are produced during the vegetative phase from germination
whereas flowers are produced later, during the reproductive
phase. Imaging the shoot meristem using confocal microscopy
during the reproductive phase, when it is called the inflorescence
meristem (IM), has been considered relatively easy compared to
imaging the shoot meristem during the vegetative phase (vegetative
meristem) due to the proximity of the leaves which obscure a clear
view of the meristem. As a consequence, although several protocols
have been published for imaging the shoot meristem, most have
focused on the inflorescence meristem [3–5] while others involve
the dissection of many leaves [6]. Here we describe a relatively rapid
method for imaging the vegetative meristem in young seedlings of
Arabidopsis thaliana which avoids the dissection of many leaves.
This method involves use of an upright microscope and dipping
lens and allows repeated imaging over time for monitoring the
consequences of experimental manipulations.
2 Materials
1. Fluorescent stained or fluorescent transgenic plants for
imaging.
2. Clear polystyrene sterile square Petri dishes with a finder grid:
These dishes are both required for growing seedlings and
mounting them on the microscope’s stage (e.g., 90 Â 15 mm).
3. GM medium plus Vitamins: 1% sucrose, 1Â Murashige and
Skoog basal salt mixture, 0.05% MES 2-(MN-morpholino)ethane sulfonic acid, 0.8% Bacto Agar, 1% Murashige and
Skoog vitamins, 1 M potassium hydroxide solution to adjust
the pH to 5.7 and ddH 2 O
4. Sterile hood to pour the media in the Petri dishes and transfer
seedlings.
5. White racks: These are required for keeping Petri dishes and
therefore seedling vertically compared to the shelf (e.g., white
polypropylene tube racks 227 Â 100 Â 73, 17–20 mm tube
diameter).
6. 70% Ethanol (EtOH) to sterilize the equipment.
7. Dissection microscope
8. A sterile blade
9. Two tweezers.
10. A small needle attached to a syringe (e.g., 30G Â ½ on a 1 mL
syringe)
11. Sterile and desalted water.
296
Monica Pia Caggiano et al.
whereas flowers are produced later, during the reproductive
phase. Imaging the shoot meristem using confocal microscopy
during the reproductive phase, when it is called the inflorescence
meristem (IM), has been considered relatively easy compared to
imaging the shoot meristem during the vegetative phase (vegetative
meristem) due to the proximity of the leaves which obscure a clear
view of the meristem. As a consequence, although several protocols
have been published for imaging the shoot meristem, most have
focused on the inflorescence meristem [3–5] while others involve
the dissection of many leaves [6]. Here we describe a relatively rapid
method for imaging the vegetative meristem in young seedlings of
Arabidopsis thaliana which avoids the dissection of many leaves.
This method involves use of an upright microscope and dipping
lens and allows repeated imaging over time for monitoring the
consequences of experimental manipulations.
2 Materials
1. Fluorescent stained or fluorescent transgenic plants for
imaging.
2. Clear polystyrene sterile square Petri dishes with a finder grid:
These dishes are both required for growing seedlings and
mounting them on the microscope’s stage (e.g., 90 Â 15 mm).
3. GM medium plus Vitamins: 1% sucrose, 1Â Murashige and
Skoog basal salt mixture, 0.05% MES 2-(MN-morpholino)ethane sulfonic acid, 0.8% Bacto Agar, 1% Murashige and
Skoog vitamins, 1 M potassium hydroxide solution to adjust
the pH to 5.7 and ddH 2 O
4. Sterile hood to pour the media in the Petri dishes and transfer
seedlings.
5. White racks: These are required for keeping Petri dishes and
therefore seedling vertically compared to the shelf (e.g., white
polypropylene tube racks 227 Â 100 Â 73, 17–20 mm tube
diameter).
6. 70% Ethanol (EtOH) to sterilize the equipment.
7. Dissection microscope
8. A sterile blade
9. Two tweezers.
10. A small needle attached to a syringe (e.g., 30G Â ½ on a 1 mL
syringe)
11. Sterile and desalted water.
296
Monica Pia Caggiano et al.