Formation, regeneration and fusion of protoplasts of Rhizobium leguminosarum bv. viciae and genetic and symbiotic characterization of recombinants

The protoplasts of two mono-auxotrophs (trp⁻ and met⁻) of R. leguminosarum bv. viciae were formed by the tris-sorbitol-EDTA-lysozyme method, regenerated on double-layered medium and fused with 40% polyethylene glycol (PEG). The average protoplasting and regeneration frequency was 85 and 11%, respectively. The frequency of recombinants was found to be in the range of 10⁻² to 10⁻⁴. The symbiotic properties of mono-auxotrophs and recombinants (tested on pea plants) had lower values than the wild type strain Rld1 (the parental strain of mono-auxotrophs). Though the protoplast formation, regeneration and fusion by PEG treatment was feasible in R. leguminosarum bv. viciae, yet the genetic recombination between mono-auxotrophs did not lead to the enhancement of symbiotic properties.