We do the following after following the procedures and rationale described in Harrison et al 2006 (https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-2-19):
1) Sterilize seeds and plate them.
2) Incubate the plates in 4C for 3 days in dark (plates covered with foil).
3) Remove foil and place plates in the growth chamber for 5-6 hours (in light).
4) Then, place plates in dark for 3 days (a drawer is practical). Resistant seedlings would elongate further and can begin to be distinguished from susceptible seedlings.
5) Move the plates to the growth chamber. Hygromycin resistant seedlings can be more reliably scored over the next few days.
School of Plant Sciences
University of Arizona
Tucson, AZ 85721
On Sunday, August 28, 2016 at 2:24:34 PM UTC-7, MAHMUDUL HASSAN wrote:
> Hi All
> I am a PhD student. I have been trying to select transgenic Arabidopsis line in hygromycin medium for the last couple months. I followed this paper (A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation, Plant Methods. 2006. 2:19. DOI: 10.1186/1746-4811-2-19) but failed to select transgenic plants. Both untransformed (Col-0) and Hyg positive plants look like same in the selection plate. I used various concentration of Hyg (15, 20, 25) but all the time I was unable to differentiate between the susceptible and resistant plants. I am using Hyg from Sigma and it was bought recently. I am desperately looking for a working protocol for transgenic Arabidopsis selection in Hygromycin media.
>> I would be really grateful if anyone can help me in this regard by giving some suggestion for transgenic Arabidopsis selection in Hyg media and hopefully any protocol if possible?
> Thank you all
> Mahmud Hassan