Seed Biology

Department of Horticulture and Crop Science
The Ohio State University


Seed Vigor and Vigor Tests


Accelerated Aging (AA) Test

This test incorporates many of the important traits desired in a vigor test. Initially proposed as a method to evaluate seed storability, the accelerated aging test subjects unimbibed seeds to conditions of high temperature (41 C) and relative humidity (around 100%) for short periods (3 to 4 days). The seeds are then removed from the stress conditions and placed under optimum germination conditions.

Principles

The AA test exposes seeds for short periods to the two environmental variables that cause rapid seed deterioration; high temperature and high humidity. High vigor seed lots will withstand these extreme stress conditions and deteriorate at a slower rate than low vigor seed lots. Although aging temperature and seed moisture have the greatest influence on test results, several other factors which modify the results must be controlled during the test.

When conducting the AA test, seeds are weighed and placed on a screen tray, which is inserted into an inner chamber (plastic box) containing 40-50 ml of water. The inner chamber is placed into an AA (outer) chamber and the seeds aged at a specified high temperature for specified time, dependent upon the species. During the aging period, the seeds absorb moisture from the humid environment within the inner chamber and are stressed by high temperatures as seed moisture increases to a uniform level. It is important that seeds on the screen tray be kept in approximately a single layer at a uniform distance above the water surface, as the layering of seed will influence water uptake and result in variable seed moisture at the end of the test. Seed size will also influence the final seed moisture and germination following aging. Larger soybean seeds had a lower final seed moisture and lower germination than smaller seeds when the same number were placed in the aging chamber. The final seed moisture can be controlled, however, by basing the sample size on a constant seed weight rather than seed number. Recommended and suggested testing variables have now been identified for a wide range of species (Table 11.2).

A uniform temperature in the outer chamber must be maintained and the temperature controls should permit a variation of no more than +/- 0.3 C, since minor changes (0.5 C) in the aging temperature will affect germination results. High relative humidity in the outer chamber is also necessary to prevent water evaporation from the inner chambers. A water-jacketed aging chamber is recommended because it provides precise temperature control, no condensation and a uniform aging environment. The door of the outer chamber must remain closed for the entire duration of the test to maintain uniform and constant temperatures. Opening the door for one minute lowered the temperature of the inner chamber 2-3 C and it took 30 minutes to recover to the desired temperature. The most uniform temperatures of the inner chambers are maintained by allowing some air space (2.5 cm) between inner chambers placed on the same shelf in the outer chamber.

Equipment and Supplies

Balance: Analytical balance capable of weighing to 1.0 mg.

Inner aging chamber: A plastic box (11.0 x 11.0 x 3.5 cm) with a lid into which is placed a plastic or wire tray with a 10.0 x 10.0 x 0.3 cm wire mesh screen (mesh 14 x 18). These trays can be purchased commercially.

Seed moisture tins: Temperatures ranging from 103-130 C as described for each species in the International Rules for Seed Testing (ISTA, 1999).

Bottle-top Dispensette (Brinkman): Range from 0-100 ml, for dispensing 40 ml water from a standard screw-neck bottle or a 50 ml graduated cylinder.

Water: Deionized or distilled

Outer aging chamber: Water-jacketed incubator capable of maintaining a constant temperature range from 40-45 C +/- 0.3 C (alternative equipment described below).

Alternative outer aging chambers: This may include incubators that have a heating element immersed in water at the base of the chamber. These chambers frequently do not have precise temperature control and require the use of auxiliary control to maintain uniform temperature (YSI model 71, Yellow Springs Instrument Inc., Yellow Springs, OH, USA). When using these chambers, water may collect inside the top of the outer chamber due to condensation and drip on the lids of the inner chamber (boxes). If water accumulates on the lid of the inner chambers, condensation can occur inside the inner chamber (under the lid) and water will drop onto the seed that will raise seed moisture levels during the aging period. This can reduce germination and cause excess mold growth. Precautions must be taken to shield the inner chambers to prevent water droplets from accumulating on the lids during the aging period. Do not use dry incubators or ovens as outer chambers.

Germination test facilities: See Rules for Seed Testing (AOSA, 1999) or International Rules for Seed Testing (ISTA, 1999). (The brand name, model numbers and suppliers for outer and inner aging chambers and other equipment needed for the AA test can be acquired from the chairperson of the AOSA vigor test committee.)

Procedure

1. The inner chamber AA boxes and screen trays should be thoroughly washed in a 15% sodium hypochlorite (clorox) solution and dried after each use to prevent fungal contamination.

2. Place 40 ml (+/- 1 ml) of distilled or deionized water in each inner aging chamber and insert a dry screen tray, being certain not to splash water onto the screen surface.

