Name: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays (Video) | JoVE Journal
Uploaded: 2011-03-27T14:00:00.000+00:00
Duration: 13 min 37 s
Description: Learn to generate hairy root composite plants in Medicago truncatula for studying nodulation and plant-rhizobium symbiosis. This rapid Agrobacterium-mediated method produces transgenic roots within weeks without traditional tissue culture.

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The authors would like to thank Dr. Chris Taylor (Ohio State University) for introducing hairy root technology to our lab and Drs. Senthil Subramanian (South Dakota State University), Juan Zhang (Ludong University, Shandong China) for improving the protocol. We also thank Dr. Hao Cheng (Nanjing Agriculture University) for helps in making this video. This work is supported in part by grants from DOE (DE-SC0001295), NSF (MCB-0923779) and USDA (2010-65116-20514) to O.Y

The following protocol has been used to generate hairy root composite plants in a model legume species M. truncatula. Similar protocols have been adapted for at least eight plant species 1-4. We used M. truncatula hairy root composite plants to study gene functions in root and nodule development. The protocol was separated into four sections: 1) preparing plant materials; 2) generating hairy root composite plants; 3) symbiotic Rhizobia infection; and 4) transgenic root identification. We used the binary vector containing the green fluorescent protein (gfp) gene as a reporter for screening transgenic root in composite plants 3. Compared to antibiotics-based selection, GFP-based screening is fast, easy, and inexpensive. In our construct, the ER-expression-optimized gfp gene is driven by the super ubiquitin promoter, which has strong constitutive GFP signals in transgenic roots, allowing easy distinction between transgenic and non-transgenic roots.
1. Preparing Plant Materials
<ol>
	<li>M. truncautlaseeds are harvested from plants grown in a greenhouse (50% relative humidity, 16/8 h light/dark, 22/18°C day/night temperature). Mature seeds can be stored at 4°C.</li>
	<li>Approximately 100 seeds are scarified in 10 ml concentrated sulfuric acid (95-98%, Sigma-Aldrich, MO) for 10 min with periodic shaking. </li>
	<li>The seeds are rinsed 5-7 times with sterile water with gentle agitation.</li>
	<li>The seeds are then soaked in water at room temperature for 6 hours and kept at 4°C for 36-48 hours to synchronize germination.</li>
	<li>Approximately 20-30 seeds are spread on moist filter papers placed in a Petri dish. They are maintained at 22°C, with 16/8 h light/dark cycle, and 40 μmol.m-2.s-1 light intensity, for 5-6 days.</li>
	<li>Germinated seedlings are transferred into soil and placed in greenhouse for a month.</li>
</ol>
2. Generating Hairy Root Composite Plants
<ol>
	<li>Binary constructs carrying genes of interest were transformed into A. rhizogenes using standard protocols. We have engineered a set of binary vectors containing the gfp gene as a marker that facilitate screening of transgenic tissues. These vectors contain various promoters and all have Gateway cloning sites (Invitrogen, Carlsbad, CA) for over-expression or silencing of targeted genes 3,5.</li>
	<li>A. rhizogenesis cultured in 50ml LB medium with appropriate antibiotic selection at 28°C for 16-24 hours.</li>
	<li>The bacteria are collected and re-suspended to a final concentration of OD600=0.3 in a nitrogen-free plant nutrient solution (Table 1 and 6).</li>
	<li>To support hairy root regeneration, a sterilized supporting matrix ("Rock Wool", Hummert International, Earth City, MO) is cut into plugs of approximately 3 cm3. We place 4 plugs into one Petri dish.</li>
	<li>A hole is poked on top of each plug using a pipette tip to facilitate the insertion of explants. A. rhizogenes (5 mL) is added to each plug. </li>
	<li>A shoot section with 2-3 axillary buds is excised by a slanting cut. The explant is then inserted into the plug, and grown at 22°C for about three weeks. We keep the Medicago explants without watering for the first 10 days, then, water them with 5 ml nitrogen-free solution when necessary. We found that removing the apical meristem might decrease the formation of adventitious roots.</li>
</ol>
3. Symbiotic Rhizobia (Sinorhizobium Meliloti) Infection
<ol>
	<li>After three weeks, some adventitious roots will emerge from the Rock Wool. The hairy root composite plants are transferred to sand or vermiculite (sterilized), with or without the Rock Wool. They are grown in greenhouse at 22°C, 16/8 h light/dark cycle, and 300 μmol.m-2.s-1 light intensity for one more week.</li>
	<li>Prior to rhizobia infection, S. meliloti 1201 is cultured in 50 ml yeast extract-mannitol medium for 7 days at 30 °C (Table 2, and 6,7).</li>
	<li>Re-suspended rhizobia cells are diluted to a final concentration of OD600=0.08 using nitrogen free nutrition solution. A 10-ml fresh suspension ofS. meliloti was applied to each composite plant to induce nodule formation.</li>
</ol>
4. Transgenic Root Identification
<ol>
	<li>Two weeks after rhizobia inoculation, the composite plants are up-rooted by washing in water. The roots can be easily separated from sands or perlite in water.</li>
	<li>We place the hairy roots under a UV-microscope (Nikon SMZ1500, Excitation 460-500nm, Dichroic 505nm, Barrier more than 510nm). The transgenic roots are GFP positive and the adventitious roots are GFP negative. We then collect the roots according the GFP signal, and count the nodule numbers, measure the root length, and calculate lateral root density. The GFP negative roots are used as control.</li>
</ol>
5. Representative Result
In our experiment, the new hair roots regenerated from the explants in 2-3 weeks after A. rhizogenes innoculation. Under the UV-microscope, transgenic roots carrying the gfp gene show strong green fluorescence (Fig 1). The amount of regenerated roots and the portion of the GFP positive roots depend on the conditions of explants and the growth environment of composite plants.On average, 25% of the roots produced were transgenic hairy roots 3.To increase the transformation efficiency, 1) the plastic cover doom, as shown on the video, is enough to maintain the humidity in the growth tray for the first few days, and plants only require little water in the first few days. Excessive watering is detrimental to hairy root formation; 2) the concentration of Agrobacterium innoculant is important for transformation. Excessive amount of cells are not helpful to hairy root formation or nodule formation. For nodule formation, the watering solution needs to be nitrogen-free, otherwise, few nodules will form.
The hairy root composite plants can be generated using cotyledons or entact seedlings as the starting material. Only minor modifications of the above protocol are ncessary to generate hairy roots from other tissues 8. Importantly, each hairy root is an independent transformation event. Therefore, the phenotypes observed in one composite plant are the sum of several transforamtion events that needed to be confirmed by repetitions in multiple composite plants
 <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Fluorescence microscopy images; A, B showing fungal hyphae structure with highlighted regions." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. A. Transgenic roots can be sorted out from hairy roots. B. Nodules were formed in the hairy root composite plants. We placed 4-week-old M. truncatula hairy root plants under the UV-microscope and the GFP positive roots can be easily identified. (Red arrow: GFP root; Yellow arrow: non-GFP root)
<table border="1">
	<tbody>
		<tr>
			<td>Stock</td>
			<td>g/200ml</td>
			<td>ml stock/ liter</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</td>
			<td>12.3</td>
			<td>2</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>14.7</td>
			<td>4</td>
		</tr>
		<tr>
			<td>K2HPO4.3H2O</td>
			<td>6.8</td>
			<td>1</td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>11</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Fe Cl3.6H2O</td>
			<td>0.49</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Micronutrients</td>
			<td>See below</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>Micronutrients</td>
			<td>g per liter</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>0.142</td>
		</tr>
		<tr>
			<td>MnSO4.H2O</td>
			<td>0.077</td>
		</tr>
		<tr>
			<td>ZnSO4.7H2O</td>
			<td>0.1725</td>
		</tr>
		<tr>
			<td>CuSO4.5H2O</td>
			<td>0.037</td>
		</tr>
		<tr>
			<td>NaMoO4. H2O</td>
			<td>0.024</td>
		</tr>
		<tr>
			<td>CoCl2. H2O</td>
			<td>0.0025</td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>0.001</td>
		</tr>
	</tbody>
</table>
Table 1. Nitogen-free nutrition solution
<table border="1">
	<tbody>
		<tr>
			<td>K2HPO4</td>
			<td>0.5g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>0.1g</td>
		</tr>
		<tr>
			<td>MgSO4·7H2O</td>
			<td>0.2g</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>0.4g</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>pH=6.8</td>
			<td>
			</td>
		</tr>
	</tbody>
</table>
Table 2. yeast extract mannitol medium (per liter)

