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D. tenuifolia seed was counted into aliquots of 200 seeds. These were sealed inside muslin pockets to allow the EMS to penetrate without seed being lost. These were placed into a 500 ml Duran bottle containing 24 ml 10% Tween20 and 216 ml sterile H2O. The bottle was placed into a shaking incubator for 15 minutes at 25°C, 150 rpm. The seed pockets were rinsed four times with H2O before filling the bottle with 300 ml sterile H2O and put on the shaking incubator for 5 minutes at 25°C, 150 rpm.

This was repeated three times. Each seed pocket was then placed into a 250 ml Duran bottle containing sterile H2O and EMS at the appropriate concentration. One batch of 200 seeds did not receive any EMS to allow a control comparison of experimental effects. Each bottle was put at 25°C for 16 hours on a shaking platform at 150 rpm. The EMS solution was poured off and inactivated with 0.1 M NaOH/20% STS for 24 hours before pouring down the fume cupboard sink. The bottles were filled with 150 ml H2O, swirled and emptied six times before refilling with 150 ml H2O and placing in the incubator for 15 minutes at 25°C, 150 rpm. The H2O was poured off and a final continual wash of H2O was done for three hours to remove any further traces of EMS. Muslin bags were opened and seeds were sown at one per cell in 98 cell trays of wet peat and covered with vermiculite before germinating at 25°C, 16 hour photoperiod for one week.

2.1.6.2. EMS trial experiments

Two trials of EMS treatment were done to optimise the concentration of EMS to use for rocket. D. tenuifolia gene bank seed (IPK, Gaterslaben, Germany) was used because the SSD4 seed was not yet available (section 2.1.1). The same EMS protocol was used throughout as described in section 2.1.6.1.

For Trial 1, gene bank seeds DIPLO 3 (Table 2.1) were used and the following concentrations trialled: EMS concentration 0 (control) 10 mM (~0.1%) 20 mM (~0.2%) 30 mM (~0.3%)

For Trial 2, gene bank seeds DIPLO 4 (Table 2.1) were used and the following concentrations trialled: EMS concentration 0 (control) 5 mM (~0.05%) 10 mM (~0.1%) 20 mM (~0.2%)

Germination tests were performed at 25°C, 16 hour photoperiod for seven days. The number of germinated seed was then recorded. Plants were transplanted at three weeks old into 5 litre pots of Levington M2 soil and grown to maturity at Elsoms Seeds Ltd, Spalding (Latitude 52o79’) in a 16 hour photoperiod glasshouse at 22°C. The results of these trials enabled a decision to be made on what concentration of EMS to use in Experiments 1 and 2.

2.1.6.3. EMS experiments

Ten batches of 200 SSD4 seeds (section 2.1.1) were treated at a concentration of 10 mM EMS on 16/05/12 using the method described in section 2.1.6.1. Germination data was recorded as for the trial experiments. Trays were then transferred to 16 hour photoperiod at 20°C conditions for further growth. At six weeks old (03/07/12), these were put into a cold frame to acclimatise to outside conditions and at eight weeks old (17/07/12), were planted into the field (section 2.1.7.2).

A second experiment was performed on another 2000 SSD4 seeds (sections 2.1.1&2.1.6.1) with an EMS concentration at 20 mM on 27/06/12. As previously, germination data was recorded and plants transferred to 16 hour photoperiod at 20°C conditions for further growth. At four weeks old (25/07/12), these were put into a cold frame to acclimatise to outside conditions and at six weeks old (09/08/12), were planted into the field (section 2.1.7.2).

2.1.6.4. Production of EMS M2generation

Harvested seeds from 1127 plants across both M1 experiments were threshed. Of these, only 332 yielded M2 seed. Seed was also threshed from 10 untreated control lines. From each of these lines, up to six seeds were sown onto wet peat and germinated at 20°C, 16 hour photoperiod. Germination was checked after seven

days and any lines which had not germinated were re-sown. After 14 days, only 223 lines had one or more seed germinate. Three weeks after sowing, plants were put into a cold frame to acclimatise to outside conditions for one week before planting into Spanish polytunnels. Plants which were later bolting than the wild types were dug up and potted into 5 litre pots and put into the glasshouse at 20°C, 16 hour photoperiod.

2.1.6.5. Production of EMS generations M2_BC1and M2_BC1_S1

Individual flowers on late bolting M2 plants were backcrossed to wild type plants of SSD6, the purest plant line to date (section 2.1.1), using bud pollination (section 2.1.4.2). Seed was set and the plants allowed to dry out before collecting and threshing the seed (section 2.1.5). Threshed seed was sown into F2+S soil in p40 cells and covered with vermiculite. Trays were placed into a glasshouse compartment at University of Warwick Phytobiology facility at 22°C with 16 hour photoperiod. These were covered with propagator lids until one week after germination. Seedlings were transplanted into 5 inch pots of M2 soil at four weeks from sowing. Initiation of bolting and 10 cm bolt was scored for every plant. Flowers were bud pollinated and bagged to ensure selfed seed was generated.

2.1.6.6. Production of M3generation

Selfed seed was produced for the M3 generation so late bolting M2 plants were bagged to prevent pollen transfer from different plants. M3seed was threshed before up to eight seed were sown onto wet peat. Seeds were germinated at 20°C, 16 hour photoperiod and grown for three weeks. These were put into a cold frame to acclimatise for one week before transplanting into the field (section 2.1.7.2).