CN103703890A - Determination method for viability of daphne giraldii seeds - Google Patents
Determination method for viability of daphne giraldii seeds Download PDFInfo
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- CN103703890A CN103703890A CN201310705225.2A CN201310705225A CN103703890A CN 103703890 A CN103703890 A CN 103703890A CN 201310705225 A CN201310705225 A CN 201310705225A CN 103703890 A CN103703890 A CN 103703890A
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Abstract
The invention belongs to a determination method for the viability of daphne giraldii seeds and belongs to the field of quality identification and cultivation of seeds of traditional Chinese medicinal materials. The method comprises the following steps: cutting seeds, carrying out purity treatment, disinfecting seed, pre-soaking the seeds, crushing hard seeds, and treating seed coats; dyeing; and determining the viability. According to the method provided by the invention, the viability of the daphne giraldii seeds can be rapidly and effectively determined and damages and death to the daphne giraldii seeds, caused by other determination methods, are reduced; the working dangers in an operation process are reduced; the method is simple and feasible, reasonable in principle, high in working efficiency and good in repeatability. The death to of the daphne giraldii seeds is 94.5%.
Description
Technical field
The invention belongs to traditional Chinese medicine seed quality identifies and cultivation field, particularly a kind of assay method of yellow winter daphne seed vigor.
Technical background
Yellow winter daphne (Daphne giraldii Nitsche) belongs to Thymelaeceae daphne plant, another name knotting flower, Edgeworthia chrysantha.Be distributed in the provinces such as China Shaanxi, Gansu, Qinghai, Henan, Gansu Province is mainly distributed in hillside shrubbery, border, the cheuch area of the height above sea level 2000～2600m such as the Qilian mountains.Root skin and the stem skin of yellow winter daphne, the conventional Chinese medicine for treatment rheumatism among the people and pain relieving, is commonly called as girald daphne bark, has the effects such as reducing blood lipid, anti-hemolysis activity, antitumor activity and strong analgesia, also can be used for treatment of arthritis, analgesia, anti-inflammatory, surgery anesthesia etc.The yellow winter daphne of plant is wild resource at present, is traditional Chinese medicine girald daphne bark's former plant, and using position is Gen Pi and stem skin, and the excessive unreasonable root that connects is excavated mode, makes plant resources be subject to heavy damage, causes the former plant of girald daphne bark extremely in short supply; Because yellow winter daphne seed has strong property and dormancy, germination rate is very low, is difficult to meet Production requirement again; And seed vigor is the key property of seed, seed vigor is significant to germinating capacity and the fast prediction percentage of seedgermination of prediction dormant seed, identifies that the vitality of seed directly has influence on the quality of seed and vegetable material user's economic benefit; The report of yellow winter daphne seed vigor not being measured in prior art, the assay method that a kind of yellow winter daphne seed vigor is provided is the task of top priority.
Summary of the invention
The object of this invention is to provide that a kind of simple and easy to do, principle is reliable, the method for the yellow winter daphne seed vigor of the mensuration of favorable repeatability, vitality, operating efficiency that this method can the yellow winter daphne seed of Fast Measurement are high, reproducible.
Content of the present invention and step are done to specific description below:
The assay method of the yellow winter daphne seed vigor of the present invention, first skewer is got yellow winter daphne seed, then described yellow winter daphne seed is carried out to following subsequent processing steps:
A) cleanliness treatment step: be that described yellow winter daphne seed is removed to pericarp, and clean, obtain clean seed;
B) sterilisation step: by described clean seed disinfection;
C) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, soaks 6-18h;
D) the strong step of abolishing: randomly draw the seed after the immersion that step c) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
E) plant skin treatment step: in the situation that not injuring embryo, to steps d) the kind skin that obtains carries out broken skin processing, obtains seed after broken skin, then seed after broken skin is placed in to test tube;
F) staining procedure: will analyze pure 2,3,5-triphenyl oxidation tetrazolium is mixed with the phosphate buffer that pH is 7.8 the TTC solution that mass fraction is 0.4-0.8%, and joins step e) in be equipped with in the test tube of seed after broken skin, described TTC solution floods seed completely; Then test tube is placed in to water-bath, water bath with thermostatic control;
G) embryo viability test step: from dyed step f) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
" vitality " of the present invention is to represent with " percentage that has vitality seed ", has vitality seed to account for the percentage for inspection seed.
The assay method of yellow winter daphne seed of the present invention, it is the TTC staining reaction of carrying out according to the enzymatic reaction in organism, with 2,3, the colourless solution of 5-triphenyl oxidation tetrazolium (TTC) is as indicator, when TTC enters after the living cells of seed embryo, the reduction-state codehydrogenase producing in living cells respiration is reduced to red, stable and indiffusible TTF, can determine that seed has or not vitality according to the depth of the size of seed dyeing part and color.
