agronomic characters can be identified and screened, a breeding material for genetic improvement of the variety is obtained, and rich research materials are provided for analyzing forward genetics and functional genomics of alfalfa.
Description
Technical field
The present invention relates to a kind of method that plant mutant storehouse builds, particularly relate to a kind of method that alfalfa ethylmethane sulfonate (EMS) mutant library builds.
Background technology
Alfalfa (Medicago sativa L.) has wide adaptability, high yield and high quality, improvement soil, high financial profit, characteristic that ecological functions are complete, have the laudatory title of " King of Pasture ".Alfalfa is rich in protein, multivitamin and mineral matter, good protein forage grass and the energy feed of multiple poultry eating, it both can be used for green grass or young crops and had raised, and was also used to develop into product simultaneously, comprised bale, grass meal, careless block, grass particle and concentrated leaf protein etc.Alfalfa has played conclusive effect in livestock breeding, has become the first-selected forage crop of China's livestock breeding agricultural structure adjustment.In addition, alfalfa adaptability is extremely strong, branch is many, can not only improve the soil, conserves water and soil by fixed nitrogen, also has important effect to the improvement of soil and the improvement of ecotope.
Building saturated mutant library is directly one of the effective method carrying out Functional Plant Genomics and genetic breeding research.In the crops such as model plant arabidopsis, paddy rice, construct saturated mutant library and the functional genomics research of being correlated with at present, obtain a collection of valuable Innovation Germplasm resource.If after building alfalfa EMS mutant library, by qualification, the screening of biological character and economical character, obtain the breeding material for this kind genetic improvement, meanwhile, for forward genetics and functional genome's Epidemiological Analysis of alfalfa provide abundant research material.
Chemical mutation artificial utilizes chemical mutagen, bring out crop to undergo mutation, by continuous screening and identification, cultivate directly or indirectly and can directly be used in the New Crop Varieties in production, thus mutagenesis is the means developing crop breeding so far rapidly.In numerous chemical mutagens, ethylmethane sulfonate (Ethyl methanesulphonate, EMS) is most widely used general, easy and simple to handle, efficient, stable chemical mutagen.But in known ethylmethane sulfonate (EMS) method of mutagenesis for alfalfa EMS process and the optimization method building EMS mutant library have not been reported.
Summary of the invention
For the deficiencies in the prior art, the problem that the present invention mainly solves is to provide a kind of method that alfalfa EMS mutant library builds.
The method that alfalfa EMS mutant library of the present invention builds, step is:
1) seed preparation: select the alfalfa seed that plumpness is high, and take this seed as derivant process material by set amount;
2) ethylmethane sulfonate (EMS) solution prepares: prepare the phosphate buffer of 0.1mol/L, pH5.8 and be dissolved in this phosphate buffer by derivant ethylmethane sulfonate (EMS), obtained ethylmethane sulfonate (EMS) solution;
Wherein, the compound method of the 0.1mol/L phosphate buffer of described pH5.8 is: by the Na of 1mol/L
2hPO
4solution is decided to be A liquid; By the NaH of 1mol/L
2pO
4solution is decided to be B liquid.Get A liquid 92.1mL and B liquid 7.9mL respectively, join in graduated cylinder, be settled to 1L.
3) seed is processed: with ethylmethane sulfonate (EMS) solution-treated alfalfa seed;
4) sow: the alfalfa seed after process is numbered respectively, and is seeded in nursery in the nutritive cube containing seedling nutritious soil;
5) transplant: grow after four true leaves until alfalfa seedling, moved into the land for growing field crops that ridging is good; Seedling growing period strengthens rich water quality management;
6) sowing: carry out mutant qualification, screening, numbering in each stage that seedling grows, individual plant sowing also records correlated traits;
7) each mutant will be obtained number respectively and record proterties, set up alfalfa ethylmethane sulfonate (EMS) mutant library, for follow-up alfalfa selection and use and the research of gene function group;
It is characterized in that:
Step 2) described ethylmethane sulfonate (EMS) solution concentration is 1.6% by weight percentage;
Step 3) method of described process seed is: existing by alfalfa seed distilled water immersion 24 ± 4 hours, then remove distilled water and by step 2) described EMS solution immersion treatment 8 ± 2 hours, during process, the container containing seed is placed on shaking table with the rotating speed jog of 60 ± 5rpm; After EMS solution-treated terminates, outwell EMS solution, distilled water to be added in the container containing seed and submergence seed, this container is placed in the rotating speed jog of 60 ± 5rpm on shaking table, each 30 minutes, outwells distilled water afterwards, distilled water is added and submergence seed again in this container, be placed in the rotating speed jog of 60 ± 5rpm on shaking table, each 30 minutes, so repeat seed 3-4 time after with distilled water flushing EMS process afterwards until remove EMS residual in seed;
Step 4) described type of seeding is: the seed after process is sowed at diameter 6 centimetres, in the non-woven fabrics nutrition pot for seedling culture of high 10 centimetres, artificial bunch planting, seed level 1-2 centimetre; One day water spray 3-4 time; The component proportion of wherein said seedling nutritious soil with parts by weight is: vermiculite: peat: perlite: become thoroughly decomposed organic compost=1:2:2:3.
