3. Remove the Agrobacterium solution from the plates. It is
important to remove all the bacteria from the seedlings in
order to avoid contamination (see Note 25).
4. Place the plates back to the growth chamber and cover them
with aluminum foil for 45 min-1 h (see Note 26).
5. After 3 days, choose the healthy seedlings (usually the ones that
are still erect) and image cotyledons by the abaxial side on a
confocal microscope (see Notes 27 and 28).
3.4 Image
Acquisition
1. Excise cotyledons with scissors and mount them on a microscope slide with the lower epidermis facing upward. Add water
before mounting the cover slip to keep the sample moisturized.
2. Image the cotyledons with a confocal laser scanning microscope. We have used either an Olympus FV1000 confocal
microscope or a Zeiss LSM880 AxioObserver equipped with
alpha Plan-Apochromat 100Â/1.46 Oil DIC M27 Elyra. Specific settings can be used to image different fluorophores (see
Notes 29–31). For the acquisition and imaging processing we
used the fv10-asw 3.1 Viewer (Olympus) or the ZEN imaging
software (Zeiss). Figure 2 shows examples of confocal images
obtained with this transient expression method of fluorescent
protein markers for some compartments of the secretory
pathway.
4 Notes
1. The plates with solid MS can be kept in the cold chamber at
4
C for months.
2. %18–24 seeds can be sowed in each plate in order to have
enough space for seed germination and optimal growth
conditions.
3. It is preferred to keep the plates growing for 5 days in order to
have stronger seedlings that stand better vacuum infiltration.
However, 4-day-old seedlings can also be used.
4. A single colony or a previous culture of Agrobacterium kept at
4
C can also be used to prepare the pre-culture. Nevertheless it
has been reported that viability of Agrobacterium cultures kept
at 4
C decreased over time.
5. An independent culture is prepared for each fluorescent
marker.
6. This culture is prepared the day before the experiment, in order
to obtain a fresh Agrobacterium culture for the vacuum infiltration experiment.
Seedling Transformation
151
important to remove all the bacteria from the seedlings in
order to avoid contamination (see Note 25).
4. Place the plates back to the growth chamber and cover them
with aluminum foil for 45 min-1 h (see Note 26).
5. After 3 days, choose the healthy seedlings (usually the ones that
are still erect) and image cotyledons by the abaxial side on a
confocal microscope (see Notes 27 and 28).
3.4 Image
Acquisition
1. Excise cotyledons with scissors and mount them on a microscope slide with the lower epidermis facing upward. Add water
before mounting the cover slip to keep the sample moisturized.
2. Image the cotyledons with a confocal laser scanning microscope. We have used either an Olympus FV1000 confocal
microscope or a Zeiss LSM880 AxioObserver equipped with
alpha Plan-Apochromat 100Â/1.46 Oil DIC M27 Elyra. Specific settings can be used to image different fluorophores (see
Notes 29–31). For the acquisition and imaging processing we
used the fv10-asw 3.1 Viewer (Olympus) or the ZEN imaging
software (Zeiss). Figure 2 shows examples of confocal images
obtained with this transient expression method of fluorescent
protein markers for some compartments of the secretory
pathway.
4 Notes
1. The plates with solid MS can be kept in the cold chamber at
4
C for months.
2. %18–24 seeds can be sowed in each plate in order to have
enough space for seed germination and optimal growth
conditions.
3. It is preferred to keep the plates growing for 5 days in order to
have stronger seedlings that stand better vacuum infiltration.
However, 4-day-old seedlings can also be used.
4. A single colony or a previous culture of Agrobacterium kept at
4
C can also be used to prepare the pre-culture. Nevertheless it
has been reported that viability of Agrobacterium cultures kept
at 4
C decreased over time.
5. An independent culture is prepared for each fluorescent
marker.
6. This culture is prepared the day before the experiment, in order
to obtain a fresh Agrobacterium culture for the vacuum infiltration experiment.
Seedling Transformation
151