CN105510319A - Method for quickly identifying color of seed coats of rape seeds in early development period - Google Patents
Method for quickly identifying color of seed coats of rape seeds in early development period Download PDFInfo
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- CN105510319A CN105510319A CN201510887008.9A CN201510887008A CN105510319A CN 105510319 A CN105510319 A CN 105510319A CN 201510887008 A CN201510887008 A CN 201510887008A CN 105510319 A CN105510319 A CN 105510319A
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- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention relates to a method for quickly identifying the color of seed coats of rape seeds in the early development period. A chemical dyeing method is adopted, dye specifically combined with flavonoid is added for dyeing the seed coats of yellow and black rape seeds, and the seed coats of rape seeds eight days after pollination are taken, put into transparent containers containing methanol, acetone, trifluoroacetic acid normal butanol and an acid solution of ferric ammonium alum, left to stand for 15-20 minutes at the room temperature, then boiled for 15-30 minutes and cooled on ice for 20-25 minutes. A solution in the container accommodating the seed coats of the black rape seeds turns into purple red while the solution in the container accommodating the seed coats of the yellow rape seeds does not turn purple red, and the purpose of identifying the color of the seed coats of the rape seeds is achieved.
Description
Technical field
The present invention relates to agriculture Applied Biotechnology field, particularly relate to a kind of method that rape seed grows early stage kernel seed coat colour Rapid identification.
Background technology
Research shows, under identical genetic background, yellow seed coat rape (abbreviation yellow seed rape) has compared with Black seed coat rape (black seed rape) that seed coat is thin, oleaginousness is high, pigment and the advantage such as impurity is few, content of lignin is low in oil.At present, yellow seed rape breeding is the focus of current rapeseed breeding.It is just can display that rape seed skin color will arrive maturation, no matter is black seed or yellow seed, and after seed maturity, embryo is all yellow.The color of seed is determined by kernel seed coat colour, and the seed coat of black seed rape is black; The seed coat of yellow seed rape is transparent, and the seed of yellow seed rape shows the yellow of embryo.No matter be that yellow seed or black seed are early stage in seed development, seed coat is all green, breeder can not select kernel seed coat colour in early days in seed development, needs to select kernel seed coat colour proterties after seed maturity, also exist so time-consuming, take a lot of work, the problem such as expensive.Therefore, find a kind of method of Rapid identification seed kernel seed coat colour, the authentication method of kernel seed coat colour can be carried out in seed development in early days, rape yellow seed breeding is of great significance.
Proanthocyanidin is the key substance causing arabidopsis seed color difference, and rape and arabidopsis belong to Cruciferae together, and we infer that proanthocyanidin may be the decision material of rape seed color distortion.We find when studying biological approach and the constituent of the yellow black seed formation of rape, dihydroflavonol 4-reductase gene (DFR), anthocyanidin synthase gene (ANS) and anthocyanidin reductase gene (ANR) are variant at the yellow black seeds growth early expression of rape, the genes such as DFR are not expressed in yellow seed seed coat, we infer cause yellow black seed difference may be the genes such as DFR institute control synthesis material (flavones 3, the analogs such as 4-glycol, the precursor substance of proanthocyanidin synthesis) cause, the materials such as flavones 3,4-glycol may be there is no in yellow seed seed coat.Research finds that the material such as trifluoroacetic acid, ferriammonium sulfate is under acid and hot conditions by experiment, and can the precursor substance of proanthocyanidin synthesis in canola seed coat be combined, generation aubergine material, solution colour becomes aubergine.
Summary of the invention
The object of the present invention is to provide a kind of directly perceived, the method that economic, easy rape seed grows early stage Rapid identification seed coat colour, to be applied to the yellow-seed breeding of rape, and being applied to the qualification of cross-bred rape seed purity, the cenospecies for yellow seed coat rape carries out the Forepart identification of purity.
The technical solution used in the present invention: a kind of method of growing early stage Rapid identification kernel seed coat colour at rape seed, the seed coat of pollination after 8 days is it is characterized in that to put into transparent vessel, add solution A and soak 15-20 minute at normal temperatures, then staining reagent B is added, after boiling 15-30 minute, at cooled on ice 20-25 minute, the color of rape seed is identified according to the change of liquid color after reaction, the liquid in containers color putting into black seed rape seed coat becomes aubergine, and the liquid in containers color putting into yellow seed rape seed coat does not have aubergine to produce.
The volume ratio of described solution A and staining reagent B is 1:5-7.
Described solution A is made up of methyl alcohol, acetone, water, trifluoroacetic acid, and its volume ratio is 38-42:30-34:26-30:1.
