during last 2 year, i have not obtain root from my transgenic poplar plantlets. I previously found the regeneration protocol and obtained regenerated transgenic plants. however, when i tried again, the root initiation stoped in plantlets.

I use TDZ 0.006mg/l and IBA 0.1 mg/l for shoot induction of leaf explants. and IBA 0.2mg/l and NAA 0.1 mg/l for root induction in plantlets.

I thought that the reason of problem should be a phytohormone effect, but changing the concentrations or even removing them from media didn't affect the results. I observed shoot multiplication with callus induction in shoots or leaves. I can't fix it anymore.

So, I decided to obtain donor in vitro plants. I used lateral buds from field poplar tree and cultured nodes in the shoot induction media containing MS +BAP 0.1 mg/l for three weeks. the plantlets was transfered to rooting media containing MS+IBA 0.2mg/l and NAA 0.1 mg/L.

after 4 weeks, unfortunately the root initiation was not observed. the plants are rossete with small stem and multiple leaves and lage callus.

I even changed the medium ingredients and freshed them, but the problem has not been solved.

I attached the plants pictures in rooting media.

I dont know how to fix it. please let me know your suggestions.

Thanks