Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

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Abstract
Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl2, and 5.0 mM MgCl2. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl2, and 5.0 mM MgCl2 which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl2, and 5.0 mM CaCl2. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1AG, 27% for strain EAN1pec, and 20% for strain EuI1c.