CN103081731B - Method for identifying glyphosate resistant cotton rapidly - Google Patents
Method for identifying glyphosate resistant cotton rapidly Download PDFInfo
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- CN103081731B CN103081731B CN201310065022.1A CN201310065022A CN103081731B CN 103081731 B CN103081731 B CN 103081731B CN 201310065022 A CN201310065022 A CN 201310065022A CN 103081731 B CN103081731 B CN 103081731B
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Abstract
A method for identifying glyphosate resistant cotton rapidly includes that cotton seeds to be tested are soaked in glyphosate solution, wherein white color is exposed on the cotton seeds already; the soaked cotton seeds are rinsed with clear water and then placed in an illumination incubator for sand culture; the number of emerged seedlings is counted, and the emergence rate is obtained through calculation; and whether the cotton to be tested is the glyphosate resistant cotton is judged according to the emergence rate. Through the method for identifying the glyphosate resistant cotton rapidly, the defects that a field identification method or a greenhouse identification method is long in cycle, heavy in workload, prone to be restricted by seasons and the like can be overcome, and the method for identifying the glyphosate resistant cotton rapidly has high practicability and operability.
Description
Technical field
The present invention relates to technical field of plant protection, specifically, relate to bioassay for herbicide technology, more particularly, relate to a kind of rapid identification method of glyphosate resistant cotton.
Background technology
Cotton is the important economic crops of China, long-term cultivated area 5,000,000 hm
2, accounting for 26% of the economic crops gross area, is key pillars of the national economy industries.But China's weed in cotton field harm is heavier, causes damage 14% ~ 16%, seriously constrains the high-quality of cotton, High-efficient Production throughout the year.Glyphosate inhales steriland herbicide as in a kind of wide spectrum, plays an important role in the preventing and kill off of weed in cotton field.But, because glyphosate does not have the selectivity between weeds and cotton, when using glyphosate, often easy poisoning is caused to non-Bt cotton kind, even dead.
The application of transgenosis glyphosate resistant cotton not only can avoid the generation of poisoning, can also expand the scope of application and the flexibility of glyphosate.Since the U.S. in 1997 takes the lead in starting to plant transgenosis glyphosate resistant cotton, glyphosate resistant cotton accounts for the total cultivated area of cotton ratio in the cultivated area of the U.S. is increased to about 70% in 2009 from 3.2% in 1997.At present, China is implementing the plan of transgenosis glyphosate resistant cotton rearing new variety, and the multiple glyphosate high resistance gene having independent intellectual property right of separation andpreconcentration, and obtain the monoclonal antibody glyphosate of a collection of stably express and there is the transgene cotton new material of the pest-resistant complex character of resistance glyphosate.Along with the raising of agricultural modernization level, a large amount of urbanization transfer of rural laborer, the demand of Cotton Production Glyphosate-Resistant kind is more and more stronger.Transgenosis glyphosate resistant cotton certainly will by become China be expected to after Insect Resistant Cotton promote another kind of transgene cotton industrialization kind.In glyphosate resistant cotton breed breeding process, the basis that cotton breeding and Industry Promotion are not only in glyphosate resistance evaluation is carried out to cotton material, also be one of content of carrying out the evaluation of transgene cotton environmental safety, therefore, detection and the authentication method of studying a kind of glyphosate resistant cotton just seem particularly important.
At present, the authentication method of glyphosate resistant cotton is mainly field or greenhouse plant Resistance Identification method, namely in field or chamber planting cotton to be measured, whether after cotton grows to certain leaf age, spray certain density glyphosate, be that glyphosate resistant cotton judges according to the dead degree of cotton plants after dispenser to it.The method is intuitively accurate, but length consuming time, take up room large, seasonal strong.Molecule and protein determination are also a kind of methods identifying transgenosis glyphosate resistant cotton, but instrument and expensive reagents needed for Molecular Detection, require higher to the professional technique of testing staff, most company and scientific research institutions are difficult to carry out, in addition, the not yet commercialization of current domestic glyphosate resistant cotton, foreign gene is not yet announced, and molecular detecting method is restricted.Therefore, set up a kind of quick, easy, low cost and accurately and effectively glyphosate resistant cotton authentication method for glyphosate resistant cotton from now on popularization and detect significant.
