Protoplasts from Oidiodendron maius strains: isolation, regeneration and sensitiveness to heavy metals.

BARDI, LAURA¹, SILVIA PEROTTO², PAOLA BONFANTE².

¹Istituto Sperimentale per la Nutrizione delle Piante, Via Ormea,47,10125 Torino, ITALY, ²Centro di Studio sulla Micologia del Terreno, Viale Mattioli, 25, 10125 Torino, ITALY.

Protoplasts are a useful tool for genetical and physiological studies on filamentous fungi. Within a project devoted to the analysis of the genetic control of tolerance against heavy metals in Oidiodendron strains, we have set up a protocol to efficiently isolate and regenerate protoplasts from an Oidiodendron maius strain and from a strain isolated from soils polluted with heavy metals. The protocol is based on cell wall digestion with lytic enzymes. Optimal conditions for growth of mycelium and for enzymatic digestion of the cell wall have been examined. Isolation of protoplasts from mycelium is obtained by gradient centrifugation. Protoplasts regenerate in hypertonic medium containing sorbitol as an osmotic stabilizer. Viability of protoplasts stored at 4°C and at -80°C has also been determined. In order to use protoplasts for genetic transformation, we have examined the sensitivity of both mycelium and protoplasts to molecules commonly used as markers for transformations. M.I.C. of benomyl and hygromycine B has been identified. Protoplasts of the Oidiodendron strain tolerant to heavy metals are more sensitive than mycelium to Cd²⁺.

Key words: Protoplasts, Oidiodendron, heavy metals.