3 Methods
3.1 Growth
of Seedlings Before
Infiltration
1. Pour 4 mL hot sterile MS agar medium into the plates in a
sterile chamber and let the plates cool until the medium is solid
(see Note 1).
2. Sterilize Arabidopsis seeds for plating in a sterile chamber. Put
seeds in an Eppendorf tube and add %1 mL of Solution I. Incubate for 3 min by inverting the tube several times to keep seeds
in solution. Let seeds settle. Remove Solution I.
3. Add 1 mL of Solution II. Incubate for 1 minute by inverting
the tubes as above. Take the seeds with the minimum possible
volume of Solution II with a micropipette (you may need to cut
the tip) and put them on a sterilized filter paper. Let seeds dry.
4. Sow Arabidopsis sterilized seeds on the medium of the plates by
using a sterile toothpick (see Note 2).
5. Seal plates with gas-permeable surgical tape.
6. Store the plates at 4
C in the dark for 2–4 days to stratify the
seeds and synchronize germination.
7. Place the plates in the growth chamber for 4–5 days (see Note
3). After germination, expanded cotyledons will be observed in
the resultant seedlings.
Fig. 1 Equipment used in the Agrobacterium-mediated transformation of Arabidopsis seedlings by vacuum
infiltration. Left panel shows the vacuum pump connected to the vacuum desiccator via a pressure
manometer. Right panels show a typical Petri dish containing the seedlings used for transformation and the
arrangement of 6 dishes within the vacuum desiccator
Seedling Transformation
149
3.1 Growth
of Seedlings Before
Infiltration
1. Pour 4 mL hot sterile MS agar medium into the plates in a
sterile chamber and let the plates cool until the medium is solid
(see Note 1).
2. Sterilize Arabidopsis seeds for plating in a sterile chamber. Put
seeds in an Eppendorf tube and add %1 mL of Solution I. Incubate for 3 min by inverting the tube several times to keep seeds
in solution. Let seeds settle. Remove Solution I.
3. Add 1 mL of Solution II. Incubate for 1 minute by inverting
the tubes as above. Take the seeds with the minimum possible
volume of Solution II with a micropipette (you may need to cut
the tip) and put them on a sterilized filter paper. Let seeds dry.
4. Sow Arabidopsis sterilized seeds on the medium of the plates by
using a sterile toothpick (see Note 2).
5. Seal plates with gas-permeable surgical tape.
6. Store the plates at 4
C in the dark for 2–4 days to stratify the
seeds and synchronize germination.
7. Place the plates in the growth chamber for 4–5 days (see Note
3). After germination, expanded cotyledons will be observed in
the resultant seedlings.
Fig. 1 Equipment used in the Agrobacterium-mediated transformation of Arabidopsis seedlings by vacuum
infiltration. Left panel shows the vacuum pump connected to the vacuum desiccator via a pressure
manometer. Right panels show a typical Petri dish containing the seedlings used for transformation and the
arrangement of 6 dishes within the vacuum desiccator
Seedling Transformation
149