Growth and Preparation of Plants

The stages below deal specifically with preparing A. thaliana seedlings for leaf movement. Other species of plant show rhythms in leaf movement, which can be assayed using the system outlined above, but these may require different preparation procedures and growth conditions (see Note 9).

1. Surface-sterilize Arabidopsis seed by soaking in 70% ethanol for 1 to 2 min, followed by soaking in 50% bleach for 8 min.

2. Remove the bleach and rinse two to three times in sterile distilled water, before placing the seed in top agar.

Fig. 3. Figure summarizing the components of the KujaMorph leaf movement system. Arrows represent the direction of information flow. Square 25-well tissue culture dishes containing 12 10-d-old Arabidopsis seedlings are placed vertically within growth chambers. Sixteen charge-coupled device cameras controlled by a 16-channel switcher are set up facing the plates inside the chambers. Images are transferred from the video switch to the computer via a flash bus. A parallel port controller unit enables MetaMorph to select the channel on the video switcher and capture and store images from every camera at 20-min intervals.

Fig. 3. Figure summarizing the components of the KujaMorph leaf movement system. Arrows represent the direction of information flow. Square 25-well tissue culture dishes containing 12 10-d-old Arabidopsis seedlings are placed vertically within growth chambers. Sixteen charge-coupled device cameras controlled by a 16-channel switcher are set up facing the plates inside the chambers. Images are transferred from the video switch to the computer via a flash bus. A parallel port controller unit enables MetaMorph to select the channel on the video switcher and capture and store images from every camera at 20-min intervals.

3. Store the seed in top agar at 4°C in a refrigerator for 4 d to stratify (see Note 10).

4. Following stratification, plate the seed onto MS 3% sucrose plates and grow at

22°C under constant white light for 6 d (see Note 11).

5. After 6 d of growth, transfer the seedlings to 12 h:12 h light-dark (LD) cycles for

6. Using aseptic technique, transfer the seedlings into 25-well tissue culture plates as outlined here (see Note 14):

a. Using a sterile scalpel or forceps, manipulate the seedlings so that they stand up straight in the media. This can best be achieved by gently pressing the hypocotyl-root junction into the medium.

b. Cut blocks of approx 1 to 2 cm2 from the media surrounding individual seedlings and transfer them into vertically placed 25-well tissue culture plates (see Fig. 4 and Note 15).

c. Place 12 seedlings into each plate, leaving the bottom and top rows and the left-hand column empty (see Fig. 4 and Note 16).

d. Once all 12 seedlings are placed in the plate, add a drop (50-100 mL) of sterile distilled water to each of the four corner wells.

e. Place the lid on the plate and secure it with Parafilm to prevent infection and loss of moisture.

7. Once all plates are set up, they can be placed in front of the cameras.

Fig. 4. Arabidopsis seedlings prepared for leaf movement imaging. (A) 25-Well tissue culture plate placed vertically, containing 12 Arabidopsis seedlings. (B) Exploded view of an individual seedling within the plate. Note that the cotyledons face toward the front and back of the plate, whereas the emerging primary leaves face toward the sides.

Fig. 4. Arabidopsis seedlings prepared for leaf movement imaging. (A) 25-Well tissue culture plate placed vertically, containing 12 Arabidopsis seedlings. (B) Exploded view of an individual seedling within the plate. Note that the cotyledons face toward the front and back of the plate, whereas the emerging primary leaves face toward the sides.

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