Hygromycin Selection Problem

Allison McDonald allison at utsc.utoronto.ca
Mon Jan 21 13:13:25 EST 2002

Hi!  I'm working with a glucocorticoid-inducible transcription system.
The selective marker for the plasmid is kanamycin, and the selective
marker for the T-DNA insertion is hygromycin.  I cannot recover any
hygromycin resistant tobacco calli after Agrobacterium mediated
transformation (even with the empty vector)!  I've tried lowering the
hygromycin concentration from 30 micrograms/mL to 20 micrograms/mL and
still no luck!  Others have used this system successfully in tobacco,
but I haven't had any success.  The plasmid appears to be o.k. in E.
coli and the Agro strain.  I'm not sure if the problem lies in the
transformation protocol (which works fine with other constructs),
hygromycin selection (it's being driven by the NOS promoter), or
something else.  If anyone has any thoughts on how to troubleshoot this
problem I would appreciate hearing them.



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