Hi Wahyu, you can use cefotaxime or carbenicillin to minimize the growth. Try to optimize and test different concentrations and check which one will work out for the genotype you have used.

You could try the bacteriostatics nipagin and sorbic acid.

Hi, you can also use Plant Preservative Mixture (PPM).

Wahyu Nirwanto

Did you add cefotaxime or carbenicillin in the selection medium too? I usually use half of cefotaxime + half of carbenicillin (I felt the effect of inhibiting Agrobacterium is better, but no systematic analysis on it).

Yuan-Yeu Yau I add timentin in the selection medium. So maybe I will try to add cefotaxime. Thanks for your replied.

Roshni Mohan Thanks for your ideas. I will try it.

Hi Roshni,

could you provide some more details about your procedure? There are many factors which can potentially influence the growth of the agrobacteria. What strain you are working with? In our hands, different strains behave quite differently. Also, the behavior could depend on the type of explant (and plant species) you are using and the co-cultivation time. For many of our transformation experiments we include a vigorous washing step with pretty high concentrations of antibiotics to prevent a subsequent overgrowth (vortex explants for 2 minutes in Falcon tube with antibiotic soln).

Also, are you sure the overgrowing bacteria are agrobacteria? We had several cases where endophytic bacteria started to overgrow the explants after some time. Endophytes are common among plants and they usually are not a big problem. But, in stress situations (and transformation procedures certainly qualify as such) they may start to overwhelm the explant. We tried different antibiotics and pre-mixes sold by different companies. It was moderately successful. We received a tip from an in-vitro company - they use ProClin 150 (Sigma-Aldritch, no affiliation). We tried that and it worked better than the others (we use 400 µl/l for apple trees).

Hope that helps.

Regards,

Klaus