Germination / Tetrazolium (TZ)
The Colorado Seed Laboratory is an official AOSA laboratory, but we also perform tests according to ISTA and Canadian standards. The primary reason for having rules regarding seed testing is consistency. It should be possible to replicate the results of a viability test in Colorado in Maine, and vice-versa. For germination testing the primary variables are temperature, substrate, and moisture. The length of the test and dormancy breaking procedures also vary according to the species.
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Samples are immediately divided into a sub-sample for testing. Different species require different testing weights, but AOSA requires that for germination the sub-sample must be 1/4 of the prescribed purity weight (a full purity consists of 2500 seeds). The purity analyst must separate inert material, other crop, and weeds from the pure seed. Only the pure seed portion is used in the germination test. |
400 seeds are always planted in a standard germination test. Depending on seed size, four to sixteen replicates may be used. Seed is either counted by hand or the lab's vacuum planter is used. The vacuum planter has different sized heads depending on the type of substrate and seed size. The seed is poured over the planter head, and then placed onto the substrate. |
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Many different types of substrate are used for germination tests. Blotters, towels, and filter paper are the most common, but creped cellulose paper and sand are also widely used. Water is the commonly used as the moisture, but in dormant species potassium nitrate (KNO3) is also used. A safflower sample in towels is pictured here. |
Seed is then placed into a growth chamber. Chambers are set at various temperatures, and some even alternate temperature during the day to simulate natural conditions. Crops that were developed closer the equator usually have a single temperature, such as wheat (20 degrees C). Native species require alternating temperatures, such as blue grama (20C at night, 30C during the day). |
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As soon as the seeds develop discernable structures we can evaluate them. Just because a seed sprouts does not mean it is counted as viable. All the structures must be present and in 'working order' for the seed to be considered normal. When evaluating, the analyst separates the seed into four categories: normal, abnormal, dead, and dormant (a TZ test is used to determine dormancy). |
A TZ test is a quick way to determine total viability. The test usually takes 24 hours to complete, and is the primary method for determining dormancy. The steps are relatively simple, but it takes a good analyst to evaluate a TZ test correctly.
The seeds are imbibed in water overnight, and then bisected (or pierced) with a razor blade the next day. The seed is placed into TZ to stain.
As the seed respirates it soaks up the solution. The parts of the embryo that do not respirate are dead, and they will not soak up the solution. A chemical reaction takes place, and live structures of the embryo stain red. Once the seed has stained (this can take 2-48 hours, depending on the species) the analyst 'reads' the TZ.
