This method have answered a key question of plant developmental biology. That is, what is the importance of spatial distribution of stomata? Our induction system for clustered stomata using sucrose solution is fairly easy and directly applicable to transgenic or mutant lines of Arabidopsis thaliana.
To begin, add 1.1 grams of Murashige-Skoog medium salts and 15 grams of sucrose to a beaker. Add 490 milliliters of distilled water to the beaker and mix with the stir bar. Use potassium hydroxide to adjust the pH to 5.8.
Then dilute the solution with 500 milliliters of distilled water and transfer the diluted solution to a medium bottle. After this, autoclave the solution. Prepare the sterilization solution according the the text protocol.
Then, working under aseptic conditions, place about 50 transgenic A.thaliana seeds carrying a fluorescent marker into a 1.5 milliliter tube. Next add one milliliter of 70%ethanol to the tube and mix by inverting the tube five times. After allowing the seeds to sink to the bottom of the tube, gently remove the ethanol with a micropipette.
Add one milliliter of sterilization solution to the seeds and invert the tube five times to mix. Gently remove the sterilization solution from the seeds with a micropipette. Then add one milliliter of sterile water.
Add 1.5 milliliters of the Murashige-Skoog solution to each well of a 24 well plate. Then add two sterilized seeds to each well. Using two layers of parafilm, tape the lid to the plate.
Next, transfer the plate to a growth chamber to incubate for 14 days. First, transfer 30 microliters of Murashige-Skoog solution from the 24 well plate to the center of a glass slide. Using dissecting scissors, remove the cotyledon from a 14-day old seedling.
Float the cotyledon with the observation side facing up on the drop of Murashige-Skoog solution. Prepare the cotyledon according to the methods previously described in the Journal of Visualized Experiments. Set the specimen on the stage of a confocal laser microscope.
Finally, use bright field illumination to select clustered guard cells for observation. In this protocol, stomatal clustering was induced in A.thaliana seedlings. The clustered guard cells grown in the sucrose containing medium having larger chloroplasts than guard cells grown in sucrose-free conditions.
Enlargement of the chloroplasts was confirmed with a chloroplast stroma marker and chlorophyll autofluorescents, suggesting that sucrose treatment resulted in starch grain accumulation in the chloroplasts. Additionally, confocal observation of GFP tub-6 revealed that the cortical microtubules were radially oriented in the sucrose-treated cells, just like in the sucrose-free guard cells. Actually we discovered this method by accident.
After the finding, we examined the environmental response of clustered stomata. We believe our system could help to examine the significance of the stomata distribution.
