CN102175660B - Method for rapidly identifying bentazon sensitivity rice - Google Patents
Method for rapidly identifying bentazon sensitivity rice Download PDFInfo
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- CN102175660B CN102175660B CN2011100229017A CN201110022901A CN102175660B CN 102175660 B CN102175660 B CN 102175660B CN 2011100229017 A CN2011100229017 A CN 2011100229017A CN 201110022901 A CN201110022901 A CN 201110022901A CN 102175660 B CN102175660 B CN 102175660B
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Abstract
The invention relates to a method for rapidly identifying bentazon sensitivity rice, and the method can be used for solving the problems of high cost, long identification time and great identification errors of the bentazon sensitivity rice identification method in a known technique. The method provided by the invention comprises the following three steps: marking rice leaves; coating bentazon; and rapidly identifying the sensitivity rice, wherein the suppression rate of the chlorophyll fluorescence parameters Yield (PS II effective photochemical quantum yield) is utilized to identify the sensitivity of the rice after the leaves are coated with the bentazon. The method provided by the invention has the advantages of simplicity, low manpower cost and high identification accuracy; the mutant identification can be finished after the bentazon processing is carried out for 7 hours; the false positive interference can be effectively eliminated; 0.02%(V/V) bentazon is utilized as a bentazon herbicide, and the concentration of the bentazon is obviously lower than that of the 0.1%(V/V) bentazon used in the traditional identification method; and the damage to the ecological environment is effectively reduced when a large amount of herbicide is used.
Description
Technical field
The invention belongs to plant authenticate technology field, be specifically related to Bentazon susceptibility paddy rice rapid identification method.
Background technology
Bentazon (bentazon) is that (the convenience goods title has Basagra, bentazone, hundred careless grams, bentazon, Lysimachia sikokiana pellet etc. for benzothiadiazole, BTH) type herbicide for the diazosulfide of a kind of wide spectrum, efficient, low toxicity.The effective constituent of Bentazon can absorb through root, the leaf of plant; It has the effect that removes extremely to most dicotyledons [except that soybean (Glycine max Merrill L.), peanut (Arachis hypogaca L.)] and nutgrass flatsedge (Cyperus L.) section weeds, and is harmless to grass.Bentazon generally believes the D of its target site point for lightsystem as a kind of selectivity systemic herbicide
1Albumen, the photosynthetic electron transport chain of non-ring type capable of blocking suppresses photosynthesis, thereby causes that the responsive type plant is dead.
The wild type rice varieties has the resistance of Bentazon, and 5000 mgL-1 Bentazon solution spray and still show safe and harmless in rice seedling.Japanese scholar Mori was through gamma-ray irradiation ' No. 8, agricultural ' (' Norin8 ' in 1984; ' N8 '); Find mutant ' agricultural No. 8 ms ' (' Norin8m ' extremely responsive to Bentazon; ' N8m '), the Bentazon of 0.5 ~ 500 mgL-1 to 3.5 ~ 4 leaf phases ' No. 8 m ' of agricultural show as in various degree injury (leaf roll is right and wrong dead to complete stool).In addition, the Zhang Jiwen of academy of agricultural sciences of China Hubei Province etc. adopts 350 Gy
60Co gamma-rays irradiation treatment paddy rice light-study on temperature sensitive male sterility system ' W6154S ' obtains Bentazon sensitive mutant ' 8077s '.The Bentazon sensitive gene of two kinds of separate sources of research proof is the recessive single-gene of karyogene control.
Hybrid rice has received the popular welcome of vast farmers in China, for significant contribution has been made in China's grain-production.Production of hybrid seeds labour intensity is big, seed production purity is difficult to problems such as guarantee but the breeding of hybrid rice process still exists.The Bentazon sensitive gene has broad application prospects in the paddy rice cross breeding production of hybrid seeds as the deadly mark of a chemistry, is mainly reflected in following two aspects.(1) with the transformation of Bentazon sensitive gene in rice restorer, the recovery system of containing the Bentazon sensitive gene is removed through spraying Bentazon in paddy rice pollination back, thereby realizes that mixed seeding mixes the mechanization production of hybrid seeds of receiving.Simultaneously also help improving the sterile line outcrossing seed-setting rate.(2) transformation of Bentazon sensitive gene guarantees paddy rice hybrid purity to rice sterile line.Therefore, set up a kind of susceptibility of Bentazon fast and accurately paddy rice authentication method, can effectively improve paddy rice sensitive mutant and transformation offspring's thereof screening effeciency, quicken the application of Bentazon sensitive gene in breeding of hybrid rice.
