Procedures:
Propagating plants
- Tomato (Lycospersicon esculentum) seeds-A variety should be selected that is not virus resistant (e.g. 'Big Boy' from W. Atlee Burpee Co.). Susceptible varieties will develop symptoms within 7-14 days, and the severity increases with time after inoculation. Systemically infected plants can be used to increase the virus for subsequent study.
- Pinto bean (Phaseolus vulgaris 'Pinto bean') seeds-This variety of bean produces necrotic local lesions on the primary leaves and is frequently used as an assay host.
- Potting mix, pots and trays
Tomato seeds should be started in a small pot; then the seedlings are transplanted to individual 7-13 cm (3-5 inch) pots. The seedlings should be placed in a sunny window. Plants should be about 10-15 cm (4-6 inches) tall or about 2-4 weeks old at the time of inoculation; however, environmental conditions influence the time it takes plants to reach this size
Pinto beans should be planted about one week prior to inoculation. Seeds (4-6 per pot) should be sown in 12 cm (5-inch) pots. When the seedlings emerge, the pots should be placed in a sunny window. When the plants are ready to inoculate, the first or primary leaves should be expanding and subsequent leaves are not obvious. Lesions will still develop if the plants are slightly older, but the stem and leaves above the primary leaves should be removed. The plants should be between one and two weeks old, depending on their rate of growth, which again depends on the temperature, etc. of the classroom.
Plants should be watered daily and fertilized weekly. Students can be assigned this task and also asked to watch for the appearance of symptoms. At the conclusion of the experiment, infected plants should be disposed of in a way that susceptible plants cannot get the virus from the infected plants. Pots, etc. should be washed with a dilute solution of bleach.
Time for planting seeds and maintaining plants: 15 minutes for the initial planting and 10-15 minutes a day for watering/fertilizing the plants. Placing the pots in a plant tray will simplify watering and fertilizing the plants.
Inoculating plants
- TMV in living/dried tissue or in frozen sap from infected tissue (See Teacher Tips for methods for long term storage of the virus.)
- Tap water, if the pH is neutral, or 0.05 M phosophate buffer, pH 7. The buffer can be made by adding 0.6 gm of sodium phosphate dibasic (Na2HPO4) and 6.4 gm sodium phosphate monobasic (Na2HPO4) to a total volume of 1 liter.
- Mortars and pestles (must be sterilized after use-See Teacher Tips)
- Systemic host (tomato) or local-lesion host (pinto bean), depending on the experiment
- Mild abrasive-celite (diatomaceous earth) or carborundum (See Teacher Tips for cautions using these abrasives.)
- Cheesecloth pieces or cotton swabs
- Plant labels and markers
Dried leaf tissue (1 gm) should be placed in a mortar along with 10 ml of water or 0.05 M phosphate buffer, pH 7. Using a pestle, the tissue is crushed to produce a light green solution. A small amount (about 0.1 gm) of a mild abrasive (diatomaceous earth or carborundum) is added to the pestle, taking care not to inhale the dust (See Teacher Tips about caution using these two abrasives). The pestle, cheesecloth pad or cotton swab is dipped into the inoculum and then used to inoculate plants.
Before inoculating the leaf, it should be supported with the other hand (Figure 7). Then the leaf is rubbed lightly distributing the inoculum over the entire surface. Immediately following inoculation, the leaves should be rinsed with water, which can be done by placing the leaves under the tap. A common mistake is to apply too much pressure when rubbing the leaf, which will damage cells and produce large, irregularly shaped lesions.
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Figure 7. Inoculation of plants using a pestle.
Notice that the leaf is fully supported with the
hand. (Courtesy R. Ford). Click image to see
a larger view. |
Beans can be used to compare different treatments (e.g., sap extracts heated at various temperatures) by inoculating half leaves on the same plant. To distinguish between the two primary leaves, a small incision should be made to one leaf to mark that side (Figure 8). This cut will identify the same position on all plants. Two advantages of using the half-leaf method of inoculation is that the number of plants required to compare treatments is reduced and because multiple treatments can be tested on the same plant, variability due to individualized plant responses is minimized.
