Transformation Protocol for Arabidopsis

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

Transformation Protocol for Arabidopsis

Method modified from Clough and Bent (1988)

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.

Media and solutions for Arabidopsis transformation

YM Media 1L Mannitol 10g Yeast extract 0.4g K2HPO4 (10% w/v stock) 1 ml KH2PO4 (10% w/v stock) 4 ml NaCl (10% w/v stock) 1 ml MgSO4.7H2O (10% w/v stock) 2 ml

• pH 6.8 • Agar 15g/L • Autoclave • When ready to pour add: • Antibiotic selection if required

Keep poured plates for 2 days at room temperature to visualise any contamination, then store at 4°C.

Minimal A Medium (Svab, et al, 1975)

Liquid 1L Stock solution A 490mL Stock solution C 11mL Milli-Q H2O 499mL

Solid 1L Stock solution A 490mL Stock solution B 499mL Stock solution C 11mL

• Store 4°C

Minimal A Stock Solution A

 Final concentration 
   g/490mL    1 x mM 

K2HPO4 10.5 60 KH2PO4 4.5 33 (NH4)2SO4 1.0 7.6 tri-Sodium citrate.2H20 0.5 1.7

• Autoclave in 490mL aliquots • Store 4°C • Substituting chemicals • K2HPO4.3H2O 13.8g/490mL

Minimal A Stock Solution B

Dissolve 15g agar in Milli-Q H2O, make up to 500mL

• Autoclave • Store room temperature

Minimal A Stock Solution C

Add 1mL 1M MgSO4 to 10mL 40% sucrose

• Store room temperature

Infiltration media

   200ml  Final conc. 

10X MS salts 20ml 1xMS salts Sucrose 10g 5% MES 0.2g 0.1% Silwet-L77 200