Callus regeneration from Trifolium subterraneum protoplasts and enhanced protoplast division by low-voltage treatment and nurse cells

Seedling and suspension culture protoplasts of subterranean clover (Trifolium subterraneum L.) were successfully cultured in semi-solid drops of calcium alginate and ultrafiltered liquid medium. Protoplast-derived subterranean-clover colonies developed as the osmolality was lowered over three steps. Callus was established from these colonies. Calli derived from protoplasts have failed to regenerate on a range of media. The frequency of dividing subterranean-clover protoplasts was increased in the presence of lucerne (Medicago sativa L.) nurse cells. Low-voltage treatments (200 mV) for the first 16–132 hours of culture also resulted in a 100% increase in the frequency of dividing protoplasts.