Seed Vigor and Vigor Tests
Cold Test
The cold test is one of the oldest methods of stressing seeds and is most often employed for evaluating seed vigor in corn and soybeans. Seeds are placed in soil or paper towels lined with soil and exposed to cold for a specified period, during which stress from imbibition, temperature, and microorganisms occurs. Following the cold treatment, the seeds are placed under favorable growth conditions and allowed to germinate.
Principles
The cold test exposes seeds to 10 C for 7 days in non-sterile field soil at approximately 60-70% of water holding capacity prior to a 4-7 day grow out period in ideal conditions (25 C). The moisture and temperature conditions provided in the cold test simulate the adverse conditions that seeds might encounter in an early spring planting. The ability of seeds to germinate and emerge in cold wet soil is affected by genotype, seed quality (both physical and physiological), seed or soil-borne pathogens and seed treatment. The cold test attempts to measure the combined effects of all of these factors. It often represents the lowest emergence that would be expected from a seed lot when planted under reasonably satisfactory field conditions, while the germination test represents the highest emergence potential that could be expected. When germination obtained in the cold test is very close to that obtained in the standard germination test, the seed lot would be expected to emerge well over a wide range of soil moisture and temperature conditions. There are two common approaches to conducting a cold test: rolled towel method and tray method.
Rolled Towel Method
Equipment and Supplies
Germination paper towels: Paper towels of the same weight and thickness as used for standard germination, 30 x 60 cm in size.
Plastic bucket or container: To hold rolled towels in an upright position for placement in germination chamber.
Plastic bags (2 ml thickness): To cover upright towels after placing in container.
Two germinators: Two chambers capable of maintaining 10 C and 25 C (+/- 0.5 C).
Heat exchange plate: To maintain temperature to towels at 10 C.
Tray Method
Equipment and Supplies
Food service tray: Fiberglass, plastic or metal tray (45 x 66 x 2.75 cm).
Crepe-cellulose paper: Sheets of creped cellulose (40 x 61 x 0.5 cm) to fit in base or tray which is available from many paper supply houses.
Graduated cylinder (500ml) or automatic watering device: Used for adding specific amount of water to crepe-cellulose and tray.
Food cart: Closed food service cart with plexiglass back to hold trays during testing.
Two walk-in temperature controlled chambers: Two chambers which allow entry of food service cart and are capable of maintaining 10 C and 25 C (+/-0.5 C).
Alternative equipment: If laboratory does not have walk-in temperature chambers for food cart use, two germinators as described for rolled towels could be used and the trays moved manually from 10 C to 25 C.
Both Methods
Field soil: The soil component is a very important aspect of the test and the selection of an appropriate soil source is critical for the reproducibility of the test. Since the soil is expected to supply disease-causing organisms, it should be from a field site which has supported some vegetation, preferably the crop to be tested. Highly organic soils may exhibit lower germination due to excessive pathogenic activity which may cause erratic test results. If possible, adequate soil for the entire testing season should be obtained from the same source. It should be handled in a consistent manner throughout the testing season and stored under uniform conditions to maintain soil moisture. Prior to use, the soil should be screened through a 5 mm sieve to remove large debris. It may be mixed with sand, vermiculite, peat or applied directly. Since the role of the soil in these tests is to provide an innoculum source, the quantity of the soil needed for each test is relatively small (i.e., one part soil/one or two parts sand).
Seed samples: If cold tests are to be a useful guide in planting decisions, the seeds should be tested in the same condition as planted. They should not be allowed to dry out prior to testing, and they should have recieved their final fungicide treatment. Seeds at unusually low moisture levels (<8% moisture, fwb) may exhibit poor cold test performance due to imbibitional chilling injury; thus, seed lot moisture should be adjusted (if necessary) to a range of 8-13%.
Rolled Towel
Procedures
This widely used procedure was first described in the 1950s. The towel procedure has gained widespread acceptance for its reliability, minimal space requirements, low volume of soil/sand needed, and generally good correlation with other cold test procedures and with field emergence. The common features of most of the current versions of this test recommend that the towel substrate includes soil or a soil/sand or peat mixture as an inoculum source. Following a low temperature (10 C) stress period for 7 days, the rolled towels are transferred to an optimum temperature (25 C) for germination for 5 additional days.
Methods
1. At least 16 hours prior to planting, the germination paper towels should be soaked until each towel has absorbed approximately 35 ml of water. The saturated towels should be equilibrated at 10 C overnight to ensure that the entire towel mass is at 10 C.
2. A minimum of four subsamples of 50 seeds each drawn from the pure seed fraction of each seed lot are tested. The seeds of each subsample are placed on a double layer of cold saturated towels in two 25-seed rows, 6 and 12 cm from the upper edge of the top towel. The planted seeds are lightly covered with soil/sand substrate (one part soil/one part sand) ensuring that all seeds are in direct contact with the soil. A top towel is placed over the two lower towels containing seeds and soil and the three towels are loosely rolled. Care should be taken during and after planting to ensure that the towels do not warm above 10 C. This can be done by placing the chilled towels in an insulated cooler over ice or by using a heat exchange plate.
