Team:Cornell/project/wetlab/fungal toolkit

Protoplasting

Background

Transformation of plant and fungal cells is difficult due to their cell walls which blocks passage of foreign DNA. Protoplasting is the method by which the cells walls of plant and fungal cells are digested to produce cells without cell walls, called protoplasts. Through additional methods, such as electroporation, and PEG transformation, DNA can be uptaken by the protoplasts and then regrown into cells containing specific genes.

Method

Protoplasting was first attempted on Ganoderma lucidum. Usually this species is protoplasted using lywallzyme and novozyme. However, these enzymes are exclusievly available in China and thus protplasting was attempted with driselease and glucanex. After several attempts it was concluded that the enzymes were unable to digest the cell wall without also killing the cell. In addition, the Ganoderma mycelium was hard to pellet when centrifuging and made the protoplasting procedure difficult. Thus, Cochliobolus ____ was used instead. Protoplasting was sucessful using driselease and glucanex and then transformed via PEG solution.