Name: Lateral Root Inducible System in Arabidopsis and Maize (Video) | JoVE Journal
Uploaded: 2016-01-14T16:00:00.000+00:00
Duration: 9 min 23 s
Description: Learn the Lateral Root Inducible System for synchronous lateral root initiation in Arabidopsis and maize using hormone treatments. This method enables cytological and molecular study of root development.

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5 minutes

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 microarray experiment after root segment dissection.
DAY 1
<ol>
	<li>Sterilization of Arabidopsis Seeds 
	Note: Select one of the following procedures for seed sterilization. Any of them is suitable for the next steps of the protocol. Gas sterilization (1.1.2) presents two main advantages over liquid sterilization (1.1.1): it takes less time when handling a large number of seeds, since no pipetting has to occur for the individual samples; and the sterilized seeds can be stored for a longer period. However, it requires a desiccator and careful handling.
	<ol>
		<li>Liquid sterilization
		<ol>
			<li>Pour the desired number of seeds in micro-centrifuge tubes. Use up to 20 mg (~800 seeds) in a 1.5&#160;ml&#160;tube or 40 mg (~1600 seeds) in a 2 ml&#160;micro-centrifuge tubes.</li>
			<li>Add 1 ml of 70% ethanol. Invert or gently shake for 2 min.</li>
			<li>Replace the 70% ethanol with 1 ml sterilization solution for 15 min. (Prepare fresh sterilization solution: 3.85 ml sodium hypochlorite (NaOCl) stock (12%) (CAUTION: Use fume hood), 5 &#181;l&#160;Tween 20, up to 10 ml with water. Invert the tubes several times to make sure that all seeds come in contact with the solution.</li>
			<li>In a laminar air flow cabinet, replace the sterilization solution with 1 ml sterile distilled water and rinse the seeds 5 times for 5 min by repeatedly replacing with 1 ml of fresh sterile distilled water.</li>
		</ol>
		</li>
		<li>Gas Sterilization
		<ol>
			<li>Pour the desired number of seeds in a 2 ml micro-centrifuge tube. Use individual tubes when working with several independent lines, and arrange them opened in a plastic micro-centrifuge tubes box or holder. If the number of seeds in one tube exceeds 500 (12.5 mg), subdivide the seeds in the appropriate number of tubes to ensure successful sterilization.&#160;Make sure the caps of neighboring tubes do not cover each other. Fit together the lid of the box on the bottom, as it needs to be in the desiccator as well for sterilization.</li>
			<li>Place the box in the bowl of a desiccator, along with a glass beaker (CAUTION: Use fume hood).</li>
			<li>Fill the beaker with 100 ml of 12% NaOCl (CAUTION: Use fume hood). Put the lid on the desiccator, but leave a small aperture where the beaker is placed.</li>
			<li>Add 3 ml of 37% hydrochloric acid (fuming) (CAUTION: Use fume hood) to the beaker through the small aperture and quickly close the desiccator. The solution will bubble for some time due to Cl2-gas formation. Leave the system closed O/N (or at least 8 hr).</li>
			<li>Remove the lid of the desiccator. Take out the box and cover it with the lid to avoid contamination during transport.</li>
			<li>Put the box in a laminar air flow cabinet, remove the lid and leave the seeds for about 1 hr at RT&#160;to allow gas release. 
			Note: Seeds can be safely stored dry in closed micro-centrifuge tubes for up to 2 weeks at 4 &#176;C.&#160;&#160;&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lateral Root Induction in Arabidopsis
	<ol>
		<li>Lateral Root Inhibition (NPA Treatment)
		<ol>
			<li>Prepare growth medium for in vitro growth. The medium contains half strength Murashige and Skoog salt mixture15, 0.1 g/L myo-inositol, 10 g/L sucrose, and is buffered with 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES).</li>
			<li>Adjust the pH to 5.7 with 1 M KOH. Add 8 g/L of plant tissue agar and autoclave the medium for 20 min at 121 &#176;C.</li>
			<li>Prepare a 25 mM stock solution of NPA in dimethylsulfoxide (DMSO) (CAUTION: Use gloves). In a laminar air flow cabinet, cool the autoclaved medium down to approximately 65 &#176;C (which is the temperature at which the bottle can be held by hand), and add 25 mM NPA stock solution to obtain a final concentration of 10 &#181;M NPA.</li>
			<li>Homogenize upon addition by gently shaking the bottle for 10 sec. Pour 50 ml of medium per square petri dish (12 cm x 12 cm). 
			Note: In case of large scale experiment, apply a nylon mesh (20 &#181;m) to ensure easy transfer. Prepare a mesh (9 cm x 9 cm) for autoclaving (Figure 1A). When autoclaved, apply the mesh to the growth medium using sterile tweezers (Figure 1B). Gently push the mesh to the growth medium using a sterilized&#160;drigalsky to make sure it is in good contact with the growth medium (Figure 1B) (CAUTION: Work in sterile conditions).&#160;</li>
