Transient_expression_Nicotiana_tabacum.doc

Personne référente : Carine Alcon
Version : 2 (Voir la version 1)
Date : september, 2010

Agrobacterium tumefaciens (GV3101) infiltration of the leaf under-epidermis
The quality of the plant is essential for successful transformation. Plants should be approximately two month-old (short day). Seeds sown on soil should be transferred individually in pot after a month. Plants with no more than 8 leaves are ideal. Exclude plants with a flower bud. The youngest large leaves are also the best.

• Grow 1 ml Agrobacterium culture overnight in LB with antibiotics in a 28°C shaker starting from a single colony grown on a LB plate with antibiotics (Rifampycin 50µg/ml and Gentamycin 25µg/ml for GV3101 agrobacteria plus plasmid antibiotic resistance).
• Centrifuge 3 min at 5000 rpm
• Resuspend the pellet in 1 ml infiltration medium
• Determine the OD600 in a 20 fold dilution of this solution
• Centrifuge 3 min at 5000 rpm and resupend the pellet in infiltration medium at a final concentration of OD600 = 10.
• Dilute the Agrobacterium solution to an OD600 = 0.03 up to 0.3 for infiltration.
  OD 0.03 is the standard OD which works well for most of the constructs but it can be increased or reduced based on the obtained effect, taking into account potential overexpression toxicity. For the simultaneous expression of different constructs combine the different Agrobacterium solutions harbouring the different constructs in this solution with each of them at the chosen OD.
• Use a 200 µl pipet tip to slightly damage the under epidermis of the tobacco leaf
• Transfer the infiltration solution to a small (1 ml) syringe (without a needle) and push this gently on the small wound in the leaf. Put a finger on the other side of the leaf and slowly apply pressure with the syringe. Avoid air between the agrobacteria suspension in the syringe and the leaf.
• Several different constructs can be infiltrated in the same leaf as long as the veins separates the various transformed areas.
• Observe the leaf under epidermis but cutting out a piece of leaf. Expression can be visible staring 24h after infiltration up to several days (ideally 2 or three days). Note that subcellular localization may change over the length of expression if the protein is transiting through intermediate locations prior to its accumulation in its final destination.

Add up to the final volume with milliQ water and filter sterilize the mixture.

Stock solutions
MES (4-morpholinoEthaneSulfonic acid), 100 mM, pH 5.6 (KOH) stored at 4°C
Na3PO4 20 mM
Acetosyringone (3,5 Dimethoxy-4-hydroxyacetopheron HOC6H2(OCH3)2COCH3) 200 mM in 70% ethanol. Store in aliquots at -20°C and do not refreeze.