Isolation of Haustorium Protoplasts Optimized by Orthogonal Design for Transient Gene Expression in Phelipanche aegyptiaca

The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in <i>Phelipanche aegyptiaca</i>. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for <i>P. aegyptiaca</i>. Even the parasitic mechanisms of <i>P. aegyptiaca</i> and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in <i>Phelipanche aegyptiaca</i> haustorium. The optimal protoplast yield and activity were 6.2 × 10⁶ protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 μg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts' isolation and transient transformation system of <i>Phelipanche aegyptiaca</i> haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants.