Schematic diagram of maize TILLING. Fresh pollen is collected and mutagenized with ethylmethanesulfonate (EMS). Pollen is then applied to silks of wild-type plants from the same genetic background. Seeds from the resulting ears are grown into plants of the M1 generation. Plants of this generation are heterozygous for any induced mutation. Tissue is collected either from each M1 plant or from approx. 10 M2 siblings from the M1 self cross. M3 seed is generated by randomly intermating 10–12 M2 siblings. This M3 seed serves as the seed stock for future studies. DNA is extracted from collected tissue and samples are pooled to increase screening throughput. For mutation detection, sequence specific primers are used to amplify the target locus by PCR. Following amplification, samples are heat denatured and reannealed to generate heteroduplexes between mutant amplicons and their wild-type counterparts. Heteroduplexes are cleaved using CEL I endonuclease and are visualized using denaturing polyacrylamide gel electrophoresis. See reference  for further details.