I have used timentin (100 mg/L) to get rid of Agrobacterium. After 2 days of incubation of the tissue with Agrobacterium, I rinsed the plant tissue in a solution of timentin (at above concentration) and transferred the tissue to plant media containing timentin at same concentration. Worked really well and if you see Agrobacterium start to grow again, just transfer the tissue again. Also, are you sure it is Agrobacterium and not a different bacteria or even a slimy fungus?

The concentration you mentioned should be able to check bacteria. I suggest you to check your antibiotic stock (changing brand may also be helpful). It is also suggested to add the antibiotic in media at proper temperature.

I have used timentin (100 mg/L) to get rid of Agrobacterium. After 2 days of incubation of the tissue with Agrobacterium, I rinsed the plant tissue in a solution of timentin (at above concentration) and transferred the tissue to plant media containing timentin at same concentration. Worked really well and if you see Agrobacterium start to grow again, just transfer the tissue again. Also, are you sure it is Agrobacterium and not a different bacteria or even a slimy fungus?

One thing you can do is to wash the explants rigorously after cocultivation. Further you can try is to optimize the Agrobacterium suspension, infection and cocultivation time period such that it is just sufficient for T DNA delivery. You can also find another substitute for cefotaxim as adviced by Jodi Humann.

Timentin alone (at 300 mg/l) works well. Also a combination of cefotaxim and carbenicillin (each at 300 mg/l) works well too. Of course you cannot use carb for agro suppression if the vector in your Agro contains a gene for ampicillin resistance.

I am using cefotaxime @ 500 mg/l for suppressing of Agrobacterium in sugarcane. If 200-600 mg/l is not sufficient than i think you can use carbecilin or Timentin.

At first you have to optimize agrobacterium concentration which use to inoculation. For example you can dilute Agrobacterium suspension to 1/2 or less before inoculation. After cocultivation wash explants on a shaker incubator at 50-60 rpm for 10-20 min, by submerging into the sterile water or diluted liquid medium (without sugar, contained cefotaxim or other proper antibiotics (500-600 mg/l) or a mixture of two or three of them). Then blot explants on the sterile filter paper to dry. Then place explants onto selection medium.

Thank you alot

you can try using repeated transfers every two days in cefotaxime with same concentration for a week and check. Also reduce the time of exposure to Agrobacterium culture while co-cultivation

Try to reduce the OD of the culture used for co-cultivation.. u can also revive the agro-culture and use the fresh revived culture for co-cultivation

I don´t know if you are doing it already. After Agrobacteria co-caltivation I rinse the explants in sterile water a couple of time and then transferred on plants containing cefotaxime. During the next days keep an eye on the plates and as soon as you notice agro contamination transfer the explants on new dish. Cheers

You can use also 150-200 mg/liter ticarcillin disodium/clavulanate potassium (15:1): It works well.

Eliminating Agro from TC depends very much on strain´s agresiveness, OD and quality of washes after coculture . Using less agressive strains like LBA4404, at optical densities below 0,05 and washing thoroughly three times after coculture, usually works well with cefotaxime (500 mg/l). Then move your cultures to lower Cefotaxime concentrations once you see NO more Agrobacterium growing. good luck.

 Drear Dr.Imani, i just started in this field.i would like to use your suggestion 200mg/l.please how do i prepare a stock of it?  I have trcar./clav 15:1 in the lab.thank you. Question 2.how do i wash my liquid medium i just replace medium w callus?

We usually make stock with 150-200mg/ml solved in water and filter sterilize subsequently and aliquoted in Eppi caps and store them by minus 18-20 degree.

You can centrifuge by 400rpm, remove the supernatants and re suspend the pellet with the same culture media containing antibiotics. 3 times washing steps are need. You may subculture you cell every 2 weeks.

Good luck

Thank you very much

you are welcome

I want to start working with agrobacterium tumefacien to transform my explant. I want to know the most suitable coculture technique (conditions and media).

Hello Umar, cefotaxime is most suitable antibiotic to remove agrobacterium from co-cultivated explants

Dear Dr. Rastagi. I really appreciate your concern