In prior art, yellow winter daphne seed vigor is not carried out to method for measuring report, the report that does not more use TTC method to measure yellow winter daphne seed vigor, TTC assay method to yellow winter daphne seed vigor of the present invention, simple, easy operation, repeatability are high, germination rate to yellow winter daphne seed provides foundation, is conducive to breeding and the breeding of yellow winter daphne plant.
The yellow winter daphne seed adopting in mensuration, the seed of preferably gathering then.
The assay method of yellow winter daphne seed vigor of the present invention, described step a), is that described yellow winter daphne seed is removed to pericarp with the method for extruding, and cleans up, obtains clean seed.
The relative additive method of extrusion is processed and totally, is not damaged seed, more can guarantee the integrality of seed.
The assay method of yellow winter daphne seed vigor of the present invention, described step b), be that the potassium permanganate that is 1-5% by described clean seed with mass fraction soaks and carries out disinfection for 5 minutes.
Seed is carried out disinfection and can prevent damage by disease and insect and the germ contamination of seed, hinder the accuracy to its viability test.
The assay method of yellow winter daphne seed vigor of the present invention, described step c), described immersion process is that 25 ℃ of constant temperature soak 12 hours under natural daylight condition.
The assay method of yellow winter daphne seed vigor of the present invention, in described step e), it is to break in the seed coat by dissecting needle thorn that described broken skin is processed.
Kind of skin can hinder the infiltration of TTC solution, and thorn breaks in the seed coat and is conducive to TTC solution and enters and embryo is dyeed, and improves the accuracy that seed vigor is measured.
The assay method of yellow winter daphne seed vigor of the present invention, in described step e), it is first with scalpel, between the seed abdomen back of the body, longitudinally to cut and break in the seed coat that described broken skin is processed, and then with tweezers, strips out gently embryo, and embryo is placed in to test tube.
Removal kind of a skin, taking-up embryo dye completely, to the mensuration of seed vigor more accurately, rapidly.
Preferably, the assay method of yellow winter daphne seed vigor of the present invention, described step f), the mass fraction of described TTC solution is 0.5%.
Mass fraction is 0.5% TTC solution, can dye preferably, and injuring-free seed embryo, measures yellow winter daphne seed vigor in this concentration again, both can guarantee the accuracy to seed vigor mensuration, can guarantee again the germination rate of seed.
The assay method of yellow winter daphne seed vigor of the present invention, described step f), described water bath with thermostatic control is to carry out 1-24h under 15-55 ℃ of condition.
Preferably, the assay method of yellow winter daphne seed vigor of the present invention, described step f), described water bath with thermostatic control is to carry out 6h under 45 ℃ of conditions.
Further preferably, the assay method of yellow winter daphne seed vigor of the present invention, comprises the steps:
A) skewer kind step: skewer is got yellow winter daphne seed;
B) cleanliness treatment step: be that described yellow winter daphne seed is removed to pericarp by extrusion, and clean up, obtain clean seed;
C) sterilisation step: be that described clean seed is carried out disinfection with 3% potassium permanganate immersion for 5 minutes;
D) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, and 25 ℃ of constant temperature soak 12 hours under natural daylight condition;
E) the strong step of abolishing: randomly draw the seed after the immersion that step d) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
F) plant skin treatment step: in the situation that not injuring embryo, first with scalpel, between the seed abdomen back of the body, longitudinally cut and break in the seed coat, then with tweezers, strip out gently embryo, and embryo is placed in to test tube;
G) staining procedure: will analyze purely 2,3,5-triphenyl oxidation tetrazolium, being mixed with mass fraction with the phosphate buffer that pH is 7.8 is 0.5% TTC solution, and joins step f), be equipped with in the test tube of embryo, described TTC solution floods seed completely; Then test tube is placed in to water-bath, 45 ℃ of water bath with thermostatic control 6h;
H) embryo viability test step: from dyed step g) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
The beneficial effect of the assay method of yellow winter daphne seed vigor of the present invention is: provide a kind of, by TTC method, the vitality of yellow winter daphne seed has been carried out to method for measuring.This method can be compared additive method directly according to the dyeing situation judgement seed vigor of embryo, and simple and easy to do, principle is reliable, to seed not damaged, favorable repeatability; The vitality of yellow winter daphne seed is 94.5%.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
The assay method of yellow winter daphne seed vigor, comprises the steps:
A) skewer kind step: skewer is got yellow winter daphne seed 500g;
B) cleanliness treatment step: described yellow winter daphne seed is removed to pericarp by extrusion, and clean up, obtain clean seed;
C) sterilisation step: be that described clean seed is carried out disinfection with 3% potassium permanganate immersion for 5 minutes;
D) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, and 25 ℃ of constant temperature soak 12 hours under natural daylight condition;
E) the strong step of abolishing: randomly draw the seed after the immersion that step d) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
F) plant skin treatment step: in the situation that not injuring embryo, first with scalpel, between the seed abdomen back of the body, longitudinally cut and break in the seed coat, then with tweezers, strip out gently embryo, and embryo is placed in to test tube;
G) staining procedure: will analyze purely 2,3,5-triphenyl oxidation tetrazolium, being mixed with mass fraction with the phosphate buffer that pH is 7.8 is 0.5% TTC solution, and joins step f), be equipped with in the test tube of embryo, described TTC solution floods seed completely; Then test tube is placed in to water-bath, 45 ℃ of water bath with thermostatic control 6h;
H) embryo viability test step: from dyed step g) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
Measurement result: the vitality of yellow winter daphne seed is 94.5%.
