11. Lay the pots flat on the side with the inflorescence on the
plastic wrap and wrap it thoroughly without damaging the
flowers (see Note 11).
12. Lay the pots with the plastic wrapped inflorescences on the side
in trays in growth chamber. Cover with another dark tray or a
dark cover. Leave covered for 24 h. Then take off the cover and
plastic wrap and grow the plants as normal in 16 h light
chamber (T0 generation).
3.3 Transgenic
Arabidopsis Selection
and Expression
1. Obtain seeds from T0 generation plants and put them out on a
large tray of soil after cold stratification (seeds are T1 generation). Also, include a pot of WT Arabidopsis Col-0 as negative
control.
2. Spray the seedlings with 100 mL of Basta (0.06 g/L) starting
on Day 3 after transfer to light. Spray every other day until
seedlings are ready to be transplanted to fresh soil (see Notes 12
and 13).
3. Grow the plants in 8 h light at 22
C for 2–3 weeks before
harvesting a leaf from each plant to check for protein expression of the transgene.
4. Extract protein from individual plants, run western blots and
use 1:15,000 HA antibody and 1:30,000 secondary antibody
to detect protein expression.
5. Allow the plants to self-fertilize and harvest seeds from the
transgenic plants that showed good expression.
6. Sow seeds out from individual T1 generation plants on MS
plates supplemented with glufosinate-ammonium (6 mg/L).
Identify homozygous lines by identifying plates where all the
seedlings survive (T2 generation). Use homozygous plants for
future experiments (see Note 7).
3.4 Biotin Infiltration
1. Grow plants as instructed in Subheading 2.4, including the
negative controls.
2. For 3–4-week-old Arabidopsis plants, infiltrate underside of
leaves with 2 mM Biotin using a 1 mL needleless syringe (see
Notes 14 and 15).
3. Blot excess liquid with paper tissues.
3.5 DEX Induction
1. Cut off infiltrated leaves (~50 leaves/construct) at the petiole
using sharp forceps and incubate leaves in 30 μM DEX solution
in 50 mL conical tubes overnight with gentle shaking/rocking
at room temperature (see Note 16).
2. After 24 h of DEX treatment, take leaves out of the solution
and blot them with paper towels to remove all excess liquid.
3. Freeze leaves in fresh 50 mL conical tubes in liquid nitrogen
and store at À80
C until ready for use (see Note 17).
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Madiha Khan et al.
plastic wrap and wrap it thoroughly without damaging the
flowers (see Note 11).
12. Lay the pots with the plastic wrapped inflorescences on the side
in trays in growth chamber. Cover with another dark tray or a
dark cover. Leave covered for 24 h. Then take off the cover and
plastic wrap and grow the plants as normal in 16 h light
chamber (T0 generation).
3.3 Transgenic
Arabidopsis Selection
and Expression
1. Obtain seeds from T0 generation plants and put them out on a
large tray of soil after cold stratification (seeds are T1 generation). Also, include a pot of WT Arabidopsis Col-0 as negative
control.
2. Spray the seedlings with 100 mL of Basta (0.06 g/L) starting
on Day 3 after transfer to light. Spray every other day until
seedlings are ready to be transplanted to fresh soil (see Notes 12
and 13).
3. Grow the plants in 8 h light at 22
C for 2–3 weeks before
harvesting a leaf from each plant to check for protein expression of the transgene.
4. Extract protein from individual plants, run western blots and
use 1:15,000 HA antibody and 1:30,000 secondary antibody
to detect protein expression.
5. Allow the plants to self-fertilize and harvest seeds from the
transgenic plants that showed good expression.
6. Sow seeds out from individual T1 generation plants on MS
plates supplemented with glufosinate-ammonium (6 mg/L).
Identify homozygous lines by identifying plates where all the
seedlings survive (T2 generation). Use homozygous plants for
future experiments (see Note 7).
3.4 Biotin Infiltration
1. Grow plants as instructed in Subheading 2.4, including the
negative controls.
2. For 3–4-week-old Arabidopsis plants, infiltrate underside of
leaves with 2 mM Biotin using a 1 mL needleless syringe (see
Notes 14 and 15).
3. Blot excess liquid with paper tissues.
3.5 DEX Induction
1. Cut off infiltrated leaves (~50 leaves/construct) at the petiole
using sharp forceps and incubate leaves in 30 μM DEX solution
in 50 mL conical tubes overnight with gentle shaking/rocking
at room temperature (see Note 16).
2. After 24 h of DEX treatment, take leaves out of the solution
and blot them with paper towels to remove all excess liquid.
3. Freeze leaves in fresh 50 mL conical tubes in liquid nitrogen
and store at À80
C until ready for use (see Note 17).
434
Madiha Khan et al.