The authors would like to acknowledge anonymous reviewers for their feedback, as well as J.C. Cahill and Tan Bao for initial guidance on developing the rhizobox protocol. Funding was provided by the Foundation for Food and Agriculture Research, the US Department of Agriculture (USDA) National Institute of Food and Agriculture, Agricultural Experiment Station Project CA-D-PLS-2332-H, to A.G. and by the UC Davis Department of Plant Sciences through a fellowship to J.S.

<ol>
	<li>Hodge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roots:+The+Acquisition+of+Water+and+Nutrients+from+the+Heterogeneous+Soil+Environment.">Roots: The Acquisition of Water and Nutrients from the Heterogeneous Soil Environment.</a> Progress in Botany 71. , 307-337 (2010).</li><li>Grossman, J. D., Rice, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+root+plasticity+responses+to+variation+in+soil+nutrient+distribution+and+concentration.">Evolution of root plasticity responses to variation in soil nutrient distribution and concentration.</a> Evolutionary Applications. 5 (8), 850-857 (2012).</li><li>Zhang, H., Forde, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+MADS+box+gene+that+controls+nutrient-induced+changes+in+root+architecture.">An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture.</a> Science. 279 (5349), 407-409 (1998).</li><li>Hodge, A., Stewart, J., Robinson, D., Griffiths, B. S., Fitter, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+roots+and+soil+micro-organisms+for+nutrients+from+nitrogen-rich+patches+of+varying+complexity.">Competition between roots and soil micro-organisms for nutrients from nitrogen-rich patches of varying complexity.</a> Journal of Ecology. 88 (1), 150-164 (2000).</li><li>Trachsel, S., Kaeppler, S. M., Brown, K. M., Lynch, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shovelomics:+high+throughput+phenotyping+of+maize+(Zea+mays+L.)+root+architecture+in+the+field.">Shovelomics: high throughput phenotyping of maize (Zea mays L.) root architecture in the field.</a> Plant and Soil. 341 (1-2), 75-87 (2011).</li><li>Rogers, E. D., Monaenkova, D., Mijar, M., Nori, A., Goldman, D. I., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+computed+tomography+reveals+the+response+of+root+system+architecture+to+soil+texture.">X-ray computed tomography reveals the response of root system architecture to soil texture.</a> Plant Physiology. , (2016).</li><li>Groleau-Renaud, V., Plantureux, S., Guckert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mechanical+constraint+on+nodal+and+seminal+root+system+of+maize+plants.">Effect of mechanical constraint on nodal and seminal root system of maize plants.</a> Comptes Rendus De L Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences. 321 (1), 63-71 (1998).</li><li>Lin, Y., Allen, H. E., Di Toro, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barley+root+hair+growth+and+morphology+in+soil,+sand,+and+water+solution+media+and+relationship+with+nickel+toxicity.">Barley root hair growth and morphology in soil, sand, and water solution media and relationship with nickel toxicity.</a> Environmental Toxicology and Chemistry. 35 (8), 2125-2133 (2016).</li><li>Wenzel, W. W., Wieshammer, G., Fitz, W. J., Puschenreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+rhizobox+design+to+assess+rhizosphere+characteristics+at+high+spatial+resolution.">Novel rhizobox design to assess rhizosphere characteristics at high spatial resolution.</a> Plant and Soil. 237 (1), 37-45 (2001).</li><li>Spohn, M., Carminati, A., Kuzyakov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+zymography+-+A+novel+in+situ+method+for+mapping+distribution+of+enzyme+activity+in+soil.">Soil zymography - A novel in situ method for mapping distribution of enzyme activity in soil.</a> Soil Biology and Biochemistry. 58, 275-280 (2013).</li><li>Vollsnes, A. V., Futsaether, C. M., Bengough, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+rhizosphere+particle+movement+around+mutant+maize+roots+using+time-lapse+imaging+and+particle+image+velocimetry.">Quantifying rhizosphere particle movement around mutant maize roots using time-lapse imaging and particle image velocimetry.</a> European Journal of Soil Science. 61 (6), 926-939 (2010).</li><li>Hewitt, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sand and Water Culture Methods Used in the Study of Plant Nutrition. , (1966).