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 1.1-1.2% w/v purified agar10 to a depth of 3-5 mm. Each plate will be used to germinate ~0.25-0.5 g of seed (equivalent to 63-125 seeds/plate). 
	Note: It is critical to use plant cell culture-tested, purified agar for all plates described in this protocol. The vendor and catalog number information for the agar is listed in the Table of Specific Reagents and equipment.</li>
	<li>Scarify/sterilize the M. truncatula A17 seeds: Weigh sufficient seeds for the desired number of seedlings. (M. truncatula A17 seeds are ~4 mg each) Place seeds in sterile 125 ml flasks with sterile foil covers (0.25-0.5 g seed/flask; this is ~63-125 seeds) and pipette 20 ml of concentrated (96%) H2SO4 along the sides of the flask. 
	Note: Acid scarification will sterilize the seeds. Also, by pipetting the acid along the sides of the flask, it will re-sterilize the inside of the flask that has come into contact with the unsterilized seeds.</li>
	<li>Break up clumps of seed with a sterile glass pipette. Swirl each flask frequently for 10-12 min. Small, brown pits will become visible on the seeds. These are tiny holes in the seed coat11. 
	Note: Wear eye protection and gloves when working with concentrated H2SO4.</li>
	<li>Remove H2SO4 and rinse seeds: When at least 10% of the seeds have small brown pits, or 12 min have passed, whichever comes first, remove ALL acid from the flasks with a sterile glass pipette. (Place waste acid into a 1-2 L beaker containing at least 300 ml tap water. This will dilute the acid and prevent overheating.) Tilt the flask to collect the remaining acid in the curve of the flask and use a small glass pipette to remove all the remaining acid. If residual acid remains, it will react with the rinse water, heat the seeds and kill them.
	<ol>
		<li>Rinse seeds by quickly pouring 100 ml of sterile ultrapure water into each flask. The excess volume of water will dilute the acid and prevent overheating and seed damage during the reaction of the acid with the water. Pour off the water and flame-sterilize the lip of the flask.</li>
		<li>Rinse seeds 8-10x with 50 ml of sterile ultrapure water. Swirl the flask during each rinse. The flask should be considered sterile at this point and the lip of the flask should be flamed before each rinse.</li>
	</ol>
	</li>
	<li>Let seeds imbibe overnight: After the last rinse, pour 50 ml sterile ultrapure water into each flask, replace the sterile foil cover on each flask and place at 4 &#176;C in the dark overnight.</li>
	<li>Place seeds on germination plate: After letting seeds imbibe overnight, pour off water, rinse 2x with ~25 ml ultrapure water, then add 20 ml sterile ultrapure water, swirl to resuspend seeds and pour them onto a 1.1-1.2% agar germination plate from step 1.1. If seeds remain in the flask, add more water and pour again. Distribute the seeds from each flask to one plate (63-125 seeds/plate).
	<ol>
		<li>Swirl the plate to distribute the seeds. The seeds should be distributed evenly in a 4-5 cm band at one end of the plate. This will be the &#34;top&#34; of the plate. The &#34;bottom&#34; 5-6 cm should be kept free of seeds (Figure 2A).</li>
		<li>Draw off the water with a sterile pipette. Tilt the plate at a 45&#176; angle to let the remaining water drain to the bottom of the plate. Replace the lid on the tilted plate and protect from light. Plates should be allowed to drain 10-20 min. (Figure 2B).</li>
	</ol>
	</li>
	<li>Set up germination: Remove all water that has collected at the bottom of the tilted plate with a sterile pipette.
	<ol>
		<li>Replace the lid and seal the plate with parafilm. Wear gloves and keep sterile (Figure 2C).</li>
		<li>Stack the germination plates and then turn all of them up at a 90&#176; angle. The seeds will be lying on the vertical surface of the agar. Fully imbibed seeds should easily adhere to the agar. The roots will grow downward (Figure 2D).</li>
		<li>Wrap the stack of germination plates in foil to block light and keep at 25 &#176;C for 3 days.</li>
	</ol>
	</li>
</ol>
Note: If germination proceeds for less than 3 days, roots may be too short to reach the agar in microcosms. If germination proceeds more than 4 days before transfer to microcosm plates, survival of seedlings will be poor. If M. truncatula ecotypes or mutants with slower germination rates are to be compared, the slowly-germinating ecotype should be allowed to germinate longer than 3 days.
2. Preparation of Individual Plate Microcosms
<ol>
	<li>Prepare Jensen's medium12(see Table 1 for composition), allowing ~70 ml medium for each microcosm. Prepare in pitchers or flasks that are convenient for rapid pouring (no more than 1-2 L per pitcher) and leave a magnetic stir bar in the pitcher during autoclaving.
	<ol>
		<li>After medium has cooled to ~55-65 &#176;C, supplements have been added, and pH has been checked (see Table 1), pour into round 100 mm diameter, 15 mm deep plates. Plates should be poured ~0.9-1 cm deep (~70 ml/plate).</li>
		<li>Components of Jensen&#39;s medium can form a cloudy precipitate, so it is important to return the pitcher to the magnetic stir plate periodically to prevent components from settling out. Precipitates may be visible in the plate after they solidify, and do not affect performance as long as they are evenly distributed among the plates.</li>
		<li>Stack plates during pouring to prevent the formation of condensation on the plate lids.</li>
	</ol>
	</li>
	<li>After Jensen&#39;s plates have solidified, notch both plates and lids with a weighing spatula that has been bent into a U-shape5 (Figure 3A). Heat the bend of the spatula with a burner until red hot, notch 5-6 plates and then heat again. When the notched lid is placed on the notched plate, this will create an oval portal through which the shoot of the seedling will grow (Figure 3B). If notched plates are to be stored, wrap individually with paraffin film..</li>
</ol>
3. Preparation of Sinorhizobium meliloti Cultures
