Chemical mutagenesis and chimera dissolution in vegetatively propagated banana

Abstract

Random mutagenesis has been widely used for forward-genetics and crop breeding since the application of ionising radiation on cereals described in the late 1920s. The development of high-throughput and accurate mutation discovery technologies has enabled reverse-genetic screening of mutant populations in the twenty-first century. The majority of mutation-based approaches in crops have involved seed-propagated species. Large bodies of data are available on the spectrum and density of induced mutations for some mutagens. It is well established that genetic chimerism caused by random accumulation of different mutations in different cells is resolved by sexual propagation and that by the second-generation post-mutagenesis (termed the M2), plants are no longer genetically mosaic. Vegetatively propagated species, however, are quite different as they primarily undergo mitotic propagation. In the absence of meiosis, procedures must be implemented to remove mosaicism and generate plant material that is genotypically homogeneous and suitable for forward- and reverse-genetic screening and breeding. We have previously developed a Targeting Induced Local Lesions IN Genomes (TILLING) platform for the vegetatively propagated triploid banana to investigate the density and spectrum of induced mutations and mechanisms by which tissue culture materials become genotypically homogeneous. Here we provide a detailed protocol for meristematic isolation, mutation induction and dissolution of chimeric sectors focusing on the use of chemical mutagen ethyl methanesulfonate (EMS).