Ethyl methanesulfonate treatment of maize pollen for development of segregating mutant populations or targeted mutagenesis

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 pollen for development of segregating mutant populations or targeted mutagenesis. Cold Spring Harbor Protocols. https://doi.org/10.1101/pdb.prot108651.
DOI: https://doi.org/10.1101/pdb.prot108651

Interpretive Summary: Production of mutant populations are important to conduct genetic studies and gene function. Using a chemical mutagen is a highly effective method to create segregating mutant populations. We have developed a step-by-step method to perform mutagenesis of maize pollen. This includes the experimental design, preparation of chemicals, mutagen application, and interpretation of results. The experimental design for creating mutant populations or targeting a specific gene is extremely important and this protocol provides the guidance to effectively undergo these types of experiments. Safety advice is provided to insure that researchers are not exposed to these highly carcinogenic chemicals. This protocol will allow researchers to create their own mutant populations for future genetic studies.

Technical Abstract: In maize, abundant pollen production and easy controlled pollination permit the direct mutagenesis of pollen to produce populations of independent mutant lines. Pollen can be treated with alkylating agents, such as ethyl methanesulfonate (EMS), to induce point mutations. The ease of applying and decontaminating this mutagen after the mutagenesis application and the advantages provided by the mutation spectra for subsequent bioinformatic analysis make EMS an attractive mutagen. We provide a maize pollen mutagenesis protocol with a list of critical supplies, a step-by-step procedure, and troubleshooting tips. Pollen is freshly collected and suspended in an emulsion of EMS and paraffin oil. The slurry of pollen, oil, and EMS is then directly placed on prepared maize silks to perform pollinations. Mutations result during embryogenesis due to replication-dependent mispairing at alkylated residues contributed by sperm nuclei. Thus, each seed bears an independent set of mutations. These progenies can be analyzed directly, as is the case in targeted mutagenesis experiments or the exploration of dominant genetic variation. Alternatively, the progenies of self-pollinated plants can be screened in the next generation to discover novel recessive mutations. In addition to the dose of EMS and contact time, the genetic background of maize can significantly influence outcomes, and some optimization of dose and contact time may be required for a genetic background and specific use case. Although we outline good practices for safe handling of EMS and waste, researchers should consult their local safety officers to ensure safe handling, decontamination, and disposal of EMS, which is toxic.