Production and regeneration of protoplasts from gremmeniella abietina and ascocalyx abietis

Abstract

This work was undertaken to develop a system of protoplast isolation and regeneration for G. abietina and A. abietis that could be of use for the genetic manipulation of both species. Nuclear staining was performed to assess the nuclear conditions of the protoplasts. Of the 19 enzyme complexes studied, only 10 were found to have some lytic effect on either Gremmeniella, A. abietis, or both. Only snail enzyme (from Szeged University), Novozym 234, lytic enzyme L1, or mixtures of snail enzyme and Novozym 234 produced satisfactory yields of protoplasts. Regeneration of protoplasts was observed on complete and on minimal medium, and occurred from protoplasts plated out directly onto the surface and from those embedded in the agarose. In most cases, embedding increased the frequency of regeneration. Protoplasts formed after incubation at 20 degrees C regenerated at a frequency of approximately 5%, as opposed to 2% for those produced at 30 degrees C. As roughly 40% of the protoplasts were anucleate, the percentage of regeneration can be estimated as about 12.5% at 20 degrees C and 5% at 30 degrees C. Protoplasting appears to be a satisfactory method of obtaining material for genetic experiments with G. abietina and A. abietis when other methods are not directly applicable.