The authors thank Davy Opdenacker for technical assistance and photography. We greatly thank Dr. Annick Bleys for helpful suggestions to improve the manuscript. This work was financed by the Interuniversity Attraction Poles Programme IUAP P7/29 'MARS' from the Belgian Federal Science Policy Office, by the FWO grant G027313N and by the Agency for Innovation by Science and Technology, IWT (IR).

1. Arabidopsis LRIS Protocol
Note: The text refers to &#34;small&#34; or &#34;large&#34; scale experiments. Small scale experiments, such as marker line analysis and histological staining6, 14, require only a few samples. Large scale experiments, such as quantitative real-time qRT-PCR, micro-arrays9-11 or RNA sequencing, require a larger amount of samples. As such, an amount of ~1000 seedlings per sample was used by Vanneste et al.11 to perform ...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histological+study+of+lateral+root+initiation+and+development+in+Zea+mays.">A histological study of lateral root initiation and development in Zea mays.</a> Protoplasma. 70, 179-205 (1970).</li><li>Benkova, E., 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=Local,+efflux-dependent+auxin+gradients+as+a+common+module+for+plant+organ+formation.">Local, efflux-dependent auxin gradients as a common module for plant organ formation.</a> Cell. 115, 591-602 (2003).</li><li>Beeckman, T., Burssens, S., Inz&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peri-cell-cycle+in+Arabidopsis.">The peri-cell-cycle in Arabidopsis.</a> J. Exp. Bot. 52, 403-411 (2001).</li><li>Himanen, K., Boucheron, E., Vanneste, S., de Almeida Engler, J., Inz&#233;, D., Beeckman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-mediated+cell+cycle+activation+during+early+lateral+root+initiation.">Auxin-mediated cell cycle activation during early lateral root initiation.</a> Plant Cell. 14, 2339-2351 (2002).</li><li>Jansen, L., Parizot, B., Beeckman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+system+for+lateral+roots+in+Arabidopsis+thaliana+and+maize.">Inducible system for lateral roots in Arabidopsis thaliana and maize.</a> Methods Mol Biol. 959, 149-158 (2013).</li><li>Casimiro, I., 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=Auxin+transport+promotes+Arabidopsis+lateral+root+initiation.">Auxin transport promotes Arabidopsis lateral root initiation.</a> Plant Cell. 13, 843-852 (2001).</li><li>Himanen, 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=Transcript+profiling+of+early+lateral+root+initiation.">Transcript profiling of early lateral root initiation.</a> Proc. 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The authors have nothing to disclose.

In the Arabidopsis LRIS protocol, it is important to only transfer the seedlings that have grown entirely in contact with the NPA-containing growth medium. This ensures that lateral root initiation is blocked over the entire root length. In order to prevent wounding the plantlets during transfer, the arms of the curved forceps can be hooked under the cotyledons of the seedling. Upon transfer, make sure that the seedling roots are in sufficient contact with the NAA-containing agar medium. This can be achieved by ...

The root system is crucial for plant growth, since it ensures anchorage and uptake of water and nutrients from the soil. Because the expansion of a root system mainly relies on the production of lateral roots, their initiation and formation have been widely studied. Lateral roots are initiated in a specific subset of pericycle cells, called founder cells1. In most dicots, such as Arabidopsis thaliana, these cells are located at the protoxylem poles2, whereas in monocots, such as maize, they are found at the phloem poles3. Founder cells are marked by an increased auxin response4, followed by expression of specific cell cycle genes (e.g.,&#160;CYCLIN B1;1 / CYCB1;1), after which they undergo a first round of asymmetric anticlinal divisions5. After a series of coordinated anticlinal and periclinal divisions, a lateral root primordium is formed that finally will emerge as an autonomous lateral root. The location and timing of lateral root initiation are however not predictable, since these events are neither abundant nor synchronized. This impedes the use of molecular approaches such as transcriptomics to study this process.
To tackle this, a Lateral Root Inducible System (LRIS) has been developed6, 7. In this system, seedlings are first treated with N-1-naphthylphthalamic acid (NPA), which inhibits auxin transport and accumulation, consequently blocking lateral root initiation8. By subsequently transferring the seedling to medium containing the synthetic auxin 1-naphthalene acetic acid (NAA), the entire pericycle layer responds to the elevated auxin levels thereby massively inducing lateral root initiating cell divisions6. As such, this system leads to fast, synchronous and extensive lateral roots initiations, allowing easy collection of root samples enriched for a specific stage of lateral root development. Subsequently, these samples can be used to determine genome-wide expression profiles during lateral root formation. The LRIS has yielded already significant knowledge about lateral root initiation in Arabidopsis and maize9-13, but the need to apply this system to other plant species becomes more apparent as more genomes are sequenced and there is an increasing interest to transfer knowledge to economical important species.
Here, the detailed protocols of the Arabidopsis and maize LRISs are given. Next, an example of the use of the system is provided, by illustrating how transcriptomics data gained from the maize LRIS can be used to identify functional homologs that have a conserved function during lateral root initiation across different plant species. Finally, guidelines to optimize the LRIS for other plant species are proposed.

