We are working on plant tissue culture in cotton. The present bacterial contamination is seed borne (Gram -ve, cocci, in chains). We have tried to overcome it with 0.1% HgCl2, 0.1% SDS as well as with different concentrations of NaOCl, but still unable to get rid off it. Additionally we also tried 70 to 100% ethanol along with flaming it, but still the problem persists. Out of the antibiotic we tried (ampicillin, kanamycin, cephotaxime, carbenicillin, neomycin, streptomycin, erythromycin and penicillin), only neomycin we found as effective to control it, but this unnecessarily kills seed and explants also.
You can incorporate antibiotics at low concentrations in the media.Use of antibiotics is in the culture media helps to kill the systemic bacteria.Usually cefetoxime,carbe. necillin and streptomycin will do .You can try 100-500mg.l-i
I think antibiotics would be the only option if the bacteria is not on seed surface as your surface sterilisation techniques are ineffective. If you have isolated the bacteria why not send to a diagonistic lab - they will identify it for you and may also suggest the correct antibiotic.
Try combination of antibiotics and silver. Hot water (around 50oC) for 2-5 min can kill bacteria inside the seeds.
Antibiotics are not allowed in commercial seed production and sales. You might use one of the alternatives used by the seed industry, which is a hot water treatment. With Brassica oleracea seeds this can be effective in killing seed borne Xanthomonas bacteria. It is a small window in treatment. Not hot or long enough and you will not kill the bacteria but too hot or too long and you kill the seeds. Seed vigour is very important for the tolerance of the seeds. Not fully mature seeds are for instance much more sensitive. The duration and temperature are also related to the species and seed size. I doubt if 2-5 seconds at 50 °C (as suggested by Alex) is enough to eradicate internal bacterial infection. For Brassica seeds treatmetns are on the minute scale.
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you can use aside sulfuric for 5 or 10 min before Sterilizing of seed may be more effective.
Cotton seeds have some fibres attached to the testa...this may difficult the use of ant disinfectant
U have two choices: passing the seeds to open flame to burn those fibres or use sulphuric acid to destroy it. After that I think any disinfestant media will be efficent to clean the seeds. Any way, u can try Sodium dichloroisocyanurate at 5 g per liter that has a better effect than hypochlorite.
Regards
Nelson
normally I use gentamicin to control bacteria coming out of tissue culture. And this can happen suddenly while the culture had been sterile and clean for quite some transfers.