GB/T 3543.9-2025Rules for agricultural seed testing - Part 9: Sowing quality - Viability test (English PDF)

1 Scope

This document describes the tetrazolium method for determining the viability of agricultural seed.

This document applies to the testing of agricultural seed, and mainly to the following cases: seed that has to be sown as soon as possible after harvest; seed that has already begun to germinate and seed damaged during harvest; deeply dormant seed and slow-germinating seed; and seed whose germination potential has to be known quickly, together with the part of the seed at the end of a germination test that is suspected of being dormant.

3 Terms and definitions

3.1 seed viability: the potential germination capacity of the seed, or the vital force possessed by the embryo.

4 General

The tetrazolium test is a rapid method of determining seed viability. Whether a seed is judged viable depends chiefly on the position and the extent of the necrotic areas in the embryo, the endosperm, the perisperm, the gametophyte and similar tissues, and not necessarily on how deep the stain is; the depth of colour serves to judge whether the tissue is sound, weakened or dead.

The viability shown by the tetrazolium test is a property peculiar to seed in the dormant state. In dormant seed there is a marked difference between viability and germination percentage. Where the following six conditions are met, however, there is no significant difference between viability and germination percentage: the seed is neither dormant nor hard, or dormancy and hardness have been broken by suitable pre-treatment; the seed is not infected, or has been suitably cleaned; the seed was not exposed to unfavourable conditions during processing and was not treated with harmful chemicals during storage; germination has not yet begun; no deterioration occurred during the normal or the extended germination test; and the germination test was carried out under suitable conditions.

5 Principle

A colourless solution of 2,3,5-triphenyl tetrazolium chloride, or of the bromide, is used as the indicator. It takes part in the reduction reaction of the living cells within the seed tissue, accepting hydrogen ions from dehydrogenase; by this hydrogenation the colourless tetrazolium produces, in the living cells, a red, stable formazan that does not diffuse readily and is insoluble in water. According to the position and the colour of the tetrazolium staining, seeds are separated into completely stained viable seed, completely unstained non-viable seed, and seed with necrotic tissue or only partly stained.

6 Apparatus and reagents

6.1.1 Electrically heated thermostatic cabinet or germination cabinet, with a control range of 5 degrees Celsius to 40 degrees Celsius and able to hold 35 degrees Celsius plus or minus 2 degrees Celsius.

6.1.2 Refrigerator, with a control range of 5 degrees Celsius to 10 degrees Celsius.

6.1.3 Balance, readable to 0.001 g.

6.2.1 Tetrazolium solution: the concentration should be 0.1 percent to 1.0 percent by mass and the pH shall be 6.5 to 7.5. It may be made up directly with distilled or deionised water, provided the pH stays within 6.5 to 7.5; where it does not, phosphate buffer should be used in place of the distilled or deionised water. The prepared solution shall be stored cold in darkness or in a brown bottle, and under dark conditions at 5 degrees Celsius to 10 degrees Celsius it will normally keep for a year.

6.2.2 Phosphate buffer, pH 6.5 to 7.5, prepared as follows. Solution one: weigh 9.078 g of potassium dihydrogen phosphate and dissolve it in 1 000 mL of distilled or deionised water. Solution two: weigh 9.472 g of disodium hydrogen phosphate, or 11.876 g of the dihydrate, and dissolve it in 1 000 mL of distilled or deionised water. Mix two parts of solution one with three parts of solution two to obtain the phosphate buffer.

7 Test procedure

7.1 The working sample shall be taken at random from thoroughly mixed pure seed complying with GB/T 3543.3, 400 seeds in all, divided into four replicates of 100 seeds each. Where only the part of the seed suspected of being dormant at the end of a germination test is to be tested, all the fresh ungerminated seed and hard seed from the end of the germination test may be used.

7.2.1 Before staining, the seed should be pre-moistened. Seed whose coat hinders the uptake of moisture, and seed that needs to be stained more evenly, shall be treated accordingly beforehand. External appendages may first be removed, as the hulls are removed from rice; where the coat hinders imbibition, it is pierced at a point that is not vital, as in cotton and hard legume seed.

7.2.1 Pre-moistening may be done in either of two ways. Seed that splits or is damaged when soaked directly in water, old seed and dry seed should be moistened slowly, following the germination test procedure of GB/T 3543.4 and placing the seed on top of paper or between paper. Seed that cannot be fully imbibed by slow moistening and that will not have its tissue split or damaged by direct immersion should be soaked in water until fully imbibed, the water being changed in good time where soaking runs beyond 24 h. The method and duration of pre-moistening for each kind of seed shall follow Table A.1. Some seeds may give off a viscous substance during pre-moistening, which shall be removed by surface drying, by wiping between cloth or paper, or by soaking for 5 min in a 1.0 percent to 2.0 percent solution of aluminium potassium sulfate dodecahydrate.