Described staining reagent B is by the hydrochloric acid of normal butyl alcohol, 6mol/L and 3% ferriammonium sulfate solution composition, and their volume ratio is 89:10:1.
The method of growing early stage Rapid identification kernel seed coat colour at rape seed that the present invention proposes, its step is as follows:
(1) solution A is configured: be made up of methyl alcohol, acetone, water, trifluoroacetic acid, its volume ratio is followed successively by 38-42:30-34:26-30:1, mixing.
(2) staining reagent B is configured: by hydrochloric acid and the 3% ferriammonium sulfate solution composition of normal butyl alcohol, 6mol/L, their volume ratio is followed successively by 89:10:1, mixing.
(3) get 10-20 grain pollinate 8 days after seed coat, put into transparent container.
(4) add 1 ml soln A, make seed coat be fully immersed in liquid, under normal temperature, place 15-20 minute.
(5) add 5-7 milliliter staining reagent B, boil 15-30 minute.
(6) at cooled on ice 20-25 minute, the change of liquid color is observed.The liquid in containers being placed with black seed rape seed coat becomes aubergine; And the liquid be placed with in the container of yellow seed rape seed coat does not have aubergine to produce.
The approach such as the biology that the present inventor is formed from rape seed skin color are started with, have extensively studied the mechanism of action of the key enzyme in kernel seed coat colour forming feature, the product of enzyme effect and mutual relationship thereof, the material of discovery difference in the yellow black seed seed coat growth course of rape is flavones 3,4-glycol and analog etc. (these materials are colourless).The present invention's chemical dyeing method, to add in reagent and seed the substance reactions such as flavones 3,4-glycol and produces red material, thus can easy, intuitively distinguish yellow black seed in early days in seed development.The method is simple to operate, and analysis speed is fast, uses reagent cheap.
Embodiment
Embodiment 1:
(1) solution A is configured: be made up of methyl alcohol, acetone, water, trifluoroacetic acid, its volume ratio is followed successively by 38:30:26:1, mixes for subsequent use.
(2) staining reagent B is configured: by hydrochloric acid and the 3% ferriammonium sulfate solution composition of normal butyl alcohol, 6mol/L, their volume ratio is followed successively by 89:10:1, mixes for subsequent use.
(3) get respectively the purple leaf mustard of the black seed mustard type rape of 10 pollinations after 8 days, Pinglu leaf mustard, the yellow seed of No. 6, corner angle and yellow seed mustard type rape Sichuan, Inner Mongol rheum officinale seed coat put into transparent container.
(4) add 1 ml soln A, make seed coat be fully immersed in liquid, place 15 minutes under normal temperature.
(5) add 5 milliliters of staining reagent B, boil 20 minutes.
(6) cooled on ice 20 minutes, the change of liquid color is observed.
(7) found that be placed with purple leaf mustard, Pinglu leaf mustard, the black seed rape seed coat of No. 6, corner angle liquid in containers become aubergine; And be placed with the yellow seed in yellow seed rape Sichuan, Inner Mongol rheum officinale seed coat container in liquid do not have aubergine to produce.
Embodiment 2:
Get the yellow seed in Sichuan and purple leaf mustard hybridize and obtains selfing and obtain F2 and be placed in transparent test tube respectively for the seed coat 20 of plant (80 strains, number respectively, and maturation is the color of rape seed on observation plant afterwards) latter 8 days seeds of pollinating; Add the solution A in 2 milliliters of embodiments 1, make seed coat be fully immersed in liquid, place 20 minutes under normal temperature; Add 7 milliliters of staining reagent B in embodiment 1 again, boil 25 minutes; Cooled on ice 25 minutes, observe the change of each test tube color.It is all black that result to show that in test tube solution becomes seed after 71 strain seed maturities corresponding to aubergine, and in test tube, the solution seed after 9 strain seed maturities corresponding to look that do not redden is all yellow.Therefore can the color of Forepart identification rape seed according to the change of solution colour.
Embodiment 3:
(1) solution A is configured: be made up of methyl alcohol, acetone, water, trifluoroacetic acid, its volume ratio is followed successively by 42:30:34:30:1, mixes for subsequent use.
(2) staining reagent B is configured: by hydrochloric acid and the 3% ferriammonium sulfate solution composition of normal butyl alcohol, 6mol/L, their volume ratio is followed successively by 89:10:1, mixes for subsequent use.
(3) get No. 15,10 pollinations black seed cabbage type rape Hunan of latter 8 days oil, 98C40 respectively and get pollination yellow seed cabbage type rape Bny4, Bny6 of latter 8 days seed coat put into test tube.
(4) add 1 ml soln A, make seed coat be fully immersed in liquid, place 20 minutes under normal temperature.
