Mutation induction is a feasible and established breeding method for crop improvement and genetic diversity creation to introduce new plant cultivars. The present study was aimed at mutagenesis of four chrysanthemum cultivars (‘Homa’, ‘Fariba2’, ‘Arina’, and ‘Delkash’) using ethyl methanesulfonate (EMS) (0, 0.125, 0.25, and 0.5%) as mutagen and leaf disks as explants to obtain novel variants. In addition, genetic polymorphism among mutants and their parents was detected using inter simple sequence repeat (ISSR) and inter-retrotransposon amplifed polymorphism (IRAP) molecular markers. A total of 2082 plantlets were produced through EMS induced mutagenesis under in vitro conditions and at the end 58 mutants including 28 leaf and 32 fower mutants were analyzed for phenotypic and molecular variation. The explant survival rate decreased by increasing EMS concentration. A wide range of phenotypic leaf and inforescence variability was obtained in four studied chrysanthemum cultivars confrming the efciency of EMS to create genetic variation and desired mutants. All generated variants with diferent inforescence and leaf shape and color were maintained through cuttings and they expressed same traits in the next generation. The mutants were diferent in leaf size and shape, plant height, day to fowering, inforescence head size, ray foret color and ray foret size. The used ISSR and IRAP primers could classify chrysanthemum mutants based on cultivar and somewhat based on used EMS concentration confrming their efectiveness for the discrimination of real variants that allow their earlier selection and reduction of the mutant population size. The in vitro EMS-induced mutation can be a promising tool to assist breeding programs for the generation of new chrysanthemum cultivars.
