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 in large envelopes using equal amounts of seed from an equal number of identical parental cross types. For example, 100 seeds from 10 cross types with the same maternal and paternal parents. Label the envelopes with identifying (cross type) information. These large envelopes are seed masters for use in filling burial strips in Section 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Maternal Parent</td>
		</tr>
		<tr>
			<td>Paternal Parent</td>
			<td>Wild: WxW</td>
			<td>F1: WxC</td>
			<td>Crop: CxC</td>
		</tr>
		<tr>
			<td>Wild: WxW</td>
			<td>0% WxW</td>
			<td>25% F1xW &#8224;</td>
			<td>50% CxW &#8224;</td>
		</tr>
		<tr>
			<td>BC: WxF1 or F1xW</td>
			<td>12.5% WxBC</td>
			<td>37.5% F1xBC</td>
			<td>62.5% CxBC</td>
		</tr>
		<tr>
			<td>F1: WxC</td>
			<td>25% WxF1 &#8224;</td>
			<td>50% F1xF1</td>
			<td>75% CxF1 &#8224;</td>
		</tr>
		<tr>
			<td>F2: F1xF1</td>
			<td>25% WxF2</td>
			<td>50% F1xF2</td>
			<td>75% CxF2</td>
		</tr>
		<tr>
			<td>Crop: CxC</td>
			<td>50% WxC &#8224;</td>
			<td>75% F1xC &#8224;</td>
			<td>100% CxC</td>
		</tr>
	</tbody>
</table>
Table 1. Parental cross types produced from hand pollination. Sunflower crop-wild hybrid cross types were produced with hand-pollination for use in seed burial experiment. For all cross types, the maternal parent listed first and the paternal parent listed second. Cross types marked with &#8224; are part of reciprocal cross type pairs with the same % crop alleles but different maternal parents. Table has been previously published in: Pace, B. A. et al. (2015)15.
2. Create Custom Seed Burial Strips
NOTE: For this example, we had three removal date treatments and 15 replicates, so we required 45 strips total. This example uses 15 compartments per strip so, we required enough fine-meshed polyester fabric or mosquito netting to house 15, 7 x 10 cm compartments. See Figure 1.
<ol>
	<li>Start by cutting a 20 x 105 cm strip of fabric for each seed strip.</li>
	<li>Measure and mark compartments, then fold the fabric in half. Using a high-temperature glue gun, seal one short (10 cm) end. Once the glue cools slightly, but not completely, press the fabric sides together to create a seal. 
	CAUTION: High-temperature glue and glue gun tips can cause burns if contact with skin is prolonged.</li>
	<li>Glue lines from the folded edge to the open short end at each compartment mark. Press the two fabric sides together along the glue lines to ensure seals without holes between compartments. Seeds migrating between compartments contribute to experimental error. 
	NOTE: Now that the compartments have been created, the unfolded edge should remain open and ready to receive seed and their identifying information (cross type name).</li>
	<li>Randomly assign seed cross types to compartments for each strip.
	<ol>
		<li>Fill seed compartments with a uniform number of seeds (20 for each compartment are used in this example). For labels, use industrial permanent markers on cut-to-fit plastic garden darts so that they are able to withstand burial. 
		NOTE: Alternative labeling methods may be used.</li>
		<li>After each compartment is filled with seed and its label, seal the compartment immediately with the high-temperature glue gun. Proceed until the entire seed strip is completely sealed. Label overall strip in the first compartment with replicate, block and/or treatment information. 
		NOTE: For our study, since we had temporal removals, blocks, and replicate strips within blocks, we included all of this information.</li>
		<li>Check for gaps between compartments and spot glue as needed before moving on to the next strip.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54663/54663fig1.jpg" /> 
Figure 1. Burial seed strip schematic. Example of burial strip showing cross type identification for individual compartments. Maternal parent is listed first with the paternal parent listed second. Colors indicate different maternal parentage, with yellow for wild, blue for F1 hybrid, and red for crop. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Bury Seed Strips in the Field
<ol>
	<li>Dig plots to the specified depth to house strips. Assign strips to individual blocks in which strips are randomly arranged to allow for removal date to be used as the main plot and compartment identity (in this example, cross type) as the subplot in analysis.</li>
	<li>Cover strips with earth. Cut metal mesh hardware cloth to be 10 cm larger than plots and place over buried strips. Use U-pins to secure edges and cover cloth edges with earth. 
	NOTE: This covering is for excluding burrowing seed predators. As another option, Mercer et al.13 constructed and buried boxes of hardware cloth that surrounded the seed strips due to intense gopher pressure in their field location.)</li>
	<li>Place colored surveyor&#39;s flags to distinguish strip location and removal dates to complement clear written records.</li>
</ol>
4. Excavate Strips and Evaluate Seed
<ol>
	<li>Dig up seed strips assigned to a given removal date. Maintain the strips in a cool moist environment until they reach the laboratory where seed evaluation will take place. This can be accomplished by wrapping the strips in wet newspaper and placing them in a cooler for transport or overnight shipping.</li>
	<li>Rinse mud and soil from strips in water until strips are clean enough that external debris will not obscure seeds.</li>
	<li>Use scissors to cut open one compartment at a time. Count germinated seeds and remove them. Count clearly dead seeds (e.g., rotting, or putrefied seeds). Place ungerminated seeds on labeled, wet Petri-sized blotter paper to further distinguish ungerminated, dormant, and dead seeds.
	<ol>
		<li>Place Petri dishes with ungerminated seeds into a growth chamber set for standard germination conditions: 25 &#176;C /10 &#176;C 12 hr day/night. Allow seeds one week to germinate. Any that do, count these as viable, but ungerminated since they did not germinate under field conditions.</li>
	</ol>
	</li>
	<li>After the growth chamber treatment, test remaining ungerminated seeds for viability using Tetrazolium chloride (TZ)13. To accomplish this, make up a percent solution of TZ appropriate for the study species using the AOSA Tetrazolium Testing Handbook14. For this example, a 1% solution is used.
	<ol>
		<li>Cut the ungerminated seeds in half and place one half were the embryo and endosperm are visible in a Petri dish containing enough TZ solution to cover the seed (seeds may float, this is okay).</li>
		<li>Place TZ Petri dishes into an incubator set at 30 &#176;C and wait 3 hr. Note: Higher temperatures will result in a faster reaction, if desired, but overstaining is a risk.</li>
	</ol>
	</li>
	<li>Remove Petri dishes from the incubator and evaluate. Seeds that have stained red in their embryos are germinable, while unstained seeds are dead. Consult the AOSA Tetrazolium Testing Handbook for more detail on distinguishing marginal cases and particular species.</li>
</ol>