At present; Identify that Bentazon susceptibility paddy rice never has method preferably; That extensively adopts has two kinds: one, utilize Bentazon to spray the field paddy rice, perhaps smear the paddy rice index blade, observe paddy rice after 7-10 days and smear the leaf look variation of handling material; What blade was shrivelled is the susceptibility paddy rice, and the leaf look does not have the resistant rice that is of significant change.But there is following shortcoming in this method: 1, the processing time longer, need to handle more than 7-10 days.2, the accuracy of identifying is low, because the authentication process time is long, envirment factors such as rainfall, high temperature and disease and pest are difficult to change the judgement Rice Sensitivity with leaf color to the very big again influence of herbicide action effect; 3, qualification process is time-consuming.Two, utilization and the closely linked molecular labeling of Bentazon sensitive gene come the Bentazon susceptibility of assistant identification paddy rice.As; Zhu Bifeng etc. find special primer---the S20 and the S316 of performance polymorphism between responsive pond of 2 paddy rice Bentazons and the anti-sense pond from 210 random primers.Pan etc. find that through the molecule Fine Mapping this gene is positioned at the long-armed end of paddy rice trisome, and its close linkage is labeled as SSR71314, and both are at a distance of 71.835kb.But utilize the method for molecular labeling assistant identification to have following shortcoming: 1, method is complicated, the pcr amplification of needs extraction paddy DNA, gene and agarose electrophoresis etc.; 2, experiment equipment, molecular agents, consumptive material cost an arm and a leg; 3, be merely the laboratory householder method that Bentazon susceptibility is identified, be not suitable for the field and identify on a large scale.
Summary of the invention
Cost is high in the evaluation of Bentazon susceptibility paddy rice, qualification time is long in order to solve, identify the big problem of error, and the inventive method is according to the D of Bentazon target site point for PSII
1Albumen, the inhibiting rate that utilizes the blade Bentazon to handle back chlorophyll fluorescence parameters Yield (the effective photochemistry quantum yield of PS II) comes the susceptibility method of Rapid identification paddy rice.
The concrete operations step of the inventive method is following:
Bentazon susceptibility paddy rice rapid identification method is characterized in that comprising following operation steps:
(1), dosing
A, with the Bentazon treating fluid of Bentazon herbicide configuration percent by volume 0.02% (V/V), wherein contain the polysorbas20 of percent by volume 0.25% (V/V);
The polysorbas20 solution of B, percent by volume 0.25% (V/V) is polysorbas20 contrast liquid;
(2), rice leaf mark
In the 6 leaf phases of paddy rice, on same strain paddy rice, choose the blade of two health, a slice is used for Bentazon to be handled, and a slice is a blank; At index blade apart from blade tip 10-11 cm place;
(3), Bentazon is smeared processing
Dip in the Bentazon treating fluid of getting percent by volume 0.02% (V/V) with writing brush or cotton swab, evenly smear the Bentazon treating fluid, evenly smear contrast liquid at another sheet blade from mark to blade tip, as blank at mark to the blade tip of a slice blade; Place 7 h;
(4), fluorescence parameter is measured
Utilize modulation system chlorophyll fluorescence appearance, fluorescence under maximum fluorescence, the stable state when light saturation pulse light that detects two blades respectively exists calculates and uses up the effective photochemistry quantum yield of photosystem II when existing (PS II); The effective photochemistry quantum yield of said PS II computing formula is: Yield=(Fm '-Ft)/Fm ';
Yield uses up effectively photochemistry quantum yield of photosystem II when existing (PS II) in the following formula, maximum fluorescence when Fm ' exists for light saturation pulse light, and Ft is a fluorescence under the stable state;
(5), susceptibility paddy rice Rapid identification
Utilize the inhibiting rate of following formula counting Bentazon to the effective photochemistry quantum yield of blade PS II; Just judge the Bentazon susceptibility of paddy rice to be measured according to inhibiting rate, inhibiting rate is the susceptibility paddy rice greater than 50%, and inhibiting rate is a resistant rice less than 50%;
Inhibiting rate (%)=(Yieldck-Yield handles)/Yieldck * 100%,
Yield processing and Yieldck are respectively the effective photochemistry quantum yield of PS II that Bentazon is handled blade and contrast blade in the following formula.
Said Bentazon herbicide is that concentration is the Bentazon herbicide of percent by volume 25% (V/V).
Useful technique effect of the present invention embodies in the following areas:
1, the inventive method is simple, and cost of labor is low, can after Bentazon is handled 7 hours, can accomplish mutant and identify;
2, the accuracy of the inventive method evaluation is high, can effectively discharge false positive and disturb;
3, the Bentazon herbicide working concentration is merely 0.02%, significantly is lower than the working concentration (0.1%) of traditional authentication method, has effectively reduced a large amount of harm of using ecologic environment of herbicide.
Description of drawings
Fig. 1 0.02% (V/V) Bentazon is handled the figure that influences to the effective photochemistry quantum yield (Yield) of rice leaf photosystem II (PS II).
Fig. 2 Bentazon concentration of treatment and rice leaf photosystem II (PS II) be the linear regression analysis figure of photochemistry quantum yield (Yield) inhibiting rate effectively.
Embodiment
Below in conjunction with embodiment, the present invention is done explanation further.
Major equipment:
1. chlorophyll fluorescence appearance; 2. micropipettor; 3. electronic scale.
Material and reagent:
1. material: the rice leaf of different responsive types
2. main agents:
(1) 25% Bentazon herbicide: commercially available.
(2) polysorbas20: commercially available.
(3) the Bentazon treating fluid of percent by volume 0.02% (V/V): get 25% Bentazon herbicide, 0.8 mL, add 2.5 mL polysorbas20s, distilled water is settled to 1000 mL, is percent by volume 0.02% (V/V) Bentazon treating fluid.
(4) contrast liquid: add 2.5 mL polysorbas20s in the 1000 mL distilled water, be the polysorbas20 contrast liquid of the percent by volume 0.25% (V/V) that does not contain Bentazon.
Bentazon susceptibility paddy rice rapid identification method comprises following operation steps:
(1), rice leaf mark: field cultivation paddy rice, normal management.In 6 leaf phases, on same strain paddy rice, choose the blade of two health, a slice is used for Bentazon to be handled, and a slice is a blank.Using the marking pen index blade apart from the about 10 cm places of blade tip respectively;
(2), dip in the Bentazon treating fluid of getting percent by volume 0.02% (V/V), evenly smear the Bentazon treating fluid, evenly smear contrast liquid at another sheet blade from mark to blade tip, as blank at mark to the blade tip of a slice blade with writing brush or cotton swab; Place 7 h;
(3), fluorescence parameter is measured
Utilize modulation system chlorophyll fluorescence appearance, fluorescence under maximum fluorescence, the stable state when light saturation pulse light that detects two blades respectively exists calculates and uses up the effective photochemistry quantum yield of photosystem II when existing (PS II); Said photosystem II (PS II) effectively photochemistry quantum yield computing formula is: Yield=(Fm '-Ft)/Fm ';
Yield uses up the effective photochemistry quantum yield of PS II when existing in the following formula, maximum fluorescence when Fm ' exists for light saturation pulse light, and Ft is a fluorescence under the stable state;
(4), susceptibility paddy rice Rapid identification
Utilize the inhibiting rate of following formula counting Bentazon to the effective photochemistry quantum yield of blade photosystem II (PS II); Just judge the Bentazon susceptibility of paddy rice to be measured according to inhibiting rate, inhibiting rate is the susceptibility paddy rice greater than 50%, and inhibiting rate is a resistant rice less than 50%;
Inhibiting rate (%)=(Yieldck-Yield handles)/Yieldck * 100%,
Yield processing and Yieldck are respectively Bentazon and handle blade and the effective photochemistry quantum yield of the photosystem II (PS II) of contrast blade in the following formula.
Major equipment:
1. chlorophyll fluorescence appearance; 2. micropipettor; 3. electronic scale; 4. card punch.
Material and reagent:
1. material: the rice leaf of different responsive types is in store at 4 ℃;
2. main agents:
(1) 25% Bentazon herbicide: commercially available.
