This work was funded by the USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative grant 2010-65108- 20582 to K.M.J. We thank Brian K. Washburn for critical review of the manuscript.

Performing steps 1, 2, 4, and 6 in a sterile laminar flow hood is recommended.
1. Preparation of M. truncatula A17 Seedlings
Note: M. truncatula A17 seeds used in these studies are produced under cooled greenhouse conditions at ~22 &#176;C, or in a plant growth room maintained at 22-26 &#176;C with ~150-400 mmol/m-2 s-1 light.
<ol>
	<li>Pour seed germination plates: Use 100 mm square plates (15 mm deep) and pour autoclaved 1...

<ol>
	<li>Galibert, F., 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=The+composite+genome+of+the+legume+symbiont+Sinorhizobium+meliloti.">The composite genome of the legume symbiont Sinorhizobium meliloti.</a> Science. 293, 668-672 (2001).</li><li>Young, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+genome+provides+insight+into+the+evolution+of+rhizobial+symbioses.">The Medicago genome provides insight into the evolution of rhizobial symbioses.</a> Nature. 480, 520-524 (2011).</li><li>Glazebrook, J., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+techniques+in+Rhizobium+meliloti.">Genetic techniques in Rhizobium meliloti.</a> Methods Enzymol. 204, 398-418 (1991).</li><li>Cheng, X., Wen, J., Tadege, M., Ratet, P., Mysore, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetics+in+medicago+truncatula+using+Tnt1+insertion+mutants.">Reverse genetics in medicago truncatula using Tnt1 insertion mutants.</a> Methods Mol Biol. 678, 179-190 (2011).</li><li>Leigh, J. A., Signer, E. R., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-deficient+mutants+of+Rhizobium+meliloti+that+form+ineffective+nodules.">Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.</a> Proc. Natl. Acad. Sci. USA. 82, 6231-6235 (1985).</li><li>Wong, K. K. Y., Fortin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Petri+Dish+Technique+for+the+Aseptic+Synthesis+of+Ectomycorrhizae.">A Petri Dish Technique for the Aseptic Synthesis of Ectomycorrhizae.</a> Canadian Journal of Botany-Revue Canadienne De Botanique. 67, 1713-1716 (1989).</li><li>Barker, D. G., Mathesius, U., Journet, E. P., Sumner, L. W., 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=.">.</a> The Medicago truncatula handbook. , (2006).</li><li>Leonard, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Assembly+for+Use+in+the+Testing+of+Cultures+of+Rhizobia.">A Simple Assembly for Use in the Testing of Cultures of Rhizobia.</a> J Bacteriol. 45, 523-527 (1943).</li><li>Trung, B. C., Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+Leonard+jar+assembly+for+screening+of+effective+rhizobium.">Improvement of Leonard jar assembly for screening of effective rhizobium.</a> Soil Sci Plant Nutr. 29, 97-100 (1983).</li><li>Penmetsa, R. V., Cook, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+diverse+developmental+mutants+of+Medicago+truncatula.">Production and characterization of diverse developmental mutants of Medicago truncatula.</a> Plant Physiol. 123, 1387-1398 (2000).</li><li>Garcia, J., Barker, D. G., Journet, E. P., Mathesius, U., Journet, E. P., Sumner, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Medicago truncatula handbook. , (2006).</li><li>Vincent, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Manual for the Practical Study of the Root-Nodule Bacteria. , (1970).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1982).</li><li>Pladys, D., Vance, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+during+Development+and+Senescence+of+Effective+and+Plant+Gene-Controlled+Ineffective+Alfalfa+Nodules.">Proteolysis during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules.</a> Plant Physiology. 103, 379-384 (1993).</li><li>Vasse, J., de Billy, F., Truchet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abortion+of+infection+during+the+Rhizobium+meliloti-alfalfa+symbiotic+interaction+is+accompanied+by+a+hypersensitive+reaction.">Abortion of infection during the Rhizobium meliloti-alfalfa symbiotic interaction is accompanied by a hypersensitive reaction.</a> The Plant J. 4, 555-566 (1993).</li><li>Johnson, L. E. B., Vance, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+Biochemical+Comparisons+of+Plant+Induced+Ineffective+Nodules.">Histological and Biochemical Comparisons of Plant Induced Ineffective Nodules.</a> Phytopathology. 71, 884 (1981).</li><li>Vance, C. P., Johnson, L. E. B., Hardarson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+Comparisons+of+Plant+and+Rhizobium+Induced+Ineffective+Nodules+in+Alfalfa.">Histological Comparisons of Plant and Rhizobium Induced Ineffective Nodules in Alfalfa.</a> Physiological Plant Pathology. 17, 167 (1980).</li><li>Van de Velde, W., 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=Aging+in+legume+symbiosis.+A+molecular+view+on+nodule+senescence+in+Medicago+truncatula.">Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula.</a> Plant Physiol. 141, 711-720 (2006).</li><li>Jones, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+production+of+the+exopolysaccharide+succinoglycan+enhances+Sinorhizobium+meliloti+1021+symbiosis+with+the+host+plant+Medicago+truncatula.">Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula.</a> J Bacteriol. 194, 4322-4331 (2012).</li><li>Queiroux, C., 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=A+comparative+genomics+screen+identifies+a+Sinorhizobium+meliloti+1021+sodM-like+gene+strongly+expressed+within+host+plant+nodules.">A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules.</a> BMC Microbiol. 12, 74 (2012).</li></ol>