In the method that above-mentioned alfalfa EMS mutant library builds: step 3) method of described process seed preferably: existing by alfalfa seed distilled water immersion 24 hours, then remove distilled water and by step 2) described EMS solution immersion treatment 8 hours, wherein EMS solution concentration is 1.6% by weight percentage.
The invention discloses a kind of method that alfalfa EMS mutant library builds, with existing alfalfa cultivar for material, is the method obtaining this kind alfalfa EMS mutant library through certain density EMS solution-treated certain hour.Method of the present invention has the features such as easy and simple to handle, efficient stable, cost are low.Utilize the method, by qualification, the screening of biological character and economical character, obtain the breeding material for this kind genetic improvement, meanwhile, for the forward genetics of alfalfa and functional genome's Epidemiological Analysis provide abundant research material.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details.
Embodiment 1: with No. one, lucerne in alfalfa cultivar for experiment material is described.
The method that alfalfa (No. one, lucerne) EMS mutant library builds, step is:
1) seed preparation: the high lucerne alfalfa seed of hand picking plumpness is as derivant process material.For ensureing to obtain alfalfa EMS saturated mutant library, the strict alfalfa seed weighing 320 grams.
2) EMS solution prepares: by the Na of 1mol/L
2hPO
4solution is decided to be A liquid; By the NaH of 1mol/L
2pO
4solution is decided to be B liquid.Get A liquid 92.1mL and B liquid 7.9mL respectively, join in graduated cylinder, be settled to 1L, obtain the 0.1mol/L phosphate buffer of pH5.8.Derivant ethylmethane sulfonate (EMS) is dissolved in this phosphate buffer, ethylmethane sulfonate (EMS) solution of obtained variable concentrations.
Wherein, EMS concentration is respectively 0,0.4%, 0.8%, 1.2%, 1.6%, 2.0%, 2.4%, 2.8%, 3.2% by weight percentage.
3) seed is processed: alfalfa (No. one, lucerne) is planted subgroup, is first placed in 1L triangular flask respectively, with distilled water immersion 24 hours.Remove after distilled water, the container containing seed, to seed immersion treatment 8 hours, 12 hours, 18 hours, is placed on shaking table with the rotating speed jog of 60rpm during process by the EMS solution adding above-mentioned each concentration respectively; After EMS solution-treated terminates, outwell EMS solution, distilled water to be added in the container containing seed and submergence seed, this container is placed in the rotating speed jog of 60rpm on shaking table, each 30 minutes, outwells distilled water afterwards, distilled water is added and submergence seed again in this container, be placed in the rotating speed jog of 60rpm on shaking table, each 30 minutes, so repeat seed 3-4 time after with distilled water flushing EMS process afterwards until remove EMS residual in seed.
4) sow: each group of alfalfa seed after process is numbered respectively, and is seeded in nursery in the nutritive cube containing seedling nutritious soil; Wherein, described type of seeding is: the seed after process is sowed at diameter 6 centimetres, in the non-woven fabrics nutrition pot for seedling culture of high 10 centimetres, and artificial bunch planting, seed level 1-2 centimetre; Nutritive cube is placed in 40 cm x 60 cm x, 10 centimetres of nutrition pallets, one day water spray 3-4 time; The component proportion of described seedling nutritious soil with parts by weight is: vermiculite: peat: perlite: become thoroughly decomposed organic compost=1:2:2:3.
5) transplant: grow after four true leaves until alfalfa seedling, moved into the land for growing field crops that ridging is good; Seedling growing period strengthens rich water quality management.
6) sowing: carry out mutant qualification, screening, numbering in each stage that young plant grows, individual plant sowing also records correlated traits.
7) each mutant obtained numbered respectively and recorded proterties, setting up alfalfa ethylmethane sulfonate (EMS) mutant library, for follow-up alfalfa selection and use and the research of gene function group.
