AGROBACTERIUM TUMEFACIENS-MEDIATED GENETIC TRANSFORMATION OF THE ECTENDOMYCORRHIZAL FUNGUS TERFEZIA BOUDIERI

Abstract

Mycorrhizal fungi are integral component of the plant ecosystem, potentially improving the establishment and yield of many crop plants, especially in harsh environments. Terfezia boudieri is a desert truffle that produces hypogeous fruit bodies that are highly valued and consumed all over the Mediterranean basin. Although genome sequencing of this important desert truffle has been completed recently, the molecular tools available to study these fungi group are very limited. Hence, only few genetic and genomic studies were carried in these species. Our goal is to develop molecular tools for genetic manipulations in these fungi. We developed an Agrobacterium tumefaciens-mediated genetic transformation system for T. boudieri. The plasmid PBGgHg conferring hygromycin resistance (hpt) as selection marker and the green fluorescent protein gene (gfp) as a visible marker was used for transformation with the Agrobacterium strain AGL1. Optimization parameters for this procedure included: culture conditions, antibiotics concentration, bacterial density, co-cultivation time, acetosyringone concentration and different ages of the fungal hyphae. The selection of transformed hyphae cubes was carried on selection medium containing 30 mg L^‑1 hygromycin. The highest frequency of transformation was (26 %) verified after four rounds of selection in medium supplemented with hygromycin at concentration up to 200 mg L^‑1. The presence of gfp in the hygromycin resistant fungi and molecular confirmation of the transformation events are underway. The protocol developed is the first step in breaking the bottleneck of genetic manipulations in desert truffles enabling future research on the life cycle of the fungus.

Keywords: Agrobacterium tumefaciens, Desert truffles, Ectendomycorrhizal fungus, Hygromycin, Terfezia boudieri.