<ol><li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula%5BTitle%5D)+AND+%22J.+Exp.+Bot%22%5BJournal%5D)">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li>
<li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vitro+composite+plants%3A+an+inexpensive%2C+rapid+method+for+root+biology%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li>
<li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li>
<li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations%5BTitle%5D)+AND+%22Mol.+Plant-Microbe+Interact%22%5BJournal%5D)">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li>
<li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 Forthcoming.</li>
<li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots%5BTitle%5D)+AND+%22Plant+Mol+Biol%22%5BJournal%5D)">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li>
<li>Vincent, J. A manual for the practical study of root nodule bacteria International Biological Program Handbook. , Blackwell Science Ltd. Oxford. 1-13 (1970).</li>
<li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li>
</ol>

No conflicts of interest declared.

Generating hairy root composite plant is a quick and easy method to obtain large quantities of transgenic material for many dicot species. Although this method cannot produce transgenic seeds, it can produce transgenic materials in a few weeks. The method is especially suitable for plants that have difficulty establishing tissue cultures or generating stable transformants. Over the years, we have used this technology to study gene functions, promoter functions, microRNAs, root and lateral root development, defense and abiotic stress responses, nodulation and other symbiotic processes, hormone responses, metabolic profiling, gene profiling, proteomic analysis, and other biological processes. The protocol is robust and replicable.