			<li>Sow the seeds on the NPA-plates. 
			Note: In case a large scale experiment is planned, sow 2 dense and well-aligned rows of 50 seeds to facilitate the sampling (see 1.2.2.3).
			<ol>
				<li>In case of liquid sterilization, sow the seeds on NPA-plates using a 200 &#181;l pipet. Cut off 3 - 4 mm of the end of the pipetting&#160;tips in sterile conditions (or before sterilization of the tips) in order to be able to pipet the seeds. Pipet up and down a couple of times to re-suspend the seeds, then pipet approximately 150 &#181;l sterile water with 10 - 20 seeds and wait till the seeds sediment at the bottom of the tip. (CAUTION: Work in sterile conditions) 
				Note: When touching the agar or the mesh gently with the tip, a drop of water with a seed will be released from it (Figure 1C).</li>
				<li>In case of gas sterilization, sow the seeds using autoclaved toothpicks.Pinch the toothpick into the agar before taking the seeds to ensure a sticky surface. Pick up the seeds one by one using the toothpick and distribute the seeds on the plates (Figure 1D). Alternatively, add sterile water to the seeds, and sow as described in 1.2.1.5.1 (CAUTION: Work in sterile conditions).</li>
			</ol>
			</li>
			<li>Seal the plates with breathable adhesive tape and store them plates at 4 &#176;C in the dark for at least 2 days and maximum one week. This is needed for stratification and ensures synchronized and more efficient germination. 
			Note: Alternatively, seeds can be stratified before sowing, immersed in distilled water in micro-centrifuge tubes, which requires less refrigerator space. In this case, store the tubes for at least one week at 4 &#176;C, and move the plates to the growth cabinet immediately after sowing (see 1.2.1.7). 
			DAY 3</li>
			<li>Move the plates to the growth cabinet (continuous light (110 &#181;E m-2 s-1), 21 &#176;C) for 5 days (2 days of germination and 3 days of growth) in a nearly vertical position (80 to 90 &#176;) to ensure root growth on, and not in the medium. 
			DAY 8&#160;</li>
		</ol>
		</li>
		<li>Lateral Root Induction (NAA Treatment)
		<ol>
			<li>Prepare the same growth medium as described in 1.2.1.1 and 1.2.1.2. Prepare a 50 mM stock solution of NAA in DMSO (CAUTION: Use gloves). Cool the autoclaved medium down to 65 &#176;C and add NAA solution to the medium to obtain a final concentration of 10 &#181;M NAA (CAUTION: Work in sterile conditions).
			<ol>
				<li>Homogenize upon addition by gently shaking the bottle for 10 sec. Pour 50 ml of medium per square petri dish (12 cm x 12 cm).</li>
			</ol>
			</li>
			<li>Transfer the seedlings from the plates containing growth medium supplemented with 10 &#181;M NPA to those supplemented with 10 &#181;M NAA.
			<ol>
				<li>Hook the arms of curved tweezers under the cotyledons of the seedling and gently remove it from the plate. Only transfer seedlings of which the roots have grown entirely in contact with the NPA-growth medium and preferably downwards.</li>
				<li>Skim the root over the NAA-containing growth medium surface over a small distance. Make sure that the plant roots are in good contact with the growth medium. If needed, press the root gently to the growth medium surface with the tweezers. 
				Note: In case of a large-scale experiment, remove all seedlings that are not in contact with the mesh and transfer by lifting the mesh at the two upper corners with tweezers. Make sure the mesh is in good contact with the NAA-containing medium by skimming the mesh over the agar surface over a small distance (Figure 1E).&#160;</li>
			</ol>
			</li>
			<li>Seal the new plates with breathable tape and place them in the growth cabinet (continuous light (110 &#181;E m-2 s-1), 21 &#176;C) in a vertical position for the desired time after induction until sampling. 
			Note: In case of a large-scale experiment, 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.&#160;&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. LRIS Maize Protocol
<ol>
	<li>Stratification of Maize Kernels
	<ol>
		<li>Incubate the maize kernels at 4 &#176;C in the dark for at least 1 week. 
		Note: This protocol has been developed using the B73 maize inbred line.</li>
	</ol>
	</li>
	<li>Sterilization of Maize Kernels 
	DAY 1 
	Note: Although maize seedlings do not have to be grown in sterile conditions, it is recommended to sterilize the maize kernels and work with sterile material to prevent fungal growth in the paper roll system.
	<ol>
		<li>Put the necessary number of maize kernels in a sterilized glass beaker.</li>
		<li>Add 100 ml of sterilization solution (6% NaOCl in water) (CAUTION: Use fume hood).</li>
		<li>Add a magnetic stir bar and place the beaker on a magnetic stirrer at low stirring speed (approximately 250 rpm) at RT&#160;for 5 min.</li>
