1) The seeds to be used are from Nicotiana benthamiana Line 16c. This line is homozygous for one unique locus containing the following construct:
Green fluorescence is monitored with a 100 W handheld long-wave ultraviolet lamp from UV products (Black Ray model B100AP).
2) The agrobacteriurn strain used for leaf infiltration is strain C58C1 (pCH32). The pCH32 plasmid carries two vir genes (vir E and vir G) and resistance to tetracycline. It also carries the silencing-inducer binary vector (which carries the construct described in 1). This plamid is under kanamycine selection. This combination of strain/ plasmid gives the best results: systemic silencing is usually achieved within 7 days after infiltration of seedlings at the 4-leaf stage (3weeks).
3) Use the stab culture supplied as a starting material and inoculate 5 mI overnight culture (28'C for agro):
-5 ml L-broth
-5 µI tetracycline (5 µl/ml final)
-5 µI kanamycin (50 µl/ml final)
4) Use all the 1 ml of the overnight culture to inoculate 50 ml L-broth and grow overnight:
-50 ml L-broth
-50 µI tetracycline (5 µglml final)
-50 µI kanamycin (50 µglml final)
-500µI MES (10 mM final) [optional]
-I0 µI Acetosyringone (20 µM final)
5) Precipitate the bacteria and resuspend the pellet in solution containing:
-10 mM MgCI2
-10 mM MES
-100 µM Acetosyringone
6) Leave on the bench for 2 to 3 hours before agroinfiltration (or overnight).
7) Perform the infiltration with a 2ml syringe. Simply press the syringe (no needle) on the underside of the leaf, and exert a counter-pressure with your finger on the other side. Infiltrate the maximum number of leaves, avoid cotyledons. (See ‘How to Agro Infiltrate movies’, links at top of page)