8. Add 8 mL Agrobacterium culture into 100 mL LB liquid
medium and culture at 28
C for 8 h.
9. Spin down cultured Agrobacterium cells by centrifugation at
5000 g for 10 min at room temperature.
10. Prepare floral dip transformation solution during centrifuge.
11. Resuspend Agrobacterium pellet into the 100 mL floral dip
transformation solution.
12. Transform Arabidopsis by the flower dipping method
[24]. Briefly, soak Arabidopsis flowers in the resuspended Agrobacterium solution, and place under dark and humid conditions overnight.
13. Allow transformed Arabidopsis plants to grow and collect
seeds.
3.1.6 Screening of
Mutations or Base Editings
The modified target gene is detected by PCR and restriction
enzyme digestion (restriction fragment length polymorphism:
RFLP) (Figs. 3b and 7a) (see Note 5).
1. Sterilize 100 to 200 μL T1 Arabidopsis seeds (obtained in
Subheading 3.1.5, step 13) with 5% sodium hypochlorite solution in 1.5 mL microcentrifuge tube by rotation for 10 min.
2. Wash sterilized seeds five times with 1 mL ddH 2 O. Spin down
the seeds and resuspend with ddH 2 O, repeat five times.
3. Resuspend the washed seeds with 1 mL 0.05% agar, and store at
4
C for 2 days.
4. Plate sterilized seeds on 0.5x MS medium containing 30 mg/L
hygromycin with 50 mg/L carbenicillin to restrict Agrobacterium growth and screen antibiotic resistance seedlings. Seal the
petri dishes with surgical tape.
5. Transplant antibiotic resistant T1 Arabidopsis plants in soil, and
grow them in greenhouse under long day conditions (16 h
light-8 h dark) at 22
C.
6. Collect one or two rosette leaves from each T1 transgenic
plants in 1.5 mL microcentrifuge tube with 2 or 3 steel balls,
put them into liquid nitrogen, and homogenize leaf tissues by
shaking or by using homogenizer.
7. Add 500 μL CTAB buffer to the microcentrifuge tubes, then
mix well and thaw the frozen sample tissues.
8. Incubate at 65
C for 30 min, invert tubes every 10 min.
9. Add 300 μL of chloroform/isoamyl alcohol, and vortex well.
10. Centrifuge at maximum speed (e.g., 13,000 Â g) for 5 min at
room temperature.
11. Transfer 400 μL of the upper layer aliquot to a new 1.5 mL
microcentrifuge tube.
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medium and culture at 28
C for 8 h.
9. Spin down cultured Agrobacterium cells by centrifugation at
5000 g for 10 min at room temperature.
10. Prepare floral dip transformation solution during centrifuge.
11. Resuspend Agrobacterium pellet into the 100 mL floral dip
transformation solution.
12. Transform Arabidopsis by the flower dipping method
[24]. Briefly, soak Arabidopsis flowers in the resuspended Agrobacterium solution, and place under dark and humid conditions overnight.
13. Allow transformed Arabidopsis plants to grow and collect
seeds.
3.1.6 Screening of
Mutations or Base Editings
The modified target gene is detected by PCR and restriction
enzyme digestion (restriction fragment length polymorphism:
RFLP) (Figs. 3b and 7a) (see Note 5).
1. Sterilize 100 to 200 μL T1 Arabidopsis seeds (obtained in
Subheading 3.1.5, step 13) with 5% sodium hypochlorite solution in 1.5 mL microcentrifuge tube by rotation for 10 min.
2. Wash sterilized seeds five times with 1 mL ddH 2 O. Spin down
the seeds and resuspend with ddH 2 O, repeat five times.
3. Resuspend the washed seeds with 1 mL 0.05% agar, and store at
4
C for 2 days.
4. Plate sterilized seeds on 0.5x MS medium containing 30 mg/L
hygromycin with 50 mg/L carbenicillin to restrict Agrobacterium growth and screen antibiotic resistance seedlings. Seal the
petri dishes with surgical tape.
5. Transplant antibiotic resistant T1 Arabidopsis plants in soil, and
grow them in greenhouse under long day conditions (16 h
light-8 h dark) at 22
C.
6. Collect one or two rosette leaves from each T1 transgenic
plants in 1.5 mL microcentrifuge tube with 2 or 3 steel balls,
put them into liquid nitrogen, and homogenize leaf tissues by
shaking or by using homogenizer.
7. Add 500 μL CTAB buffer to the microcentrifuge tubes, then
mix well and thaw the frozen sample tissues.
8. Incubate at 65
C for 30 min, invert tubes every 10 min.
9. Add 300 μL of chloroform/isoamyl alcohol, and vortex well.
10. Centrifuge at maximum speed (e.g., 13,000 Â g) for 5 min at
room temperature.
11. Transfer 400 μL of the upper layer aliquot to a new 1.5 mL
microcentrifuge tube.
CRISPR/Cas9-Based Genome Editing in Arabidopsis
137