(2) polysorbas20: commercially available.
(3) the Bentazon treating fluid of percent by volume 0.02% (V/V): get 25% Bentazon herbicide, 0.8 mL, add 2.5 mL polysorbas20s, distilled water is settled to 1000 mL, is the Bentazon treating fluid of percent by volume 0.02% (V/V).
(4) contrast liquid: add 2.5 mL polysorbas20s in the 1000 mL distilled water, be 0.25% (V/V) polysorbas20 contrast liquid that does not contain Bentazon.
Method of operating:
(1), rice leaf mark: the field rice leaf is fetched the laboratory, behind the mark, be placed in the double dish that is covered with wet gauze, in case the dehydration of uppermost leaf sheet;
(2), Bentazon is smeared processing: dip in cotton swab and get the Bentazon treating fluid, evenly spread upon on the index blade.0.25% (V/V) polysorbas20 solution not contain Bentazon is smeared as blank;
(3), fluorescence parameter is measured: after Bentazon is handled 7 h; Utilize and adopt the modulation system luminoscope to measure chlorophyll fluorescence parameters; Under the natural light irradiation; Maximum fluorescence when detecting light saturation pulse light respectively and existing (Fm '), fluorescence under the stable state (Ft) calculates and uses up the effective photochemistry quantum yield (Yield) of photosystem II when existing (PS II);
(4), susceptibility paddy rice Rapid identification: utilize the inhibiting rate of following formula counting Bentazon to the effective photochemistry quantum yield of blade photosystem II (PS II).Just judge the Bentazon susceptibility of paddy rice to be measured according to inhibiting rate, inhibiting rate is the susceptibility paddy rice greater than 50%, and inhibiting rate is a resistant rice less than 50%.
Inhibiting rate (%)=(Yieldck-Yield handles)/Yieldck * 100%,
Yield processing and Yieldck are respectively Bentazon and handle blade and the effective photochemistry quantum yield of the photosystem II (PS II) of contrast blade in the following formula.
The technical characterstic explanation:
1, the chlorophyll fluorescence index chooses
After variable concentrations Bentazon titer is smeared the susceptibility rice leaf; Light is handled 7 h down; Hide dark place reason 20min with black cloth then; Survey fluorescence index Fo (the minimum fluorescence quantum yield that obtains after the dark adatpation), Fm (the maximum fluorescence output that obtains after the scotopic sample irradiation saturation pulse), Fv/m (the maximum suboutput of dark adatpation sample lightsystem), remake with surveying Fo ', Fm ' (the maximum fluorescence output that obtains when opening saturation pulse under the illumination), Yield behind the optical processing 5min.Can find out from table 1, the Fv/m in Bentazon concentration of treatment and the chlorophyll fluorescence index, Fo ', Fm ' and Yield significant correlation, related coefficient is respectively-0.92,0.99,0.91 and-0.99.But see that from the standard deviation of above-mentioned fluorescence index the degree of accuracy of fluorescence indexs such as Fo ', Fm ' is relatively poor, and the degree of accuracy of fluorescence indexs such as Fv/m and Yield is higher.Analyze in conjunction with above, confirm that finally chlorophyll fluorescence index Yield is as the inventive method identification of indicator.
The variation and the correlation analysis table of chlorophyll fluorescence index under the different Bentazon solution-treated of table 1.
2, the selection in Bentazon processing time
Rice leaf is handled with 0.02% (V/V) Bentazon in the field, measures and handles the dynamic change of leaf chlorophyll fluorescence index Yeild with the time of processing.Can know that by Fig. 1 at the processing initial stage, Bentazon Yield responsive and resistant rice kind processing blade all sharply descends.After handling 2 h, the Yield of resistant variety constantly recovers, and 7 h have returned to 64.67% of contrast after processing; And the forfeiture of responsive paddy rice processing leave photosynthetic ability can not recover with the passing of the time of processing.In order to ensure the reliability of qualification result, finally definite Bentazon is handled the Bentazon processing time that 7 h are the inventive method.
