EMS is an excellent mutagen and consequently is highly carcinogenic; your safety and that of others is therefore of utmost importance. EMS is volatile and all necessary safety precautions should be taken when conducting a mutagenesis experiment. Dress in full safety gear, including lab coat, appropriate double gloves, and safety glasses. The experiment should be carried out in a fully functional and inspected chemical fume hood. Inform all members of the laboratory about the experiment and post appropriate warning signs. EMS is chemically inactivated by exposure to sodium thiosulfate or 1 M NaOH. Use disposable containers and plasticware where possible and wash with 1 M NaOH before disposal. Dispose of liquids in the same way using 1 M NaOH. Use 1 M NaOH to wash all surfaces that may be contaminated with EMS and then rinse them with water. Do not let anyone use the fume hood until the area is completely decontaminated. A material safety data sheet must be obtained from the manufacturer of the EMS and read before conducting the mutagenesis experiment to ensure that all local regulations are met. The following protocol describes a typical EMS mutagenesis experiment.
1. Prepare one 10-mL syringe per 2500 seeds by cutting off the end and covering it with either plastic gauze or Miracloth (see Note 5). This will allow solutions in the syringe to be changed without losing any seed. All solutions are prepared in beakers.
2. Batches of approx 2500 Arabidopsis seeds are soaked overnight in 0.1% potassium chloride inside the syringes. Preimbibing allows good uptake of the EMS.
3. The potassium chloride solution is then replaced with 20 mL of 100 mM potassium phosphate solution, pH 5.0. To do this, expel the first solution and replace it with the new.
4. To this add 1 mL of DMSO to permeabilize the seed.
5. At this stage all equipment is transferred into the glove bag (Atmos bag, Aldrich) in a fume cupboard and the glove bag is sealed (see Note 6 and Fig. 1).
6. To each beaker containing potassium phosphate and DMSO, 135, 180, or 225 ^L of EMS is added and mixed thoroughly. This gives a final concentration of 60, 80, and 100 mM of EMS, respectively.
7. The seeds are then syringed up and down several times to ensure even exposure to the EMS. This is repeated every half hour.
8. After 3 h the solution of EMS is discarded into a beaker of sodium thiosulfate crystals to ensure chemical inactivation.
9. The seeds are then washed three times with 100 mM sodium thiosulfate solution to remove traces of EMS. Each wash is discarded into the beaker of sodium thiosulfate crystals.
10. The seeds are then washed with 100 mL of distilled water before being removed from the glove bag and washed from the syringes onto pieces of Whatman paper and left to dry for several hours (see Note 7).
11. After the seeds are dry they should be sown onto soil as soon as possible. The density of seeds is dependent on whether one is colleting in pools or individual plants (see Subheading 3.3. and Note 8). Also, sow a tray of nonmutagenized mm
seeds at the same density, which will act as a control. Keep the soil moist after planting.
12. It is valuable to know the size of the Ml population. Estimate the population size 10 to 14 d after sowing, as after the early rosette stage the plants will become too dense to allow counting.
At this stage it is important to estimate the degree of mutagenesis. Effective EMS treatment will lead to reduced germination speed and reduced seedling growth in addition to plants containing sectors with dominant color mutations and growth aberrations and sterility (see Notes 9 and 10).
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