I am performing Rice agrobacterium mediated Transformation . Facing the bacterial contamination in REIII regeneration stage. How to avoid this contamination?
After selecting the callus for 4 weeks on respective medium contain Antibiotic (kanamycine-70mg and Cefotexamine250mg ). I transfer the callus on REIII regeneration medium for shoot initiation with ( Kanamycine50mg). After transferring on this medium, i am getting the bacterial contamination in my plate . Its wasting time and callus as well. Can you guys suggest how to resolve this issue or use the contaminated callus?
Dear,
Apparently everything is normal but you can check if you can test another antibiotics.
I suggest you also to check all your practices because you can have just a silly practice mistake. Your solutions, medium and intrumentals must be totally and completely "brand new" and steriles!!!!! Good luck!
Dear Rupesh Tayade,
I do agree with Maria Cristina Rocha Cordeiro madam,
you may also utilize broad spectrum antibiotics and frequent subculture followed by surface sterilization of callus.
Dear Rupesh Tayade,
You gave "kanamycine-70mg and Cefotexamine 250mg", but what is the final concentration (mg/L) of them in the plates??
Dear, Maria Cristina Thanks for your suggestion i will avoid if any mistakes if am doing.
Dear Yuan Yeu YauThis is final concentration 70mg /L and 250mg/L in case selection Medium. I am not using cef. in REIII medium (Regeneration)
Hi,
I am not sure what you are observing is bacterial "contamination" or Agrobacterium overgrowth. Try adding 250 mg/L cefotaxime in REIII regeneration medium as well. Are you using LBA4404 strain of Agrobacterium. Adding cefotaxime throughout the tissue culture stages might help overcome this problem.
Yes sir i am using LBA4404 strain. Can i add cefotaxime in RE III as well . Does It will have any impact on regeneration process?
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