Génomique fonctionnelle des phytopathosystèmes
Agrobacterium-mediated floral transformation
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
- Inoculate colony in 4 ml culture in YEB media with appropriate antibiotics. Shake O/N @ 28C
- Use 1 ml to start large 300 mL of the same media with same antibiotic, again O/N @ 28C
- Spin the cells at 5000 rpm for 10 minutes
- Discard supernatant
- Resuspend cells in 300 mL 5% sucrose, 0.05% OFX-309 in water.
- Adjust OD600 at 0.8-1.5 in 200-300ml
- 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
- 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.
- Re-transform 1 week later, can also be done a third time if plants still look healthy.
- 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.