Isolation of biochemical mutants using haploid mesophyll protoplasts ofHyoscyamus muticus

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Abstract
Two amino-acid auxotrophic cell clones ofHyoscyamus muticus, VA5 (His-) and VIIIB9 (Trp-), isolated in a previous experiment have been characterised quantitatively. Studies of growth in the presence and absence of histidine and tryptophan and an examination of dose-response relationships for the two amino acids confirmed the strict auxotrophy of both cell lines. No revertants to prototrophy were detected in either cell line after more than two years in culture. N-methyl-N′-nitro-nitrosoguanidine did not induce reversion in VA5 cell populations. Wild-type aggregates mixed in various combinations with VA5 and VIIIB9 cells could be recovered after plating in selective conditions. No cross-feeding was detected, either between wild-type and auxotrophic cells or between the auxotrophic lines themselves. Both variants were recloned by protoplast culture. All protoplast-derived clones were auxotrophic. The auxotrophic phenotypes behaved as recessive traits in protoplast-fusion experiments.