3. Determine the inital seed moisture of the sample to be tested using the method appropriate for the species (ISTA, 1999) and if >14.0% (fresh weight basis), dry the seed to 10 - 14% before testing.

4. A minimum of 200 seeds, determined on a weight basis (Table 11.2), are placed on the surface of the screen tray (approximately one layer deep). More than one inner chamber should be used to obtain the quantity of seeds needed for larger seeded species (Table 11.2). Preferably, seeds to be aged should be untreated. However, if seeds of the crop species are primarily marketed with fungicide treatment, treated seeds may be used.

5. The lid is secured (not sealed) on each inner chamber. The inner chambers should be placed on a shelf, transported and placed in the outer aging chamber at the same time. Allow an air space of approximately 2.5 cm between inner chambers on each shelf in the outer chamber to assure temperature uniformity.

6. Precisely monitor the temperature of the outer aging chamber and maintain temperature at +/- 0.3 C of desired temperature (Table 11.2) during the aging period. Record the time when inner chambers are placed in the outer chamber.

7. The outer chambers should not be opened during the aging period specified in Table 11.2.

8. Seed in the inner chambers should be removed from the outer chamber at the exact hour (+/- 15 min) specified (Table 11.2) and planted for standard germination within one hour after removal.

9. The conditions for the standard germination test are those outlined in the AOSA Rules for Testing Seeds (AOSA, 1999) or ISTA International Rules for Seed Testing (ISTA, 1999).

10. Include a control sample with each AA test. At the conclusion of the aging period, but prior to planting for standard germination, remove a small sample of seed (10-20 seeds) from the inner chamber of the control sample and weigh immediately to test for seed moisture (fresh weight basis) using the oven method (ISTA, 1999). If seed moistures are lower or higher than those shown in Table 11.2, the test results may not be accurate and the sample should be re-tested.

11. When AA tests are initiated for many seed lots on the same day, samples should be grouped at approximately one hour intervals between two outer chambers to allow adequate time for samples to be planted immediately after the aging period.

Interpretation of AA Results

The AA test does not provide an absolute vigor or field emergence score, but simply records germination (percentage normal seedlings) after a period of stress under conditions of high temperature and seed moisture. When the AA results are compared to the standard germination results of the same seed lot prior to aging, the AA germination will be either similar to standard germination (high vigor seed) or less than standard germination (medium to low vigor seed). One use of these results can be to rank seed lots by vigor and decisions can be made regarding the storability or planting of each seed lot.

Relationship with field emergence

Early storage studies suggested that AA might have utility as a vigor test to predict field performance. Additional studies have shown that this vigor test functions well in forecasting field emergence and stand establishment in a wide range of crop species. In general, when seeds are planted under stressful field conditions, AA germination provides higher correlations with field emergence than does standard germination.

Predicting storage potential

The primary emphasis of early investigations of the AA test was predicting the relative storability of seed of several crop species. It was shown that the changes in germination of high and low quality seed lots after a period of time were consistent with those that occurred in warehouse storage. Subsequent studies have verified that accuracy of this test in predicting the life span of a number of different species under a range of storage conditions.

Limitation of AA

Accelerated aging shows great potential as a vigor test for a wide range of species. Although variability in referee results was shown initially, excellent progress has been made in the refinement of techniques and the evaluation of time, temperature and seed moisture interactions. AOSA and ISTA referee tests with soybean seed report greater uniformity among laboratories and confirm the relationship between AA germination and field emergence. Sufficient evidence has now accumulated to show that repeatable results can be achieved using this test. Thus, the AOSA and ISTA now consider this test standardized and have recommended it as a vigor test for soybean. It is essential, however, that laboratories rigidly follow the procedures and equipment outlined above and the criteria listed in Table 16. Even slight modifications in temperature control, sample size or aging time will cause variation in final seed moisture and/or germination which will limit the acceptance of the vigor test.

The accelerated aging test is rapid, inexpensive, simple and useful for all species; it can be used for individual seed evaluation and requires no additional training for correct evaluation.

Standardization of Vigor Tests

For any seed quality test to be useful, it must provide reproducible tests. To evaluate reproducibility of various vigor test results, both the AOSA and ISTA have conducted extensive referees. Generally, results of these referees have shown that seed testing laboratories can reproduce their own results on the same sample of seed within acceptable confidence limits. However, when the same tests are conducted by different laboratories, the amount of variability can be unacceptable.

Many possibilities exist to explain the lack of standardization among laboratories. For example, most vigor tests require a degree of subjectivity. The seedling vigor classification and tetrazolium vigor tests possibly involve the most subjective interpretations because seeds and seedlings or both are separated into categories based on characteristics that are difficult to describe precisely. Even the cold test and accelerated aging test require the classification of seedlings as normal or abnormal.