<table border="0" cellpadding="0" cellspacing="0" width="1258">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ARABIDOPSIS LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Arabidopsis seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>Col-0 ecotype</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Gas sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 1.5 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 125.215</td>
			<td>Eppendorf microtubes 3810X, PCR clean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 2 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 120.094</td>
			<td>Eppendorf Safe-Lock microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">hydrochloric acid</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1,003,171,000</td>
			<td>37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">glass desiccator</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;"></td>
			<td>Pyrex</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">plastic micro-centrifuge tubes box or holder</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ethanol</td>
			<td style="width:228px;">Chem-Lab nv</td>
			<td style="width:119px;">CL00.0505.1000</td>
			<td>Ethanol, abs. 100% a.r. dilute to 70%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Tween 20</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth medium</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Murashige and Skoog salt mixture</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M0221-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">myo-inositol</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">I5125-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M1503.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sucrose</td>
			<td style="width:228px;">VWR, Internation LLC</td>
			<td style="width:119px;">27483.294</td>
			<td>D(+)-Sucrose Ph. Eur.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">KOH</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1050211000</td>
			<td>pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Plant Tissue Culture Agar</td>
			<td style="width:228px;">LabM Limited</td>
			<td style="width:119px;">MC029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Making a mesh for transfer</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">nylon mesh</td>
			<td style="width:228px;">Prosep byba</td>
			<td style="width:119px;"></td>
			<td>Synthetic nylon mesh 20 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">square petri dish plates</td>
			<td style="width:228px;">GOSSELIN</td>
			<td style="width:119px;">BP124-05</td>
			<td>12 x 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">50 ml DURAN tubes</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">CLS430304</td>
			<td>Corning&#160;50 mL centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">drigalski</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">K732.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">pipette</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">cut pipette tips</td>
			<td style="width:228px;">Daslab</td>
			<td style="width:119px;">162001X</td>
			<td>Universal 200, cut off 5 mm of tip before autoclaving</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">breathable tape&#160;</td>
			<td style="width:228px;">3M Deutschland GmbH</td>
			<td style="width:119px;">cat. no. 1530-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">growth room</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>21 &#176;C, continuous light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Materials</td>
			<td style="width:228px;">Company</td>
			<td style="width:119px;">Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">MAIZE LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Maize kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>B-73</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stirrer&#160;</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">C267.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>50 &#181;M (maize primary root), 25 &#181;M (maize adventitious root)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>50 &#181;M (maize)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">paper hand towels</td>
			<td style="width:228px;">Kimberly-Clark Professional*</td>
			<td style="width:119px;">6681</td>
			<td>SCOTT Hand Towels - Roll / White; sheet size (24 x 46 cm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">seed germination paper</td>
			<td style="width:228px;">Anchor Paper Company</td>
			<td style="width:119px;"></td>
			<td>10 X 15 38# seed germination paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">250 ml (centrifuge) tubes</td>
			<td style="width:228px;">SCHOTT DURAN</td>
			<td style="width:119px;">2160136</td>
			<td>approx. 5.6 cm diameter and 14.7 cm height&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">700 ml tubes</td>
			<td style="width:228px;">DURAN GROUP</td>
			<td style="width:119px;">213994609</td>
			<td>cylinders, round foot tube, D 60&#160; x 250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rack</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>for maize tubes, home made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">growth cabinet</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>27 &#176;C, continuous light, 70% relative humidity</td>
		</tr>
	</tbody>
</table>

lateral root inducible system in arabidopsis and maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Application of the LRIS to Perform Comparative Transcriptomics of the Lateral Root Initiation Process
One application of the LRIS is the comparison and correlation of gene expression profiles during lateral root formation in different species. Comparative transcriptomics approaches create the possibility to pinpoint orthologous genes involved in the lateral root development process in different species. Lateral ...

Watch this Scientific Journal Video about Lateral Root Inducible System in Arabidopsis and Maize at JoVE.com