Embodiment 2
The assay method of yellow winter daphne seed vigor, comprises the steps:
A) skewer kind step: skewer is got yellow winter daphne seed 500g;
B) cleanliness treatment step: described yellow winter daphne seed is removed to pericarp with paddling process, and clean up, obtain clean seed;
C) sterilisation step: be that described clean seed is carried out disinfection with 1% potassium permanganate immersion for 5 minutes;
D) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, and 25 ℃ of constant temperature soak 6 hours under natural daylight condition;
E) the strong step of abolishing: randomly draw the seed after the immersion that step d) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
F) plant skin treatment step: in the situation that not injuring embryo, by dissecting needle thorn, break in the seed coat, and seed after broken skin is placed in to test tube;
G) staining procedure: will analyze pure 2,3,5-triphenyl oxidation tetrazolium, with the phosphate buffer that pH is 7.8, being mixed with mass fraction is 0.8% TTC solution, and join step f) in be equipped with in the test tube of seed after broken skin, described TTC solution floods seed completely; Then test tube is placed in to water-bath, 55 ℃ of water bath with thermostatic control 1h;
H) embryo viability test step: from dyed step g) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
Measurement result: the vitality of yellow winter daphne seed is 91.5%.
Embodiment 3
The assay method of yellow winter daphne seed vigor, comprises the steps:
A) skewer kind step: skewer is got yellow winter daphne seed 500g;
B) cleanliness treatment step: described yellow winter daphne seed is removed to pericarp by extrusion, and clean up, obtain clean seed;
C) sterilisation step: be that described clean seed is carried out disinfection with 5% potassium permanganate immersion for 5 minutes;
D) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, and 25 ℃ of constant temperature soak 18 hours under natural daylight condition;
E) the strong step of abolishing: randomly draw the seed after the immersion that step d) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
F) plant skin treatment step: in the situation that not injuring embryo, first with scalpel, between the seed abdomen back of the body, longitudinally cut and break in the seed coat, then with tweezers, strip out gently embryo, and embryo is placed in to test tube;
G) staining procedure: will analyze purely 2,3,5-triphenyl oxidation tetrazolium, being mixed with mass fraction with the phosphate buffer that pH is 7.8 is 0.4% TTC solution, and joins step f), be equipped with in the test tube of embryo, described TTC solution floods seed completely; Then test tube is placed in to water-bath, 15 ℃ of water bath with thermostatic control 24h;
H) embryo viability test step: from dyed step g) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
Measurement result: the vitality of yellow winter daphne seed is 93%.
Claims (10)
1. an assay method for yellow winter daphne seed vigor, first carries out skewer and gets yellow winter daphne kind sub-step, it is characterized in that, described assay method also comprises described yellow winter daphne seed is carried out to following subsequent processing steps:
A) cleanliness treatment step: be that described yellow winter daphne seed is removed to pericarp, and clean, obtain clean seed;
B) sterilisation step: by described clean seed disinfection;
C) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, soaks 6-18h;
D) the strong step of abolishing: randomly draw the seed after the immersion that step c) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
E) plant skin treatment step: in the situation that not injuring embryo, to steps d) the kind skin that obtains carries out broken skin processing, obtains seed after broken skin, then seed after broken skin is placed in to test tube;
F) staining procedure: will analyze pure 2,3,5-triphenyl oxidation tetrazolium is mixed with the phosphate buffer that pH is 7.8 the TTC solution that mass fraction is 0.4-0.8%, and joins step e) in be equipped with in the test tube of seed after broken skin, described TTC solution floods seed completely; Then test tube is placed in to water-bath, water bath with thermostatic control;
G) embryo viability test step: from dyed step f) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
2. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, described step a), is that described yellow winter daphne seed is removed to pericarp with the method for extruding, and cleans up, obtains clean seed.
3. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, described step b), be that the potassium permanganate that is 1-5% by described clean seed with mass fraction soaks and carries out disinfection for 5 minutes.
4. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, described step c), described immersion process is that 25 ℃ of constant temperature soak 12 hours under natural daylight condition.
5. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, in described step e), it is to break in the seed coat by dissecting needle thorn that described broken skin is processed.
6. the assay method of yellow winter daphne seed vigor according to claim 1, it is characterized in that, in described step e), it is first with scalpel, between the seed abdomen back of the body, longitudinally to cut and break in the seed coat that described broken skin is processed, then with tweezers, strip out gently embryo, and embryo is placed in to test tube.
7. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, described step f), the mass fraction of described TTC solution is 0.5%.
8. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, described step f), described water bath with thermostatic control is to carry out 1-24h under 15-55 ℃ of condition.
9. the assay method of yellow winter daphne seed vigor according to claim 6, is characterized in that, described step f), described water bath with thermostatic control is to carry out 6h under 45 ℃ of conditions.
10. the assay method of yellow winter daphne seed vigor according to claim 1, is characterized in that, comprises the steps:
A) skewer kind step: skewer is got yellow winter daphne seed;
B) cleanliness treatment step: be that described yellow winter daphne seed is removed to pericarp by extrusion, and clean up, obtain clean seed;
C) sterilisation step: be that described clean seed is carried out disinfection with 3% potassium permanganate immersion for 5 minutes;
D) pre-soaking treatment step: the clean seed after sterilization is placed in to the beaker that fills distilled water, and 25 ℃ of constant temperature soak 12 hours under natural daylight condition;
E) the strong step of abolishing: randomly draw the seed after the immersion that step d) obtains, with the hard kind skin of sand papering, carry out several times and repeat experiment;
F) plant skin treatment step: in the situation that not injuring embryo, first with scalpel, between the seed abdomen back of the body, longitudinally cut and break in the seed coat, then with tweezers, strip out gently embryo, and embryo is placed in to test tube;
G) staining procedure: will analyze purely 2,3,5-triphenyl oxidation tetrazolium, being mixed with mass fraction with the phosphate buffer that pH is 7.8 is 0.5% TTC solution, and joins step f), be equipped with in the test tube of embryo, described TTC solution floods seed completely; Then test tube is placed in to water-bath, 45 ℃ of water bath with thermostatic control 6h;
H) embryo viability test step: from dyed step g) obtaining, pick out wine-colored embryo, as viable seed, and calculate the percentage that has vitality seed.
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Cited By (5)
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| CN104686011A (en) * | 2015-03-19 | 2015-06-10 | 北京市农林科学院 | Tetrazole dyeing method for measuring eggplant seed viability |
| CN106211865A (en) * | 2016-07-28 | 2016-12-14 | 甘肃省治沙研究所 | A kind of method measuring husky rice seed vigor fast and accurately |
| CN106234146A (en) * | 2016-09-28 | 2016-12-21 | 合肥沁牧生态农业有限公司 | A kind of implantation methods of ligustrum lucidum ait |
| CN107087455A (en) * | 2017-05-11 | 2017-08-25 | 湖北省农业科学院中药材研究所 | A kind of assay method of levisticum seed vigor |
| CN109402290A (en) * | 2018-12-05 | 2019-03-01 | 秦皇岛市山海关药业有限责任公司 | A kind of girald daphne bark's identification method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104686011A (en) * | 2015-03-19 | 2015-06-10 | 北京市农林科学院 | Tetrazole dyeing method for measuring eggplant seed viability |
| CN106211865A (en) * | 2016-07-28 | 2016-12-14 | 甘肃省治沙研究所 | A kind of method measuring husky rice seed vigor fast and accurately |
| CN106234146A (en) * | 2016-09-28 | 2016-12-21 | 合肥沁牧生态农业有限公司 | A kind of implantation methods of ligustrum lucidum ait |
| CN107087455A (en) * | 2017-05-11 | 2017-08-25 | 湖北省农业科学院中药材研究所 | A kind of assay method of levisticum seed vigor |
| CN109402290A (en) * | 2018-12-05 | 2019-03-01 | 秦皇岛市山海关药业有限责任公司 | A kind of girald daphne bark's identification method |
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