Hypersensitive response covert resistance is an effective defense response determined by the N-resistant genes. It manifests in the formation of cell death zones on inoculated lifts. We are studying the rate of cell death initiation by imaging inoculated lifts in the time between the cell death initiation and the cell death appearance.
We established a simple method for determination of cell death initiation rates in different planned HR pathosystems, which is not time consuming, not expensive, and is easy to use. Our protocol enables continuous observation of leaf in desire intellets, which means that we can determine the exact time of lesion formation and therefore the send that initiation rate up to minutes exactly, as opposed to hours with the use of other methods. If they describe protocol is used on transgenic plants with other components of interest, hypothesized to be involved in cell death initiation.
The protocol enables the user to determine if the decreased level of a studied component affects the rate of cell death initiation. By that components involved in cell death initiation can be identified. Begin by propagating healthy potato plants and test tubes containing stem node tissue culture media.
After six to eight weeks, use sterile tweezers and a scalpel to cut out ten one centimeter long X plants containing nodes. Transfer the X plants to plastic boxes containing Murashige and Skoog medium. And grow them in controlled environmental conditions in a growth chamber.
After two weeks, fill some pots with soil to prepare for plant transfer. Use a finger to create a three to four centimeter deep hole in the soil at the center of the pot. Now pour water into the hole and allow it to absorb into the soil.
Next, place each plant into the hole ensuring that the leaves remain above the surface. Carefully cover the roots with soil. Cultivate these plants in a growth chamber in a controlled environment.
In three to four weeks, the plants are ready to be inoculated. To begin use extraction bags with filter net to harvest six to eight week old potato virus infected pentland potato plants. Add four milliliters of phosphate buffer supplemented with DIECA for every gram of tissue.
Then homogenize the plants with a hand homogenizer for one to two minutes. Next, lightly coat the first three fully developed leaves of three to four weeks old healthy potato plants with carborundum powder, gently rubbed the leaves with the prepared viral inoculum. After 10 minutes, rinse the leaves clean with tap water.
Finally transfer the plants to a growth chamber to allow them to grow under controlled environmental conditions at 28 degrees Celsius for three days. To begin transfer the virus infected potato plants to a growth chamber maintained at 22 degrees Celsius. Pick a plant for observation and use adhesive tape to fully immobilize the second inoculated leaf.
Next, connect the digital microscope to the computer and launch the image capture software. Place the leaf under the microscope. Then use the dial to focus on the surface of the leaf.
To adjust the camera settings, click the settings button. Now set the brightness to 64, the contrast to 14, and the hue to zero. Next, adjust the white balance, then set the saturation to 47, sharpness to zero and gamma to five.
Click on the image capture icon and select time lapsed video option. Next, set image capture for every 15 minutes for a total of 24 hours, press the start button to begin the image capture. To save the captured images, select all images and click the save icon.
Now choose the export options, then set dots per inch to maximum. Once saved, delete all images from the program. To edit the images, launch an editing software like Image J.Click on file then select import, and choose image sequence to import the time sequences of images from a single field of the vision, paste the path of the directory of saved pictures and press okay to initiate the conversation.
Once the conversion is complete, Image J will automatically launch an internal video player displaying the finished video. Click on file, then save as and select the AVI format to export the video file. When a small window opens, set the frame rate to 0.3 FPS then press okay to save the video as an AVI file.
Three to four week old inoculated potato plants with at least three to four fully developed leaves were used for digital microscopic analysis. The same area on the inoculated leaf was observed at 15 minute intervals to determine the lesion occurrence and expansion over time. At 14 hours of observation, no lesion was visible.
About 90 minutes later, a visible lesion was recorded. The lesions started to expand two hours later and continued expansion over the next eight and a half hours.
