Dear sir,

some strains of agro bacterium are high multiplication rate( copy number), or  some what resistant and /or compatible   to some antibiotics..  so u may use/ tray other antibiotics like Carbanicilin, augumaintin etc ..for  GV strain

Continue to earlier...sir u may use same antibiotic with change in  alter the infection O.D (use less as that u use earlier) .....

Dear Arambam.

In parallel you may also try blotting of extra medium on explant surfaces during washing step and transferring it for incubation on plate.

You could also try the combination of different antibiotics, e.g. we had used cerbenicillin, rifampicina nd spectinomycin; at a different occasion we were successfull with ticarcillin. 

I had best results using timentin. In addition, lowering the OD of the bacterial suspension, washing leaf discs after the co-cultivation period with plenty of liquid medium including antibiotics, followed by dry blotting might also help to solve your problem.

Hi, I strongly suggest you to use Augmentin (300mg/l) to remove agrobacterium infection. I have tried various antibiotics to remove agrobacterium infection and found Augmentin is the best among all. What you can do is, after co-infecting your callus with agrobacterium and incubating for 48 hours in dark for co-cultivation on co-cultivation media, wash those callus with autoclaved water containing 300mg/l Augmentin and placed those callus on selection media containing 300mg/l Augmentin. You will not get the Agrobacterium contamination. This may solve your problem for sure. 

I am working on Agrobacterium mediated transformation. as far i understand, the problem will be in autoclave. use autoclave forceps flasks and even culture plates, Same drug (Cefo) i am using, but contamination occurs still. Clean and dry the embryos carefully with autoclave tissue paper or filter paper to remove any broth or agro contamination. hope so you will find better results

1. Once you see the 'overgrowth' of Agrobacterium on explants (leaf discs), chances are there is no way to turn back. I wouldn't put too much effort trying to remove them. Start another batch.

2. GV strain, such as GV3101, is more virulent than other strains, such as LBA4404. When you use GV strain, you need to keep an eye on 'overgrowth' issues.

3. Transformation:  When I used GV3101 for tobacco leaf-disc transformation (I suppose that you use 'leaf-disc' method), I diluted the Agrobacterium culture 1/10x (diluted with sterilized MS liquid medium) before I dipped the explants into the solution. After dipping, blot them dry.

4. Selection: Two antibiotics were used for selection. For example, if 250 mg cefotaxime needed, I would use 125 mg cefotaxime + 125 mg carbenicillin. I found that such treatment has a better result compared to use only one antibiotic (although no formal study to confirm it has carried out).

5. Selection: Explants on the solid selection medium will become 'curvy' and prevent the explants completely contact with the surface of the selection medium. When you see this occurs, push those explants into contacting the surface of the medium. This needs to be taken care of especially at the beginning of selection because the potency of antibiotics will decrease later, at room temperature.

Thank you to all, I appreciate the efforts and the various suggestions

I would advice you to test your Agrobacterium GV strain before transformation to be sure that you do not have an occasional resistant contaminating strain mixed with your culture, and to select appropriate antibiotic for Agrobacterium elimination after transformation.

Grow night culture from your Agrobacterium GV stock. For example, strain GV3101 should be resistant to Rifampicin 100 µg/ml (chromosomal resistance) and Gentamycin 50 µg/ml (Ti-helper plasmid resistance). Use these antibiotics additionally to selective agent for your binary vector - it will help you to eliminate foreign bacteria if they are in your stock.  

Spread your night culture on Petri dish  to get single colony, then take a colony and grow it again in liquid medium with the same antibiotics.

Prepare Petry dishes with the antibiotics which were advised above (Cefotaxime,  Timentin usually up to 600 mg/l, Augmentin up to 300, Carbenicillin) or with their combinations and test your night culture from single colony with these antibiotics. Find an agent which efficiently suppresses your Agrobacterium growth and use it for further transformation experiments. Cefotaxime at high concentration could cause some inhibition effect to shoot regeneration although tobacco is not so sensitive to it.  

In your original question you didn't happen to mention the selection marker you are utilizing for the plant. We have found that the combination of Hygromycin 40 mg/L (selection for the plant tissue, so your T-DNA would need to include a plant expressed hygromycin resistance cassette) and Timentin 150 mg/L is quite effective at limiting Agrobacterium overgrowth, as long as you have taken steps to limit excess water (per all of the great suggestions made above).