The authors have nothing to disclose.

There are several steps of the protocol that are critical for success: 1) The necessity of using purified, plant cell-culture tested agar cannot be overemphasized. This is not critical for alfalfa seedlings, but it is critical for growth of M. truncatula A17. 2) It is important to germinate seedlings on vertical agar plates as described in Step 1. The widely-used technique of germinating seedlings on an inverted horizontal surface in a 15 mm deep plate7 is not advised for this protocol because ...

The interaction between the legume host plant Medicago truncatula A17 and the rhizobial bacterium Sinorhizobium meliloti 1021 is one of the most tractable model systems for the study of root nodule development and nitrogen-fixing symbiosis. The genomes of both symbiotic partners have been sequenced1,2 and both plant and bacterium are amenable to genetic manipulation3,4 . Analysis of the phenotypes of both plant and bacterial mutants requires the ability to observe the stages of nodule development and to quantify the symbiotic productivity over time. Here we describe a method for observation of root nodule development of M. truncatula cv. Jemalong A17 inoculated with S. meliloti 1021 within individual microcosms made from standard, round 100 mm diameter, 15 mm deep laboratory plates (Figure 1A). The shoot is exposed and grows through a notched portal in the side of the plate (Figures 1B, 1C, and 1E). Roots are contained within the microcosms and kept sterile, while allowing observation through the lid of the plate (Figure 1D). Since the shoot is accessible, its growth is not constrained and it can be measured at periodic intervals without compromising the sterility of the root. The method of creating notched-plate microcosms was originally developed by Leigh, et al.5 for growing alfalfa plants, but it was not widely adopted in spite of its many advantages over other methods. A variation on this method was also developed for the analysis of the interaction between plant roots and mycorrhizae6. We have now adapted and optimized this method for the growth of M. truncatula plants. The advantages of this protocol over more commonly used methods are described below.
There are several methods that are currently in wide use for nodulation studies of M. truncatula inoculated with S. meliloti7. The most commonly used method for large-scale preparation of nodulated roots is growth in aeroponic caissons7. In this method, plants are suspended over a large vessel and roots are aerated with a mixture of S. meliloti and nutrient solution7. This method is practical if only one genotype of S. meliloti is to be tested. Since it requires aerosolization of high concentrations of bacteria, there is a high probability of cross-contamination between caissons inoculated with different bacterial strains. Another method that is often used to prepare large quantities of inoculated plants is growth in tubs or pots of perlite, vermiculite, sand or calcined clay that are infused with nutrient solution and inoculated with S. meliloti7. This method requires the use of open tubs or pots, and requires watering and replenishment of the nutrient solution. Another disadvantage of pots is that plants must be removed from this particulate matrix for examination of the roots and nodules. Another drawback of this method is that a large area of incubator space is required when many different S. meliloti genotypes are to be compared, because a separate pot must be used for each bacterial genotype. The &#34;Leonard jar&#34; is a variation on this method8,9 . A Leonard jar is composed of two sterilized vessels stacked one on top of the other and connected by a wick. Growth medium is placed in the lower vessel and drawn through the wick by capillary action into a growth matrix of perlite, vermiculite, sand or calcined clay in the upper vessel. The seedling(s) are placed in the growth matrix and inoculated with S. meliloti. This method does not require watering, but examination of roots and nodules requires that the seedling be removed from the particulate growth matrix.
