The use of cryopreserved apical protoplasts from Curvularia lunata for etectrotransformation

An electroporation method, utilizing cryopreserved protoplasts, has been de veloped for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901. Protoplasts released from the apical parts of 24- and 48-h-old mycel ia were suspended in cryopreservation buffer and stored at -75 degrees C fo r several weeks. The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromyc in B resistance gene (hph) under the control of Aspergillus nidulans sequen ces. The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per mu g DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively). Protoplasts released from th e younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency. After 16 weeks of cryopreserv ation the yield of electroporation was 61.3 % of the control value. All iso lated electrotransformants proved to be stable for a period of >4 months ev en without selective pressure.