In above-mentioned test, EMS concentration used is respectively 0,0.4%, 0.8%, 1.2%, 1.6%, 2.0%, 2.4%, 2.8%, 3.2%, in process alfalfa, the time gradient of a lucerne seed is 8 hours, 12 hours, 18 hours, each process repetition 3 times, the seed of each reprocessing is 200.
Draw by after the concentration of above-mentioned EMS process, time and experimental result statistical analysis: select lucerne seed in induction alfalfa to produce ethylmethane sulfonate (EMS) the medial lethal dose concentration 1.6% of sudden change and the processing time of 8 hours, therefore the present invention is defined as the optimal treatment condition (see table 1) building alfalfa EMS mutant library.
Table 1: the impact (%) of different EMS concentration, a different time gradient centering lucerne seed planting percent
Claims (2)
1. a method for alfalfa EMS mutant library structure, step is:
1) seed preparation: select the alfalfa seed that plumpness is high, and take this seed as derivant process material by set amount;
2) ethylmethane sulfonate (EMS) solution prepares: prepare the phosphate buffer of 0.1mol/L, pH5.8 and be dissolved in this phosphate buffer by derivant ethylmethane sulfonate (EMS), obtained ethylmethane sulfonate (EMS) solution;
3) seed is processed: with ethylmethane sulfonate (EMS) solution-treated alfalfa seed;
4) sow: the alfalfa seed after process is numbered respectively, and is seeded in nursery in the nutritive cube containing seedling nutritious soil;
5) transplant: grow after four true leaves until alfalfa seedling, moved into the land for growing field crops that ridging is good; Seedling growing period strengthens rich water quality management;
6) sowing: carry out mutant qualification, screening, numbering in each stage that seedling grows, individual plant sowing also records correlated traits;
7) each mutant obtained numbered respectively and recorded proterties, setting up alfalfa ethylmethane sulfonate (EMS) mutant library, for follow-up alfalfa selection and use and the research of gene function group;
It is characterized in that:
Step 2) described ethylmethane sulfonate (EMS) solution concentration is 1.6% by weight percentage;
Step 3) method of described process seed is: existing by alfalfa seed distilled water immersion 24 ± 4 hours, then remove distilled water and by step 2) described EMS solution immersion treatment 8 ± 2 hours, during process, the container containing seed is placed on shaking table with the rotating speed jog of 60 ± 5rpm; After EMS solution-treated terminates, outwell EMS solution, distilled water to be added in the container containing seed and submergence seed, this container is placed in the rotating speed jog of 60 ± 5rpm on shaking table, each 30 minutes, outwells distilled water afterwards, distilled water is added and submergence seed again in this container, be placed in the rotating speed jog of 60 ± 5rpm on shaking table, each 30 minutes, so repeat seed 3-4 time after with distilled water flushing EMS process afterwards until remove EMS residual in seed;
Step 4) described type of seeding is: the seed after process is sowed at diameter 6 centimetres, in the non-woven fabrics nutrition pot for seedling culture of high 10 centimetres, artificial bunch planting, seed level 1-2 centimetre; One day water spray 3-4 time; The component proportion of wherein said seedling nutritious soil with parts by weight is: vermiculite: peat: perlite: become thoroughly decomposed organic compost=1:2:2:3.
2. according to claim 1 alfalfa EMS mutant library build method, it is characterized in that: step 3) method of described process seed is: existing by alfalfa seed distilled water immersion 24 hours, then remove distilled water and by step 2) described EMS solution immersion treatment 8 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108419669A (en) * | 2018-02-28 | 2018-08-21 | 山东农业大学 | A kind of EMS directed mutagenesis, the method and device for screening salt tolerant alfalfa |
| CN108617502A (en) * | 2018-04-26 | 2018-10-09 | 贵州省蚕业研究所 | A method of building capsicum mutant library using ethylmethane sulfonate |
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| WO2010042865A2 (en) * | 2008-10-10 | 2010-04-15 | Arcadia Biosciences, Inc. | Non-transgenic tomato varieties having increased shelf life post-harvest |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108419669A (en) * | 2018-02-28 | 2018-08-21 | 山东农业大学 | A kind of EMS directed mutagenesis, the method and device for screening salt tolerant alfalfa |
| CN108419669B (en) * | 2018-02-28 | 2023-11-17 | 山东农业大学 | Method and device for EMS (enhanced message service) directional mutagenesis and screening salt-tolerant alfalfa |
| CN108617502A (en) * | 2018-04-26 | 2018-10-09 | 贵州省蚕业研究所 | A method of building capsicum mutant library using ethylmethane sulfonate |
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