</li><li>Choudhary, M. I., Shalaby, A. A., Al-Omran, 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=Water+holding+capacity+and+evaporation+of+calcareous+soils+as+affected+by+four+synthetic+polymers.">Water holding capacity and evaporation of calcareous soils as affected by four synthetic polymers.</a> Communications in Soil Science and Plant Analysis. 26 (13-14), 2205-2215 (1995).</li><li>Bakker, P. A. H. M., Berendsen, R. L., Doornbos, R. F., Wintermans, P. C. A., Pieterse, C. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rhizosphere+revisited:+root+microbiomics.">The rhizosphere revisited: root microbiomics.</a> Frontiers in Plant Science. 4, 2013 (2013).</li><li>McNear, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rhizosphere+-+Roots,+Soil,+and+Everything+In+Between.">The Rhizosphere - Roots, Soil, and Everything In Between.</a> Nature Education Knowledge. 4 (3), 1 (2013).</li><li>Ortas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+extent+of+rhizosphere+soil.">Determination of the extent of rhizosphere soil.</a> Communications in Soil Science and Plant Analysis. 28 (19-20), 1767-1776 (1997).</li><li>. Carbon (13C) and Nitrogen (15N) Sample Preparation Available from: <a target="_blank" href="https://stableisotopefacility.ucdavis.edu/13cand15nsamplepreparation.html">https://stableisotopefacility.ucdavis.edu/13cand15nsamplepreparation.html</a> (2018)</li><li>Barraclough, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=15N+isotope+dilution+techniques+to+study+soil+nitrogen+transformations+and+plant+uptake.">15N isotope dilution techniques to study soil nitrogen transformations and plant uptake.</a> Fertilizer research. 42 (1-3), 185-192 (1995).</li><li>Belter, P. R., Cahill, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disentangling+root+system+responses+to+neighbours:+identification+of+novel+root+behavioural+strategies.">Disentangling root system responses to neighbours: identification of novel root behavioural strategies.</a> AoB PLANTS. 7, (2015).</li><li>Nagel, K. A., 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=GROWSCREEN-Rhizo+is+a+novel+phenotyping+robot+enabling+simultaneous+measurements+of+root+and+shoot+growth+for+plants+grown+in+soil-filled+rhizotrons.">GROWSCREEN-Rhizo is a novel phenotyping robot enabling simultaneous measurements of root and shoot growth for plants grown in soil-filled rhizotrons.</a> Functional Plant Biology. 39 (11), 891-904 (2012).</li><li>Adu, M. O., Yawson, D. O., Bennett, M. J., Broadley, M. R., Dupuy, L. X., White, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scanner-based+rhizobox+system+enabling+the+quantification+of+root+system+development+and+response+of+Brassica+rapa+seedlings+to+external+P+availability.">A scanner-based rhizobox system enabling the quantification of root system development and response of Brassica rapa seedlings to external P availability.</a> Plant Root. 11, 16-32 (2017).</li><li>Neumann, G., George, T. S., Plassard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+methods+for+studying+the+rhizosphere-the+plant+science+toolbox.">Strategies and methods for studying the rhizosphere-the plant science toolbox.</a> Plant and Soil. 321 (1-2), 431-456 (2009).</li><li>Bodner, G., Alsalem, M., Nakhforoosh, A., Arnold, T., Leitner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGB+and+Spectral+Root+Imaging+for+Plant+Phenotyping+and+Physiological+Research:+Experimental+Setup+and+Imaging+Protocols.">RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols.</a> JoVE (Journal of Visualized Experiments). (126), e56251-e56251 (2017).</li><li>Kuchenbuch, R. O., Ingram, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+analysis+for+non-destructive+and+non-invasive+quantification+of+root+growth+and+soil+water+content+in+rhizotrons.">Image analysis for non-destructive and non-invasive quantification of root growth and soil water content in rhizotrons.</a> Journal of Plant Nutrition and Soil Science. 165 (5), 573-581 (2002).</li><li>Dresb&#248;ll, D. B., Thorup-Kristensen, K., McKenzie, B. M., Dupuy, L. X., Bengough, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timelapse+scanning+reveals+spatial+variation+in+tomato+(Solanum+lycopersicum+L.)+root+elongation+rates+during+partial+waterlogging.">Timelapse scanning reveals spatial variation in tomato (Solanum lycopersicum L.) root elongation rates during partial waterlogging.</a> Plant and Soil. 369 (1-2), 467-477 (2013).