CN114586496A - Identification method for glyphosate resistance of cotton seeds - Google Patents
Identification method for glyphosate resistance of cotton seeds Download PDFInfo
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- CN114586496A CN114586496A CN202210148495.7A CN202210148495A CN114586496A CN 114586496 A CN114586496 A CN 114586496A CN 202210148495 A CN202210148495 A CN 202210148495A CN 114586496 A CN114586496 A CN 114586496A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like
- A01C1/025—Testing seeds for determining their viability or germination capacity
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention discloses a method for identifying glyphosate resistance of cotton seeds, which comprises the following steps: (1) carrying out delinting treatment and air drying on the cotton seeds, carrying out sterilization treatment after delinting and air drying, and repeatedly washing the cotton seeds with sterile water for later use; (2) respectively soaking the cotton seeds treated in the step (1) in glyphosate solutions with different concentrations, taking sterile water soaked seeds as a control, and counting and calculating the germination conditions of the cotton seeds; (3) and dividing the glyphosate-resistant performance grade of the cotton seeds according to the germination condition of the cotton seeds. The method adopts a seed soaking method to identify the glyphosate resistance of the cotton seeds, has short experimental period, is not influenced by external climatic conditions, does not need to spend a large amount of test cost, and is an economic, rapid and convenient identification method for the glyphosate resistance of the cotton seeds.
Description
The technical field is as follows:
the invention relates to the technical field of seed variety evaluation in agricultural products, in particular to a method for identifying glyphosate resistance of cotton seeds.
Background art:
cotton is an important economic crop in China, and weeds in a cotton field are seriously harmful in the planting process, so that the high-quality and high-efficiency production of the cotton is severely restricted. The glyphosate is used as a broad-spectrum systemic biocidal herbicide and plays an important role in preventing and killing weeds in cotton fields. However, glyphosate is not selective between weeds and cotton, and when glyphosate is used, the glyphosate is easy to cause phytotoxicity and even death to conventional cotton varieties.
The application of the transgenic glyphosate-resistant cotton can not only avoid the generation of phytotoxicity, but also enlarge the application range and flexibility of glyphosate. The transgenic glyphosate-resistant cotton is bound to become another type of transgenic cotton industrialized variety expected to be popularized after insect-resistant cotton in China. In the process of breeding glyphosate-resistant cotton varieties, the evaluation of glyphosate resistance on cotton materials is not only the basis of cotton breeding and industrial popularization, but also one of the contents of the evaluation of environmental safety of transgenic cotton, so that the research of a method for identifying glyphosate resistance of cotton seeds is very important.
At present, the identification method of glyphosate-resistant cotton is mainly a field or greenhouse plant resistance identification method, namely, cotton to be detected is planted in a field or a greenhouse, glyphosate with a certain concentration is sprayed when the cotton grows to a certain leaf age, and whether the cotton is glyphosate-resistant cotton is judged according to the death degree of the cotton plant after application. The method is visual and accurate, but has the defects of long time consumption, large occupied space, strong seasonality and the like.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The invention content is as follows:
the invention aims to provide a method for identifying glyphosate resistance of cotton seeds, thereby overcoming the defects in the prior art.
In order to achieve the aim, the invention provides an identification method for glyphosate resistance of cotton seeds, which comprises the following steps:
(1) carrying out delinting treatment and air drying on the cotton seeds, carrying out sterilization treatment after delinting and air drying, and repeatedly washing the cotton seeds with sterile water for later use;
(2) respectively soaking the cotton seeds treated in the step (1) in glyphosate solutions with different concentrations, counting and calculating the germination conditions of the cotton seeds by taking sterile water soaked seeds as a reference;
(3) and dividing the glyphosate-resistant performance grade of the cotton seeds according to the germination condition of the cotton seeds.
And (2) adopting a concentrated sulfuric acid delinting method during the delinting treatment in the step (1).
In the step (1), sodium hypochlorite with the mass concentration of 10% is adopted for disinfection for 5-10min, and after disinfection, the solution is repeatedly washed with sterile water for 5-6 times, wherein each time of washing is 30-90 s.
And (3) diluting a 33% glyphosate isopropylamine solution and sterile water by using 40 times of solution, 80 times of solution, 100 times of solution, 120 times of solution and 140 times of solution respectively during seed soaking in the step (2).
The seed soaking operation in the step (2) comprises the following steps:
s1: respectively putting the seeds in mesh bags, soaking the seeds in glyphosate solutions with different concentrations and sterile water, and placing the seeds in an illumination incubator for dark culture for 24 hours at the temperature of 30 ℃;
s2: transferring the seeds to a culture dish for germination acceleration, tightly attaching single filter paper to the culture dish during germination acceleration, then putting the culture dish into pure water for soaking until the filter paper is completely wet, putting the seeds into the culture dish after the filter paper is wet, covering a cover on the culture dish, and putting the seeds into an illumination culture box for germination acceleration for 24 hours.
Counting the germination condition of the cotton seeds in the step (2) by using relative germination rate, wherein the germination rate is equal to germination number/50 multiplied by 100%; relative germination percentage%.
The glyphosate-resistant performance grading standard of the cotton seeds in the step (3) is as follows: when the relative germination rate of the cotton seeds is more than 90.0%, the cotton seeds are in a high glyphosate-resistant grade, the germination rate is 75% -89.9% in a glyphosate-resistant grade, 50.0% -74.9% in a glyphosate-resistant grade, and 0.0% -49.9% in an intolerant grade.
Compared with the prior art, the invention has the following beneficial effects:
the method adopts a seed soaking method to identify the glyphosate resistance of the cotton seeds, has short experimental period, is not influenced by external climatic conditions, does not need to spend a large amount of test cost, and is an economic, rapid and convenient identification method for the glyphosate resistance of the cotton seeds.
Description of the drawings:
FIG. 1 is a schematic diagram of the germination of cotton seeds of different glyphosate tolerance levels of the present invention before 3 days, wherein a is the germination of the seeds of number 7, b is the germination of the seeds of number 16, c is the germination of the seeds of number 26, and d is the germination of the seeds of number 37, all of which are treated to germinate under the condition of 100 times sterile water dilution of 33% glyphosate isopropylamine salt solution concentration;
the specific implementation mode is as follows:
the following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example (b):
the identification method for glyphosate resistance of cotton seeds comprises the following steps:
(1) selecting 60 parts of cotton seeds as materials, wherein 35 parts of resistant materials and 25 parts of common materials are subjected to delinting treatment on the cotton seeds by a concentrated sulfuric acid delinting method, and airing for later use after the delinting treatment;
selecting 50 seeds from each part of material as a treatment, sterilizing the selected seeds with sodium hypochlorite solution with the mass concentration of 10% for 5-10min, and repeatedly washing with sterile water for 5-6 times, wherein the washing time is about 1min each time;
(2) prepare 5L glyphosate solution, 33% glyphosate isopropylamine salt diluted 40 times liquid, 80 times liquid, 100 times liquid, 120 times liquid, 140 times liquid and sterile water according to the following table.
| 40 times of | 80 times of | 100 times of | 120 times of | 140 times of | Sterile water | |
| 33% Glyphosate isopropylamine salt (ml) | 122 | 62 | 50 | 42 | 35 | - |
| Sterilization pure water (ml) | 4878 | 4938 | 4950 | 4958 | 4965 | 5000 |
Sterile water seed soak as control experiment:
respectively filling 60 parts of cotton seeds to be detected into mesh bags, soaking the cotton seeds in sterile water, placing the cotton seeds in a light incubator for dark culture for 24 hours, controlling the temperature around 30 ℃ day and night, transferring the seeds to a culture dish for germination acceleration after soaking for 24 hours, and adopting a filter paper germination method;
the germination accelerating operation is as follows: preparing filter paper, tightly attaching a single piece of filter paper to a culture dish, then soaking the filter paper in pure water until the filter paper is completely wet, then placing 50 seeds of each material into the culture dish, covering the culture dish with a cover, placing the culture dish into an illumination incubator for culturing for 24 hours, counting the germination rate of each material, considering the germination when the germination length reaches 2mm, then counting the germination conditions for 1, 2, 3, 5 and 7 days, and counting the germination number after 3 days, so that the relative germination rate is calculated by selecting the germination number of the first 3 days. Selecting materials with the germination rate of more than 90 percent and 37 parts of materials, wherein 21 parts of resistant materials and 16 parts of common materials are used for carrying out glyphosate seed soaking experiments.
