I recently tried this protocol, and the sap-inoculated leaves...

Yes it is possible.
However for our own experiments, we never do this because virus genome theoretically accumulates mutations while replicating and that 's why we prefer always use leaf sap derived from leaves of primary inoculated plants.

Hello Stéphanie, thank you for your reply. I forgot to ask as well, is it possible to save tissue from th secondary inoculation to use as inoculum for further inoculations?

Thank you,
Evan

Possible explanations :
1/ Too much carborundum.
Be carefull not to powder too much carborundum on the leaves before rubbing. If you have many plants to inoculate with the same sap, powder only the first 6-10 plants with carborundum. The miracloth will be charged with carborundum and you can inoculate all the rest of your plants without powdering their leaves. In all cases, always rinse the leaves with tap water and dry them carefully ! The inoculated leaves will appear "crumpled" the first 24 h but should recover their normal shape after.

2/ Too hard rubbing.
Did you observe some necrosis areas on the inoculated leaves ? If so, it is possible that you rubbed too hard. The rubbing intensity is also genotype-dependant. Just try to rub more gently.

The survival of the inoculated leaf is crucial for virus infection, multiplication and spreading in the hole plant and VIGS induction is dependent on these first steps.

Good luck for your next inoculations.