Transformation Protocol for Arabidopsis
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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.