Glyphosate seed soaking experiment:
respectively selecting 50 seeds from 37 parts of screened materials to carry out a glyphosate seed soaking experiment, respectively filling 37 parts of cotton seeds to be tested into net bags, respectively soaking the cotton seeds in 33 percent glyphosate isopropylamine salt solution diluted by 40 times of solution, 80 times of solution, 100 times of solution, 120 times of solution and 140 times of solution, placing the cotton seeds in a light incubator for 24 hours to carry out dark culture, and controlling the temperature around 30 ℃ day and night. After soaking for 24 hours, transferring the seeds to a culture dish for germination acceleration, and adopting a filter paper germination method;
and (3) accelerating germination, namely, tightly attaching single filter paper to a culture dish, soaking the culture dish in pure water until the filter paper is completely wet, then putting 50 seeds of each material into the culture dish, covering the culture dish with a cover, putting the culture dish into an illumination incubator for culturing for 24 hours, counting the germination rate of each material, considering the germination when the germination length reaches 2mm, then counting the germination conditions for 1, 2, 3, 5 and 7d, counting and finding that the germination number is extremely small after 3 days, and calculating the relative germination rate by selecting the germination number of the first 3 days. Wherein the germination rate is the germination number/50 multiplied by 100%, and the relative germination rate is the germination rate of the material to be detected/the germination rate of the reference material multiplied by 100%;
through 5 times of experimental statistics, the germinated seeds of the cotton seeds account for more than 90% of the total germination number when the cotton seeds are treated for 3 days under the condition that the sterile water is diluted by 100 times and the concentration of the glyphosate isopropylamine salt solution is 33%, and the difference between the resistant material and the common material is extremely obvious. The germination rate at 3 days of treatment at a concentration of 33% glyphosate isopropylamine salt solution diluted 100-fold with sterile water was therefore selected as an indicator for the selection of resistant materials.
(3) Dividing the glyphosate-resistant performance grade of the cotton seeds according to the experimental result:
when the relative germination rate of the cotton seeds is more than 90.0%, the cotton seeds are in a high glyphosate-resistant grade, the germination rate is 75% -89.9% in a glyphosate-resistant grade, 50.0% -74.9% in a glyphosate-resistant grade, and 0.0% -49.9% in an intolerant grade.
The specific experimental data and results are as follows:
the names and the numbers of 37 parts of materials of the glyphosate seed soaking experiment are shown in the table I:
table one: materials and names of experiments
The statistical results of the relative germination percentage% and the glyphosate-resistant performance grade after 3 days of treatment with sterile water diluted by 100 times and 33% glyphosate isopropylamine salt solution concentration are divided as shown in table two, wherein I, II and III represent 3 repeated experiments:
a second table: relative germination (%)
A third table: two-factor analysis of variance
Analysis of variance shows that the difference between the blocks is not obvious, namely the repeatability between the three blocks is better; the difference between the multiples is extremely obvious, which shows that the glyphosate soaking seeds with different concentrations have great influence on the germination rate of cotton seeds, and the difference is extremely obvious; the interaction between the varieties x is very obvious in difference, which shows that the difference exists in glyphosate resistance between different varieties.
Table four: glyphosate resistance rating standard
The evaluation of glyphosate resistance to 37 parts of material according to the above-mentioned grading criteria resulted in the following:
the foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (7)
1. A method for identifying glyphosate resistance of cotton seeds is characterized by comprising the following steps: the method comprises the following steps:
(1) carrying out delinting treatment and air drying on the cotton seeds, carrying out sterilization treatment after delinting and air drying, and repeatedly washing the cotton seeds with sterile water for later use;
(2) respectively soaking the cotton seeds treated in the step (1) in glyphosate solutions with different concentrations, taking sterile water soaked seeds as a control, and counting and calculating the germination conditions of the cotton seeds;
(3) and dividing the glyphosate-resistant performance grade of the cotton seeds according to the germination condition of the cotton seeds.
2. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: and (2) a concentrated sulfuric acid delinting method is adopted during delinting treatment in the step (1).
3. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: and (2) adopting sodium hypochlorite with the mass concentration of 10% to disinfect for 5-10min during disinfection treatment in the step (1), and repeatedly washing with sterile water for 5-6 times after disinfection, wherein the washing time is 30-90 s each time.
4. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: and (3) diluting a 33% glyphosate isopropylamine solution and sterile water by respectively adopting 40 times of solution, 80 times of solution, 100 times of solution, 120 times of solution and 140 times of solution during seed soaking in the step (2).
5. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: the seed soaking operation in the step (2) comprises the following steps:
s1: respectively putting the seeds in mesh bags, soaking the seeds in glyphosate solutions with different concentrations and sterile water, and placing the seeds in an illumination incubator for dark culture for 24 hours at the temperature of 30 ℃;
s2: transferring the seeds to a culture dish for germination acceleration, tightly attaching single filter paper to the culture dish during germination acceleration, then putting the culture dish into pure water to soak the filter paper until the filter paper is completely wet, putting the seeds into the culture dish after the filter paper is wet, covering a cover on the culture dish, and putting the seeds into an illumination incubator for germination acceleration for 24 hours.
6. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: counting the germination condition of the cotton seeds in the step (2) by using relative germination rate, wherein the germination rate is equal to germination number/50 multiplied by 100%; relative germination percentage is the germination percentage of the material to be detected/the germination percentage of the control material multiplied by 100%.
7. The method for identifying glyphosate tolerance of cotton seeds of claim 1, wherein the method comprises the following steps: the glyphosate-resistant performance grading standard of the cotton seeds in the step (3) is as follows: when the relative germination rate of the cotton seeds is more than 90.0%, the cotton seeds are in a high glyphosate-resistant grade, the germination rate is 75% -89.9% in a glyphosate-resistant grade, 50.0% -74.9% in a glyphosate-resistant grade, and 0.0% -49.9% in an intolerant grade.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102165885A (en) * | 2010-12-15 | 2011-08-31 | 中国农业科学院植物保护研究所 | Method for identifying glyphosate resistance of cotton |
| CN103081731A (en) * | 2013-02-28 | 2013-05-08 | 中国农业科学院棉花研究所 | Method for identifying glyphosate resistant cotton rapidly |
| CN103364355A (en) * | 2013-07-19 | 2013-10-23 | 中国农业科学院棉花研究所 | Method for rapidly evaluating glyphosate tolerance of cotton |
| CN103411971A (en) * | 2013-08-19 | 2013-11-27 | 中国农业科学院棉花研究所 | Method for quickly evaluating glyphosate tolerance of cotton in field |
| CN105340743A (en) * | 2015-11-11 | 2016-02-24 | 山西省农业科学院棉花研究所 | Method for rapidly detecting transgenic glyphosate-resistant cotton |
| 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 |
-
2022
- 2022-02-17 CN CN202210148495.7A patent/CN114586496A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102165885A (en) * | 2010-12-15 | 2011-08-31 | 中国农业科学院植物保护研究所 | Method for identifying glyphosate resistance of cotton |
| CN103081731A (en) * | 2013-02-28 | 2013-05-08 | 中国农业科学院棉花研究所 | Method for identifying glyphosate resistant cotton rapidly |
| CN103364355A (en) * | 2013-07-19 | 2013-10-23 | 中国农业科学院棉花研究所 | Method for rapidly evaluating glyphosate tolerance of cotton |
| CN103411971A (en) * | 2013-08-19 | 2013-11-27 | 中国农业科学院棉花研究所 | Method for quickly evaluating glyphosate tolerance of cotton in field |
| CN105340743A (en) * | 2015-11-11 | 2016-02-24 | 山西省农业科学院棉花研究所 | Method for rapidly detecting transgenic glyphosate-resistant cotton |
| 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 |
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