different genetic backgrounds in a few days and requiring a small
growth room space. Few approaches of Agrobacterium-mediated
transient expression in Arabidopsis seedlings have been developed
[2–5]. This chapter describes a protocol for Arabidopsis seedling
transformation by vacuum infiltration of Agrobacterium, based on a
previous report [2] with modifications. In addition, we present
examples of confocal images obtained with this transient expression
method of fluorescent protein markers for some compartments of
the secretory pathway, including endoplasmic reticulum (ER),
Golgi apparatus, plasma membrane, and vacuole.
2 Materials
2.1 Buffers,
Solutions and Media
1. Seed sterilization solution I: 70% ethanol, 0.05% Triton-X 100.
2. Seed sterilization solution II: 96% ethanol.
3. MS agar medium: 0.5Â Murashige and Skoog (MS) mineral
salt (0–1% sucrose), 2.5 mM 2-(N-Morpholino) ethanesulfonic
acid (MES), 0.6% phytoagar. Adjust pH to 5.7 with KOH.
Autoclave and store at 65
C until pouring it into plates.
4. Liquid MS medium: 0.5Â MS mineral salt, 1% sucrose, 5 mM
2-(N-Morpholino) ethanesulfonic acid (MES). Adjust pH to
5.7 with KOH. Autoclave and store at 4
C.
5. Luria-Bertani (LB) medium. Autoclave and store at room temperature. After addition of the appropriate antibiotic for Agrobacterium selection, store at 4
C.
6. 1 M Acetosyringone in DMSO.
2.2 Equipment
1. Plant growth chamber at 22
C, 16 h/8 h photoperiod with
16 h of white, cold, and fluorescent light (150 μE m
À2 s
À2 ,
Sylvania Standard F58W/133-T8).
2. Incubator for growth of bacterial cultures at 28
C.
3. Vacuum pump (Fig. 1).
4. Vacuum desiccator, 15 cm interior diameter (Fig. 1).
5. Plates: 35 Â 10 mm Petri dishes (Fig. 1).
6. Pressure manometer (Fig. 1).
7. Spectrophotometer.
2.3 Plants,
Agrobacterium
Strains, and Vectors
1. Seeds of Arabidopsis ecotype Columbia (Col-0).
2. A. tumefaciens GV3101::pMP90 strain [6] transformed with
the T-DNA vector containing the fluorescence marker of
interest.
148
Ce ´ sar Bernat-Silvestre et al.
growth room space. Few approaches of Agrobacterium-mediated
transient expression in Arabidopsis seedlings have been developed
[2–5]. This chapter describes a protocol for Arabidopsis seedling
transformation by vacuum infiltration of Agrobacterium, based on a
previous report [2] with modifications. In addition, we present
examples of confocal images obtained with this transient expression
method of fluorescent protein markers for some compartments of
the secretory pathway, including endoplasmic reticulum (ER),
Golgi apparatus, plasma membrane, and vacuole.
2 Materials
2.1 Buffers,
Solutions and Media
1. Seed sterilization solution I: 70% ethanol, 0.05% Triton-X 100.
2. Seed sterilization solution II: 96% ethanol.
3. MS agar medium: 0.5Â Murashige and Skoog (MS) mineral
salt (0–1% sucrose), 2.5 mM 2-(N-Morpholino) ethanesulfonic
acid (MES), 0.6% phytoagar. Adjust pH to 5.7 with KOH.
Autoclave and store at 65
C until pouring it into plates.
4. Liquid MS medium: 0.5Â MS mineral salt, 1% sucrose, 5 mM
2-(N-Morpholino) ethanesulfonic acid (MES). Adjust pH to
5.7 with KOH. Autoclave and store at 4
C.
5. Luria-Bertani (LB) medium. Autoclave and store at room temperature. After addition of the appropriate antibiotic for Agrobacterium selection, store at 4
C.
6. 1 M Acetosyringone in DMSO.
2.2 Equipment
1. Plant growth chamber at 22
C, 16 h/8 h photoperiod with
16 h of white, cold, and fluorescent light (150 μE m
À2 s
À2 ,
Sylvania Standard F58W/133-T8).
2. Incubator for growth of bacterial cultures at 28
C.
3. Vacuum pump (Fig. 1).
4. Vacuum desiccator, 15 cm interior diameter (Fig. 1).
5. Plates: 35 Â 10 mm Petri dishes (Fig. 1).
6. Pressure manometer (Fig. 1).
7. Spectrophotometer.
2.3 Plants,
Agrobacterium
Strains, and Vectors
1. Seeds of Arabidopsis ecotype Columbia (Col-0).
2. A. tumefaciens GV3101::pMP90 strain [6] transformed with
the T-DNA vector containing the fluorescence marker of
interest.
148
Ce ´ sar Bernat-Silvestre et al.