Hello I am new in this forum and in plant biology in general. Recently I am trying to establish axenic root cultures from Lotus japonicus hairy roots (A. rhizogenes mediated transformation).
I have 2 questions:
1) After cefotaxime step in A. rhizogenes mediated transformation I am keeping my explants in 1/2xB5 medium (containing vitamins, sucrose..) without antibiotic until hairy roots are ~ 1 cm. Then I can cut roots in order to initiate the root culture. But I am getting large amounts of A. rhizogenes contamination, probably because the explants do not fully come in contact with the medium which contains antibiotic. Recently I have tried to grow explants o/n in liquid B5 medium containing antibiotic. I do not know yet if that works. Any ideas?
2) After I cut-off the hairy roots and place them in fresh B5 medium in order to start root cultures, do I place petri dishes in the dark vertically or horizontally?
Thank you in advance!