Agrobacterium-mediated floral transformation

Floral dipping

This protocol is derived from:

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735-743.

 

however we observed that transformation efficiency could be increased when Silwet L-77 is substituted by OFX-309

 

Mireault, C., Paris, L.-P., Germain, H. 2014. Enhancement of the Arabidopsis floral dip method with XIAMETER OFX-0309 as alternative to Silwet L-77 surfactant. Botany. 92:1-3  

 

YEB medium
5g/l beef-extract
1g/l yeast-extract
5g/l peptone
5g/l sucrose

2 mM MgSO4

 

 

Procedure:

 

1)      Inoculate colony in 4 ml culture in YEB media with appropriate antibiotics. Shake O/N @ 28C

2)      Use 1 ml to start large 300 mL of the same media with same antibiotic, again O/N @ 28C

3)      Spin the cells at 5000 rpm for 10 minutes.

4)      Discard supernatant

5)      Resuspend cells in 300 mL 5% sucrose, 0.05% OFX-309 in water.

6)    Adjust OD600 at 0.8-1.5 in 200-300ml

7)      Dip or spray the flowers of 4-5 weeks plants (stems half of full size is better than full size plants), cover with a dome. Crack dome after 24h, remove after 48h.

 

Tips to improve efficiency:

 

1)      Cut the main stem of the plants a few days after bolting to allow several axillary shoots to come emerge. This will cause a little delay but more flowers will be available for transformation.

2)      Re-transform 1 week later, can also be done a third time if plants still look healthy.

3)      If using a lousy selection like hygromycin or kanamycin (by opposition to Basta) all siliques can be removed from plants prior to transformation to decrease the number of seeds to be plated.