Botrytis cinerea Protoplasts Production and Inoculation.

The absence of a cell wall turns fungal protoplasts very receptive to the uptake of exogenous genetic material and, consequently, they can be used for genetic transformation or transfection experiments. Additionally, isolated monosporic fungal strains, naturally infected or previously transfected with a mycovirus, can be used in assays of fungal protoplasts fusion. This technology will overcome the barriers of vegetative incompatibility with other Botrytis cinerea strains, or even with other fungi, and successfully allow the transference of mycoviruses from an infected to a non-infected fungal strain. We describe in this chapter the protocols to isolate B. cinerea protoplasts; to perform fungal protoplasts transfection or transformation; and to do protoplasts fusion assays to transfer mycoviruses from infected to non-infected B. cinerea strains.