3. When ready for transfer to the cold (10 C) chamber, the rolled towels are placed upright in a plastic bucket or container. The containers have wire dividers to keep the towels separated and are covered with a plastic bag to prevent loss of moisture.
4. The covered containers are placed in a cold room at 10 C (+/- 0.5 C) in darkness for 7 days.
5. After the cold treatment, the containers are transferred to a germination chamber at 25 C for 5 days in darkness.
6. Seedlings are evaluated using the same criteria as for the standard germination test (ISTA, 1999). If desirable, seedlings may be classified as strong or weak after 7-10 days as described by Fiala (ISTA, 1987).
Tray
Common features of this test include a food service tray, a base of creped cellulose paper which acts as the water reservoir for the seed and a covering of a mixture of soil/sand. Test conditions are similar to the rolled towel test in that the first 7 days are at 10 C, but the growth period is shorter, 4 days at 25 C. Although the test requires considerably more space than the rolled towel procedure, it has several attractive features including: mechanisation and standardization of substrate watering, vacuum-head or trip-board planting, uniform distribution of seed onto the substrate allowing ease of interpretation, natural emergence of seedlings through a soil/sand substrate allowing faster evaluation and greater efficiency (less time per sample). The results of the Tray Method are correlated with field emergence and with other widely used cold test procedures. The primary limitations are the initial expense of the equipment and the relatively large amount of soil and floor space required to conduct the test, which may reduce the number of samples tested.
Procedures
1. Prior to planting, the water, trays and carts and sand/soil should be chilled to 10 C.
2. One sheet of crepe-cellulose is placed into a standard food service tray and soaked with 1,100 ml of chilled water. The addition of water is most conveniently done with an automatic watering system which ensures uniform water distribution across the tray. Water can also be distributed in the tray with a graduated cylinder or other measuring device if desired.
3. Each tray accommodates 4 x 100 seed subsamples. Thus, two seed lots of 2 x 100 seeds each are planted in each tray. The 100 seeds in each subsample may be planted with a vacuum head or a trip planting board directly onto the saturated crepe-cellulose in each one-quarter section of the tray. The seeds are pressed uniformly into the crepe-cellulose using the bottom of an unused tray so that seed contact is constant and uniform distribution is maintained during the addition of the soil/sand substrate.
4. An excess of the chilled soil/sand mixture (1 part soil/2 parts sand) is spread over the seeds and crepe-cellulose. A board, notched to provide a level surface 0.4 cm below the lip of the tray, is used to level the soil/sand mixture and remove any excess. After the soil/sand absorbs water from the wet crepe-cellulose, the resulting seedbed should approach 70% of the water holding capacity.
5. After planting, the test trays are placed at 7.5 cm intervals in a closed food service cart with the plexiglass back. The cart is moved into a 10 C (+/- 0.5 C) chamber in darkness for 7 days. The carts are then moved to a 25 C chamber with an alternating light source (8-12 h light per day) facing the plexiglass back side of the cart for 4 days.
6. Seedlings are evaluated following the criteria outlined by ISTA (1999) for the standard germination test.
Interpretation of Results
The cold test does not provide an absolute vigor score, but records germination after 7 days stress under cold temperatures and high soil moisture in the presence of soil microbial activity. Since germination can be influenced by both the soil source and the cold test method used, the interpretation of results should be facilitated by the use of a control seed sample of moderate cold test performance with each seed lot or group of seed lots being tested. The relationship between the germination of the control sample and that of the samples being tested is probably more important than the actual germination of the test samples. Within a laboratory and/or a seed company, the level of cold test germination can be related to field emergence and seed quality decisions can be made regarding potential seed lot performance. Until cold test methods are standardized, the results cannot be interpreted at a given vigor level among laboratories. The cold test is therefore limited primarily as an "in-house" vigor test within a laboratory and/or seed company.
Scope
The cold test has been used extensively for many years for testing corn and sweet corn seeds. The relationship between cold test results and field emergence has ranged from excellent to variable depending on the method used and the level of stress in the field. When seeds are planted under stressful field conditions, however, there have been many comparisons which show higher correlations between cold test germination and field emergence than standard germination and field emergence. The general principles of the cold test have also been applied successfully to soybean and sorghum seed and could probably be applied to other kinds of seed.
The greatest difficulty with the cold test is the lack of uniformity in field soil. Soils differ in moisture, pH, particle composition, and pathogen levels, all of which contribute to divergent results. Use of vermiculite, a more uniform medium, has been suggested as a possible solution to the variability of soil conditions. However, it is widely believed that a cold test requires field soil to be successful. Regardless of these difficulties, cold test vigor rankings of seed lots tend to remain consistent within laboratories, which support this test as a most useful in-house vigor test.