</li><li>Wu, J., 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=RhizoChamber-Monitor:+a+robotic+platform+and+software+enabling+characterization+of+root+growth.">RhizoChamber-Monitor: a robotic platform and software enabling characterization of root growth.</a> Plant Methods. 14 (1), 44 (2018).</li><li>Rogers, S. W., Moorman, T. B., Ong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+In+Situ+Hybridization+and+Micro-autoradiography+Applied+to+Ecophysiology+in+Soil.">Fluorescent In Situ Hybridization and Micro-autoradiography Applied to Ecophysiology in Soil.</a> Soil Science Society of America Journal. 71 (2), 620-631 (2007).</li><li>Eickhorst, T., Tippk&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microorganisms+in+undisturbed+soil+by+combining+fluorescence+in+situ+hybridization+(FISH)+and+micropedological+methods.">Detection of microorganisms in undisturbed soil by combining fluorescence in situ hybridization (FISH) and micropedological methods.</a> Soil Biology and Biochemistry. 40 (6), 1284-1293 (2008).</li><li>Spohn, M., Kuzyakov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+microbial-+and+root-derived+phosphatase+activities+in+the+rhizosphere+depending+on+P+availability+and+C+allocation+-+Coupling+soil+zymography+with+14C+imaging.">Distribution of microbial- and root-derived phosphatase activities in the rhizosphere depending on P availability and C allocation - Coupling soil zymography with 14C imaging.</a> Soil Biology and Biochemistry. 67, 106-113 (2013).</li><li>Lv, G., Kang, Y., Li, L., Wan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+irrigation+methods+on+root+development+and+profile+soil+water+uptake+in+winter+wheat.">Effect of irrigation methods on root development and profile soil water uptake in winter wheat.</a> Irrigation Science. 28 (5), 387-398 (2010).</li><li>Asseng, S., Ritchie, J. T., Smucker, A. J. M., Robertson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+growth+and+water+uptake+during+water+deficit+and+recovering+in+wheat.">Root growth and water uptake during water deficit and recovering in wheat.</a> Plant and Soil. 201 (2), 265-273 (1998).</li><li>Hernandez-Ramirez, G., 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=Root+Responses+to+Alterations+in+Macroporosity+and+Penetrability+in+a+Silt+Loam+Soil.">Root Responses to Alterations in Macroporosity and Penetrability in a Silt Loam Soil.</a> Soil Science Society of America Journal. 78 (4), 1392-1403 (2014).</li><li>Zhang, Y. L., Wang, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+enzyme+activities+with+greenhouse+subsurface+irrigation.">Soil enzyme activities with greenhouse subsurface irrigation.</a> Pedosphere. 16 (4), 512-518 (2006).</li><li>Robinson, D., Hodge, A., Griffiths, B. S., Fitter, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+root+proliferation+in+nitrogen-rich+patches+confers+competitive+advantage.">Plant root proliferation in nitrogen-rich patches confers competitive advantage.</a> Proceedings of the Royal Society of London B: Biological Sciences. 266 (1418), 431-435 (1999).</li><li>Lobet, G., Draye, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+scanning+procedure+enabling+the+vectorization+of+entire+rhizotron-grown+root+systems.">Novel scanning procedure enabling the vectorization of entire rhizotron-grown root systems.</a> Plant Methods. 9, 1 (2013).</li><li>Swarup, R., Wells, D. M., Bennett, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+Gravitropism.">Root Gravitropism.</a> Plant Roots: The Hidden Half. , (2013).</li><li>Smit, A. L., Bengough, A. G., Engels, C., van Noordwijk, M., Pellerin, S., van de Geijn, S. 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> Root Methods: A Handbook. , (2000).</li><li>van Dusschoten, D., 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=Quantitative+3D+Analysis+of+Plant+Roots+Growing+in+Soil+Using+Magnetic+Resonance+Imaging1[OPEN].">Quantitative 3D Analysis of Plant Roots Growing in Soil Using Magnetic Resonance Imaging1[OPEN].</a> Plant Physiology. 170 (3), 1176-1188 (2016).</li><li>Metzner, R., 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=Direct+comparison+of+MRI+and+X-ray+CT+technologies+for+3D+imaging+of+root+systems+in+soil:+potential+and+challenges+for+root+trait+quantification.">Direct comparison of MRI and X-ray CT technologies for 3D imaging of root systems in soil: potential and challenges for root trait quantification.</a> Plant Methods. 11, 17 (2015).</li></ol>

