Appropriate tissue collection and subsequent purification of genomic DNA from each M2 plant is essential for successful recovery of the mutations induced during mutagenesis. In many plant species, tissue collection must be done at a young developmental stage to optimize quantity, quality, and purity of DNA. Sampling of older or damaged tissue should be avoided as it can adversely affect the quality and long-term stability of the extracted DNA. In tomato, high quality DNA can be extracted from tissue samples of the first two or three true leaves at approximately 1 month after planting. The apical meristem is left intact so that the plant can continue to grow and produce fruit for M3 seed collection.
A high-throughput DNA extraction method is preferable, since TILLING is practiced on large DNA populations. The use of commercial DNA purification kits in combination with a liquid handling robot and a 96-well format allows a single researcher to produce over 1,000 DNA preparations per day. Liquid handlers also help with the normalization of large numbers of DNA samples to a standard DNA concentration. This in turn allows for the efficient pooling of DNA prior to mutation screening, ensuring that each individual in a pool is equally represented. Collection of the M2 DNA samples into bar-coded tubes allows the DNA library to be linked to M3 seed progeny of the plant. The DNA library then becomes a stable asset that can be used for the TILLING of many gene targets over many years.
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