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Figure 8. Preparation of half leaves.
(Courtesy R. Ford)
Click image to see a larger view. |
Because TMV is highly stable, it is easy to transmit. To reduce the likelihood of inadvertent transmission, students should either wear gloves while working or wash their hands thoroughly with soap after handling the virus inoculum or infected plants. Even touching the plants while observing them (e.g., opening the newly unfolding leaves to see if they exhibit symptoms) may result in picking up the virus on their hands and transmitting it to other plants.
Observing TMV-infected leaves
- Slide containing cross sections of TMV-infected and healthy leaves
- Compound microscope (100X and 400X)
- Cross sections of TMV-infected and healthy leaves are included on a single slide. The two can be distinguished by comparing the mesophyll or middle section of the leaf. Healthy mesophyll consists of well-formed palisade and spongy layers with compact cells containing chloroplasts. In comparison with cells from healthy tissue, TMV-infected cells are large and round with few chloroplasts, and the mesophyll does not contain the two distinct layers.
Inactivating the virus using heat treatment
- TMV-infected tissue
- Water or 0.05 M phosphate, pH 7, buffer for grinding tissue
- Cheesecloth for straining tissue and for inoculating leaves
- Water baths or hot plates, 500-ml beakers, and thermometers. A range of temperatures from room temperature to boiling should be tested. For example, four temperatures: 25, 50, 75, and 100°C could be used. If water baths are not available, water can be heated in beakers placed on hot plates.
- Test tubes, holders, and rack
- Ice
- Pinto beans--the assay host (See sections on Propagating plants and Inoculating plants)
- Pot labels
Procedure:
An extract of TMV is prepared as described previously (See Inoculating plants). The extract should be strained through four layers of cheesecloth to remove large pieces of tissue. The teacher can prepare a large amount of plant extract before class and then divide it into portions (about 3 ml) for students to treat with heat.
Each extract is heated at the desired temperature for 10 minutes (Figure 9), then immediately cooled by placing it on ice. The primary leaves of pinto bean are inoculated with the cooled extracts in a half-leaf design (See Inoculating plants).
Figure 9. Heating the sample in a boiling water bath.
Glass beads or boiling chips in the water will prevent
"bumping" of the water. (Courtesy R. Ford)
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Filtering infected sap with a bacteria-proof filter
- TMV-infected leaves
- Water or 0.05 M phosphate buffer, pH 7
- Test tubes, 3 per preparation, and a test-tube rack
- Clinical centrifuge
- Transfer pipettes, 2 per preparation
- Micron filters (pore size 220 nm or 0.22 µm) with Luer-lock fitting for attachment onto a syringe
- Syringe (5- or 10-ml) with a Luer-lock fitting
- Marker
- Pinto beans
- Cheesecloth or cotton swabs for inoculating leaves
Procedures
The inoculum is prepared as described previously (Inoculating plants) then strained through four layers of cheesecloth. The extract is centrifuged at highest speed for 10 minutes in a clinical centrifuge to remove pieces of plant materials that will pass through the cheesecloth and clog the filter. The top layer (supernatant) is removed using a transfer pipette, and placed in a new tube. This extract is divided into two equal portions. One should be set aside as the control.
The plant extract in one tube should be placed into the barrel of a syringe that is fitted with a 220 nm filter (Figure 10). The filtrate should be collected in a new tube. Primary leaves of pinto bean are inoculated with the two solutions (1) unfiltered sap and (2) filtered sap.
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Figure 10. Forcing the extract through 220 nm
syringe filter to remove particles the size of
bacteria and larger. (Courtesy R. Ford)
Click image to see a larger view. |
Teacher Tips:
Sterilizing mortars and pestles and other TMV-contaminated glassware
Because TMV is stable, mortars and pestles and other materials that are contaminated with TMV must be sterilized. They can be sterilized by autoclaving or by baking for 3-4 hours at 110°C. An alternative is to soak the contaminated materials in dilute bleach (10%) for 24 hours, rinse thoroughly, then dry.
Reducing contamination of TMV with other plants, during and after inoculation
The ease of transmitting the virus makes it important for the students to wash their hands with soap after working with the TMV-infected plants or plant extracts. Another way to reduce contamination is for students to wear gloves while working with TMV-infected tissue or extracts. Even if the plants are touched while observing them (e.g., opening the newly unfolding leaves to see if they exhibit symptoms), students could pick up the virus on their hands and transmit it to other plants.
Preserving the virus by preparing a viral stock
A stock of TMV can easily be made from infected tissue and will be infective for many years. Stocks can be made using frozen sap or leaves or dried tissue. To dry the leaves, they should be first cut into small pieces or strips (about 5 mm in width) to promote rapid drying. This tissue is laid on paper towels, which cover the bottom of a baking dish. The dish should be covered with plastic wrap and placed in a refrigerator for several weeks, which will allow the tissue to dry slowly. The dried tissue can be crushed, placed in a vial, and sealed.
Troubleshooting:
Problems with inoculation?
No symptoms: The tap water may be too acidic, so distilled water (available at grocery stores) or a phosphate buffer can be used. If lesions are not observed on bean or tomato, the virus concentration could also be low. More tissue should be added to the extraction buffer. This problem will not occur in tomato since that host allows the virus to reach high concentrations within the plant.
Large, irregularly shaped lesions: The leaves were damaged during the inoculation process. Less pressure should be applied to the leaves during the inoculation.
Sources of Materials:
Abrasives: Carborundum (silicon carbide) can be purchased from Wards (www.wardsci.com) (CAS#4 09-21-2). Caution: The dust may cause eye and skin irritation. Be sure to read the MSDS before using. Celite or diatomaceous earth can be purchased from a local pet store. It, too, may cause eye and skin irritation.
Micron filters and syringes are available both from Carolina Biological Supply (www.carolina.com) (BA-19-9593 for a 0.22 µm or 220 nm syringe filter and BA-69-7771 for 5- and BA-69-7774 for 10-ml syringes) and from Wards (18 W 1580 for 0.2 µm or 200 nm filters and 14 W 1616 for 5- and 14 W 1617 for 10-ml syringe).
Plants: The seeds for the plants are available from Carolina Biological Supply or Wards. Many also can be obtained at local stores. 'Big Boy' can be obtained from W. Atlee Burpee Company (http://www.burpee.com).
Slides of TMV-infected leaf tissue: Slides containing both TMV-infected and healthy leaf sections are available from Carolina Biological Supply (WW-29-3866).
Virus: The common or vulgare strain of TMV is available at a reasonable price from Carolina Biological Supply, either in a kit which includes material for infectivity assays (D8-12-6000) or in dried tissue (D8-12-6005). TMV is also available from the American Type Culture Collection (ATCC Number PV-598) (http://www.atcc.org/).
NOTE: Teachers must first obtain a permit from the U.S. Department of Agriculture (USDA) to allow the interstate transport of virus before the virus is shipped. It takes about 60 days to obtain a permit, which will be valid for 4 years. The Application to Move Live Pests, Noxious Weeds, or Imported Bees, PPQ Form 526 is available from the web site of the Animal and Plant Health Inspection Service (APHIS) (http://www.aphis.usda.gov/ppq/permits/plantpest/pathogen.html). Inquiries can be directed to the office of PPQ: Permits and Risk Assessments, phone 301-734-5609 and fax 301-734-8700. This regulation, which applies to all plant pests and noxious weeds, is important to safeguard plants in the United States.
Tobacco products (e.g., cigarettes) can also be used as a source of virus although the strains and, consequently, the symptoms may differ from those produced by the common strain.