The authors have nothing to disclose.

The rhizoboxes described in this protocol can be used to answer varied questions in root and rhizosphere science, and have found diverse uses elsewhere10,20,21,22,23,24,25. Other researchers have captured time-lapse images of rhizoboxes21,...

Although often overlooked compared to their aboveground counterparts, roots play a critical role in plant nutrient acquisition. Given the substantial carbon cost of root construction and maintenance, plants have evolved mechanisms to develop roots only where foraging is worth the investment. Root systems can thus efficiently and dynamically mine resource patches by proliferating in hotspots, upregulating rates of uptake, and rapidly translocating nutrients to the phloem for further transport1. Plasticity responses can vary widely among plant species or genotypes2,3 and depending on the chemical form of the nutrient involved4,5. Variation in root plasticity should be explored further, as understanding complex root responses to heterogeneous soil resources could inform breeding and management strategies to increase nutrient use efficiency in agriculture.
Despite its necessity and relevance for understanding plant systems, visualizing and quantifying root plasticity at relevant scales poses technical challenges. Excavating the root crown from the soil (&#34;shovelomics&#34;6) is a common method, but fine roots exploit small pores between soil aggregates, and excavation inevitably leads to some degree of loss of these fragile roots. Furthermore, destructive harvest makes it impossible to follow changes in one root system over time. In situ imaging methods such as X-ray computed tomography allow direct visualization of roots and soil resources at high spatial resolution7, but are expensive and require specialized equipment. Hydroponic experiments avoid constraints associated with extracting roots from soil, but root morphology and architecture differ in aqueous media as compared to the mechanical constraints and biophysical complexity of soils8,9. Finally, rhizosphere processes and functions cannot be integrated with developmental plasticity in these artificial media.
We present a protocol for the construction and use of rhizoboxes (narrow, clear-sided rectangular containers) as a low-cost, customizable method to characterize root growth in soil over time. Specially designed frames encourage roots to grow preferentially against the back&#160;panel due to gravitropism, increasing the accuracy of root length measurements. Rhizoboxes are commonly used to study root growth and rhizosphere interactions10,11,12, but the method presented here offers an advantage in simplicity with its single-compartment design and inexpensive materials, and is designed to study root responses to localized nutrients. However, the method could also be adapted to study a range of other root and rhizosphere processes such as intra/interspecies competition, spatial distribution of chemical compounds, microbes, or enzyme activity. Here, we investigate genotypic differences among maize hybrids in response to patches of 15N-labeled legume residue and highlight representative results to validate the rhizobox method.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.27 cm diameter PVC pipe</td>
			<td style="width:123px;">JM Eagle</td>
			<td style="width:119px;">530048</td>
			<td style="width:407px;">305 cm per box, cut into lengths as specified in the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVC side elbows</td>
			<td style="width:123px;">Lasco</td>
			<td>315498</td>
			<td>2 per box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVC 90-degree elbows</td>
			<td style="width:123px;">Charlotte</td>
			<td style="width:119px;">PVC 02300 0600</td>
			<td>4 per box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVC T joints</td>
			<td style="width:123px;">Charlotte</td>
			<td style="width:119px;">PVC 02402 0600</td>
			<td>4 per box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extruded acrylic panes</td>
			<td style="width:123px;">TAP Plastics</td>
			<td style="width:119px;">N/A</td>
			<td>2 per box, 0.64 cm thick x 40.5 cm wide x 61 cm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HDPE spacers (sides)</td>
			<td style="width:123px;">TAP Plastics</td>
			<td style="width:119px;">N/A</td>
			<td>2 per box, 0.64 cm thick x 2.5 cm wide x 57 cm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HDPE spacers (bottom)</td>
			<td style="width:123px;">TAP Plastics</td>
			<td style="width:119px;">N/A</td>
			<td>1 per box, 0.64 cm thick x 2.5 cm wide x 40.5 cm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HDPE spacers (patch)</td>
			<td style="width:123px;">TAP Plastics</td>
			<td style="width:119px;">N/A</td>
			<td>2 per box, 0.64 cm thick x 3.8 cm wide x 28 cm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyester batting</td>
			<td style="width:123px;">Fairfield</td>
			<td>#A-X90</td>
			<td>2.5 cm x 40.5 cm strip per box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-thread screws</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>3.2 cm long, 0.64 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Washers</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>0.64 cm internal diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hex nuts</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sized to fit the screws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light deprivation fabric</td>
			<td style="width:123px;">Americover, Inc.</td>
			<td>Bold 8WB26.5</td>
			<td>1 piece 95 cm wide and 69 cm long per box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sand</td>
			<td style="width:123px;">Quikrete</td>
			<td>No. 1113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Field soil</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transparencies for tracing</td>
			<td style="width:123px;">FXN</td>
			<td>FXNT1319100S</td>
			<td>One per side of the box to be traced</td>
		</tr>
	</tbody>
</table>

an optimized rhizobox protocol to visualize root growth and responsiveness to localized nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Roots grew preferentially against the back of the box, as anticipated. Total traced root length on the back of the box ranged from 400 to 1,956 cm, as compared to 93-758 cm on the front of the box. Pairwise Pearson correlation coefficients were calculated between scanned root length and traced root length on the front of the box, back of the box, and the sum of front and back was used to determine whether tracing accurately reflected total root length (n = 23, as the plant in one box died...

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An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

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15.6K Views.  University of California at Davis. Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Video: An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...