(5) add 5 milliliters of staining reagent B, boil 20 minutes.
(6) cooled on ice 20 minutes, the change of liquid color is observed.
(7) found that be placed with No. 15, black seed rape cabbage type rape Hunan oil, the liquid in containers of 98C40 seed coat becomes aubergine; And the liquid be placed with in the container of yellow seed rape Bny4, Bny6 seed coat does not have aubergine to produce.
Embodiment 4:
The seed coat 15 getting the black seed of the brassicacarinata of pollination after 8 days and the yellow seed of brassicacarinata respectively puts into test tube; Add the solution A in 2 milliliters of embodiments 3, make seed coat be fully immersed in liquid, place 25 minutes under normal temperature; Add the 5 milliliters of staining reagent B executed in example 3 again, boil 30 minutes; Cooled on ice 15 minutes, observe the change of test tube color.Result shows that the solution being placed with the black seed of brassicacarinata in test tube becomes aubergine, and aubergine does not appear in the solution being placed with the yellow seed of brassicacarinata in test tube.
Claims (4)
1. grow the method for early stage Rapid identification kernel seed coat colour at rape seed for one kind, the seed coat of pollination after 8 days is it is characterized in that to put into transparent vessel, add solution A and soak 15-20 minute at normal temperatures, then staining reagent B is added, after boiling 15-30 minute, at cooled on ice 20-25 minute, the color of rape seed is identified according to the change of liquid color after reaction, the liquid in containers color putting into black seed rape seed coat becomes aubergine, and the liquid in containers color putting into yellow seed rape seed coat does not have aubergine to produce.
2. method of growing early stage Rapid identification kernel seed coat colour at rape seed according to claim 1, is characterized in that, the volume ratio of described solution A and staining reagent B is 1:5-7.
3. method of growing early stage Rapid identification kernel seed coat colour at rape seed according to claim 1, it is characterized in that, described solution A is made up of methyl alcohol, acetone, water, trifluoroacetic acid, and its volume ratio is 38-42:30-34:26-30:1.
4. method of growing early stage Rapid identification kernel seed coat colour at rape seed according to claim 1, is characterized in that, described staining reagent B is by the hydrochloric acid of normal butyl alcohol, 6mol/L and 3% ferriammonium sulfate solution composition, and their volume ratio is 89:10:1.
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| CN201510887008.9A CN105510319A (en) | 2015-12-07 | 2015-12-07 | Method for quickly identifying color of seed coats of rape seeds in early development period |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112161974A (en) * | 2020-09-01 | 2021-01-01 | 陕西新视明医药生物科技有限公司 | Method for rapidly identifying blueberry-pseudocomponent-containing health care product |
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| CN101149334A (en) * | 2007-11-06 | 2008-03-26 | 湖南农业大学 | Rape seed skin color quick identification method |
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| CN102134587A (en) * | 2010-11-01 | 2011-07-27 | 湖南科技大学 | Kit and method for identifying seed coat color of rape seed in very early development stage |
| CN102141519A (en) * | 2010-12-30 | 2011-08-03 | 浙江现代中药与天然药物研究院有限公司 | Method for detecting procyanidins content |
| CN104860916A (en) * | 2015-04-07 | 2015-08-26 | 南京晓庄学院 | Method for extracting proanthocyanidins from apple peel |
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2015
- 2015-12-07 CN CN201510887008.9A patent/CN105510319A/en active Pending
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| CN101153031A (en) * | 2006-09-27 | 2008-04-02 | 徐奎根 | Method for extracting proanthocyanidins |
| CN101149334A (en) * | 2007-11-06 | 2008-03-26 | 湖南农业大学 | Rape seed skin color quick identification method |
| CN102134587A (en) * | 2010-11-01 | 2011-07-27 | 湖南科技大学 | Kit and method for identifying seed coat color of rape seed in very early development stage |
| CN102141519A (en) * | 2010-12-30 | 2011-08-03 | 浙江现代中药与天然药物研究院有限公司 | Method for detecting procyanidins content |
| CN104860916A (en) * | 2015-04-07 | 2015-08-26 | 南京晓庄学院 | Method for extracting proanthocyanidins from apple peel |
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| AUGER BATHILDE ET AL.: "A Detailed Survey of Seed Coat Flavonoids in Developing Seeds of Brassica napus L", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
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| 刘丽莉 等: "3种显色反应对稻米籽粒中原花色素含量的检测分析", 《湖南工业大学学报》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112161974A (en) * | 2020-09-01 | 2021-01-01 | 陕西新视明医药生物科技有限公司 | Method for rapidly identifying blueberry-pseudocomponent-containing health care product |
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Application publication date: 20160420 |