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

The overall goal of the lateral root inducible system is to synchronously induce the initiation of lateral roots in plants using hormone treatments. Plant roots are the hidden and are known to be extremely difficult to study. We have developed an in vitro system that allows to study the process of lateral root initiation on a cytological, histological, and molecule level.Generally, scientists new to this method might struggle because the plants have not grown into proper contact with the medium, preventing a homogenous induction of the lateral roots. Visual demonstration of this method helps showing the critical steps that should be taken into account to ensure a homogenous induction. Although this system was originally developed in a little plant Arabidopsis, it can also be applied to other systems such as maize, rice, or barley.To begin, apply a 20-micron nylon mesh to NPA-supplemented growth medium using a pair of sterile tweezers. Gently push the mesh onto the growth medium using a drigalski to make sure it is in good contact with the medium. Next, pinch a sterile toothpick into the agar to create a sticky surface on the toothpick.Use the toothpick to evenly distribute a batch of gas-sterilized seeds onto the prepared plates. Seal the plates with breathable adhesive tape and store them at four degrees Celsius in the dark for at least two days and a maximum of one week. This is needed for stratification and ensures synchronized and efficient germination.On the third day, move the plates to the growth room for two days of germination followed by three days of growth at an angle of 80 to 90 degrees. On day eight, identify the roots that have grown entirely in contact with the mesh and on the contrary, roots that are not in contact with the mesh. Remove all the seedlings which roots are not in contact with the mesh.Transfer the seedling-containing meshes to plates supplemented with 10 micromolar NAA. Once on the plate, skim the mesh over the medium surface if necessary to make sure that it is in good contact with the growth medium. To ensure a homogenous induction in all seedlings, it is important to discard the seedlings that have not grown into contact with the mesh on NPA-supplemented medium and to properly skim the mesh if necessary on NAA-supplemented medium.Seal the new plates with breathable tape and place them in the growth room in a vertical position for the desired amount of time after induction. When ready to collect the samples, use a scalpel to cut the root segments all at once directly on the mesh and sample them by gently scraping the surface of the mesh. We will now show some steps of the lateral root inducible system in maize.Begin by sterilizing the maize kernels by first placing the necessary number of kernels into a sterile glass beaker. Then, add 100 milliliters of 6%sodium hypochlorite in water to the beaker and add a magnetic stir bar. Place the beaker on a magnetic stirrer and stir at a low speed for five mintues at room temperature.Gently remove the bleach solution. Replace it with 100 milliliters of fresh sterile water and rinse the kernels for five minutes. Repeat this step five times.Put on a pair of gloves and tear off two stretches of hand paper towels, placing them on top of each other on a clean surface. Then, fold the sheets double over the length. Use tweezers to distribute 10 kernels approximately two centimeters from the top, over the entire length of the paper while keeping an interspace distance of eight centimeters between each kernel and eight centimeters free at both ends.Make sure the radical of the kernel is facing down and toward the paper. Gently roll the paper over the length while keeping the seeds in place. Spraying the paper with sterile water can facilitate this step if necessary.Put the rolls into sterilized glass tubes and combine additional tubes into the rack. Pour 125 milliliters of a 50 micromolar NPA solution over the paper roll in the tube. The paper roll will absorb the solution and become completely soaked.Place the paper roll system in a growth cabinet for three days. Make sure that the paper rolls remain soaked during incubation by adding extra NPA solution as needed. To ensure homogenous induction of lateral root initiation, it is important to check that the paper rolls remain moisturized in NPA solution, but that the roots are not submerged in the liquid itself.After three days on NPA, the maize seedling has a small shoot and a root of approximately two to three centimeters. On day four, gently squeeze out most of the remaining NPA solution from the paper rolls and place the rolls in sterile water until they become saturated. After about five minutes, gently squeeze out most of the water from the paper rolls.Repeat the wash step three times. Following the final rinse, squeeze out most of the water and place it back into the tube. Pour a 50 micromolar solution of NAA over the paper rolls in the tubes until they become completely soaked.Then, place the paper roll system in the growth cabinet for the desired duration after induction. In comparison to oxen response marker line pDR5-GUS seedlings that haven't grown on NAA or have grown for one hour on NAA, the seedlings grown for two hours on NAA show an oxen response which is one of the first events triggering lateral root initiation. These divisions start moving up from the tip starting at around two hours, and increase with prolonged exposure to NAA.Using the cell cycle marker line pCYCB1;1-Gus, the first cell divisions that lead to lateral root formation can be seen starting at around six hours after the start of NAA treatment. Microarrays and quantitative rt-PCR can be used to evaluate expression of various genes throughout the different regions of the Arabidopsis and maize roots during lateral root induction. In both Arabidposis and maize, when the plants grow for a longer time, the induced divisions will give rise to the development and outgrowth of lateral roots.As proof of concept for the system on maize, here are seedlings grown under different conditions for comparison. Seedlings grown for 10 days on water have lateral roots that didn't initiate synchronously and are widely spaced along the axis. Seedlings grown for 10 days on NPA contain no lateral roots at all.Seedlings grown for three days on NPA, followed by an NAA treatment, show a massive and synchronized induction of lateral roots. In this video we have shown you how to set up a lateral root inducible system that has turned out to be very essential in the discovery of fundamental new insights into the process of root branching. We have shown you how it works for Arabidopsis and maize, but it can easily be adapted to other plant species of choice.Good luck.

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13.6K Görüntüleme.  Ghent. The overall goal of the lateral root inducible system is to synchronously induce the initiation of lateral roots in plants using hormone treatments. Plant roots are the hidden and are known to be extremely difficult to study. We have developed an in vitro system that allows to study the process of lateral root initiation on a cytological, histological, and molecule level.Generally, scientists new to this method might struggle because the plants have not grown into proper contact with t...