Isolation of an isoleucine-valine-requiring auxotroph fromDatura innoxia cell cultures by arsenate counterselection

Abstract

Cells of adenine(Ad1)- and pantothenate(Pn1)-requiring cells lines ofDatura innoxia Mill. were killed by 2 mM arsenate in the medium when growing but were not killed when in a non-growing state which was induced by starvation for required metabolites. This observation confirmed that arsenate might be useful as a counterselection agent to isolate new auxotrophs from plant cell cultures. Arsenate counterselection using predominantly haploidDatura cell suspensions led to the isolation of a cell line (I-IV) for which isoleucine and valine were both necessary and sufficient for growth. The cell line has been maintained in solid and liquid culture for about three years. The arsenate counterselection technique devised requires improvement. In some experiments, many colonies survived arsenate treatment; in others, there were no survivors. In the former experiments, putative but unstable auxotrophs were regularly observed but all were shown not to be auxotrophic after several retests.