Isolation of biochemical mutants using haploid mesophyll protoplasts of Hyoscyamus muticus

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Abstract
The total isolation procedure for isolation of auxotrophic and temperature-sensitive mutants was applied to haploid mesophyll protoplasts of Hyoscyamus muticus after treatment with N-methyl-N′-nitro-N-nitrosoguanidine. Twelve variant clones were isolated after screening a total of 29,000 clones. Two are auxotrophic for histidine, one clone for tryptophan and three clones for nicotinamide. Two clones that grow only in presence of a group of amino acids including glutamine and asparagine are also ClO 3 - resistant. Two further clones have as yet undefined amino-acid requirements. Two temperature-sensitive clones were found, one of which stops growing at the restrictive temperature of 32°C and the other undergoes chlorosis and accumulates an insoluble brown pigment. All clones expressed consistently the variant phenotypes in many retests and characterisation experiments over more than one year. Shoots have been regenerated from the nicotinamide- and histidine-requiring clones and from one temperaturesensitive clone. Two control (wild-type) morphogenic clones were used: one green and the other more variably pigmented and showing some growth stimulation in presence of medium supplements.