Transformation Protocol for Arabidopsis – Abbreviated
Germinate seed in pots
↓ 4 weeks
Streak bacteria onto YM/MinA
↓ 2-3 days 28°C
Spray/dip bacterial suspension onto plants
↓ 1 day in box
Let plants set seed
↓ 3-4 weeks
Plate seed to GM + selection
↓
Transfer transformed plants to the glasshouse
Plant material: ? Thinly sow Arabidopsis thaliana cv C24 seed into soil. Grow at 20°C under 16h light until plants are beginning to flower. Cut off any seed pods that have formed prior to transformation.
Prepare bacteria: ? Streak YM plus antibiotic plates with bacteria, or Minimal A plus antibiotic plates for Agrobacterium. ? Incubate plates for 2-3 days at 28°C.
Transformation: ? Measure about 20mL Infiltration Medium for each bacterial strain. ? Scrape or wash bacteria from plate with sterile loop and suspend in 20mL of Infiltration media. ? Adjust density to an OD600 0.9-1.0. ? Dip flowers and unopened buds of Arabidopsis into the bacterial suspension, or alternatively the plants can be sprayed with bacterial suspension using a plant mister. ? Cover plants to maintain humidity overnight. Grow at 20°C in light. ? Repeat dipping/spraying after 7 days. ? Collect seed, store at 4°C
Selection: ? Weigh seed to estimate number (100 seed ≈ 21.8mg) ? Sterilise for 2 mins in 70% (v/v) ethanol followed by 20 mins in 20% (v/v) bleach (Zixo brand) + 0.02% Triton X-100. Keep the seeds moving in the bleach solution by putting tubes on a rotator wheel. ? Wash seeds 5 times with sterile distilled water (in laminar flow hood) ? Plate to Germination Medium + selection plates, seal with Micropore tape. ? Incubate overnight at 4°C then in light at 20°C. After 10 days select transformants, transplant to soil.
References:
Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.
Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55-77.