Two days before plant inoculations, start cultures of all S. meliloti strains to be inoculated onto seedlings. Cultures should be grown in LBMC (Luria-Bertani [Miller]) medium13 supplemented with 2.5 mM MgSO4, 2.5 mM CaCl2, and appropriate antibiotics (TY medium also works well). As little as 2-3 ml of each culture will be sufficient.
<ol>
	<li>Grow at 30 &#176;C with shaking for 2 days until cells are dense.</li>
	<li>For most plant inoculations, the growth phase of the S. meliloti is not critical. Typically, late log or early stationary phase cells are used.</li>
</ol>
4. Laying out M. truncatula Seedlings on Individual Microcosms
<ol>
	<li>After 3 days, open a germination plate (prepared in step 1) and immediately flood with sterile ultrapure water. Dip forceps in ethanol and flame briefly to sterilize. Use sterile forceps to gently push the root tips of all seedlings under the water to prevent drying. Roots will range from 2-6 cm long. The majority will be 3-4 cm. Select seedlings with straight roots and no obvious defects.</li>
	<li>Check the lids of the Jensen's medium microcosms for the build-up of condensation. Flick the lid to remove the condensation or replace it with a new notched lid. If there is an excess of condensation on the lid, the seedling root may adhere to the lid and then die after the condensation dries.</li>
	<li>Using the forceps, gently pick a seedling from the germination plate by the cotyledons and lay the root on a Jensen&#39;s medium microcosm. Make sure the root tip is in contact with the agar.</li>
	<li>Gently remove the seed coat if it is still present. The seed coats should be loose after soaking in water for 5-10 min. Gently tease the seed coat with the forceps until it gives way, and discard. The cotyledons and approximately 0.5 cm of shoot should protrude out of the U-notch in the plate (Figure 4A). Carefully replace the lid of the plate with the U-notch of the lid over the seedling, creating a portal for the seedling (Figure 4B). Set plates aside in horizontal stacks until all seedlings have been placed on microcosms.</li>
	<li>Use all seedlings from each germination plate that look viable and have a straight root. (If possible, leave one plate of seedlings unopened to use as replacements for wilted seedlings just before inoculation.)</li>
	<li>After laying out all seedlings, allow them to sit on the horizontal Jensen&#39;s microcosms while the S. meliloti inoculum suspensions are being prepared.</li>
</ol>
5. Preparation of S. meliloti Inoculum Suspensions
<ol>
	<li>Check S. meliloti cultures periodically after inoculation to be sure that they are growing. After 2 days growth, the OD600 should be between 2 and 4.</li>
	<li>Pipette 0.5 ml of each culture into a sterile 1.5 ml tube and centrifuge to pellet. Remove supernatant.</li>
	<li>Wash cells two times in either 0.85% sterile NaCl or sterile ultrapure water. Resuspend cells in 0.5 ml 0.85% sterile NaCl or ultrapure water.</li>
	<li>Measure the OD600 of a 1/10 dilution of resuspended S. meliloti cultures from step 5.3. Based on this reading, make a suspension of each culture in sterile ultrapure water at OD600 = 0.05. If the cell density is too high, nodulation may be inhibited. Make enough of the dilute suspension so that 100 &#956;l can be inoculated onto each seedling. The cloudiness of the OD600 = 0.05 suspension should be just barely perceptible by eye.</li>
</ol>
6. Inoculation and Sealing of Microcosms
<ol>
	<li>Immediately prior to beginning inoculations, check for wilted seedlings that have not survived the transfer to the Jensen&#39;s medium microcosms. Replace those seedlings with fresh seedlings from germination plates.</li>
	<li>Open each microcosm and inoculate 100 &#956;l of the appropriate S. meliloti suspension onto the seedling root. Replace the lid and set aside in horizontal stacks of 6-10 microcosms. For each S. meliloti strain to be compared, inoculate at least 15 plants. For strains that have subtle differences in symbiotic productivity, inoculate 30-35 plants. Leave at least 10 plants uninoculated as a negative control. Inoculate 30-35 plants with S. meliloti 1021 or other appropriate wild type strain to serve as the positive control.</li>
	<li>After the S. meliloti suspension has had time to adsorb onto the root surface and the suspension liquid has soaked into the agar (at least 20 min.), wrap each plate individually with paraffin film. Plates should be wrapped up to the very edge of the notch, but the paraffin film should not block the growth of the seedling (Figure 4C).</li>
	<li>At the time of wrapping, make a final check for roots adhered to the inside of the plate lid. To remove adhered roots from the lid, lay the plate flat on the bench and tap or flick the lid to knock the root back down onto the agar surface.</li>
	<li>Line up the seedling portals of each stack of 6-10 plates. Lay the stack at a 90&#176; angle with the seedlings pointing upward (Figure 4D). The stickiness of the paraffin film will hold the plates in place long enough to wrap the entire stack in foil, leaving a 1-2 cm strip unwrapped where the seedlings emerge (Figure 4E). The foil will protect the roots from light.</li>
	<li>Place the foil wrapped stacks in a lighted growth chamber or growth room at 21-25 &#176;C, 60-70% relative humidity and 100-175 &#956;mol/m-2 s-1 light on a 16 hr light/8 hr dark cycle (Figure 1A). Under these growth conditions, prolonged incubation at light levels higher than 200 &#956;mol/m-2 s-1 can have a detrimental effect on plant growth.</li>
</ol>
7. Examination of Root Systems and Quantitation of Symbiotic Phenotypes
Plants can be maintained in microcosms for up to 9 weeks.
<ol>
	<li>Nodule number can be quantified at a series of time points by unwrapping the foil from each stack and counting nodules. Developing nodules on plants inoculated with S. meliloti 1021 wild type are apparent by 10-14 days after inoculation.</li>
	<li>Symbiotic productivity can also be measured at each time point by measuring the length of the emerging shoot.</li>
	<li>Final quantitation of symbiotic productivity is usually performed at 7 weeks after inoculation by detaching the shoot and measuring the fresh weight of the shoot. This step can be performed as late as 9 weeks if longer growth is desired.</li>
</ol>

