After determining the mutagenic treatment that gives ahigh mutation frequency suitable for screening without widespread lethality or sterility, a large population (>10,000) of M1 plants is typically cultivated to provide sufficient numbers of plants for efficient TILLING (McCallum et al. 2000b). In the specific case of tomato, growing the M1 plants in the field at commercial densities, instead of in the greenhouse, is usually more economical. This is especially true because the M1 generation is often prolonged by 1 or 2 months, apparently due to delays in germination and to developmental setbacks experienced by the mutagenized plants. To produce plants with stably inherited mutations, the M1 tomato plants are grown to maturity and allowed to self-fertilize. A single fruit is harvested from each fertile M1 plant and the collected M2 seed is considered to be equivalent to a new line or family, reflecting the unique collection of heritable genetic alterations in each M1 plant (Caldwell et al. 2004; Hohmann et al. 2005). A single M2 seed is planted from each line and labeled with a unique plant identifier and sampled for DNA. Once the plants reach maturity, M3 seed is harvested and catalogued such that M2 plant identifier, genomic DNA, and M3 seed packet are linked in a database information management system. The result is a seed library comprising thousands of unique lines with corresponding genomic DNA that can be continually accessed for TILLING and replenished over time without repeating mutagenesis.
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