Induction of systemic GFP gene silencing by Agrobacterium

Download 'How to agro infiltrate movie 1'
Download 'How to agro infiltrate movie 2'

1) The seeds to be used are from Nicotiana benthamiana Line 16c. This line is homozygous for one unique locus containing the following construct:

fig1

Green fluorescence is monitored with a 100 W handheld long-wave ultraviolet lamp from UV products (Black Ray model B100AP).

For silencing,

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).

figure2

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)