Summary of the invention
The invention discloses a kind of rapid identification method of glyphosate resistant cotton, comprising: the cotton seeds glyphosate solution to be measured showed money or valuables one carries unintentionally is soaked; After clear water rinses, by the seed that soaked be placed in illumination box and carry out sand culture; Add up number of emerging, calculate emergence rate; Judge whether cotton to be measured is glyphosate resistant cotton according to emergence rate, wherein emergence rate be more than or equal to 70% for glyphosate resistant cotton, and emergence rate be less than or equal to 30% be non-glyphosate resistant cotton.
In the rapid identification method of above-mentioned glyphosate resistant cotton, in the step of described immersion, the cotton seeds glyphosate solution to be measured that part has showed money or valuables one carries unintentionally can be soaked, the cotton seeds clear water to be measured that another part can have been showed money or valuables one carries unintentionally soaks, and the seed that wherein clear water soaks is contrast; In the step of described statistics and calculating, can add up emerge number and radicle long, emergence rate and radicle growth inhibiting rate can be calculated; In the step of described judgement, can judge whether cotton to be measured is glyphosate resistant cotton according to emergence rate and radicle growth inhibiting rate, wherein emergence rate be more than or equal to 70%, radicle growth inhibiting rate be less than or equal to 30% be glyphosate resistant cotton, and emergence rate is less than or equal to 30%, radicle growth inhibiting rate be more than or equal to 70% be non-glyphosate resistant cotton.
In the rapid identification method of above-mentioned glyphosate resistant cotton, in the step of described statistics and calculating, go back observable radicle growth situation; In the step of described judgement, also can judge whether cotton to be measured is glyphosate resistant cotton, and wherein radicle growth is glyphosate resistant cotton normally according to radicle growth situation, and radicle growth is suppressed, be non-glyphosate resistant cotton without side root.
In the rapid identification method of above-mentioned glyphosate resistant cotton, if the emergence rate of the seed of wherein clear water immersion is more than or equal to 70%, then qualification effectively; If the emergence rate of the seed that clear water soaks is less than 70%, then it is invalid to identify.
In the rapid identification method of above-mentioned glyphosate resistant cotton, in the step of described immersion, described immersion can comprise soaks 1 ~ 2 hour with the glyphosate solution of 4.1 ~ 8.2ga.i./L, preferably soaks 1 hour with the glyphosate solution of 8.2ga.i./L.
In the rapid identification method of above-mentioned glyphosate resistant cotton, the described cotton seeds to be measured showed money or valuables one carries unintentionally can be obtained by vernalization process, wherein, described vernalization process can comprise: get cotton seeds to be measured and soak 24 ~ 48 hours in 25 DEG C ~ 30 DEG C clear water, period changes water 0 ~ 1 time.
In the rapid identification method of above-mentioned glyphosate resistant cotton, in the step of described sand culture, described sand culture can comprise: by the fine sand of sterilizing and water mixing, load in germination box, the seed soaked in the step of described immersion is evenly put in germination box, then on seed, cover loosen loose sand, cover lid is placed in illumination box and cultivates, and wherein preferably germination box is of a size of 13cm*19cm*12cm; Preferably the fine sand of sterilizing and water are mixed according to the ratio of weight ratio 50:8 ~ 10, most preferably mix according to the ratio of weight ratio 50:8; The wherein thick 1.5 ~ 2cm of the preferred loosen loose sand covered on seed; Wherein, the time of described sand culture is preferably 7 days.
In the rapid identification method of above-mentioned glyphosate resistant cotton, imposing a condition of described illumination box can be: temperature 28 ~ 30 DEG C, humidity 60% ~ 80%, illumination ratio be 8 ~ 14 little time: 16 ~ 10 hours, be preferably 8 hours: 16 hours, intensity of illumination 6000 ~ 8000lx.
In the rapid identification method of above-mentioned glyphosate resistant cotton, described fine sand sterilizing methods can comprise: being sieved by fine sand is placed on high-temperature sterilization in drying box, preferably hot air sterilization 4 ~ 6 hours at 120 ~ 180 DEG C.
In the rapid identification method of above-mentioned glyphosate resistant cotton, described glyphosate is preferably agriculture and reaches (Roundup), and it contains 41% glyphosate isopropyl amine salt, is produced by About Monsanto Chemicals.
