Transformation Protocol for Arabidopsis

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Transformation Protocol for Arabidopsis – Abbreviated  
Transformation Protocol for Arabidopsis – Abbreviated  
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''Germinate seed in pots  
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Germinate seed in pots  
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↓ 4 weeks  
↓ 4 weeks  
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Transfer transformed plants to the glasshouse  
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Transfer transformed plants to the glasshouse''
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  '''
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Transformation Protocol for Arabidopsis'''            Method modified from Clough and Bent (1988)  
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Transformation Protocol for Arabidopsis
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Method modified from Clough and Bent (1988)  
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Plant material:  
Plant material:  
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• Incubate overnight at 4°C then in light at 20°C.  After 10 days select  
• Incubate overnight at 4°C then in light at 20°C.  After 10 days select  
transformants, transplant to soil.  
transformants, transplant to soil.  
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References:
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Clough SJ and Bent AF, 1998. Floral dip: a simplified method for
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Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
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16:735-43. 
   
   
   
   
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Media and solutions for Arabidopsis transformation
 
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YM Media   
 
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1L
 
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Mannitol                        10g                 
 
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Yeast extract                  0.4g
 
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K2HPO4 (10% w/v stock)            1 ml
 
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KH2PO4 (10% w/v stock)            4 ml
 
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NaCl (10% w/v stock)                1 ml
 
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MgSO4.7H2O (10% w/v stock)    2 ml
 
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• pH 6.8
 
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• Agar 15g/L 
 
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• Autoclave
 
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• When ready to pour add:
 
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• Antibiotic selection if required
 
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Keep poured plates for 2 days at room temperature to visualise any
 
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contamination, then store at 4°C.
 
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Minimal A Medium  (Svab, et al, 1975)
 
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Liquid  1L
 
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Stock solution A  490mL 
 
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Stock solution C  11mL 
 
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Milli-Q H2O  499mL 
 
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Solid  1L
 
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Stock solution A  490mL 
 
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Stock solution B  499mL 
 
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Stock solution C  11mL 
 
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• Store 4°C
 
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Minimal A Stock Solution A   
 
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  Final concentration
 
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    g/490mL    1 x mM
 
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K2HPO4  10.5    60
 
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KH2PO4  4.5    33
 
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(NH4)2SO4  1.0    7.6
 
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tri-Sodium citrate.2H20 0.5    1.7
 
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• Autoclave in 490mL aliquots
 
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• Store 4°C
 
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• Substituting chemicals
 
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• K2HPO4.3H2O    13.8g/490mL
 
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Minimal A Stock Solution B   
 
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Dissolve 15g agar in Milli-Q H2O, make up to 500mL
 
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• Autoclave
 
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• Store room temperature
 
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Minimal A Stock Solution C   
 
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Add 1mL 1M MgSO4 to 10mL 40% sucrose 
 
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• Store room temperature
 
   
   
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Infiltration media
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Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by
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    200ml  Final conc.  
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co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In
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10X MS salts  20ml  1xMS salts
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“Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga,
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Sucrose  10g  5% 
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D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold
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MES    0.2g  0.1% 
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Spring Harbor Press: 55-77.
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Silwet-L77  200
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Revision as of 23:06, 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.

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