<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 ...

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16.9K Views.  Florida State University. 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.

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

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

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

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

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

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

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone as a cheap and readily available CO2 sorbent. While the technology has been widely studied, there are a few available options that could be applied to make it more economically viable. One of these is to increase the oxygen concentration in the calciner to reduce or eliminate the amount of recycled gas (CO2, H2O and impurities); therefore, decreasing or removing the energy necessary to heat the recycled gas stream. Moreover, there is a resulting increase in the energy input due to the change in the combustion intensity; this energy is used to enable the endothermic calcination reaction to occur in the absence of recycled flue gases. This paper presents the operation and first results of a CaL pilot plant with 100% oxygen combustion of natural gas in the calciner. The gas coming into the carbonator was a simulated flue gas from a coal-fired power plant or cement industry. Several limestone particle size distributions are also tested to further explore the effect of this parameter on the overall performance of this operating mode. The configuration of the reactor system, the operating procedures, and the results are described in detail in this paper. The reactor showed good hydrodynamic stability and stable CO2 capture, with capture efficiencies of up to 70% with a gas mixture simulating the flue gas of a coal-fired power plant.

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

This manuscript describes a procedure for operating a calcium looping pilot-plant for post-combustion carbon capture with high oxygen concentrations in the calciner in order to reduce or eliminate the flue gas recycle.

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

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

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

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

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