The authentication method Glyphosate-Resistant cotton of application the application is identified, has the following advantages:
(1) cycle is short, within general about 8 days, can obtain qualification result.Compared with field or greenhouse Resistance Identification method, shorten about 35 days qualification cycle.
(2) workload is little.The authentication method of the application completes in indoor, and without the need to carrying out the operations such as whole, sowing, dispenser, a people just can complete whole qualification process.And it is little to take up room, can identify multiple material simultaneously.
(3) be not subject to seasonal restrictions.The authentication method of the application is at in-house operation, and seed is cultivated in illumination box.Compared with field or greenhouse Resistance Identification method, the authentication method of the application avoids the impact of bad climate.Therefore, the authentication method of the application can repeat, and result uniformity is better.
Embodiment
Below by the present invention is described in further detail by way of example, put into practice the present invention to enable those skilled in the art.Should be appreciated that and can adopt other embodiments, and suitable change can be made and do not depart from the spirit or scope of the present invention.In order to avoid for enabling those skilled in the art put into practice details unnecessary the present invention, specification may eliminate some known to those skilled in the art information.Therefore, below describe in detail and should not understand with restrictive meaning, and scope of the present invention is only defined by claims.
Following embodiment is convenient to understand the present invention better, but is not used for limiting the scope of the invention.
The each raw material used in following embodiment, except particularly pointing out, all can by commercially available acquisition.Quantitative test in following embodiment, all arranges and repeats experiment for three times, results averaged.Ga.i. implication is active constituent content.
Embodiment
materials and methods
Material:
The glyphosate resistant cotton material totally 8 kinds of test, is respectively KCM1, KCM2, KCM3, KCM4, KCM5, KCM6, KCM7, KCM8; Non-resistance glyphosate cotton material totally 7 kinds, be respectively CCRI 41, CCRI 43, CCRI 49, CCRI 52, CCRI 63, CCRI 69, CCRI 79, above-mentioned material provides by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute's breeding chamber.
The glyphosate used reaches (Roundup) for agriculture, and it contains 41% glyphosate isopropyl amine salt, is produced by About Monsanto Chemicals.
Method:
Vernalization
All cotton seeds are carried out sulfuric acid lint, and photon process is for subsequent use.
The seed getting often kind of detected materials soaks 24 hours in clear water, gets the seed showed money or valuables one carries unintentionally for subsequent use.
Soak
Each sample to be tested selects that vernalization shows money or valuables one carries unintentionally, full cotton seeds, soaks, rinse subsequently with water with glyphosate solution.Meanwhile, if clear water control treatment: soak sample to be tested with clear water.
Sand culture
Fine sand is sieved and to be placed in drying box at 180 DEG C hot air sterilization 4 hours, dry in the air cool after fine sand and water are mixed according to the ratio of weight ratio 50kg:8kg, load and be of a size of in the germination box of 13cm*19cm*12cm, to 2/3 height.The cotton seeds processed above is evenly put in germination box, every box 30 seeds, repeat 3 times.Then on seed, cover the loosen loose sand of about 1.5cm, cover lid is placed in illumination box and cultivates.Illumination box impose a condition as, temperature 30 DEG C, humidity 60% ~ 80%, illumination ratio be 8 little time: 16 hours, intensity of illumination 6000lx.
Statistics and calculating
Sand culture is after 7 days, and the number of emerging of the cotton seeds of statistics clear water process and glyphosate process, calculates emergence rate, and statistics radicle is long, and calculates radicle growth inhibiting rate.Wherein the criterion of seed sprouting is that cotyledon breaks ground.
The computing formula of radicle growth inhibiting rate % is:
Radicle growth inhibiting rate %=
(radicle of the radicle length-glyphosate processed group of clear water control group is long) × 100/the radicle of clear water control group is long
Embodiment 1
Glyphosate resistant cotton material (KCM1, KCM2, KCM3) and non-resistance glyphosate cotton material (CCRI 49, CCRI 79), soak 1 hour and 2 hours with the glyphosate solution that concentration is 4.1ga.i./L, 6.15ga.i./L and 8.2ga.i./L respectively, experimental result lists in table 1.
The emergence rate of table 1 cotton seeds to be measured after the process of glyphosate variable concentrations different time and radicle growth inhibiting rate
Radicle growth inhibiting rate is that negative value represents that the radicle of glyphosate processed group is grown up in clear water control group, glyphosate process is described on radicle growth without impact.
As shown in Table 1, soak cotton seed to be measured after 1 ~ 2 hour with 4.1g a.i./L glyphosate solution, the emergence rate of 3 kinds of glyphosate resistant cotton is 82.2%-93.3%, contrasts difference compared with (94.4%-97.8%) little with clear water; Radicle growth inhibiting rate is lower than 16.9%; And the emergence rate of 2 kinds of non-glyphosate resistant cotton is only 8.9%-27.8%, contrast difference compared with (77.8%-98.9%) with clear water large; Radicle growth inhibiting rate reaches more than 80.8%.
Soak cotton seed to be measured after 1 ~ 2 hour with 6.15ga.i./L glyphosate solution, the emergence rate of 3 kinds of glyphosate resistant cotton is 75.6%-82.2%, contrasts difference compared with (94.4%-97.8%) little with clear water; Radicle growth inhibiting rate is lower than 25.8%; And the emergence rate of 2 kinds of non-glyphosate resistant cotton is only 1.1%-16.7%, contrast difference compared with (77.8%-98.9%) with clear water large; Radicle growth inhibiting rate reaches more than 80.1%.
Soak cotton seed to be measured after 1 ~ 2 hour with 8.2g a.i./L glyphosate solution, the emergence rate of 3 kinds of glyphosate resistant cotton is 73.3%-86.7%, contrasts difference compared with (94.4%-97.8%) little with clear water; Radicle growth inhibiting rate is lower than 18.6%; And the emergence rate of 2 kinds of non-glyphosate resistant cotton is only 1.1%-10.0%, contrast difference compared with (77.8%-98.9%) with clear water large; Radicle growth inhibiting rate reaches more than 82.6%.
As can be seen here, under the glyphosate solution concentration of 4.1 ~ 8.2g a.i./L, the impact of glyphosate process used in this application on non-glyphosate resistant cotton and glyphosate resistant cotton has obvious difference, thus the discrimination method of the application can distinguish non-glyphosate resistant cotton and glyphosate resistant cotton effectively.
Embodiment 2
Glyphosate resistant cotton material (KCM4, KCM5, KCM6, KCM7, KCM8) and non-resistance glyphosate cotton material (CCRI 41, CCRI 43, CCRI 52, CCRI 63, CCRI 69), by 8.2g a.i./L glyphosate solution immersion treatment 1 hour, experimental result listed in table 2.
Table 2 processes the long and radicle growth inhibiting rate of the emergence rate of rear cotton seeds to be measured, radicle
Data according to table 2 can be found out, to non-resistance glyphosate cotton material, glyphosate process significantly affects emergence rate (emergence rate is reduced to 4.4% ~ 14.4% from 72.2% ~ 92.2%), and radicle growth inhibiting rate is up to 75.5% ~ 81.3%; And Glyphosate-Resistant cotton material, glyphosate process is on emergence rate impact little (emergence rate is reduced to 75.6% ~ 93.3% from 95.6% ~ 100%), and radicle growth inhibiting rate is only 0.2% ~ 7.6%.As can be seen here, the impact of glyphosate process used in this application on non-glyphosate resistant cotton and glyphosate resistant cotton has remarkable difference, thus the discrimination method of the application can distinguish non-glyphosate resistant cotton and glyphosate resistant cotton effectively.