7.2.2.1 The seed of most crop species needs pre-treatment; where the coat hinders staining, piercing, longitudinal cutting, transverse cutting, transverse sectioning, excision of the embryo or removal of the coat may be used to expose the tissue. The pre-treatment for each kind of seed follows Table A.1. Pre-treated seed shall be kept moist until every replicate has been dealt with.

7.2.2.2 Piercing may be done with a dissecting needle or scalpel; in pre-moistened seed and in hard seed the piercing shall be done at a part of the tissue that is not vital.

7.2.2.3 Longitudinal cutting may follow one of three patterns. In cereal seed, cut lengthwise from the base of the seed along the midline of the embryonic axis, the cut running to about three quarters of the length of the endosperm. In seed of dicotyledons without endosperm and with an erect embryo, cut lengthwise through about half of the cotyledons, slightly to one side of the central axis, without injuring the embryonic axis. In seed whose embryo is surrounded by living tissue, cut lengthwise close to the embryo.

7.2.2.4 Transverse cutting may follow one of three patterns. In grass seed, cut across at a part of the tissue that is not vital, immediately above the embryo, and immerse the part containing the embryo in the tetrazolium solution. In dicotyledon seed with a straight embryo and no endosperm, cut off one third to two fifths of the distal end of the cotyledons. In small seed, a transverse section that is cut into but not cut through may be used instead of a full transverse cut.

7.2.2.5 In seed of Hordeum, Secale and Triticum the embryo may be separated with a dissecting needle: pierce the endosperm slightly off centre above the scutellum and twist gently so that the endosperm splits lengthwise, then lift out the embryo with the scutellum attached.

7.2.2.6 In seed unsuited to piercing or cutting, the whole coat, and certain other covering tissues, may be removed. Seed with a hard outer covering, such as nuts and drupes, may be split or cracked open dry or after pre-moistening, care being taken not to injure the embryo. After pre-moistening the coat is carefully torn away with a dissecting needle or scalpel.

7.3 The prepared seeds or embryos shall be fully immersed in the tetrazolium solution and stained in darkness or dim light in the thermostatic cabinet, the germination cabinet or the refrigerator. Small seed that is hard to handle may first be pre-moistened and exposed on a paper strip, which is then folded or rolled and immersed in the solution. The concentration, temperature and time given in Table A.1 should be used for each type of seed. Staining time varies with the kind of seed, the way it was prepared, the strength of its viability, the concentration of the solution and the temperature; in general, the higher the concentration and the temperature, the shorter the staining time. The temperature may be varied around the value given in Table A.1 but shall stay between 20 degrees Celsius and 40 degrees Celsius. Where the optimum of 30 degrees Celsius cannot be used, the duration should be adjusted accordingly: an increase or decrease of 5 degrees Celsius corresponds to halving or doubling the staining time. Seed that is still incompletely stained within the prescribed time may be left longer, but it shall not be overstained, since overstaining can mask the different staining patterns produced by particular injuries such as frost damage, or by inherent weakness, and so affect the evaluation. Foam with a black deposit may appear during staining, and a trace of bactericide or antibiotic may be added to the solution to remove the interference. When staining is over the solution is poured off and the seed is rinsed with clean water and examined before evaluation.

7.4.1 Evaluation shall follow immediately after staining. Before evaluation the stained seed may be prepared so that the main structures of the embryo and the other living nutritive tissues are exposed and can be examined and counted; the preparation for each kind of seed follows Table A.1.

7.4.2 Viable and non-viable seeds are separated by observation. Depending on the species, the judgement rests chiefly on the staining of those functional tissues that can develop into a normal seedling and on how complete those tissues are. The whole structure of the seed has to be considered. Most seeds have main structures and nutritive tissue, the main structures being the meristem and the structures necessary for development into a normal seedling. In a well developed and well differentiated seed or embryo, small areas of necrosis on the surface of the main structures may still be judged viable where the capacity to repair remains; the permitted extent of such small necrotic areas shall meet Table A.1.