3, the selection of Bentazon concentration of treatment
Variable concentrations Bentazon titer is smeared the susceptibility rice leaf, handles and measures chlorophyll fluorescence index Yield behind 7 h, and the typical curve of making is seen Fig. 2.Experiment is carried out three times and is repeated; Be respectively y=0.44 x+2.49, y=0.44x+2.39, y=0.44 x+2.30; ED50 (inhibiting rate is 50% o'clock a concentration) is respectively 0.0119% (V/V), 0.0119% (V/V), 0.0118% (V/V); The facies relationship number average reaches 0.99, explains that this method has good repeatability.The concentration of the Bentazon solution when reaching 50% inhibiting rate confirms that finally Bentazon concentration of treatment 0.02% (V/V) Bentazon is the Bentazon concentration of treatment of the inventive method.
Claims (2)
1. Bentazon susceptibility paddy rice rapid identification method is characterized in that comprising following operation steps:
(1), dosing
A, with the Bentazon treating fluid of Bentazon herbicide configuration percent by volume 0.02%, wherein contain the polysorbas20 of percent by volume 0.25%;
The polysorbas20 solution of B, percent by volume 0.25% is polysorbas20 contrast liquid;
(2), rice leaf mark
In the 6 leaf phases of paddy rice, on same strain paddy rice, choose the blade of two health, a slice is used for Bentazon to be handled, and a slice is a blank; At index blade apart from blade tip 10-11cm place;
(3), Bentazon is smeared processing
Dip in the Bentazon treating fluid of getting percent by volume 0.02% with writing brush or cotton swab, evenly smear the Bentazon treating fluid, evenly smear contrast liquid at another sheet blade from mark to blade tip, as blank at mark to the blade tip of a slice blade; Place 7h;
(4), fluorescence parameter is measured
Utilize modulation system chlorophyll fluorescence appearance, fluorescence under maximum fluorescence, the stable state calculated and uses up the effective photochemistry quantum yield of photosystem II when existing when the light saturation pulse light that detects two blades respectively existed; The effective photochemistry quantum yield of said photosystem II computing formula is: YIeld=(Fm '-Ft)/Fm ';
Yield uses up the effective photochemistry quantum yield of photosystem II when existing in the following formula, maximum fluorescence when Fm ' exists for light saturation pulse light, and Ft is a fluorescence under the stable state;
(5), susceptibility paddy rice Rapid identification
Utilize the inhibiting rate of following formula counting Bentazon to the effective photochemistry quantum yield of photosystem II of blade; Just judge the Bentazon susceptibility of paddy rice to be measured according to inhibiting rate, inhibiting rate is the susceptibility paddy rice greater than 50%, and inhibiting rate is a resistant rice less than 50%;
Inhibiting rate (%)=(Yieldck-Yield
Handle)/Yieldck*100%,
Yield in the following formula
HandleBe respectively the effective photochemistry quantum yield of photosystem II that Bentazon is handled blade and contrast blade with Yieldck.
2. Bentazon susceptibility paddy rice rapid identification method according to claim 1 is characterized in that: said Bentazon herbicide is that concentration is the Bentazon herbicide of percent by volume 25%.
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| CN102660463B (en) * | 2012-05-09 | 2013-10-16 | 上海师范大学 | SGB buffer solution for efficiently improving energy transfer of phycobilisomes of blue algae to photosystem II in vitro |
| CN103650939B (en) * | 2012-09-10 | 2015-06-03 | 中国科学院亚热带农业生态研究所 | Method for measuring sensibility of rice plants to bentazone and method for screening rice plants |
| CN106636188A (en) * | 2016-12-29 | 2017-05-10 | 浙江大学 | Smearing agent and application thereof in screening rice T0 transgenic mutant plant |
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| DE19910436A1 (en) * | 1999-03-10 | 2000-10-12 | Ulrich Schreiber | Apparatus for highly sensitive detection of photosynthesis herbicides in water, e.g. for toxicity testing of drinking water, comprising fluorometer for determining effect on chlorophyll fluorescence in biological samples |
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| JP2007320902A (en) * | 2006-05-31 | 2007-12-13 | Utsunomiya Univ | Chlorophyll nanoparticle and method for producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19910436A1 (en) * | 1999-03-10 | 2000-10-12 | Ulrich Schreiber | Apparatus for highly sensitive detection of photosynthesis herbicides in water, e.g. for toxicity testing of drinking water, comprising fluorometer for determining effect on chlorophyll fluorescence in biological samples |
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| Title |
|---|
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