		<li>Rinse five times for 5 min by replacing the solution with 100 ml of fresh sterile water.</li>
	</ol>
	</li>
	<li>&#160;Sowing Maize Kernels
	<ol>
		<li>Put on gloves to reduce the risk of contamination, and take a roll of paper towels.</li>
		<li>Tear off two stretches of hand towel paper with a total length of two sheets (approximately 92 cm x 24 cm) and place them on top of each other on a clean surface (Figure 2A).</li>
		<li>Fold the sheets double over the length (approximately 92 cm x 12 cm) (Figure 2A).</li>
		<li>Use tweezers to distribute 10 kernels at approximately 2 cm from the top over the entire length of the paper, while keeping an interspace of 8 cm between each kernel and 8 cm free at both ends (Figure 2B). Make sure the radicle of the kernel is facing down and toward the paper (Figure 2B).</li>
		<li>Gently roll the paper over the length, while keeping the seeds in place (Figure 2B). To facilitate the rolling, it is optional to first spray the paper with sterile water.</li>
		<li>Put the paper rolls in sterilized glass tubes of approximately 6 cm diameter and 14 cm height (e.g., 250 ml centrifuge tubes) and place them in a rack (Figure 2C).</li>
	</ol>
	</li>
	<li>Lateral Root Induction in Maize
	<ol>
		<li>Prepare a 50 mM NPA stock solution (dissolved in DMSO) (CAUTION: Use gloves).</li>
		<li>For each tube, make a 50 &#181;M NPA solution by adding 125 &#181;l from the 50 mM NPA stock solution to 125 ml sterile water in a sterilized glass beaker.</li>
		<li>Mix well and pour 125 ml of the 50 &#181;M NPA solution over the paper roll in the tube. The paper roll will absorb the solution and become completely soaked. 
		Note: When using paper rolls and tubes with other dimensions, adjust the volume accordingly. The liquid should reach halfway the tube to ensure sufficient uptake.</li>
		<li>Place the paper roll system in a growth cabinet for three days (27 &#176;C, continuous light, 70% relative humidity). Make sure that the paper rolls remain soaked by adding extra 50 &#181;M NPA solution, since the solution evaporates over time (Figure 2D). 
		DAY 4</li>
		<li>Prepare a 50 mM NAA stock solution (dissolved in DMSO) (CAUTION: Use gloves).</li>
		<li>For each tube, prepare a 50 &#181;M NAA solution by adding 125 &#181;l from the 50 mM NAA stock to 125 ml sterile water in a sterilized glass beaker.</li>
		<li>Gently squeeze out most of the remaining NPA solution from the paper rolls and place them in sterile water for 5 min to wash it out (CAUTION: Use gloves). Gently squeeze out most of the water from the paper rolls. Repeat this washing step three times.</li>
		<li>Mix well and pour the 50 &#181;M NAA solution over the paper rolls in the tubes. Make sure they are completely soaked.</li>
		<li>Place the paper roll system in the growth cabinet (27 &#176;C, continuous light, 70% relative humidity) for the desired duration after induction.</li>
	</ol>
	</li>
	<li>Lateral Root Induction in Adventitious Crown Roots of Maize 
	Note: The LRIS as described above induces lateral roots in the primary root. However, in maize and other monocotyledons, the primary root and the embryonic seminal roots, together with their lateral roots, are mainly important during seedling development. At a later stage in plant growth, the post-embryonic adventitious roots arise on the stem and also develop lateral roots to form a new root system16. The LRIS can also be used to induce lateral roots on these post-embryonic adventitious crown roots by following some minor adjustments to the LRIS on the embryonic primary root.
	<ol>
		<li>To make the paper rolls, create a sandwich of papers with respectively two layers of hand towel paper on the outer side, and two layers of germination paper within. Place the sterilized kernels (see steps 2.1 to 2.2) in between the two sheets of germination paper. Germination paper, compared to hand towel paper, will be less prone to be penetrated by root hairs and lateral roots, which will make the opening of the paper rolls easier later on. On the other hand, the two external layers of hand towel paper will facilitate absorption of the liquid.</li>
		<li>Insert the rolls in 700 ml glass tubes and pour sterile water without NPA over them. Make sure they are completely soaked.</li>
		<li>Germinate the sterilized kernels in the growth cabinet (see step 2.4.9).</li>
		<li>Grow the seedlings till the adventitious crown roots start to emerge (6 days for B73). Make sure the paper rolls are kept wet at all times by adding sterile water when necessary.</li>
		<li>Transfer to a 25 &#181;M NPA solution for 4 days (see steps 2.4.1 to 2.4.4), followed by a similar induction of lateral root initiation by replacing the NPA solution with a 50 &#181;M NAA solution after a washing step (see step 2.4.5 to 2.4.9).</li>
	</ol>
	</li>
</ol>

