Mutagenesis of in vitro cultures of Miscanthus × giganteus cultivar Freedom and detecting polymorphisms of regenerated plants using ISSR markers

Giant Miscanthus (Miscanthus × giganteus; M × g) is a key candidate energy crop for use in biomass to liquid fuel production. It is a naturally seed sterile triploid lacking genetic variation, the basis for selection. Therefore, induced mutation techniques are particularly important in M × g. The objective of this research was to induce variation in Freedom, an elite cultivar, through in vitro chemical mutagenesis. A previously-optimized in vitro propagation protocol for Freedom was used following the in vitro mutagenesis procedure. Immature inflorescence explants (2-3 mm) and calli (2-3 mm3) were treated with five mutagenic dose treatments [0.01%, 0.1%, 0.5%, 1%, and 3% (v/v) of ethyl methanesulfonate (EMS) for 90 min] along with 2% (v/v) dimethyl sulfoxide (DMSO) as a carrier agent to determine the optimum mutagen dosage. The dose at which 50% of the calli/explants recovered (RP50) after the mutagen treatment was used as the optimum EMS dose. Calli and explants of M × g were treated with RP50, 2 × RP50, and 3 × RP50 EMS dosages, and subsequent regenerants that arose from explants/calli were transferred to soil. Inter simple sequence repeat (ISSR) markers were used to identify variants in the regenerated plants. Results showed variability among regenerants originating from EMS mutagenic treatments. Putative mutants of M × g may be useful for bioenergy research and functional genomics.