I am using a marker-free Agrobacterium tumefaciens LBA4404 strain gene construct for infecting somatic embryos of papaya after diluting the overnight bacterium in 1/10x MS liquid medium. I have started washing of Agrobacterium with 250 mg/l of cefotaxime but as it could not stop the overgrowth, now I am using cefotaxime 500 mg/l + augmentin 250 mg/l + streptomycin 125 mg/l. I have tried keeping sterilized filter paper below the tissues but after removing it after few days the bacteria growth was prominent again. I have started infecting fresh cultures as well but I am experiencing the same problem of Agrobacterium overgrowth in the new set of experiments too. Kindly guide me how to control Agrobacteirum recurrent overgrowth?
P.S.- All antibiotics are freshly prepared, media is kept @ 20°C and tissues are showing no mortality but delayed conversion.
How long you have been keeping your medium at 20C? I keep them at 4C if I need to store them for a while.
Been a week I am keeping the media at 20°C. I have controlled Agrobacterium using the same media and at a much lower concentration of antibiotics earlier. But this time it's not showing any control at all, despite of keeping all conditions same as earlier.
Hi, after how many days you subculture your explants? Subculture them every 1 week to maintain the antibiotic concentration in the medium.
One more thing, after agrobacterium infection, wash your explants with sterile water 5-6 times until milky white color disappears. This dilutes the amount of agrobacterium in your cultures. Then wash your explants with cefotaxime.
Since, you are using somatic embryos, the shape could be a factor for residual agrobacterium which is not the case when using leaves as explants.
Are you using cefotaxime purchased from chemists? Try once from duchefa which is of course costly but works perfectly fine.
One more thing, what is the duration of your co-culture and OD of your agrobacterium cultures? Generally, lower OD of agrobacterium gives higher transformation efficiency and also the concentration of agrobacterium is low.
I wash the embryos as soon as I see Agrobacterium growth in it, precisely every 3rd day. I am already doing all steps suggested by you from months, but it shows no result in controlling the bacteria. Somatic embryos are proven to be the best explants for genetic transformation of papaya so taking leaves is of no use because of their well organized structures. The co-cultivation period is of 72 hours although I have tried reducing it to 48 h as well but it showed no effect. The OD of culture is 1 @ 600 nm.
Well, for me the OD of your culture is too high. I am asking you to wash the explants after Agrobacterium infection of the explants. You can experimenting with the OD of the infecting agrobacterium culture.
@ Dr. Yuan, NOT yet, despite trying all above suggested methods and a lot more. :(
Hi, I had the same problem. I used less bacteria concentration (60 ul of culture OD 0.5 to 20 ml of infection media in one petri dish with 30 explants), incubation time of 48 h. After that I washed the explants with cefotaxime 400 ppm, dry on filter paper, put the explants on regeneration media with 300 ppm cefotaxime plus selective antibiotic for 3-4 days, then I used only 200 ppm cefotaxime plus selective antibiotic. Change plates every 10 days aprox.
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