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<li>Wang, S., Taketa, S., Ichii, M., Xu, L., Xia, K., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lateral+root+formation+in+rice+%28Oryza+Sativa.+L.%29%3A+differential+effects+of+indole-3-acetic+acid+and+indole-3-butyric+acid%5BTitle%5D)+AND+%22Plant+Growth+Regul%22%5BJournal%5D)">Lateral root formation in rice (Oryza Sativa. L.): differential effects of indole-3-acetic acid and indole-3-butyric acid.</a> Plant Growth Regul. 41, 41-47 (2003).</li>
<li>Martinez-de la Cruz, E., Garcia-Ramirez, E., Vazquez-Ramos, J. M., Reyes de la Cruz, H., Lopez-Bucio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auxins+differentially+regulate+root+system+architecture+and+cell+cycle+protein+levels+in+maize+seedlings%5BTitle%5D)+AND+%22J+Plant+Physiol%22%5BJournal%5D)">Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.</a> J Plant Physiol. 176, 147-156 (2015).</li>
<li>Beyer, E. M., Morgan, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+ethylene+on+uptake%2C+distribution%2C+and+metabolism+of+indoleacetic+acid-1-14C+and+-2-14C+and+naphthaleneacetic+acid-1-14C%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Effect of ethylene on uptake, distribution, and metabolism of indoleacetic acid-1-14C and -2-14C and naphthaleneacetic acid-1-14C.</a> Plant Physiol. 46, 157-162 (1970).</li>
<li>Delbarre, A., Muller, P., Imhoff, V., Guern, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+mechanisms+controlling+uptake+and+accumulation+of+2%2C4-dichlorophenoxy+acetic+acid%2C+naphthalene-1-acetic+acid%2C+and+indole-3-acetic+acid+in+suspension-cultured+tobacco+cells%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells.</a> Planta. 198, 532-541 (1996).</li>
<li>Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microgenomics%3A+genome-scale%2C+cell-specific+monitoring+of+multiple+gene+regulation+tiers%5BTitle%5D)+AND+%22Annu+Rev+Plant+Biol%22%5BJournal%5D)">Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers.</a> Annu Rev Plant Biol. 64, 293-325 (2013).</li>
<li>Perez-Torres, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphate+availability+alters+lateral+root+development+in+Arabidopsis+by+modulating+auxin+sensitivity+via+a+mechanism+involving+the+TIR1+auxin+receptor%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor.</a> Plant Cell. 20, 3258-3272 (2008).</li>
</ol>

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

lateral root inducible system in arabidopsis 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 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 ...

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

lateral-root-inducible-system-in-arabidopsis-and-maize

Research

JoVE Journal

Biology

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

A Method for Characterizing Embryogenesis in Arabidopsis

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Learn the Lateral Root Inducible System for synchronous lateral root initiation in Arabidopsis and maize using hormone treatments. This method enables cytological and molecular study of root development.

Lateral Root Inducible System in Arabidopsis and Maize

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