Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

Nisha Govender, Mui-Yun Wong

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explants and Agrobacterium strain LBA 4404 with successful expression of hygromycin (hyg) marker gene and gus-gfp fusion gene, under the control of heterologous p416 gpd promoter. Optimal transformation conditions included 1:100 Agrobacterium to explant ratio, vir genes induction of the Agrobacterium in presence of 250 µm of acetosyringone, co-cultivation at 22 °C for 2 days on nitrocellulose membrane overlaid on an induction medium and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Transformants evaluated were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.