Here is a protocol that I worked out for ems mutagenesis of
Arabidopsis seeds. It has worked well for me many times.
This protocol was gives heavy mutagenesis (50% of siliques of M1
plants were segregating at least one embryo defective), but little or
no sterility. I think the key thing is the extensive rinsing after
the ems treatment.
1) Agitate 100mg of seed in 25mL water with 3uL Tween20 for 15
minutes. Then rinse with dH2O until there are no more Tween20 bubbles.
2) Leave seed overnight at 4C on a rocker in 40mL dH2O.
3) The next day, pour off the water and add 10mLs of 0.4% EMS in 100mM
Sodium Phosphate buffer, pH 7. Leave in hood for 9 hours on a rocker
or rotator so that seed are gently agitated.
4) After 9 hours, rinse the seed 10 times (this is very important)
with 25 mLs of water each time.
5) Sow the seed on soil. To do this I resuspend them in 0.1% Agar and
sow them out with a 25mL pipet.
What I have done is sow out about 20 flats of seed, leave them in the
cold room, and put out 5 flats per week (Seeds will start to
germinate in the cold room after about 3 weeks). That way you don't
need to look at all of the flats at the same time.
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On Aug 10, 2009, at 10:20 PM, Xu, Xianfeng wrote: