This work was supported by Biotech Risk Assessment Grant Program competitive grand no. 2006-39454-17438 to A. Snow, K. Mercer, and H. Alexander from the United States Department of Agriculture, National Institute of Food and Agriculture. Experiments using this method were conducted at and supported by the University of Kansas Field Station, a research unit of the Kansas Biological Survey and the University of Kansas. The authors would like to thank P. Jourdan and E. Regnier for helpful reviews on earlier versions of this manuscript. Additionally, this work was aided by the contributions of the staff at the University of Kansas Field Station, Waterman Farm at the Ohio State University (OSU), the USDA Ornamental Plant Germplasm Center at OSU, and the Seed Biology Lab in the Department of Horticulture and Crop Science at OSU, especially E. Renze, S. Stieve, A. Evans, and E. Grassbaugh, for technical support.

1. Gather Seed from Multiple Species or Controlled Crosses of a Single Species
Note: This example used seed from 15 cross types within the species Helianthus annuus (sunflower) using wild, hybrid, and crop types as the maternal (seed producing) parent.
<ol>
	<li>At the end of the growing season, collect mature seed heads in labeled bags. Clean seed from chaff and place seed in envelopes labeled with parental cross type in standard format (i.e., maternal x paternal).</li>
	<li>Bulk seed together...

<ol>
	<li>Walck, J. L., Baskin, J. M., Baskin, C. C., Hidayati, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+transient+and+persistent+seed+banks+in+species+with+pronounced+seasonal+dormancy+and+germination+patterns.">Defining transient and persistent seed banks in species with pronounced seasonal dormancy and germination patterns.</a> Seed Sci Res. 15 (3), 189-196 (2005).</li><li>Alexander, H. A., Schrag, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+soil+seed+banks+and+newly+dispersed+seeds+in+population+dynamics+of+the+annual+sunflower.+Helianthus+annuus.">Role of soil seed banks and newly dispersed seeds in population dynamics of the annual sunflower. Helianthus annuus.</a> J Ecol. 91, 987-998 (2003).</li><li>Burnside, O. C., Wilson, R. G., Weiseberg, S., Hubbard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+longevity+of+41+weed+species+buried+17+years+in+eastern+and+western.">Seed longevity of 41 weed species buried 17 years in eastern and western.</a> Weed Sci. 44 (1), 74-86 (1996).</li><li>Baskin, C. C., Baskin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seeds: Ecology, biogeography, and evolution of dormancy and Germination. , (2001).</li><li>Finch-Savage, W. E., Leubner-Metzger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+the+control+of+germination.">Seed dormancy and the control of germination.</a> New Phytol. 171, 501-523 (2006).</li><li>Baskin, J. M., Baskin, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+classification+system+for+seed+dormancy.">A classification system for seed dormancy.</a> Seed Sci Res. 14 (1), 1-16 (2004).</li><li>Donohue, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+genetic+influences+on+the+germination+of+Arabidopsis+thaliana+in+the+field.">Environmental and genetic influences on the germination of Arabidopsis thaliana in the field.</a> Evolution. 54 (4), 740-757 (2005).</li><li>Roach, D. A., Wulff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+effects+in+plants.">Maternal effects in plants.</a> Ann Rev Ecol Syst. 18, 209-235 (1987).</li><li>Pace, B. A., Alexander, H. M., Emry, J. D., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+fates+in+crop-wild+hybrid+sunflower:+crop+allele+and+maternal+effects.">Seed fates in crop-wild hybrid sunflower: crop allele and maternal effects.</a> Evol Appl. 8 (2), 121-132 (2015).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+maternal+effects+and+nuclear+genetic+composition+change+across+the+life+cycle+of+crop-wild+hybrids.">Roles of maternal effects and nuclear genetic composition change across the life cycle of crop-wild hybrids.</a> Am J Bot. 101 (7), 1176-1188 (2014).</li><li>Stewart, N. C., Matthew, J., Halfhill, D., Warwick, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+introgression+from+genetically+modified+crops+into+their+wild+relatives.">Transgene introgression from genetically modified crops into their wild relatives.</a> Genetics. 4, 806-817 (2003).</li><li>Mercer, K. L., Shaw, R. G., Wyse, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+germination+of+diverse+crop-wild+hybrid+sunflower+seeds.">Increased germination of diverse crop-wild hybrid sunflower seeds.</a> Ecol Appl. 16, 845-854 (2006).</li><li>Delouche, J. C., Still, T. W., Rapset, M., Lienhard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tetrazolium+test+for+seed+viability.">The tetrazolium test for seed viability.</a> Mississippi Sta Uni Ag Exp Sta Techn Bull. 51, 1-63 (1962).</li><li>Association of Official Seed Analysts. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tetrazolium Testing Handbook. , (2010).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+maternal+effects+and+nuclear+genetic+composition+change+across+the+life+cycle+of+crop-wild+hybrids.">Roles of maternal effects and nuclear genetic composition change across the life cycle of crop-wild hybrids.</a> Am J Bot. 10 (7), 1176-1188 (2014).</li><li>Weiss, A. N., Primer, S. B., Pace, B. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+effects+and+embryo+genetics:+germination+and+dormancy+of+crop-wild+sunflower+hybrids.">Maternal effects and embryo genetics: germination and dormancy of crop-wild sunflower hybrids.</a> Seed Sci Res. 23, 241-255 (2013).</li></ol>

Here we present methods for using seed burial strips to observe seed germination, dormancy and mortality of diverse seed stocks at preselected time periods in the field. The advantages of using strips rather than individual packets lie in (1) the speed of strip and compartment construction over creation of individual packets; and (2) the ease and speed of removing multiple compartments in one motion without the danger of omitting a packet or removing one unintentionally. As two of the removal dates in the example present...