<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>

The authors have nothing to disclose.

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 presented here were during winter months when the soil is hard and field conditions are cold, this method reduces both labor and the potential for human error. Seed strips also aid experimental logistics by allowing researchers to randomize compartmental locations in the laboratory in a manner that cannot be altered by human error in the field. Through selection of removal times that encompassed the breadth of predicted natural germination times we were able to make inferences about seed bank dynamics-in our example the potential for hybrid seed to persist in seed bank and contribute to future populations. One limitation of this method is that some migration of seed between compartments may be unavoidable, depending on the size of the seed. For this reason, (and as stated in the protocol) it is important that investigators double-check the high temperature glue seals between compartments to minimize seed migration.
Some problems with seed burial experiments can be avoided by thinking shrewdly about timing. Select burial and removal dates at times relevant to the natural cycles of the study species and the study objectives. It is best to plant as soon after natural seed shed as possible. Dates of removal should be selected based on observation of peak seedling emergence time for assessment of normal germination behavior; the addition of earlier dates also allows researchers to capture early germination. Because our previous work had indicated that some maladaptive germination was occurring due to complete lack of dormancy in some cross types, we selected a very early removal date only a few weeks after burial (removal 1, late fall)15, 16.
The advantage of strips over individual packets is that a dozen or more compartments can be buried, removed, and evaluated as one, allowing for complex experimental designs involving everything from multiple removal dates to irrigation or nutrient treatments. Investigators should take care to construct blocks that allow the treatments they are most concerned with understanding to be the main effect. Our example emphasized seed behavior over time, but other treatments can applied that would require consideration of field layout and replicate arrangement within blocks during the experimental design phase. By randomly assigning strip burial position within blocks, removal time can be analyzed as the main effect.
After removal of seed strips, preparing a pre-labeled stack of Petri-sized blotter paper for the entire strip before assessment begins reduces human error. Similarly, opening and evaluating a single compartment at a time helps maintain accuracy. When placing ungerminated seeds into the growth chambers, be sure that the day and night temperatures match optimal seed germination conditions for the study species. While standard germination conditions (25 &#176;C /10 &#176;C 12 hour day/night) may be appropriate for a wide variety of species, this is not the case for all species.
Consider that the TZ treatment application and evaluation of seed staining patterns strongly influences study outcome. When evaluating the ungerminated seeds after the growth chamber treatment, consult the literature (or the AOSA Tetrazolium Testing Handbook14) for the correct concentration of TZ for a given species. While judging individual TZ stained seeds, it is important to remember the difference between seed viability and a seed&#39;s ability to germinate-weakly stained seeds may indicate respiring tissue, which may be unable to emerge from the soil and produce a seedling. That stated, truly dormant seeds are those that remain ungerminated under optimal germination conditions, yet when tested are still strongly viable. These seeds are the only seeds likely to contribute to a persistent seed bank and germinate in future years.
A number of applications for these methods are possible beyond those outlined here. Seed burial strips can be customized for virtually any number of experimental units and experimental designs. They can be applied to compare different species, seed sizes, or conditions/treatments. Building on these methods has the potential to deepen knowledge of the basic seed biology of many species in an ecologically relevant context.

