The overall goal of this procedure is to directly observe and quantify seed germination, mortality, and dormancy in field conditions over time. This method can help answer key questions in the seed ecology field, such as how different species or ecotypes of species contribute individual seedlings to the population of the next generation. Or, individual seeds to the persistent soil seed bank.
The main advantage of this technique is that any number of experimental designs, species, treatments or removal dates can easily be deployed in the field and retrieved as a complete replicate after weeks to months of burial. To begin, cut a 20 x 105 cm strip of fabric for each seed strip. Using a tape measure, mark out compartments onto the strip.
Fold the fabric in half, and use a high temperature glue gun to apply glue at one short end. Allow the glue to cool slightly and then press the two sides together to create a seal. Place lines of glue along each compartment mark from the folded edge to the open short end.
Press the two fabric sides together along the glue lines to make seals without holes between compartments. Randomly assign seed cross types to compartments for each strip. Next, count and place a uniform number of seeds into each compartment.
Using a plastic garden dart, label the compartment with a seed type. Use the glue gun to seal the compartment securely. Proceed along the strip, filling, labeling, and sealing the seed pockets one by one until the entire strip is filled and sealed.
Check for gaps between compartments and spot glue as needed before proceeding. To conduct the burial trials, first dig plots to the specified depth to house strips based on the experimental design. Place the seed strips in the ground and cover them with earth.
Next, cut metal mesh hardware cloth into a panel 10 cm larger on each side than the buried seed plots. Seal the edges of the mesh cloth with soil. This covering should exclude seed burrowing predators.
Finally, place colored plastic plot labels at the site location, labeled with removal date and color coded to complement the written records. At the assigned removal date, dig up the subject seeds. Wrap the strips in wet newspaper to keep them moist, and place them into a cooler for transport.
Once in the laboratory, rinse the strips in water to remove extraneous dirt and debris, until seeds are visible in the compartments. Using scissors, cut open one compartment at a time. Count all germinated seeds present and remove them.
Count and remove any clearly dead seeds. Finally, collect all of the remaining ungerminated seeds and place them onto a labeled wet piece of blotting paper in a Petri dish. Place the dishes into a growth chamber for one week to allow for germination.
Examine the seeds and identify and count any that have germinated. Record these as viable but ungerminated, as these seeds did not germinate under field conditions. Cut the remaining seeds in half to test for viability using tetrazolium chloride.
Place one half in a Petri dish with the embryo and endosperm visible. Add enough tetrazolium chloride to cover the seed halves. Place the dish into an incubator set at 30 degrees Celsius for three hours.
Finally, evaluate the seed halves. Seeds that display red staining of the embryo are germinable, while unstained seeds are dead. This graph shows the germination of sunflower crop wild cross types at the first removal date after burial in late fall.
Cross types are arranged by increasing crop allele percentages, and the maternal parent is indicated for each cross. In general, lower crop allele percentages reduced maladaptive germination in late fall. At early spring removal, all genotypes had high germination, but differed significantly in the numbers of ungerminated or dead seeds.
In particular, cross types with a crop maternal parentage had higher mortality, while maternal wild cross types were more likely to remain ungerminated. By spring, germination declined for some cross types. This is likely due to seed mortality within the burial strips for earlier germinating cross types.
Accordingly, mortality was higher for more crop-like cross types produced by crop maternal parents. Once mastered, this technique can be assembled in a few hours, depending on the design. While the evaluation of strips typically takes only a few minutes per compartment, plus time in the growth chamber and incubation in TZ.While attempting this procedure, it's important to remember that only seeds that have been through the growth chamber and were subsequently determined by TZ testing to be viable may be considered truly dormant.
Seeds that germinate in the growth chamber are considered ungerminated. Following this procedure, other methods run in parallel, like seedling studies can be performed in order to answer additional questions, like whether or not seed survival translates to seed emergence and life cycle completion. After watching this video, you should have a good understanding of how to conduct a field evaluation of seed germination, mortality, and dormancy.
Don't forget that tetrazolium chloride is an irritant, and precautions, such as wearing gloves, should always be taken while performing this procedure.
