SUMMARY: We have attempted to undertake genetic analysis in using the technique of protoplast fusion that has been successfully applied in and Efficient production of protoplasts, fusion and regeneration techniques have been established. However, variability in numbers and types of recombinants using two-, three-, and four-factor crosses was observed throughout these studies. No linkages were detected, even between loci known to be linked by cotransduction with bacteriophage MP 13. These results were similar to those reported by Alföldi and coworkers using strain 216, even though the experimental design was significantly changed. During initial subculturing, segregants were observed in a 1:2:2 ratio of noncomplementing diploids:parental-l:parental-2. The ratio changed dramatically after seven subcultures. Double recombinants appeared after nine subcultures. These results corroborate those reported in and suggest that there is a locus-inactivation phenomenon present in which is not evident in or Until the mechanism is elucidated, protoplast fusion should not be used for chromosomal mapping in However, it can be used to transfer plasmids among the bacilli at a frequency of 10-10 per regenerated protoplast.


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