The overall goal of this method is to reliably assess seed survival over time under field conditions.
Soil seed banks are a reserve of dispersed, viable yet ungerminated seeds distributed either on the soil surface, within surface litter, or within the soil profile, which may persist transiently or for many years1,2. When seed burial methods similar to those presented here were applied to a 17-year study using several dozen species, viable seeds were found in many of the species tested3. Seed dormancy is a block to seed germination until the appropriate combination of conditions for seedling survival arise4. Remaining dormant can allow seeds to survive harsh conditions, such as low winter temperatures, nutrient limitation, or seasonal drought, until an external trigger for dormancy-release allows for germination. Triggers for dormancy-release can vary from exposure to extended cold, compounds left by fire, or physical attack on the seed coat through abrasion or contact with animal stomach acids5. As germination cues can be genera or species specific and often result from past natural selection, maladaptive seed germination is that which occurs at an inappropriate time, and may result in seed or seedling mortality or poor seedling growth. While dormancy has been classified into a number of types based on the mechanisms of dormancy release (e.g., physical dormancy, physiological dormancy),6 seed dormancy remains one of the least understood topics in plant biology. Thus, field studies that allow for assessment of the status of individual seeds or groups of seeds under relevant ecological conditions have higher explanatory power than those that simply rely on standard germination tests in the laboratory.
Exploitation of known seed characteristics can provide insight into the mechanisms of dormancy. Control of seed dormancy is complex, including genetic control of physiological and morphological factors. While a full understanding of the breadth of dormancy mechanisms has yet to be elucidated, a general model has emerged, involving a feedback relationship between the two plant hormones Gibberellic Acid (GA) and Abscisic Acid (ABA)7. In this generalized model for seeds with a physiological component to their dormancy, GA serves as signal for dormancy release, while ABA serves to maintain the dormant state. Maternal genetic effects as well as the maternal growth environment can influence dormancy and other seed traits, such as size, through maternally generated tissues and developmental signals8. Maternally generated external structures (or seed coverings) may maintain dormancy, at times in combination with physiological cues. Since maternally derived seed coverings are controlled by the mother plant&#39;s genes, they may not reflect the seed&#39;s actual nuclear genetic make-up. We have used the Helianthus annuus achenes from an array of crop-wild hybrid crosses to tease out these maternal vs. embryo genetic effects on seed characteristics9,10. Thus, study designs that include diverse species, cross types, or genotypes can glean information about the ecology and genetics of seed dormancy, germination and survival.
An important example of how seed germination and survival phenotypes can affect population dynamics can be seen in crop-wild hybrid zones. Selection during domestication of cultivated plants eliminates most dormancy and reduces a seed&#39;s ability to survive outside of the growing season. Yet gene flow, or hybridization, between the cultivated and wild types in crop-wild hybrid zones can reintroduce crop alleles (or genetic variants) into a wild population, with potential effects on seed bank dynamics. Hybrids between cultivated and wild relatives potentially found in crop-wild hybrid zones may possess a variety of intermediate dormancy phenotypes, with only a few phenotypes expected to survive conditions outside of cultivation (e.g., winter months)11.
The aim of this manuscript is to show how, using the seed burial strip method, we can evaluate germination, dormancy, and survival of a range of seed types at different time periods to investigate their natural variation under field conditions. In our example, we employed sunflower seeds from 15 crop-wild hybrid cross types since we are interested in maternal and embryo genetic effects on seed characteristics.

<table border="0" cellpadding="0" cellspacing="0" width="440">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Small coin envelopes</td>
			<td style="width:72px;">Any</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Large coin envelopes</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fine meshed polyester mosquito netting</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">high-temperature glue gun</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">high-temperature glue stick refills</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Industrial permenant markers</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic garden labels</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scissors</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shovel</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal mesh hardward cloth</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surveyor&#39;s flags, multiple colors</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wet newspaper</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cooler</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">blotter paper</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petri dishes</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temp. controlled growth chamber</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">razor blades</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petri dishes</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrazolium chloride</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">water</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat incubator</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

reliable method for assessing seed germination, dormancy, and mortality under field conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Cross types with varied maternal parentage and crop allele percentage (Table 1) differed across removal dates in percent germinated, ungerminated, and dead seed (Fig. 2 and 3). Using TZ testing of ungerminated seeds, we found some truly dormant seeds at the second removal (early spring) (Table 2), while all seeds ungerminated by the third removal (spring) were found to be truly dormant.
<p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions at JoVE.com

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

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.

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10.4K vistas.  Ohio State University. 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, treatm...