Transformation Protocol for Arabidopsis: Difference between revisions

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Transformation Protocol for Arabidopsis – Abbreviated  
Transformation Protocol for Arabidopsis – Abbreviated  
 
''Germinate seed in pots  
Germinate seed in pots  
   
   
↓ 4 weeks  
↓ 4 weeks  
Line 24: Line 23:
↓   
↓   
   
   
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:  
Line 38: Line 34:
pods that have formed prior to transformation.  
pods that have formed prior to transformation.  
   
   
Prepare bacteria:  
Prepare bacteria:  
• Streak YM plus antibiotic plates with bacteria, or Minimal A plus  
• Streak YM plus antibiotic plates with bacteria, or Minimal A plus  
antibiotic plates for Agrobacterium.  
antibiotic plates for Agrobacterium.  
• Incubate plates for 2-3 days at 28°C.  
• Incubate plates for 2-3 days at 28°C.  
   
   
Transformation:  
Transformation:  
Line 55: Line 53:
• Collect seed, store at 4°C  
• Collect seed, store at 4°C  
   
   
Selection:  
Selection:  
• Weigh seed to estimate number (100 seed ≈ 21.8mg)  
• Weigh seed to estimate number (100 seed ≈ 21.8mg)  
Line 65: Line 64:
• 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.  
* [[Media and solution preparation for this protocol]]
   
   
References:
 
Media and solutions for Arabidopsis transformation
Clough SJ and Bent AF, 1998. Floral dip: a simplified method for  
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
16:735-43.
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 
Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by
• Autoclave
co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In
• When ready to pour add:
“Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga,
• Antibiotic selection if required
D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold
Spring Harbor Press: 55-77.
Keep poured plates for 2 days at room temperature to visualise any
 
contamination, then store at 4°C.  
[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]] [[Category:Arabidopsis thaliana]]
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

Latest revision as of 09:57, 23 February 2009

back to protocols

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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