Variations in temperature, moisture, and other environmental conditions are more critical for tests in which rate of growth or rate of a biochemical process is measured than for those such as the standard germination test which measures the completion of a process. For example, a 1 C difference in temperature during the course of the standard laboratory germination test probably has little effect on the final percent germination, but a 1 C temperature difference may have a considerable effect on results in the seedling growth rate test or seed deterioration in the accelerated aging test, as would minor variations in the moisture content of germination substrata or the relative humidity of the air. Consequently, conditions and equipment that are suitable for the standard laboratory germination test may not be suitable for vigor tests.

The cold test would appear to be difficult to standardize because it exposes seeds to soil microorganisms to measure their ability to resist attack under cold, wet conditions. Thus, the standardization of the cold test procedure in its present state still requires standardization of the substrate (soil) microflora, a task that would be difficult, if not impossible. The use of sterile media inoculated with specific microorganisms has been suggested, an approach that is probably too simplistic, since it is difficult to culture microorganisms and maintain a constant level of pathogenicity. Also, pathogens behave differently in soil, where there is a complex population of microorganisms, from how they behave in the absence of such an interacting population.

Despite the problems, a recent study by Byrum and Copeland (1995) indicated that the cold test for corn in the U.S. corn belt is as repeatable as the standard germination test. This study was conducted on four corn seed lots with varying levels of quality and each sample was tested by 10 different laboratories throughout the Midwest and Midsouth representing official, commercial, and crop improvement associations. This study illustrates the present state of the art of cold testing in the United States and demonstrates the potential for the eventual standardization of all vigor tests.

It may be important to determine whether such factors as dormancy affect vigor test results. Many species (e.g., legumes) have seeds that are impermeable or only slowly permeable to water. This can affect the percentage of seeds that germinate as well as the length of seedling growth in stress tests and can cause a biased index of vigor in a seedling growth rate test. It can also affect the leaching of electrolytes from seeds in a conductivity test.

Dormancy can also confound the interpretation of vigor test results. For example, dormancy can cause lettuce and cereal seeds to germinate poorly in tests conducted at high temperatures. If dormancy is suspected of affecting vigor results, the seed should be checked by a standard warm germination test or a tetrazolium test.

CONTROL SAMPLES IN VIGOR TESTING

An essential component of any laboratory testing program is the use of control or check samples. The primary purpose of control samples is to provide internal quality control to enable seed technologists to detect variations in test procedures. Seed vigor tests require precise time, temperature, and moisture that must be monitored and controlled during testing. Even small variations in these components can produce dramatic fluctuations in test results. The selection, maintenance, and use of control samples for each species tested is essential for laboratories engaged in vigor testing.

Control seed lot selection

Seed lots of each crop species tested should be selected annually by screening several samples for both germination and vigor. The sample selected should be relatively free of physical injury and disease, high in germination and moderate in vigor. Seed lots with extremely high vigor will not allow detection of minor differences in methodology and, therefore, would not make good control samples. For example, an acceptable control sample for corn or soybean may have a germination exceeding 95 or 90%, respectively, and vigor level of 70 to 80% and 80 to 90% for the accelerated aging test for soybeans and the cold test for corn, respectively.

Seed Storage and Maintenance

Store enough seed of the control sample to provide an adequate supply for an entire year or testing season. Store the control samples in conditions that will preserve both germination and vigor. Prepackaging the control sample into sub-sumples needed each day may be advantageous, removing from storage the number of samples needed for a week of testing. Two alternatives should be considered for storage and use of control (in order of preference):

Seed moisture of control samples

The initial seed moisture of the control seed lot must be an acceptable seed moisture for the seed being tested. The moisture of the seed lot must be measured periodically to be certain that the seed has not decreased or increased in moisture during storage. Adjust the moisture to the initial level if necesary, as extremely dry seed can cause imbibitional injury and inaccurate vigor readings during testing.

Use of control samples

Control samples should be included as routine samples in every vigor test run and should not be identified as control or check seed until the test is completed. At the conclusion of the test run, the seed technologist or laboratory manager should determine if the vigor test results of the check are within the set parameters. If the sample falls outside the parameters, the problem should be identified and corrected and the test repeated. At frequent intervals, the vigor test results of the control samples should be summarized to evaluate uniformity of the samples and testing procedures.

Clearly, vigor testing for all species and regions has not yet achieved the same level of standardization possessed by the standard germination test. However, both seed testing organizations (AOSA and ISTA) and the seed trade are focusing on improving the reproducibility of vigor tests. Considering the important role that vigor tests have in plant breeding, seed production, quality control, and marketing programs, standardization is badly needed. Although future research and testing are required before vigor testing becomes a routine phase of seed testing, the promise of vigor testing in the future is bright.