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.
In general, lower crop allele percentages reduced maladaptive germination in late fall (Fig. 2). At the early spring removal, all cross types had high germination, but differed in their proportion dead and ungerminated (Fig. 3). In particular, cross types with 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. While counter-intuitive, 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 (i.e., those with higher crop allele percentages) produced by crop maternal parents (Fig. 3)
<img alt="Figure 2" src="/files/ftp_upload/54663/54663fig2.jpg" /> 
Figure 2. Germination at the first removal in late fall. Germination of sunflower crop-wild cross types at the first removal date, in late fall. Cross types are organized on the y-axis by increasing crop allele percentages as marked. Maternal parent is listed first for each cross type. Germination least squares means (with s.e. bars) followed by the same letter are not significantly different using a Tukey-Kramer adjustment for multiple comparisons. Figure has been previously published in: Pace, et al. (2015)15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54663/54663fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54663/54663fig3.jpg" /> 
Figure 3. Percent Germinated, ungerminated, and mortality for the second removal (early spring) and third removal (spring). Percent germinated for early spring (A), spring (B), percent ungerminated for early spring (C), spring (D), and percent dead for early spring (E) and spring (F) removal dates. Sunflower crop-wild hybrid cross types are organized by increasing crop allele percentages, with the most crop-like on the top of the y-axis and the most wild-like at the bottom of the y-axis. Cross type maternal parent is listed first. Least squares means for germinated, ungerminated and dead seed (with s.e. bars) followed by the same letter are not significantly different using a Tukey-Kramer adjustment for multiple comparisons. Figure has been previously published in: Pace, et al. (2015)15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54663/54663fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cross type</td>
			<td>TZ Dormant</td>
			<td>Standard Error</td>
		</tr>
		<tr>
			<td>WxW</td>
			<td>0.071</td>
			<td>0.0131</td>
		</tr>
		<tr>
			<td>WxBC</td>
			<td>0.041</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>WxF1</td>
			<td>0.089</td>
			<td>0.0136</td>
		</tr>
		<tr>
			<td>WxF2</td>
			<td>0.054</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>F1xW</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>F1xBC</td>
			<td>0.013</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>WxC</td>
			<td>0.075</td>
			<td>0.0131</td>
		</tr>
		<tr>
			<td>F1xF1</td>
			<td>0.007</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>F1xF2</td>
			<td>0.013</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>CxW</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>CxBC</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>F1xC</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>CxF1</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>CxF2</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
		<tr>
			<td>CxC</td>
			<td>0</td>
			<td>0.0126</td>
		</tr>
	</tbody>
</table>
Table 2. Seeds likely to contribute to a persistent seed bank. Least squares means for the percent dormant fraction of ungerminated seeds at the second (early spring) removal. Seeds were determined to be viable using tetrazolium chloride after incubation in favorable germination conditions. Table has been previously published in: Pace, et al. (2015)15.

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 ...

reliable-method-for-assessing-seed-germination-dormancy-mortality

Nanyang Technological University

Research

JoVE Journal

Environment

10.4K Views.  Ohio State University. 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.

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

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Preparation and Testing of Plant Seed Meal-based Wood Adhesives

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable and environment-friendly. These natural products may serve as alternatives to petroleum-based adhesives to ease environmental and sustainability concerns. This work demonstrates the preparation and testing of the plant seed-based wood adhesives using cottonseed and soy meal as raw materials. In addition to untreated meals, water washed meals and protein isolates are prepared and tested. Adhesive slurries are prepared by mixing a freeze-dried meal product with deionized water (3:25 w/w) for 2 hr. Each adhesive preparation is applied to one end of 2 wood veneer strips using a brush. The tacky adhesive coated areas of the wood veneer strips are lapped and glued by hot-pressing. Adhesive strength is reported as the shear strength of the bonded wood specimen at break. Water resistance of the adhesives is measured by the change in shear strength of the bonded wood specimens at break after water soaking. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for substitution of synthetic-based wood adhesives. Adjustments to the adhesive formulation with or without additives and bonding conditions could optimize their adhesive properties for various practical applications.

To facilitate the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives, this work demonstrates the preparation and testing of plant seed-based wood adhesives. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for the substitution of synthetic-based wood adhesives.

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...