[Arabidopsis] EMS mutagenesis protocol

Stewart Gillmor via arab-gen%40net.bio.net (by sgillmor from sas.upenn.edu)
Sun Aug 16 10:14:38 EST 2009


Dear Xianfeng,

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.

best wishes,

Stewart


Stewart Gillmor
   Department of Biology
    University of Pennsylvania
     433 S University Ave.
      Philadelphia PA 19104 USA
       tel 215 898 8916
        fax 215 898 8780



On Aug 10, 2009, at 10:20 PM, Xu, Xianfeng wrote:

> Dear All,
>
> I am writing to seek suggestions on dealing with EMS-induced  
> sterility in Arabidopsis. I have been doing EMS mutagenesis with  
> Arabidopsis seeds and found fertility of the M1 plants severely  
> dropped. Fertility problems persisted into subsequent generations as  
> well. A seed germination problem was also spotted.
>
> There are various protocols for Arabidopsis seed EMS mutagenesis,  
> differing in the EMS concentration and the length of EMS incubation.  
> I followed one which pre-soaks the seeds in water @4C for 2 days and  
> then soaks in 0.3% fresh EMS diluted in water overnight.
>
> A report from Euphytica 17 (1968): 363-370 suggested that sterility  
> and compromised germination may be simply considered as the price  
> one has to pay for the high mutant frequency after EMS.
>
> I am wondering anyone might have or might be having similar  
> problems. Any suggestions or sharing will be greatly appreciated!  
> Thanks,
>
>
> Morgan (Xianfeng) Xu
>
> Postdoc Fellow
> Cold Spring Harbor Laboratory
> Delbruck Building
> 1 Bungtown Rd
> Cold Spring Harbor, NY, 11724, USA
> 516-367-8827



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