GB/T 3543.5-1995 Inspection procedures for crop seeds - Authenticity and variety purity identification
Some standard content:
National Standard of the People's Republic of China
Rules for agricultural seed testing- Verification of genuineness and cultivar1 Subject content and scope of application
This standard specifies the methods for determining the authenticity and purity of seeds. This standard applies to the quality testing of crop seeds. 2 Referenced standards
GB/T3543.2 Rules for testing agricultural seed- Sampling GB/T3543.4 Rules for testing agricultural seed- Germination test 3 Terminology
3.1 Genuineness of seed Whether the variety to be tested is consistent with the document record (such as label, etc.). 3.2 Varietal purity
GB/T 3543. 5-1995
Replaces GB3543-83
The degree of typical consistency of a variety in terms of characteristics, expressed as the percentage of seeds of this variety to the number of seeds of the sample of this crop for inspection. 3.3 Off-type
A plant whose one or more traits (characteristics) are significantly different from those described by the breeder of the original variety. 3.4 Breeder seed
The first batch of seeds of a variety with stable genetic traits or parental seeds bred by breeders, used for further propagation of basic seed. 3.5 Basic seed
The first to third generation propagated by breeder seeds, or seeds that meet the basic seed quality standards produced according to the basic seed production technical regulations, used for further propagation of improved seed.
3. 6 Certified seed
Seeds of the third to third generations and hybrids propagated from conventional seed stock that meet the quality standards of certified seeds are used for field production. 4 Reagents
Phenol, guaiacol, hydrogen peroxide, potassium hydroxide, sodium hydroxide, hydrogen chloride. 5 Instruments and equipment
Instruments and equipment also vary with the methods. a. Test in the laboratory
Approved by the State Administration of Technical Supervision on August 18, 1995 7.
Implemented on June 1, 1996
GB/T3543.5--1995
Equipped with appropriate instruments (such as stereo microscopes, magnifying glasses, dissecting microscopes) and reagents for seed morphology, physiological and cytological examinations, chemical determinations and seed germination.
b. Testing in greenhouses and culture rooms
Equipped with equipment that can adjust environmental conditions (such as growth chambers) to facilitate the development of identification traits. c. Identification in field plots
There must be climate, soil and cultivation conditions that can allow the identification traits to develop normally, and there must be relative protection measures for the prevention and control of pests and diseases. 6 Testing procedures
6.1 Weight of samples to be tested
The minimum weight of samples to be tested for variety purity testing should comply with the provisions of Table 1. Table 1 Weight of samples for variety purity determination Type
Pea, Phaseolus, Vicia, Zea, Glycine max and other genera with similar seed size
Rice, Hordeum, Avena, Wheat, Secale and other genera with similar seed size
Beet and other genera with similar seed size All other genera
6.2 Seed identification
6.2.1 Morphological identification method
Limited to laboratory determination
Field plot and laboratory determination
Randomly select 400 seeds from the samples for inspection. Repeat the identification, and each repeat shall not exceed 100 seeds. g
Based on the morphological characteristics of the seeds, each seed may be observed with the help of a magnifying glass when necessary. Standard samples or identification pictures and relevant information must be available.
Rice seeds are judged based on grain shape, aspect ratio, size, shell and tip color, hair length, density, stigma clamping rate, etc.; barley seeds are judged based on grain shape, outer two base wrinkles, grain color, ventral base thorns, ventral expansion degree, outer sacral side dorsal vein pattern teeth and vein color, outer base shell wrinkles depression, spikelet axis hair quantity, scale (lodicule) shape and hair density, etc.; soybean seeds can be judged based on seed size, shape, color, gloss, smoothness, wax powder quantity and hilum shape and color, etc.; onions can be judged based on seed size, shape, color, surface structure and hilum characteristics, etc. 6.2.2 Rapid determination method
Randomly select 400 seeds from the samples to be tested, and repeat the identification, with no more than 100 seeds in each repeat. a. Phenol staining method
Wheat, barley, oats: Soak the seeds in clean water for 18 to 24 hours, use filter paper to absorb the surface moisture, and put them into a culture medium (ventral groove facing down) with filter paper moistened with 1% phenol solution. At room temperature, keep wheat for 4 hours, oats for 2 hours, and barley for 24 hours before identifying the depth of staining. Observe the staining of caryopsis for wheat, and evaluate the staining of seeds inside and outside for barley and oats. Usually the color is divided into five levels, namely light color, light brown, brown, dark brown and black. Take out seeds that are different from the basic color as different varieties. Rice: Soak the seeds in clean water for 6 hours, pour out the clean water, inject 1% (m/V) phenol solution, soak at room temperature for 12 hours, take out and wash with clean water, put on filter paper for 24 hours, and observe the degree of staining of grains or rice grains. Grain staining is divided into five levels: unstained, light brown, brown, dark brown and black, and rice grain staining is divided into three levels: unstained, light brown, brown or purple. b. Soybean seed coat guaiacol staining method
Peel off the seed coat of each soybean seed, place it in a small test tube, then inject 1mL of distilled water, extract at 30℃ for 1h, then add 10 drops of 0.5% guaiacol solution to each test tube, 10min later, add 1 drop of 0.1% hydrogen peroxide solution to each test tube. After 1min, count the number of seeds in the test tube whose seed coat extract is reddish brown and the number of seeds whose extract is colorless. c. Potassium hydroxide-bleaching powder determination method for sorghum seeds 75
GB/T3543.5--1995
Prepare a mixture of 1:5 (m㎡/V) potassium hydroxide and fresh ordinary bleaching powder (5.25% bleaching powder) [i.e. 1g potassium hydroxide (KOH) is added to 5.0ml of bleaching liquid, usually 100ml of solution is prepared and stored in a refrigerator for later use. Place the seeds in a culture medium and add potassium hydroxide-bleaching solution (cover the temperature for a while before measuring) until the seeds are submerged. Soak the brown seed coat for 10 minutes. Gently shake the seeds regularly during soaking to ensure good contact between the solution and the seeds, then pour the seeds onto the gauze and slowly rinse them with tap water. After rinsing, place the seeds on paper and let them air dry. After the seeds are dry, record the number of black seeds and light seeds. d. Oat seeds\fluorescence determination method
Use ultraviolet light with a wavelength of 360A and identify in a dark room. Arrange the seeds on black paper and place them 10 to 15 cm away from the ultraviolet light. After irradiation for a few seconds to a few minutes, identify them based on whether there is fluorescence inside and outside. e. Oat seed hydrogen nitride determination method
Put the oat seeds in a glass container containing a previously prepared hydrogen fluoride solution [1 part 38% (V/V) hydrochloric acid (HCl) and 4 parts water] and soak for 6 hours, then take them out and place them on filter paper and let them air dry for 1 hour. Identify seeds based on brown (fluorescent seeds) or yellow (non-fluorescent seeds). f. Sodium hydroxide method for wheat seeds
When wheat seeds are difficult to distinguish between red and white skins (especially seeds treated with fungicides), the sodium hydroxide method can be used to distinguish them. Count 400 or more seeds, soak them in 95% (V/V) methanol for 15 minutes, then let the seeds dry for 30 minutes, and soak the seeds in 5M/I.NaH solution at room temperature for 5 minutes. Then move the seeds to a culture dish without covering them, let them dry at room temperature, and count the seeds based on their light and dark colors.
6.2.3 Acid polyacrylamide electrophoresis method for wheat and barley alcohol-soluble proteins is shown in Appendix A (reference). 6.3 Identification of young seeds
Randomly count 400 seeds from the samples to be tested, and repeat the identification, with 100 seeds per repeat. In a culture room or greenhouse, 100 seeds can be used, with 100 repeats.
Young seedlings can be identified in two main ways: one is to provide the plants with conditions that accelerate their development (similar to field plot identification, but the time required is shorter), and when the seedlings reach a developmental stage suitable for evaluation, all or part of the seedlings are identified; the other is to let the plants grow under special adverse conditions, and measure the different responses of different varieties to adversity to identify different varieties. Cereals: The bud sheaths and mesocotyls of cereal crops are purple and green, which are controlled by genetic genes. Sow the seeds in the sand (corn and sorghum seeds are spaced 1.0cm×4.5cm, oats and wheat seeds are spaced 2.0cm×4.0cm, and the sowing depth is 1.0cm), cultured at a constant temperature of 25°C and 24h light. Water was added to corn and sorghum every day, and phosphorus-deficient Hoagland No. 1 culture solution was applied to wheat and oats every 4 days. When the seedlings developed to the appropriate stage, sorghum and corn were 14 days, wheat was 7 days, and oats were 10-14 days, and the color of the bud sheath was identified. Note: The formula of phosphorus-deficient Hoagland No. 1 culture solution is: add 4mLIM/L calcium nitrate solution (Ca(NO2+),), 2mL1M/L magnesium sulfate solution (MgSO2) and 6mL1M/L potassium nitrate solution (KNO2+) to 1L distilled water. Minister: Sow the seeds in sand (seed spacing 2.5cm×2.5cm, sowing depth 2.5cm), culture at 25℃, 24h light, apply Hoagland No. 1 culture solution every 4 days, until the various characteristics of the seedlings are obvious, identify them according to the color of the hypocotyl (growth 10~14d), the color of the hairs (21d), the angle of the hairs on the hypocotyl (21d), the shape of the leaflets (21d), etc. Note: The formula of Hoagland No. 1 culture solution: add 1mL 1M/L potassium dihydrogen phosphate solution (KH2PO4), 5mL 1M/L potassium nitrate solution (KN2O3), 5mL 1M/L calcium nitrate solution (Ca(NO2)2) and 2mL 1M/L magnesium sulfate solution (MgSO4) to 1L distilled water. Lettuce: Sow lettuce seeds in sand (seed spacing 1.0cm×4.0cm, sowing depth 1cm), culture at a constant temperature of 25℃, apply Hoagland No. 1 culture solution every 4 days, and identify them according to the color of the hypocotyl, leaf color, leaf curling degree and cotyledon shape after 3 weeks (with 3 to 4 leaves).
Beet: Some cultivars can be distinguished according to the color of the seedlings (white, yellow, dark red or red). Sow the bulbs on the wet sand of the culture dish. In the soft sunlight of the greenhouse, check the color of the hypocotyl of the seedlings after 7 days. The authenticity of sugar beet and white fodder beet cultivars can be indicated to a certain extent according to the ratio of white to dark red seedlings. 6.4 Inter-plot planting identification
Inter-plot planting is the most reliable and accurate method to identify the authenticity of varieties and determine the purity of varieties. In order to identify the authenticity of varieties, comparison with standard samples should be made at all stages of identification. The reference standard samples provide a comprehensive and systematic description of the characteristics of the cultivated varieties. The standard samples should represent the original characteristics of the varieties, preferably breeders' seeds. The number of standard samples should be sufficient to be used for many years and stored under low temperature and dry conditions. It is best to obtain them from breeders when they are replaced. Note: 1) This section is equivalent to the OECD variety certification scheme for seed circulation in international trade (Guidelines for Plot Identification Methods, OECD 1982). In order to fully express the characteristics of the varieties, the design and layout of the experiment should select fields with suitable climatic and environmental conditions, uniform soil, consistent fertility, no similar crops and weeds in the previous crop, and appropriate cultivation and management measures. There should be enough distance between rows and plants. The row-plant distance can be appropriately increased for large crops. Spot sowing and spot planting can be used when necessary. In order to determine the percentage of variety purity, it must be linked to the current national standard seed quality standards issued and implemented. The number of plants to be planted in the test design should be determined according to the requirements of the national standard seed quality standard. Generally speaking, if the standard is (N-1)×100%/N, a satisfactory result can be obtained by planting 4N plants. If the standard stipulates that the purity is 98%, that is, N is 50, planting 200 plants can meet the requirements. The inspector should have rich experience, be familiar with the characteristics of the inspected variety, and be able to correctly judge whether the plant belongs to the original variety or a variant. The variant should be a genetic variation, not a variation caused by environmental influences. For many species, it is possible to identify the authenticity and purity of the variety at the seedling stage, but the maturity period (conventional species), flowering period (hybrid species) and edible organ maturity period (vegetable species) are the periods when the variety characteristics are manifested, and identification must be carried out. Whether the purity of the improved variety meets the requirements of the national standard seed quality standard, contract and label can be judged using Table 2. Table 2 Tolerable difference of variety purity
(One-tail test at 5% significance level)
Standardized value
Above 50%
Below 50%
Number of sample plants, seedlings or seeds
Standardized value
Above 50%
Below 50%
GB/T3543.5-1995
Continued Table 2
Number of sample plants, seedlings or seeds
National standard Seed quality standards stipulate that the seeds with very high purity requirements, such as breeder seeds and original seeds, can use the elimination value to determine whether they meet the requirements. The elimination value is based on the consideration of the interests of seed producers and the possibility of less misjudgment. The number of variant plants observed in a sample is compared with the quality standard to make a decision to accept or eliminate the seed batch that meets the requirements. Its reliability is closely related to the sample size (see Table 3).
Table 3 Reliability of accepting seed batches containing variants at 99.9% with different sample sizes Sample size
(number of plants)
Elimination value
Note: 1) refers to the number of variants detected in every 1000 plants 1.5/10001)
Reliability of accepting seed batches, %
3/10001)
2/10001)
Elimination values under different specified standards and different sample sizes are shown in Table 4. If the variant is greater than or equal to the specified elimination value, the seed batch should be eliminated.
Specified standard
GB/T 3543.5-1995
4 Elimination values for different standards and different sample sizes Table 4
Elimination values for different sample (number of plants) sizes
Note: The numbers with "一\" or "
一" below indicate that the number of samples is too small.
7 Calculation and expression of results
7.1 Seeds and seedlings
When using seeds or seedlings for identification, the purity percentage of the variety is used for expression. Number of seeds for inspection (number of seedlings) × Number of seeds of different varieties (number of seedlings) × 100 Variety purity (%) =
7.2 Field plot identification
Number of seeds for inspection (number of seedlings)
The identified variety, different varieties, different crops and weeds are all expressed as the percentage of the identified plants. 8 Result report
The results measured in the laboratory or culture room must be reported in terms of the number of seeds, seedlings or plants. 200
In addition to the purity of the variety, the results of field plot planting identification should also report the percentage of foreign crops, weeds and other cultivated varieties found when possible.
A1 Principle
GB/T3543.5—1995
Appendix A
Determination of barley and wheat seed purity by polyacrylamide electrophoresis (reference)
The alcohol-soluble proteins extracted from the seeds are well separated under the molecular sieving effect of the gel and the charge effect of electrophoretic separation, and the protein band type is displayed by color development. Due to different genetic compositions, different varieties contain different types of proteins in their seeds. Differences can be identified using electrophoresis patterns, thereby identifying the authenticity and purity of the variety. A2 Instruments and reagents
A2.1 Instruments
Electrophoresis instrument (meeting the voltage regulation of 500V), centrifuge, vertical plate electrophoresis tank, tweezers, 5mL, 10mL pipettes, microinjector, polypropylene centrifuge tubes. bzxz.net
A2.2 Reagents
Urea, ethanol, glycine, methyl green, trichloroacetic acid, glacial acetic acid, hydrogen peroxide, ferrous sulfate, ascorbic acid, α-mercaptoethanol, acrylamide, Coomassie Brilliant Blue R-250, methylene bisacrylamide, α-chloroethanol. A3 Procedures
A3.1 Preparation of reagents
A3.1.1 Protein extract
Wheat 0.05 1. Dissolve 0.05 g methyl green in 25 mL α-chloroethanol, add distilled water to 100 mL. Store at low temperature. Barley: Dissolve 0.05 g methyl green in 20 mL α-fluoroethanol, add 18 g urea, then add 1 mL α-mercaptoethanol, add distilled water to 100 mL. Store at low temperature.
