Other procedures

The development of an efficient transformation protocol for L. laccata suggests that other ectomycorrhizal fungi can be transformed, provided sufficient numbers of viable protoplasts can be formed. Incubation of intact hyphae in lithium ion-containing buffers (Dhawale et al., 1984; Binninger et al., 1986; Bej et al., 1989) has provided an alternative, although not always efficient, means of introducing DNA into fungal cells. This or other procedures may prove feasible for transforming species of ectomycorrhizal fungi not suited for efficient protoplast formation.

One strategy for transformation used recently with fungal systems is electroporation (Delmore, 1989; Richey et al., 1989; Goldman et al.,

1990). Electroporation is non-destructive permeabilization of biological membranes in short-duration, high-amplitude electric fields. This technique may provide a method of introducing DNA into species of fungi not amenable to or inefficient for transformation by the traditional calcium ion and PEG treatments. Yet another technique, a ballistic process involving DNA bound to microprojectiles (Sanford, 1988), can introduce DNA into cells by high velocity propulsion in a partial vacuum. This procedure has been effective in transformation studies involving mammalian cells as well as intact plant cells (Sanford, 1988, 1990) and might be especially useful if adapted to fungal systems where cell wall removal and efficient protoplast generation have not been successful.

IV. Concluding remarks

Current research with ectomycorrhizal fungi involves the use of protoplasts as starting material for genetic manipulation through DNAmediated transformation (Barrett et al., 1990). Transformation procedures are expected to extend more traditional approaches to genetic study and thus enhance the potential for selective breeding and strain improvement of these fungi. Through the use of protoplasts and procedures of molecular genetics it should be possible to decipher the complex genetic behaviour of ectomycorrhizal formation and to identify traits which affect that symbiosis. The goal of such research is to develop improved fungal strains for use as inocula in sylvicultural practices related to tree seedling production, forest productivity and reafforestation. To accomplish this goal it will be necessary to obtain fundamental information on relevant genes and their expression in order to carry out genetic manipulations leading to improved ectomycorrhiza. In considering control strategies to improve symbiosis, it will be important to avoid non-target effects that could disrupt the balance among rhizosphere components. The overall intention of the research is to optimize symbiosis through genetic manipulation of the fungal component without impairment of components of rhizosphere ecology that may be supportive of that symbiosis.

Regardless of the specific gene(s) introduced, and perhaps in some cases impaired, by transformation, transformed phenotypes of ectomycorrhizal fungi should be stable due to integration of the foreign DNA into the fungal chromosome. This stability adds to the desirability of developing transgenic fungi as inocula to improve symbioses in order to ameliorate stress factors that adversely affect plants.


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