Ethyl methanesulfonate treatment of maize seed for recovery of vegetative mutant sectors and segregating germinal mutations

Authors: KHANGURA, RAJDEEP - Purdue University; Best, Norman; DILKES, BRIAN - Purdue University

Submitted to: Cold Spring Harbor Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2025
Publication Date: 6/3/2025
Citation: Khangura, R.S., Best, N.B., Dilkes, B.P. 2025. Ethyl methanesulfonate treatment of maize seed for recovery of vegetative mutant sectors and segregating germinal mutations. Cold Spring Harbor Protocols. https://doi.org/10.1101/pdb.prot108650.
DOI: https://doi.org/10.1101/pdb.prot108650

Interpretive Summary: Identifying additional alleles of a genetic mutation can be difficult and time consuming. We have developed a method using ethyl methanesulfonate, a chemical mutagen, that is applied to maize seeds to induce new mutations. This approach is advantageous as it decreases the potential exposure to the carcinogenic mutagen and allows for highly efficient identification of new mutants. This method is highly effective for dominant alleles that are extremely difficult to genetically characterize. This protocol provides a thorough guide for researchers on how to design their experiment, prepare the chemical mutagen, mutagenesis application, and interpretation of results.

Technical Abstract: Seed mutagenesis using alkylating chemical agents such as ethyl methanesulfonate (EMS) can generate somatic and germinal mutations in many plant species. In monoecious plants like maize, the sperm- and egg-producing reproductive germlines are derived from distinct cell lineages in the embryo. This separation results in independent mutations inherited via the egg and sperm lineages and prevents the recovery of recessive mutant phenotypes in diploid progeny after the first round of self-pollination. Thus, two generations of self-pollination are required to screen for recessive mutations when conducting seed mutagenesis. The additional time and manual self-pollination make this approach laborious. However, a high mutation rate and the ability to screen for somatic sectors in heterozygous mutant plants and other defined genetic backgrounds make seed mutagenesis an effective but underutilized mutagenesis tool for maize research. This protocol provides the directions and optimization steps to perform effective seed mutagenesis in maize. A high frequency of somatic mutations from seed mutagenesis can be achieved, but comes at the expense of poor and disordered growth, failure to form reproductive structures, and low or no seed production at high EMS concentrations or long contact times. In experiments where germinal mutations are a goal, an optimum dose of EMS is required in the first generation. Maize genetic backgrounds vary in their sensitivity to EMS, requiring some pilot testing in new genetic backgrounds. Researchers using this protocol can carry out seed mutagenesis safely and effectively to develop libraries of mutants or alleles for various experiments.