7.4.2 A seed or embryo that is completely stained, or that is partly stained but whose maximum non-viable area does not exceed what Table A.1 allows, is judged viable. A seed that does not meet the requirements for a viable seed, and that shows no uncharacteristic colouring or weakness of the main structures, is judged non-viable. Furthermore, a seed whose embryo or main structures are clearly abnormal shall be judged non-viable whether it is stained or not. Each seed is examined and assessed according to the staining pattern shown and the firmness of the tissue; where necessary a magnifying glass may be used in a good light.

8 Calculation and expression of results

The number of viable seeds in each replicate is counted separately and the average viability percentage calculated and rounded to the nearest whole number. The maximum tolerated difference between replicates shall not exceed Table B.1.

To compare whether the results of two independent samples from the same laboratory are compatible, the tolerances of Table B.2 are used; to compare two independent samples from different laboratories, those of Table B.3. In either case the arithmetic mean of the two determinations is calculated first, and where the difference between the two results does not exceed the maximum tolerance corresponding to that mean, the two results are taken as reliable.

9 Report of results

The report shall comply with GB/T 3543.1 and shall state the result as follows. For a routine seed viability determination, as 'tetrazolium test: percentage of viable seed', any departure from the technical requirements of Table A.1 being noted. For the viability of seed left ungerminated at the end of a germination test, as 'tetrazolium test: so many viable seeds out of so many seeds'. For legume seed, where the viability of hard seed has not been determined, as 'tetrazolium test: percentage of viable seed, percentage of hard seed'; where it has been determined, as 'tetrazolium test: percentage of viable seed, including the percentage of hard seed'. For coated seed, the type of coating and the result shall be stated, as 'tetrazolium test of coated seed: percentage of viable seed'.

At the request of the sender, the percentage of empty seed, of seed containing larvae, and of broken or rotten seed may be reported as appropriate.

A Annex A (normative) Technical requirements for the tetrazolium test of seed viability

Table A.1 runs to nine pages and covers the grasses, the cereals, the legumes, the cucurbits, the brassicas and a range of vegetable and oil crops, each entered under its Chinese name and its Latin name. For each genus or species the table fixes the method and duration of pre-moistening in hours, the pre-treatment before staining, the concentration of the tetrazolium solution as a mass fraction, the optimum staining time in hours, the preparation before evaluation, the maximum area that may remain non-viable, and any additional note. The OCR has run the columns of this table together and the individual figures cannot be attached to their species with certainty, so they are not reproduced here. A note to the table states that where no temperature is given for the pre-moistening method, the temperature is 20 degrees Celsius.

A.2 Figure A.1 shows the different ways of preparing the seed before staining, with cereal seed cut lengthwise and oat seed cut transversely, cut lengthwise and pierced, and lettuce and other composite seed cut along the cotyledons, cut along the embryo and cut transversely at both ends. The key to the figure identifies the cut through the embryo and about three quarters of the endosperm, the transverse cut immediately above the embryo, the longitudinal cut through the distal part of the endosperm, the piercing of the endosperm at a mid point near the embryo, the longitudinal cut through the distal half of the cotyledons, the position of the scalpel for that cut, the longitudinal cut alongside the embryo, and the transverse cuts at both ends that remove a small part of the endosperm and open the embryo cavity.

A.3 Figure A.2 shows the evaluation of viable cereal seed: column one holds seed that is completely stained and viable, and columns two to five show the maximum permitted area of unstained, slack or necrotic tissue in viable seed; row A is for wheat, triticale, rye, barley and oat seed cut lengthwise, row B for oat seed cut transversely, row C for barley with the embryo separated, row D for rye with the embryo separated, and row E for wheat and triticale with the embryo separated. Figure A.3 shows non-viable cereal seed in columns one to five, row A being seed cut lengthwise and row B seed with the embryo separated, the second item of row B being a wheat scutellum seen from the back.

B Annex B (normative) Tolerances applicable to the tetrazolium viability test

The maximum tolerated difference between the four replicates of one tetrazolium test, the maximum tolerated difference between results on the same or different submitted samples of 400 seeds each within one laboratory, and the maximum tolerated difference between results on different submitted samples of 400 seeds each from different laboratories shall comply with Tables B.1, B.2 and B.3 respectively.

In each of the three tables the arithmetic mean of the determinations is calculated first and rounded to a whole number, the corresponding maximum tolerance is read from the table against that mean, and it is then compared with the actual difference, which for Table B.1 is the difference between the highest and the lowest percentage among the replicates. Table B.1 and Table B.2 are two-tailed tests at the 2.5 percent level of significance; Table B.3 is a one-tailed test at the 5 percent level.