Usually we do co-cultivation for 2 days followed by transfer to plain media where Agrobacterium outgrowth starts to appear and goes on increasing. Even if we have treated contaminated (with agrobacterium) explants with Cefotaxime/Carbenicillin, we are unable to get rid of it. More of the treatment with these antibiotics may affect the explants. Though finally we remained with the option of discarding those explants and start a new experiment.
Hi, this is a common problem and can be controlled by a few simple changes to your protocols.
When you say 'transferred to plain media' I assume you mean nutrient agar and not liquid, although this should help with liquid contamination also.
When you transfer your explants after co-cultivation, you could try rinsing them thoroughly in sterile H2O containing 500ug/ml cefotaxime, and then make sure that in the nutrient media you also have 500ug/ml cefotaxime. (Note I have observed some desperate people washing their explants in 50% ethanol for a few seconds and rinsing them with water, however this has had varying success and has caused death and stress to the explant).
This should be repeated after another 2 days, in-order to minimize the contamination.
After a few transfers like this, the agrobacterium should be under control and not cause more trouble.
If you are still having trouble I would suggest combining cefotaxine with carbenacillin/ampicillin and if all else fails use timentin.
if you are making transgenics and your binary vector contains a selection resistance eg for kanamycin, you can use that antibiotic as well as the cefotaxime/penicillin type antibiotics.
The concentration of antibiotics is important, yes they may affect the growth of the explanat, (although in my experience I haven't had trouble with the penicillin type antibiotics) so it's best to hit the agrobacterium early on with a high dose of antibiotic, and then after a few transfers you can reduce their concentration.
Hi, this is a common problem and can be controlled by a few simple changes to your protocols. When you say 'transferred to plain media' I assume you mean nutrient agar and not liquid, although this should help with liquid contamination also. When you transfer your explants after co-cultivation, you could try rinsing them thoroughly in sterile H2O containing 500ug/ml cefotaxime, and then make sure that in the nutrient media you also have 500ug/ml cefotaxime. (Note I have observed some desperate people washing their explants in 50% ethanol for a few seconds and rinsing them with water, however this has had varying success and has caused death and stress to the explant). This should be repeated after another 2 days, in-order to minimize the contamination. After a few transfers like this, the agrobacterium should be under control and not cause more trouble. If you are still having trouble I would suggest combining cefotaxine with carbenacillin/ampicillin and if all else fails use timentin. if you are making transgenics and your binary vector contains a selection resistance eg for kanamycin, you can use that antibiotic as well as the cefotaxime/penicillin type antibiotics. The concentration of antibiotics is important, yes they may affect the growth of the explanat, (although in my experience I haven't had trouble with the penicillin type antibiotics) so it's best to hit the agrobacterium early on with a high dose of antibiotic, and then after a few transfers you can reduce their concentration.
Thanx Giorgio for your reply. May I know in which crop species you tried this?
Hi, I have done this with many different plant sp and tissue types, mostly with Solanaceous plants, including many Nicotiana sp, solanum lycopersicum, solanum tuberosum and petunia sp. and other dicot plants like Arabidopsis and carrot, but I've also had success with various monocot sp. like rice sugarcane and sorghum.
Growth medium is also a factor that can aid agro bacterium growth, try cutting back the amount of sugar you add to the medium :)
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