A3.1.2 Electrode buffer
Dissolve 0.4 g glycine in distilled water, add 4 mL glacial acetic acid, add distilled water to 1000 mL. Store at low temperature. A3.1.3 Gel buffer
Dissolve 1.0 g glycine in distilled water, add 20 mL glacial acetic acid, and make up to 1000 mL. Store at low temperature. A3.1.40.6% hydrogen peroxide
2 mL 30% hydrogen peroxide, add distilled water to 100 mL. Store at low temperature. A3.1.5 Staining solution
Dissolve 0.25g Coomassie brilliant blue in 25mL anhydrous ethanol, add 50g trichloroacetic acid, and add water to 500mL. A3.1.6 Gel solution
20 g acrylamide, 0.8 g methylene bisacrylamide, 12 g urea, 0.01 g ferrous sulfate, 0.2 g ascorbic acid, dissolved in gel buffer and fixed to 200 ml. Store at low temperature. A3.2 Sample extraction
Generally, 100 seeds are tested for each sample. If the purity of the variety is estimated more accurately, more seeds are needed. If the analysis results are to be compared with a certain purity standard value, sequential testing can be used to determine it, that is, 50 seeds are used as a group. If necessary, groups can be tested continuously to reduce the workload. If only authenticity is to be identified, 50 seeds can be used. Take wheat or barley seeds, crush them one by one with pliers (when clamping seeds, it is best to put a small piece of clean paper on the pliers to facilitate cleaning of the pliers head and prevent contamination between samples), put them in a 1.5mL centrifuge tube, add protein extract (0.2mL for wheat and 0.3mL for barley), shake them thoroughly, extract them at room temperature for 24 hours, and then centrifuge them at 18000×g for 15 minutes. Take the supernatant for electrophoresis. 80
A3.3 Gel preparation
GB/T 3543.5—1995
Take out the gel solution and hydrogen peroxide solution from the refrigerator, absorb 10ml of gel solution, add 1 drop of 0.6% hydrogen peroxide, shake well, and quickly pour it into the sealing part, shake it slightly to fill the entire seam with glue, and let it polymerize and seal it in 5~10 minutes. Absorb 45mL of gel solution, add 3 drops of 0.6% hydrogen peroxide, shake quickly, pour between the gel plates, insert the sample comb immediately, and let it polymerize within 5~~10 minutes.
A3.4 Injection
Carefully pull out the sample comb, clamp the glass plate on the electrophoresis tank, use filter paper or syringe to absorb excess water in the sample tank, and then use a microinjector to absorb 10~20μL of sample and add it to the sample tank. A3.5 Electrophoresis
Inject electrode liquid into the front and rear tanks, connect the front tank to the positive electrode, and the rear tank to the negative electrode. Then turn on the power supply and gradually increase the voltage to 500V. During electrophoresis, it is required to be carried out at a temperature of 15~20℃. The electrophoresis time is generally 60~80min. The specific time can be calculated according to the migration time of methyl green. The electrophoresis time is 2~2.5 times the time required for methyl green to move to the front. A3.6 Staining
Carefully remove the gelatin plate and stain it in the staining solution for 1 to 2 days. Generally, decolorization is not required, but it can be rinsed with clean water to make the bands clear.
A3.7 Identification
Band naming can be done by relative mobility method or electrophoresis program method. According to the composition and consistency of the alcohol-soluble protein bands and by comparing them with the electrophoresis patterns of standard samples, the authenticity of seeds and the purity of varieties can be identified. Additional remarks:
This standard was proposed by the Ministry of Agriculture of the People's Republic of China. This standard is under the jurisdiction of the National Technical Committee for Standardization of Crop Seeds. This standard was drafted by the National Seed Station, Zhejiang Agricultural University, Sichuan Province, Heilongjiang Province, Tianjin Seed Company (Station), Nanjing Agricultural University, Beijing, Hunan Seed Company. The main drafters of this standard are Zhi Juzhen, Bi Xinhua, Du Kemin, Chang Xiulan, Yang Shuhui, Ren Shuping, Wu Zhixing, Li Renfeng, Zhao Juying. This standard was first issued in March 1983.
This standard refers to the International Seed Inspection Procedure (ISTA, 1993 edition) Part 8 Identification of Seeds and Cultivar Varieties.
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