In sum; these are only preferred embodiment of the present invention, be not intended to limit protection scope of the present invention, therefore; all any amendments done within the spirit and principles in the present invention, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. a rapid identification method for glyphosate resistant cotton, comprising:
Soaked by the cotton seeds glyphosate solution to be measured showed money or valuables one carries unintentionally, described immersion comprises soaks 1 ~ 2 hour with the glyphosate solution of 4.1 ~ 8.2g a.i./L;
After clear water rinses, by the seed that soaked be placed in illumination box and carry out sand culture, described sand culture is made up of following steps: by the fine sand of sterilizing and water mixing, load in germination box, the seed soaked in the step of described immersion is evenly put in germination box, then on seed, cover loosen loose sand, cover lid is placed in illumination box and cultivates, and wherein germination box is of a size of 13cm × 19cm × 12cm; The fine sand of sterilizing and water are mixed according to the ratio of weight ratio 50:8 ~ 10; Thick 1.5 ~ the 2cm of the loosen loose sand wherein covered on seed; Wherein, the time of described sand culture is 7 days;
Add up number of emerging, calculate emergence rate;
Judge whether cotton to be measured is glyphosate resistant cotton according to emergence rate, wherein emergence rate be more than or equal to 70% for glyphosate resistant cotton, and emergence rate be less than or equal to 30% be non-glyphosate resistant cotton.
2. the method for claim 1, wherein
In the step of described immersion, soaked by the cotton seeds glyphosate solution to be measured that part has showed money or valuables one carries unintentionally, the cotton seeds clear water to be measured showed money or valuables one carries unintentionally by another part soaks, and the seed that wherein clear water soaks is contrast;
In the step of described statistics and calculating, statistics emerge number and radicle long, calculating emergence rate and radicle growth inhibiting rate;
In the step of described judgement, judge whether cotton to be measured is glyphosate resistant cotton according to emergence rate and radicle growth inhibiting rate, wherein emergence rate be more than or equal to 70%, radicle growth inhibiting rate be less than or equal to 30% be glyphosate resistant cotton, and emergence rate is less than or equal to 30%, radicle growth inhibiting rate be more than or equal to 70% be non-glyphosate resistant cotton.
3. method as claimed in claim 1 or 2, wherein,
In the step of described statistics and calculating, also observe radicle growth situation;
In the step of described judgement, also judge whether cotton to be measured is glyphosate resistant cotton, and wherein radicle growth is glyphosate resistant cotton normally according to radicle growth situation, and radicle growth is suppressed, be non-glyphosate resistant cotton without side root.
4. method as claimed in claim 2, if the emergence rate of the seed of wherein clear water immersion is more than or equal to 70%, then qualification is effectively; If the emergence rate of the seed that clear water soaks is less than 70%, then it is invalid to identify.
5. method as claimed in claim 1 or 2, wherein, in the step of described immersion, described immersion comprises soaks 1 hour with the glyphosate solution of 8.2g a.i./L.
6. method as claimed in claim 1 or 2, wherein, the described cotton seeds to be measured showed money or valuables one carries unintentionally is obtained by vernalization process, wherein, described vernalization process comprises: get cotton seeds to be measured and soak 24 ~ 48 hours in 25 DEG C ~ 30 DEG C clear water, period changes water 0 ~ 1 time.
7. the method for claim 1, wherein the fine sand of sterilizing and water are mixed according to the ratio of weight ratio 50:8.
8. method as claimed in claim 1 or 2, wherein, described illumination box impose a condition as temperature 28 ~ 30 DEG C, humidity 60% ~ 80%, illumination ratio be 8 ~ 14 little time: 16 ~ 10 hours, intensity of illumination 6000 ~ 8000lx.
9. method as claimed in claim 8, wherein, illumination ratio be 8 little time: 16 hours.
10. the method for claim 1, wherein described fine sand sterilizing methods comprises: being sieved by fine sand is placed on high-temperature sterilization in drying box, hot air sterilization 4 ~ 6 hours at 120 ~ 180 DEG C.
11. methods as claimed in claim 1 or 2, wherein, described glyphosate is that agriculture reaches, and it contains 41% glyphosate isopropyl amine salt.
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| CN105379570A (en) * | 2015-11-16 | 2016-03-09 | 中国农业科学院棉花研究所 | Method for rapidly identifying glufosinate-ammonium resisting cotton |
| CN106576829A (en) * | 2016-12-13 | 2017-04-26 | 河南省农业科学院 | Identification method for glyphosate-resistant maize variety |
| CN113484513A (en) * | 2021-06-29 | 2021-10-08 | 贵州国芯生物科技有限公司 | Immunochromatography test strip for rapidly detecting glyphosate and preparation method thereof |
| CN114586496A (en) * | 2022-02-17 | 2022-06-07 | 江西省棉花研究所 | Identification method for glyphosate resistance of cotton seeds |
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