generation of composite plants in medicago truncatula used for nodulation assays

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Hi, my name is Oliver Yu at the Dan Plant Science Center. Today we're going to show you how to generate Ute Compote Plants by Agrobacterium Ogn. This technique was brought to Dan Center by Dr.Chris Taylor, currently at Ohio State University.Several of my former group members improved these techniques over the years and today my current group member, Dr.Stone and Mao, put together this video to show how Harry Root Composite Plans is produced. Agrobacterium ZOS are very similar to agrobacterium tumor fis in that they introduce foreign DNA into plants, but the ZOS can induce higher root formation of wild type tissues forming composite plants as this one. So on the, on these campy plants, some of the roots are transgenic while the shoots are still wild tap.Therefore, this technology will not produce transgenic seeds. However, there are three major advantages of high root campy plants that make this technology very useful. First, many plant species, especially dicho species, can be transformed by agro bacterial ous.Second, we do not need to establish tissue culture prior to transformation. We can take many parts of plants such as seedlings, shoots and co to generate campy plants directly. And finally, we can get transgenic root tissue in a matter of weeks.For soybean and medic cargo trans, we can get transgenic roots in as short as three weeks faster than a OPIS transformation. Overall, the higher root cyte plant technology is very useful tool to study gene functions and the root related phenotypes. No.Hello, my name is Ong.Hello, my name is, There is my cameraman bill. Today we'll introduce Harry root Composite Plant protocol. Yeah, we use this protocol in our several projects.I already, we Started, yeah.Yeah. For our demonstration, we'll use meta cargo Tula as an example. These are meta cargo mature pods.Put the pods on a grinding box and then grind the pods with a long firm Motion. Separate the seeds from the seed pods by gently shaking place Seeds in a 50 milliliter centrifuge tube. Then add five milliliters of concentrated sulfuric acid.Very Carefully scarring. Takes about 10 minutes. An acid Vortex the tube two to three times.During the acid treatment, the seed coat will change to a yellow color. Remove sulfuric acid from the tube and rinse the seeds at least five times with sterile water. Soak The seeds in deionized water at room temperature for six hours, and then keep them at four degrees centigrade for two days.To synchronize germination. Spread 20 to 30 seeds on moist filter paper in the Petri dish. After five to six days, transfer the seed links to soil and then grow them in the greenhouse for four weeks.For composite plant transformation, we normally use the agrobacterium Zony K 5 99 strain. The shuttle vector or binary vector can be transformed into the K 5 99 following the standard agrobacterium tumor facit transformation protocol Inoculate the agrobacterium, Grow them at 30 Degrees centigrade for 16 to 24 hours after the growth period. Collect the cells by placing the culture solution in a 50 milliliter centrifuge tube.Run it in the centrifuge at 5, 000 G for 30 minutes. Resuspend the pellet with nitrogen free nutrient solution. Make sure to dilute the agrobacterium cells to 0.3 at OD 600.This concentration is important for maximum transformation. We use rock wall as a supporting matrix. Using this material is important for high frequency transformation.Cut the rock wall into the right size.Plug. Poke a hole at the top about one and a half to two centimeters in diameter. Using a pipette tip, add five milliliters of Resuspended Agrobacterium Zony solution into each plug.Select a stem section with two to three auxiliary buds and make a slanting cut. Insert the plant sections into the hole in the rock wall. Place the Cut plants into a growth chamber or lab bench for three weeks.Add five milliliters of nutrient solution into each plug every 10 days. After three weeks, the roots are regenerated from the cut side. Soak the plant in water.Remove the softened rock wall carefully and do not damage the roots. Be sure to remove all of the rock Wall. You can transfer the hairy root composite plants to sand or pearlite For symbiosis experiments, grow the rhizobium in a yeast extract manito medium for seven days at 30 degrees Celsius.Collect the zoia cells Resus. Suspend the pellet with the nitrogen free nutrient solution. Dilute the solution to 0.08 at OD 600.Add 10 milliliters of ZOA solution to each composite plant culture, the plant for an additional two to three weeks to allow nodules to form. Remove sand from the composite plants by uprooting and washing them. Use green fluorescent protein expression to check the transgenic root.You'll use a fluorescent microscope to do this. All our hairy root constructs have a constitutive GFP cassette for screening transgenic lines. This picture shows the GFP transgenic hairy root composite plants observed in the fluorescent microscope.The transgenic roots with the brighter green fluorescence are easy to distinguish from the non transgenic roots. Nodules are formed in the hairy root of the composite plants. Here is the complete protocol.Once again, generating hairy root composite plants is a quick and easy method to obtain large quantities of transgenic tissue, especially for diop species. Although this method cannot give you transgenic seeds, it can produce transgenic material in a few weeks. It's especially suitable for plants that have difficulty in establishing tissue culture and transformation.Over the years, we have used this technology to study gene functions, promoter functions, microRNAs, root and lateral root developments, defense and antibiotic stress response, nodulation and other symbiotic process. We studied hormone response, metabolic profiling, gene profiling, and the proteomic analysis using the higher root composite plants. We hope this video will help you adapt this system to your favorite species.You are welcome to contact us for questions related to this technology and Good luck.

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Learn to generate hairy root composite plants in Medicago truncatula for studying nodulation and plant-rhizobium symbiosis. This rapid Agrobacterium-mediated method produces transgenic roots within weeks without traditional tissue culture.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

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