Optimisation of in vitro ethyl methanesulfonate- induced mutagenesis for future application in development of mutants in aromatic rice (Oryza sativa L.)

This study optimised in vitro culture conditions and ethyl methanesulfonate (EMS)-induced mutagenesis in the aromatic rice landrace Tulasikanti for optimising the regeneration protocol and determining the optimal EMS treatment. Mature seeds were cultured on Murashige and Skoog (MS) medium with different plant growth regulator combinations to optimise callus induction and regeneration. The resulting calli were treated with EMS at 0.1 %, 0.2 % and 0.3 % along with an untreated control (0 % EMS), for exposure durations of 2, 4 and 6 hr to determine the median lethal dose (50 % lethality (LD50)) and evaluate callus survival and regeneration efficiency. Plant growth regulators (PGRs) significantly influenced callus induction, with the highest embryogenic callus formation (85 %) recorded on T4 (MS + 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) + 0.1 mg L-1 6-benzylaminopurine (BAP) + 0.25 mg L-1 naphthaleneacetic acid (NAA)) medium. Further, EMS treatment caused a concentration- and exposure time-dependent decline in callus survival, with 0.2 % EMS for 6 hr identified as the LD50. The highest shoot regeneration (48.2 %) occurred on R2 (MS + 0.01 mg L-1 NAA + 0.25 mg L-1 Kn + 2.0 mg L-1 BAP) medium following 0.2 % EMS treatment, whereas higher EMS concentrations exerted pronounced cytotoxic effects. Rooting was highly efficient, with approximately 90 % of regenerated shoots forming healthy roots. Thus, the study provided a reproducible in vitro EMS mutagenesis framework that can efficiently balance mutagenic intensity and regeneration, thereby providing a foundation for future mutant development for aromatic rice improvement and the future identification of semi-dwarf mutants.