Please look at the genes on T-DNA of Ri plasmid. If it does not have any of rol gene then you are not likely to get hairy roots.

Thanks for the answer. I know they are hairy roots because I checked it by Rol_B

First you have to confirm all the genes including the rol gene on the plasmid transformed in Agrobacterium, then to optimize the transformation conditions again with strong selective agent. Better incubate the explants in dark after co-cultivation period. Good luck

Thanx, dear Rham Sher Khan. I already got hairy roots and still, I can get hairy roots but the problem started when I transformed my recombinant plasmid into that A.rhizogenous, I want to express my gene into hairy roots. I need to get transgenic hairy root, and everything is the same except that I transformed a recombinant plasmid into A.rhizogenous and because of that, I cant get hairy roots anymore.

Not quite sure what the question is.

But I got hairy roots from a transgenic plant very easily.

Ahmad, can you describe in detail a) what genes are in the plasmid and b) the exact procedure you are using to try to generate the hairy roots? I have an idea of what might be happening, but would like to first see your procedure before speculating further.

Well, It was a long long time ago dear Ray Collier when I had that problem. It was because of the way I transferred my recombinant plasmid to Agrobacterium, And due to that way of transformation, the Ti plasmid of Agrobacterium was lost. I've had past that problem by changing the transformation Method. Tnx to all of you.@Ray Collier