Shoot apical meristems control plant growth and reproduction. This method helps us investigate the morphology and internal structures of shoot apical meristems not only in the model species but also in different crops. This technique is a very efficient procedure.
Shoot apical meristem can be directly visualized in a high cellular resolution without any labor-intensive sample fixation and tissue sectioning steps. To begin, grow Arabidopsis, tomato, and soybean plants as detailed in the text protocol. Cut the inflorescence shoot apex from the bolted Arabidopsis plants.
Hold the basal part of the main stem, and use forceps to remove as many older flower organs as possible from the main stem. Continue removing the rest of the flowers in the field of the stereo microscope until the shoot apical meristem can be viewed from the eyepieces. To view the vegetative shoot apical meristems from either tomato or soybean, dissect out the cotyledons, leaves, and roots from the plant.
Hold the hypocotyls of the plant under the stereo microscope, and use forceps to further dissect out the leaves and leaf primordia covering the vegetative shoot apical meristems. Pipette 50 microliters of propidium iodide solution in a clean and empty Petri dish. Dip the whole dissected shoot apex into dye for two minutes.
During the staining process, immerse the whole inflorescence shoot apical meristem or vegetative shoot apical meristem into the propidium iodide solution to achieve uniform staining. Rinse the stained shoot apex twice in sterile, deionized water. Now, pierce a hole at the center of a prepared imaging dish using forceps, and stick the stained shoot apex upright in the medium.
Fill the imaging dish with sterile, deionized water to completely immerse the sample. View the sample through the stereo microscope. Pipette up and down to remove air bubbles trapped around the meristem.
Then, adjust the angle of the stem in the agar to make sure that the shoot apical meristem is fully visible from directly above. Now, place the imaging dish on the sample stage of the confocal microscope. Lower the water-dipping lens, and raise the microscope sample stage to let the tip of lens dip into the water.
Open the confocal microscope software, and use the eyepieces to locate and focus on the shoot apical meristem in the bright field. Operate the Acquisition function in the confocal microscope software. Start the live mode to view the sample from the computer screen, and set up all parameters for the laser scanning experiment.
When adjusting the parameters, use the Range Indicator function to define whether the signal is saturated or not. The same software that was used for imaging acquisition can be used for visualizing a three-dimensional transparent projection. Open the original confocal file, click 3D menu, and select Transparent to generate a 3D projection view.
Optionally, select Transparency to adjust three parameters of the projection, including Threshold, Ramp, and Maximum for the transparency of the 3D image. Now, select Light to adjust the brightness of the 3D image. Export the projected images, and save them as TIFF files.
For visualizing the depth coding view of the 3D images, use the same software. Again, click 3D menu, and select Appearance. Select Special, and select Depth Coding.
Export the projected images, and save them as TIFF files. This image from the orthogonal section through the middle of the Arabidopsis shoot apical meristem shows that the horizontal walls are stained with propidium iodide in almost all the cells at multiple cell layers. A view of one transverse section through the corpus of the inflorescence shoot apical meristem shows that the cells from the XY plane are also clearly imaged.
The 3D projection view reveals that the inflorescence meristem forms a dome-like structure and is surrounded by the developing flower primordia. This image from the orthogonal section through the middle of the tomato shoot apical meristem also shows that the horizontal walls are stained with propidium iodide at multiple cell layers, although the propidium iodide signal from the deep interior region is slightly lower. An image from one transverse section through the deep layers of the vegetative shoot apical meristem reveals the cells from the XY planes and the boundary formed between the vegetative meristem and leaf primordia.
The 3D projection view can further provide a comprehensive view of the shape and organization of the vegetative meristem from tomato. Shown here is an orthogonal view through the middle of the soybean shoot apical meristem and a 3D projection of the same shoot apical meristem. The dome-like vegetative meristem and its derived new leaf primordia are also observable.
Propidium iodide can easily stain damaged or dead cells, which will greatly influence the quality of images, so it's important to avoid any physical damage while preparing shoot apical meristem samples.
