Difference between revisions of "Transformation Protocol for Arabidopsis"

From OpenWetWare
Jump to: navigation, search
Line 22: Line 22:
Transfer transformed plants to the glasshouse''  
Transfer transformed plants to the glasshouse''  
Transformation Protocol for Arabidopsis'''            Method modified from Clough and Bent (1988)  
'''Transformation Protocol for Arabidopsis'''            Method modified from Clough and Bent (1988)  
Plant material:  
Plant material:  

Revision as of 20:07, 19 April 2007

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.

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.