There are several methods that do permit easy examination of roots. One of these is the transparent plastic &#34;growth pouch&#34;7. A disadvantage of this method is that frequent watering is required since only &#8804;10 ml liquid medium is optimal for growing M. truncatula in pouches7. Pouch experiments are also usually limited to ~2 weeks7, due to breakdown of the paper wick within the pouch. Two other methods that are commonly used are similar to our method in that the plants are grown on agar and the roots are visible, but these methods also have disadvantages that our procedure avoids. In these methods, plants are completely contained within 24.5 cm x 24.5 cm agar plates sealed around the top with porous surgical tape or grown in agar-slant tubes stoppered with cotton plugs or plastic caps7. Both of these methods allow easy examination of roots and can be kept sterile. However, the agar slant tubes are usually grown with only 20 ml agar medium7 and require watering and nutrient addition for long-term growth of plants. Plants grown within the 24.5 cm x 24.5 cm agar plate microcosms have sufficient moisture and nutrients for long-term growth, but the growing shoot quickly becomes constrained within the enclosed plate microcosm and ethylene gas can build up inhibiting nodulation7. In the procedure described here, the shoot is exposed and grows freely outside the microcosm which contains ~70 ml of media, allowing long-term growth.
The procedure described here may be useful not only for the study of nodulation of legume plants, but also for the study of root phenotypes of other mid-size plants. The difference between these microcosms and a traditional polycarbonate plant tissue-culture jar is that roots in the plate microcosms described here grow on the vertical surface of the agar rather than down into a horizontal layer of agar at the bottom of the jar. This allows the root to be lifted off of the agar surface with minimal damage to root hairs and minimal adhesion of agar to the root surface, which facilitates the examination of root hairs by microscopy.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Agar purified, plant cell culture-tested</td>
			<td>Sigma</td>
			<td>A7921</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

single-plant, sterile microcosms for nodulation and growth of the legume plant medicago truncatula with the rhizobial symbiont sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

The preparation and inoculation of these plate microcosms is relatively simple compared to most other methods described in the Introduction. Use of these microcosms also permits prolonged plant growth (up to 9 weeks) without watering or nutrient supplementation, maintenance of root sterility, both easy examination of roots and protection of roots from light, unconstrained growth of plant shoots, easy access to the shoot for length measurement, and removal of plants from the microcosms with minimal damage to root hairs. <...

Watch this Scientific Journal Video about Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti at JoVE.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

single-plant-sterile-microcosms-for-nodulation-growth-legume-plant

Research

JoVE Journal

Environment

16.8K Görüntüleme.  Florida State University.  Standart laboratuvar plakalardan yapılmış bireysel, steril mikrokosmlarından yılında azot bağlayıcı bakteriler Sinorhizobium meliloti ile simbiyoz Medicago truncatula bitkilerin büyüme ödün vermeden, sterilite kök sistemleri ve nodüller sık muayene izin verir. Bitkiler kadar 9 hafta boyunca bu büyüme odalarına korunabilir. 

Video: Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti