Sweetclover Methods
Plant Culture Methods/Inoculation with Rhizobium
We use several different methods to grow sweetclover and alfalfa for inoculation studies. Two of these (the agar-based culture medium and growth pouches technique) enable you to follow the progress of nodule development under the dissecting microscope. The other two methods (Magenta jars or plastic dishpans) keep the roots in the dark. Some legumes such as pea are sensitive to light and will not nodulate if the roots are exposed to light (sweetclover and alfalfa donot fall into this category).
These plant culture methods are axenic (agar-based culture medium and Magenta jars) or semi-axenic (growth pouches and dishpans). In all cases, the seeds are sterilized before planting.
Seed sterilization . There are a number of different methods to sterilize the seeds of small legumes. For sweetclover (after scarification) and alfalfa, the seeds are soaked in 95% ethanol for 5 minutes and then in full-strength commercial bleach for 30-60 minutes. We usually do this procedure in a sterile plastic Petri dish. After the bleach treatment, the seeds are copiously rinsed (5-6 times) with sterile distilled water. They are now ready for directly planting into vermiculite/perlite (Magenta jars or dishpans) or for germination on water-agar. Water-agar is 1% agar autoclaved in distilled water. The seeds are germinated in the dark for 72 hours and then transferred either to growth pouches or solidified culture medium.
A. Agar-Based Culture Methods
For nodulation studies, a culture medium without nitrogen is used. We use either Jensen's medium or one-quarter strength Hoagland's medium without nitrogen. The medium is solidified with 1% agar and poured in square Petri dishes that are available from Labtek. Three days after germination, five to six seedlings are transferred from the water-agar plates to the middle of the Labtek plates.
Figure A. Sweetclover plants growing on an agar-based medium in square Labtek Petri dishes.
Photo, courtesy of A. M. Hirsch.The plants can be inoculated immediately or within the next day or two. After inoculation, the plate edges are wrapped with Parafilm and then the plates are incubated vertically with the plant roots pointing downwards. The plants are grown in a growth chamber under long-day conditions. We sometimes cover the bottom of the plate with aluminum foil if we want the roots to have less exposure to the light. In the light, sweetclover and alfalfa roots develop anthocyanin, and although the accumulation of this pigment does not appear to interfere with nodulation, covering the roots more closely approximates natural conditions.
1) Flood inoculation. The Rhizobium strain is grown in RDM medium until an OD600 of 1.0 is reached. The culture is pelleted and rinsed with sterile distilled water, and finally resuspended in enough distilled water to give an OD600 of 0.1-0.2; this is approximately 105 to 106 cells/mL. Two-hundred fifty to 500 µL are used to inoculate each root. Alternatively, if working from a plate, you can resuspend a single colony in 1 mL of sterile distilled water, vortexing to make certain that the rhizobia are well dispersed. Then inoculate each root with 500 µL of the suspension.
2) Spot inoculation . Spot-inoculation is used to follow the progress at a single infection site. Here the suspension of rhizobia should be denser, approximately 109 to 1010 cells/mL. We pull capillary tubes to make a narrow-diameter instrument for inoculating a single drop of the rhizobial suspension onto the root. Under the dissecting microscope, we look for the zone where the root hairs are just beginning to emerge. A single drop of Rhizobium is inoculated at this site. Just above (or below if you prefer) the site, we place a single drop of India ink using a separate pulled capillary tube. The India ink clogs the capillary so it is necessary to change it frequently.
We also use test tubes to examine nodulation responses. Generally, we utilize Jensen's medium and pour approximately 10 mL of melted agar culture medium into each test tube. The tubes are autoclaved and put on a slant while cooling. Two sterilized seeds are placed in each test tube and 3 days after germination, the tubes are flood-inoculated with 500 µL of rhizobia. The tubes can be covered with cotton plugs or with plastic caps to keep everything axenic.
Figure B. Alfalfa plants grown for 4 weeks in test tubes containing Jensen's nitrogen-free medium. The plants were inoculated with different combinations of Rhizobium meliloti mutants.
Photo, courtesy of A. M. Hirsch.B. Growth Pouch Method (Contributed by Keith Wycoff)
Sweetclover, alfalfa and other small-seeded legumes can be grown and nodulated semi-axenically in growth pouches that are essentially seal-a-meal bags with a specially folded paper towel inside. They may be watered easily through a straw placed on one edge of the pouch. They are also ideal for the growth and nodulation of plants with hairy roots caused by Agrobacterium rhizogenes.
The pouches we use are obtained from:
Mega International
3208 W. Lake St. #22
Minneapolis, MN 55416
Tel. 612-924-0863
Fax 612-924-0701
http://www.mega-international.comPlastic straws can be obtained at most supermarkets.
Figure C. Sweetclover plants grown in pouches were inoculated with wild-type Rhizobium meliloti. The majority of nodules have formed along the primary root.
Photo, courtesy of T.A. LaRue.Pouches should be held upright by the use of a wire rack. This can be simple or fancy, but basically consists of wires (~1.5 mm thick) formed in the shape of an upside-down U's, spaced about 1 cm apart and fixed to some sturdy base such as wood or lucite. The top of the wire loops should be no more than 15 cm high.
Pouches seem to autoclave best dry, but they have a tendency to deform and should be held upright in such a way that they cannot slouch downwards during autoclaving. Push the paper towel to one side of the pouch and place a straw down the space on the other side.
After autoclaving, place the pouches in the rack to be used and add 20 mL of plant medium through the straw. The paper will wet by capillary action. Some legumes such as alfalfa will nodulate better with 2 mM KNO3 than with no nitrogen source. 20 mM KNO3 or higher will inhibit nodulation.
Surface-sterilized seeds will germinate in the pouches if the top is covered so that it does not dry out. However, seed coats seem to be a good substrate for the growth of fungus, and better sterility can be maintained if the seeds are pre-germinated on water agar or between filter papers and the seedlings transplanted (without seed coat) after about 3 days. The root will grow down through the perforations in the fold at the top of the pouch.
Pouches should be watered as needed through the straw with sterile distilled water. Once roots are growing down in the lower part of the pouch (about a week after germination) they can be inoculated with Rhizobium. We inoculate with a concentration of approximately 106 cells/mL, a final OD600 of 0.1 to 0.2. We grow the rhizobia in RDM, then centrifuge and wash the pellet twice with PBS or sterile water to eliminate the culture medium. Generally, we add 1 mL of resuspended bacteria per pouch directly onto the roots. Nodules are usually visible within a week.
C. Magenta jars
Magenta jars offer the advantage that the roots can be maintained in the dark and that plants that would quickly outgrow the square Labtek Petri dishes can be maintained for a longer period of time. The difficulty with Magenta jars as well as the closed Petri dishes is that ethylene gas, which inhibits nodulation, can accumulate. It is possible to leave the top of the Magenta jar slightly askew so that the jar is open, but there is a greater chance of contamination when this occurs.
The Magenta jars (Magenta Corporation, Chicago, IL) are half-filled with a 1:2 mixture of perlite and vermiculite. The matrix is saturated with nitrogen-free Jensen's medium or with one-quarter strength Hoagland's medium minus nitrogen and the jars are autoclaved. Seedlings or seeds can be planted directly in the Magenta jars. The plants are inoculated as described for flood inoculation. You can also use an agar-based culture medium in the Magenta jars.
D. Dishpans
Plastic dishpans enable you to grow a large number of plants for nodulation studies. We generally use black plastic pans that are used for changing the oil in cars; they are available from automotive stores. Make certain that the type of plastic you are using is autoclavable. Dishpans made of high density polyethylene or polycarbonate are stable in the autoclave.
Fill the dishpan half-fill with a mixture of vermiculite:perlite (2:1) and cover with aluminum foil. Autoclave this and a large quantity of one-quarter strength Hoagland's medium minus nitrogen. When you are ready, add approximately 1.5 L of the nitrogen-free Hoagland's medium to the sterilized substrate in a transfer hood. Use a sterilized weighing spatula to make rows in the moistened vermiculite-perlite for planting the surface-sterilized seeds. Carefully cover the seeds; they should be at a depth of approximately 0.5 cm. You can flood-inoculate immediately or 2-3 days post-germination. Until the seedlings are well established, we cover the plastic dishpan with Saran wrap to keep in the moisture. Water when necessary with one-quarter strength Hoagland's medium minus nitrogen or with sterile distilled water. Be careful not to over-water. If the dishpan feels very heavy upon picking it up, you probably do not have to add any liquid.
It is also possible to use the dishpans to grow plants hydroponically. In this case, the roots hang from a screen into liquid Hoagland's medium that is in the pan. Use an aquarium pump to make certain that the roots are sufficiently aerated.