USE OF SEED VIGOR TESTS

Many farmers know from experience that seed lots with equal germination levels may emerge from the soil quite differently, resulting in erratic field stands, replanting (on some occasions), or both. These same farmers recognize that this problem is usually more severe when adverse environmental conditions occur at or immediately after planting. Thus, if farmers know that they will be planting seed in adverse soil conditions or can predict an adverse field environment following planting, high vigor seeds should provide higher field emergence than low vigor seeds. If this prevents replanting, and the associated delayed maturity or yield reductions due to poor stands, additional money spent on high vigor seeds may be a worthwhile investment. It cannot be assumed, however, that high vigor seeds will produce excellent emergence and stands in any soil environment. It should improve the chances for satisfactory emergence, but it will not guarantee it. Conditions prevailing after planting may be so severe that even the most vigorous seeds cannot perform satisfactorily.

INTERPRETATION OF SEED VIGOR TEST RESULTS

It is extremely important to recognize that the results of a seed vigor test do not predict percentage field emergence. This is because environmental conditions differ from field to field, day to day and year to year, and a particular seed lot will reach a different emergence percentage under each set of environmental conditions. Seed vigor tests supply only relative values. This means that tests must be conducted on a number of seed lots simultaneously with the inclusion of a control seed lot with known seed vigor to serve as a reference point. This allows the arrangement of seed lots in ranking order, from high to low vigor. Because plant stand will vary from field to field, and because vigor tests, in some cases, vary among laboratories, it is very difficult to establish cut-off points between acceptable and unacceptable levels of seed vigor across many crop species.

Many seed companies in the central United States conduct a series of vigor tests on each soybean seed lot and combine this information into a seed vigor index. They establish an acceptable vigor level for a specific production season and seed lots are evaluated (in-house) prior to conditioning, treatment, and marketing. In a few cases, those seed lots that exceed a specified vigor level after conditioning are advertised and promoted in general terms as vigor-proven, vigor-rated, or high vigor seed. If the seed is sold with strong emphasis on vigor, a buyer should ask the dealer for specific information on how many vigor tests were conducted and the criteria used to assess vigor potential.

Does high-vigor seed mean higher yields? This question is asked by many farmers following implications concerning yield by some seed producers. Unfortunately, there have been fewer comparisons of seed vigor to final yield than to field emergence. As with other investigations relating to seed vigor, the yield comparisons are variable depending on the crop planted, the stand achieved, and the original vigor level of the seed. Many factors that may not relate to the vigor of the seed can influence yield during the growing season. Also, certain crops and genotypes have the ability to compensate for minor stand differences following emergence, and this may result in little difference in final yield. As would be expected, if adequate field stands resulted from high vigor seeds, higher yields should occur. However, less evidence supports improved yields using high vigor seeds than supports equal stands achieved from planting medium or low vigor seeds. To date, insufficient evidence is available to show a strong relationship between yield and vigor in the absence of stand differences. Future research on plant growth and development or the physiological or biochemical basis for seed vigor may improve our understanding of this relationship.


Table 1. Recommended and suggested variables for accelerated aging test using an inner chamber (plastic box) with 40 ml water.

Inner ChamberOuter Chamber
Genus/Species Crop Seed
weight
(g)
Number
of
chambers
Aging
temp.
°C
Aging
duration
(h)
Seed moisture
(fwb)
following aging2
(%)
Recommended
Glycine max (L.) MerrillSoybean421417227-30
Suggested
Medicago sativa L.Alfalfa
(lucerne)
3.51417240-44
Phaseolus vulgaris L.Bean, dry421417228-30
Bean, French502454826-30
Bean, garden302417231-32
Brassica napus L.Canola
(oil seed rape)
11417239-44
Zea mays L.Field corn402457226-29
Sweet corn241417231-35
Lactuca sativa L.Lettuce0.51417238-41
Phaseolus vulgaris L.Mungbean401459627-32
Allium cepa L.Onion11417240-45
Capsicum spp.Pepper21417240-45
Trifolium pratense L.Red clover11417239-44
Lolium perenne L.Ryegrass11414836-38
Sorghum bicolor
Moench.
Sorghum151437228-30
Helianthus annuus L.Sunflower201457223-27
Festuca arundiancea L.Tall Fescue11417247-53
Nicotiana tabacum L.Tobacco0.21437240-50
Lycopersicon
lycopersicon
Tomato11417244-46
Triticum aestivum L.Wheat201417228-30
1 For samples with larger seed size, the weight per box and/or the number of boxes may need to be increased.
2 If seed moistures are above or below this range, the test should be repeated.


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